Mclaughlin 2015

Edited by
Karl Esser
THE MYCOTA
A Comprehensive Treatise on Fungi
as Experimental Systems for Basic and Applied Research
Systematics and Evolution

Part B
VII
Second Edition
David J. McLaughlin
Joseph W. Spatafora
Volume Editors
The Mycota
Edited by
K. Esser
The Mycota
I Growth, Differentiation and Sexuality

1st edition ed. by J.G.H. Wessels and F. Meinhardt
2nd edition ed. by U. Kües and R. Fischer
3rd edition ed. by J. Wendland
II Genetics and Biotechnology
1st and 2nd edition ed. by U. Kück
III Biochemistry and Molecular Biology
1st and 2nd edition ed. by R. Brambl and G. Marzluf
3rd edition ed. by D. Hoffmeister
IV Environmental and Microbial Relationships
1st edition ed. by D. Wicklow and B. Söderström
2nd edition ed. by C.P. Kubicek and I.S. Druzhinina
3rd edition ed. by I.S. Druzhinina and C.P. Kubicek
V Plant Relationships
1st edition ed. by G. Carroll and P. Tudzynski
2nd edition ed. by H.B. Deising
VI Human and Animal Relationships
1st edition ed. by D.H. Howard and J.D. Miller
2nd edition ed. by A.A. Brakhage and P.F. Zipfel
VII Systematics and Evolution
1st edition ed. by D.J. McLaughlin, E.G. McLaughlin, and P.A. Lemke{
2nd edition ed. by D.J. McLaughlin and J.W. Spatafora
VIII Biology of the Fungal Cell
1st and 2nd edition ed. by R.J. Howard and N.A.R. Gow
IX Fungal Associations
1st and 2nd edition ed. by B. Hock
X Industrial Applications
1st edition ed. by H.D. Osiewacz
2nd edition ed. by M. Hofrichter
XI Agricultural Applications
1st and 2nd edition ed. by F. Kempken
XII Human Fungal Pathogens
1st edition ed. by J.E. Domer and G.S. Kobayashi
2nd edition ed. by O. Kurzai
XIII Fungal Genomics
1st edition ed. by A.J.P. Brown
2nd edition ed. by M. Nowrousian
XIV Evolution of Fungi and Fungal-Like Organisms
Ed. by S. Pöggeler and J. Wöstemeyer
XV Physiology and Genetics: Selected Basic and Applied Aspects
Ed. by T. Anke and D. Weber
The Mycota
A Comprehensive Treatise on Fungi as
Experimental Systems for Basic and Applied
Research
Edited by K. Esser
VII
Volume Editors:
Systematics and Evolution
Part B
2nd Edition
D.J. McLaughlin and J.W. Spatafora
Series Editor
Professor Dr. Dr. h.c. mult. Karl Esser
Allgemeine Botanik
Ruhr-Universität
44780 Bochum, Germany
Tel.: +49 (234)32-22211
Fax.: +49 (234)32-14211
e-mail: Karl.Esser@rub.de
Volume Editors
Professor Dr. David J. McLaughlin

Department of Plant Biology
University of Minnesota
1445 Gortner Avenue
St. Paul, MN 55108-1095, USA
Tel.: +1(612)625-5736
Fax: +1(612)625-1738
e-mail: davem@umn.edu
Professor Dr. Joseph W. Spatafora
Department of Botany and Plant Pathology
Oregon State University
Corvallis, OR 97331, USA
Tel.: +1(541)737-5304/5305
e-mail: spatafoj@science.oregonstate.edu
ISBN 978-3-662-46010-8 ISBN 978-3-662-46011-5 (eBook)

DOI 10.1007/978-3-662-46011-5
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com)
Karl Esser
(born 1924) is retired Professor of General Botany
and Director of the Botanical Garden at the Ruhr-
Universität Bochum (Germany). His scientific work
focused on basic research in classical and molecular
genetics in relation to practical application. His stud-
ies were carried out mostly on fungi. Together with
his collaborators he was the first to detect plasmids in
higher fungi. This has led to the integration of fungal
genetics in biotechnology. His scientific work was
distinguished by many national and international
honors, especially three honorary doctoral degrees.
David J. McLaughlin
(born 1940) is retired Professor of Plant Biology and
Curator of Fungi of the Bell Museum and cofounder
of the Mycological Culture Collection at the Univer-
sity of Minnesota, St. Paul (USA). His scientific work
has focused on the evolution of subcellular structure
in Fungi, and phylogeny and systematics of basido-
mycetes, ectomycorrhizal community structure, and
the Minnesota macrofungal flora with a view to estab-
lishing baseline data for mid continental macro-
fungi. He was formerly Editor-in-chief of Mycologia,
and has been President of the Mycological Society of
America and is a Fellow and Distinguished Mycolo-
gist of that Society.
Joseph S. Spatafora
(born 1964) is Professor of Botany and Plant Pathol-
ogy and Curator of Oregon State University Mycolog-
ical Collection at Oregon State University, Corvallis,
OR (USA). His scientific work has focused on evolu-
tion of the Fungi, with emphases on phylogenetics of
Ascomycota, insect pathogenic fungi and compara-
tive genomics. He is a Fellow of, and has served as
President of, the Mycological Society of America, and
he is currently a Senior Editor of the journal Fungal
Biology.
ThiS is a FM Blank Page
Series Preface
Mycology, the study of fungi, originated as a sub discipline of botany and was a
descriptive discipline, largely neglected as an experimental science until the early
years of this century. A seminal paper by Blakeslee in 1904 provided evidence for
self incompatibility, termed “heterothallism”, and stimulated interest in studies
related to the control of sexual reproduction in fungi by mating-type
specificities. Soon to follow was the demonstration that sexually reproducing
fungi exhibit Mendelian inheritance and that it was possible to conduct formal
genetic analysis with fungi. The names Burgeff, Kniep and Lindegren are all
associated with this early period of fungal genetics research.
These studies and the discovery of penicillin by Fleming, who shared a Nobel
Prize in 1945, provided further impetus for experimental research with fungi.
Thus began a period of interest in mutation induction and analysis of mutants
for biochemical traits. Such fundamental research, conducted largely with
Neurospora crassa, led to the one gene: one enzyme hypothesis and to a second
Nobel Prize for fungal research awarded to Beadle and Tatum in 1958.
Fundamental research in biochemical genetics was extended to other fungi,
especially to Saccharomyces cerevisiae, and by the mid-1960s fungal systems
were much favored for studies in eukaryotic molecular biology and were soon
able to compete with bacterial systems in the molecular arena.
The experimental achievements in research on the genetics and molecular
biology of fungi have benefited more generally studies in the related fields of
fungal biochemistry, plant pathology, medical mycology, and systematics. Today,
there is much interest in the genetic manipulation of fungi for applied research.
This current interest in biotechnical genetics has been augmented by the develop-
ment of DNA-mediated transformation systems in fungi and by an understanding
of gene expression and regulation at the molecular level. Applied research initia-
tives involving fungi extend broadly to areas of interest not only to industry but to
agricultural and environmental sciences as well.
It is this burgeoning interest in fungi as experimental systems for applied as
well as basic research that has prompted publication of this series of books
under the title The Mycota. This title knowingly relegates fungi into a separate
realm, distinct from that of either plants, animals, or protozoa. For consistency
throughout this Series of Volumes the names adopted for major groups of fungi
(representative genera in parentheses) areas follows:
Pseudomycota
Division: Oomycota (Achlya, Phytophthora, Pythium)
Division: Hyphochytriomycota
viii Series Preface
Eumycota
Division: Chytridiomycota (Allomyces)
Division: Zygomycota (Mucor, Phycomyces, Blakeslea)
Division: Dikaryomycota
Subdivision: Ascomycotina
Class: Saccharomycetes (Saccharomyces, Schizosaccharomyces)
Class: Ascomycetes (Neurospora, Podospora, Aspergillus)
Subdivision: Basidiomycotina
Class: Heterobasidiomycetes (Ustilago, Tremella)
Class: Homobasidiomycetes (Schizophyllum, Coprinus)
We have made the decision to exclude from The Mycota the slime molds which,
although they have traditional and strong ties to mycology, truly represent
nonfungal forms insofar as they ingest nutrients by phagocytosis, lack a cell
wall during the assimilative phase, and clearly show affinities with certain proto-
zoan taxa.
The Series throughout will address three basic questions: what are the fungi,
what do they do, and what is their relevance to human affairs? Such a focused and
comprehensive treatment of the fungi is long overdue in the opinion of the
editors.
A volume devoted to systematics would ordinarily have been the first to
appear in this Series. However, the scope of such a volume, coupled with the
need to give serious and sustained consideration to any reclassification of major
fungal groups, has delayed early publication. We wish, however, to provide a
preamble on the nature of fungi, to acquaint readers who are unfamiliar with
fungi with certain characteristics that are representative of these organisms and
which make them attractive subjects for experimentation.
The fungi represent a heterogeneous assemblage of eukaryotic microorgan-
isms. Fungal metabolism is characteristically heterotrophic or assimilative for
organic carbon and some nonelemental source of nitrogen. Fungal cells charac-
teristically imbibe or absorb, rather than ingest, nutrients and they have rigid cell
walls. The vast majority of fungi are haploid organisms reproducing either
sexually or asexually through spores. The spore forms and details on their
method of production have been used to delineate most fungal taxa. Although
there is a multitude of spore forms, fungal spores are basically only of two types:
(i) asexual spores are formed following mitosis (mitospores) and culminate
vegetative growth, and (ii) sexual spores are formed following meiosis (meios-
pores) and are borne in or upon specialized generative structures, the latter
frequently clustered in a fruit body. The vegetative forms of fungi are either
unicellular, yeasts are an example, or hyphal; the latter may be branched to
form an extensive mycelium.
Regardless of these details, it is the accessibility of spores, especially the direct
recovery of meiospores coupled with extended vegetative haploidy, that have
made fungi especially attractive as objects for experimental research.
Series Preface ix
The ability of fungi, especially the saprobic fungi, to absorb and grow on rather
simple and defined substrates and to convert these substances, not only into
essential metabolites but into important secondary metabolites, is also notewor-
thy.The metabolic capacities of fungi have attracted much interest in natural
products chemistry and in the production of antibiotics and other bioactive com-
pounds. Fungi, especially yeasts, are important in fermentation processes. Other
fungi are important in the production of enzymes, citric acid and other organic
compounds as well as in the fermentation of foods.
Fungi have invaded every conceivable ecological niche. Saprobic forms
abound, especially in the decay of organic debris. Pathogenic forms exist with
both plant and animal hosts. Fungi even grow on other fungi. They are found in
aquatic as well as soil environments, and their spores may pollute the air. Some
are edible; others are poisonous. Many are variously associated with plants as
copartners in the formation of lichens and mycorrhizae, as symbiotic endophytes
or as overt pathogens. Association with animal systems varies; examples include
the predaceous fungi that trap nematodes, the microfungi that grow in the
anaerobic environment of the rumen, the many insect associated fungi and the
medically important pathogens afflicting humans. Yes, fungi are ubiquitous and
important. There are many fungi, conservative estimates are in the order of
100,000 species, and there are many ways to study them, from descriptive
accounts of organisms found in nature to laboratory experimentation at the
cellular and molecular level. All such studies expand our knowledge of fungi
and of fungal processes and improve our ability to utilize and to control fungi for
the benefit of humankind.
We have invited leading research specialists in the field of mycology to
contribute to this Series. We are especially indebted and grateful for the initiative
and leadership shown by the Volume Editors in selecting topics and assembling
the experts. We have all been a bit ambitious in producing these Volumes on a
timely basis and therein lies the possibility of mistakes and oversights in this first
edition. We encourage the readership to draw our attention to any error, omis-
sion or inconsistency in this Series in order that improvements can be made in
any subsequent edition.
Finally, we wish to acknowledge the willingness of Springer-Verlag to host
this project, which is envisioned to require more than 5 years of effort and the
publication of at least nine Volumes.
Bochum, Germany KARL ESSER

Auburn, AL, USA PAUL A. LEMKE
April 1994 Series Editors
.
Volume Preface to the Second Edition
There have been major changes in our knowledge of the systematics and evolu-
tion of fungi since the first edition of the Mycota, Vol. VII. These changes have
been driven by an outpouring of molecular phylogenetic analyses at first based on
one or a few genes but now by multiple conserved genes. The Assembling the
Fungal Tree of Life projects have been a major contributor to the data needed to
construct the molecular phylogenies along with work from many additional labs.
The resulting phylogenies have made possible a new taxonomic outline for the
Fungi (Hibbett D.S. et al., 2007, Mycol. Res. 111: 509–547), which has provided a
more stable systematic treatment for this kingdom, although some of the basal
groups of Fungi remain incompletely resolved (Table 1). Agreement among many
mycologists on nomenclature is providing a stable framework for Fungi that has
been incorporated into reference works and online databases (McLaughlin D. J.
et al., 2009, Trends Microbiol. 17: 488–497), and has provided an escape from the
conflicting phenetic classifications of the past. These nomenclatural changes are
incorporated into these volumes along with much new information on the
evolution and ecology of these organisms made possible by a variety of methods,
including environmental sequencing and reevaluation of character evolution
using molecular phylogenies.
While there is agreement on nomenclature within Kingdom Fungi, there is
less agreement on the names for groups of fungus-like organisms, although these
organisms remain a major interest of those who study fungi. Some of the
confusion arises from the treatment of fungus-like organisms under two nomen-
clatural codes (Table 1). Of special concern has been the treatment of the
oomycetes and their relatives with variant spellings of the kingdom and common
name. The solution adopted by Beakes (Chap. 3, Vol. VII, Part A) reserves
Straminipila for the kingdom and uses the widely cited stramenopiles for the
common name.
Chapters in this edition of the Mycota, Vol. VII, vary from updates of chapters
published in the first edition to new chapters. All systematic chapters treat
monophyletic groups; clearly polyphyletic groups, such as those based on yeasts
or asexual stages (anamorphs), have been omitted. While authors have been
encouraged to provide illustrations of the diversity within each group, the results
are somewhat uneven. Some authors have extensively illustrated the organisms,
while others for reasons of time or access have provided limited illustrations. In
the interest of getting these chapters to press in a not too tardy manner, the
authors have not been unduly pressed to add illustrations. The reader’s under-
standing is requested for the omissions, which is caused in part by the difficulty of
getting all of the chapters needed to cover a wide spectrum of organisms.
xii Volume Preface to the Second Edition
Table 1 Taxonomic outline for Fungi and fungus-like organismsa
Fungus-like organisms
Supergroup: Amoebozoa
Phylum: Dictyosteliomycota
Phylum: Myxomycota
Supergroup: Excavata
Phylum: Acrasiomycota
Supergroup: Sarb
Subgroup: Rhizaria
Phylum: Phytomyxea
Kingdom: Straminipilac
Phylum: Labyrinthulomycota
Phylum: Hyphochytriomycota
Phylum: Oomycota
Fungi
Supergroup: Opisthokonta
Kingdom: Fungi
Basal fungi
Phylum: Cryptomycotad
Phylum: Microsporidia
Traditional Chytridomycota
Phylum: Chytridiomycota
Phylum: Monoblepharidomycota
Phylum: Neocallimastigomycota
Phylum: Blastocladiomycota
Zygomycotan (Zygomycetous) Fungi
Phylum: Entomophthoromycota
Phylum/a incertae sedis:
Subphylum: Kickxellomycotina
Subphylum: Mortierellomycotina
Subphylum: Mucoromycotina
Subphylum: Zoopagomycotina
Phylum: Glomeromycota
Subkingdom Dikarya
Phylum: Basidiomycota
Subphylum: Pucciniomycotina
Subphylum: Ustilaginomycotina
Subphylum: Agaricomycotina
Phylum: Ascomycota
Subphylum: Taphrinomycotina
Subphylum: Saccharomycotina
Subphylum: Pezizomycotina
a
Names for Fungi and fungus-like organisms traditionally studied by botanists are governed by the
International Code for Nomenclature of algae, fungi and plants (Melbourne Code) (McNeil J. et al., 2012,
Regnum Vegetabile 154, Koeltz Scientific Books). Multiple names exist for eukaryotic microorganisms that
are treated under both the Melbourne Code and the International Code of Zoological Nomenclature, except
for Microsporidia, which are classified under the zoological code
b
Sar (Stramenopiles, Alveolata, and Rhizaria)
c
Also known as Stramenopila or Stramenopiles. The latter is used by Adl et al. (2012, J. Eukaryot. Microbiol.
59: 429–493) and as a common name, stramenopiles, for Straminipila
d
Also known as Rozellida and Rozellomycota
The Mycota, Vol. VII, includes treatments of the systematics and related topics
for Fungi and fungus-like organisms in four eukaryotic supergroups (Table 1) as
well as specialized chapters on nomenclature, techniques, and evolution. Most
Fungi and fungus-like organisms are covered, including the Microsporidia.
Chapter 1, Vol. VII, Part A, provides an overview of fungal origins and evolution.
Volume Preface to the Second Edition xiii
Chapters 2–4, Vol. VII, Part A, cover the fungus-like organisms, and Chaps. 5
to 14, Vol. VII, Part A, and Chaps. 1–6, Vol. VII, Part B, cover the Fungi. Each of
these chapters covers approximately the following topics: occurrence and distri-
bution, economic importance, morphology and ultrastructure, development of
the taxonomic theory, classification, and maintenance and culture. The fungus-
like organisms are distributed in three distantly related supergroups (Table 1).
The basal fungi and traditional Chytridiomycota are treated as six phyla and
covered in four chapters, including Chap. 1, Vol. VII, Part A. The zygomycetous
fungi, whose deeper relationships remain unresolved, and Glomeromycota are
covered in two chapters. The Basidiomycota and Ascomycota, the largest groups
of fungi, are treated in five or six chapters each. In the Basidiomycota two
chapters cover Pucciniomycotina and Ustilaginomycotina, respectively, while
three chapters are devoted to classes of the Agaricomycotina. In the Ascomycota
a single chapter covers Taphrinomycotina and Saccharomycotina, while eight
classes of the Pezizomycotina are covered in five chapters.
The following topics are treated in Chaps. 7–11 in Vol. VII, Part B: Chap. 7
deals with the nomenclatural changes necessitated by the recent changes to the
International Code for Nomenclature of algae, fungi, and plants (Table 1), includ-
ing the elimination of separate names for anamorphic fungi. Chapter 8 deals with
methods for preservation of cultures and specimens, while Chap. 9 reviews the
phylogenetic implications of subcellular and biochemical characters and methods
for ultrastructural study. Chapter 10 deals with the fungal fossil record and Chap.
11 with the impact of the availability of whole genomes on studies of Fungi.
We are entering a new era in the study of fungi with whole genomes becoming
available for an increasing number of species across all the known clades of
Fungi. This genome-enabled mycology will utilize large numbers of genes in
phylogenomic analyses to resolve difficult to determine relationships in fungi
and to provide insights into fungal biology (Hibbett D.S. et al., 2013, Mycologia
106: 1339–1349). Initial studies are already having a significant impact on our
understanding of biochemical processes and their ecological impacts. In time
genomic studies may shed light on the genetic processes and the genes that
control the great morphological diversity in Fungi from the subcellular to the
macroscopic level. Thus, there is much new information on the systematics and
evolution of fungi to be expected in the future.
We thank Meredith Blackwell for sharing unpublished manuscripts and
discussions on the classification system, Esther G. McLaughlin for advice
throughout the work, and the U.S. National Science Foundation for support to
many labs for the AFTOL 1 and AFTOL 2 projects (including DEB-0732550 to
DJM, and DEB-0732993 to JWS), and numerous scientists who have contributed
to the work which has made the advances in these volumes possible.
St. Paul, MN David J. McLaughlin

Corvallis, OR Joseph W. Spatafora
22 May 2014
.
Volume Preface to the First Edition
This is an exciting time to produce an overview of the systematics and evolution of

the fungi. Homoplasy is evident in all lineages, e.g., those based on the gross
morphology of the chytrid zoospore, the perithecium and apothecium, the smut
teliospore and the agaric fruiting body, and some classifications based on light
microscope morphology have been shown to be unsound. Molecular and subcellu-
lar characters, aided by new methods of phylogenetic analysis, have allowed us to
see through the conflicts between various phenetic classification schemes and have
given us some confidence that we are beginning to achieve a true phylogeny of the
fungi. Molecular data have both supported ultrastructural characters that first
began to unravel the homoplasies unrecognized at the light microscopic level,
and have also revealed the relationships of fungi to other eukaryotes. They continue
to enlarge the scope of the fungi, e.g., with the recent addition of the Microsporidia
(see Cavalier-Smith, Chap. 1, Vol. VII, Part A), and they have shown the need for
more detailed chemical, subcellular, and developmental studies for a fuller under-
standing of these organisms and their relationships.
This volume is a mixture of phylogenetic and more classical systematics.
Progress in knowledge of species and development of taxonomic characters is
mixed. Groups with few species have been studied in great detail, while in groups
with large numbers of species much effort is still needed to find and determine the
taxa. Classical systematics groups organisms on a phenetic basis, then sets up a
classification; phylogeny is a secondary consideration. Phylogenetic systematics
first determines organism relationships, then constructs a systematic classification
that reflects the phylogeny. Molecular characters have made possible the establish-
ment of a monophyletic and, hopefully, more permanent classification for the
fungi. Thus, Volume VII of The Mycota contains both classical and phylogenetic
classifications, reflecting the available data and the orientation of different authors.
The incompleteness of some classifications, e.g., those for the Urediniomycetes
(Swann, Frieders, and McLaughlin, Chap. 2, Vol. VII, Part B) and Homobasidio-
mycetes (Hibbett and Thorn, Chap. 5, Vol. VII, Part B), demonstrates that we are in
the early stages of a phylogenetic systematics for these groups.
The taxonomic outline used in The Mycota, Vol. VII, differs somewhat from
that of other volumes in the series (Table 1), reflecting current mycological
systematics. There is a lack of agreement on the naming of higher taxa, and the
rules of nomenclature permit more than one name for these taxa. Cavalier-Smith
(Chap. 1, Vol. VII, Part A) presents an alternative view to the taxonomic outline
used for the remainder of the volume (Table 2). Some of the nomenclatural
problems stem from a lack of resolution of deep branches in molecular evolu-
tionary trees, a problem that appears likely to be resolved only with additional
xvi Volume Preface to the First Edition
Table 1 Taxonomic outline at the kingdom, phylum, and class levels as used in other volumes
in the series and in this volume. The classification in this volume is necessarily confusing at this
time because authors are using their own classifications rather than an imposed classification
Mycota, Vol. I Mycota, Vol. VII

PSEUDOMYCOTA PSEUDOMYCOTAa,b
Oomycota Oomycotac
Peronosporomycetes
Hyphochytriomycota Hyphochytriomycota
Hyphochytriomycetes
Plasmodiophoromycota
Plasmodiophoromycetes
EUMYCOTA EUMYCOTA
Chytridiomycota Chytridiomycotad
Chytridiomycetes
Zygomycota Zygomycotad
Zygomycetes
Trichomycetes
Dikaryomycota
Ascomycotina Ascomycotae
Saccharomycetes Saccharomycetes
Ascomycetes Plectomycetes
Hymenoascomycetesa
Loculoascomycetesa
Basidiomycotina Basidiomycota
Heterobasidiomycetes Urediniomycetes
Ustilaginomycetes
Heterobasidiomycetesa,f
Homobasidiomycetes Homobasidiomycetesa,f
a
Artificial taxon
b
For a natural classification for Oomycota and Hyphochytriomycota, kingdom Stramenopila (Strameni-
pila, Dick, Chap. 2, Vol. VII, Part A) or Chromista have been proposed, and for Plasmodiophoromycota,
kingdom Protozoa (see Cavalier-Smith, Chap. 1, Vol. VII, Part A)
c
Or Heterokonta (see Cavalier-Smith, Chap. 1, and Dick, Chap. 2, Vol. VII, Part A)
d
Probably paraphyletic (see Cavalier-Smith, Chap. 1, Vol. VII, Part A, and Berbee and Taylor, Chap. 10,
Vol. VII, Part B)
e
A phylogenetic classification for Ascomycota is not available. Current thinking among ascomycete scholars is
that three classes should be recognized, as follows: “Archiascomycetes”, which may not be monophyletic,
Hemiascomycetes (see Kurtzman and Sugiyama, Chap. 9, Vol. VII, Part A), and a filamentous group, Euasco-
mycetes, that eventually will be subdividable, perhaps at the subclass level [M.E. Berbee and J.W. Taylor, 1995,
Can J Bot 73 (Suppl. 1):S677, and Chap. 10, Vol. VII, Part B; J.W. Spatafora, 1995, Can J Bot 73 (Suppl. 1):S811].
Saccharomycetes as used here (see Barr, Chap. 8, Vol. VII, Part A) includes “Archiascomycetes” and Hemi-
ascomycetes. See the relevant chapters for further speculation on the ultimate disposition of these groups
f
Heterobasidiomycetes as used in Vol. VIIB cannot be separated from Homobasidiomycetes. Hymenomy-
cetes [E.C. Swann and J.W. Taylor, 1995, Can J Bot 73 (Suppl. 1):S862] has been proposed as a class for these
groups (see Berbee and Taylor, Chap. 10, Vol. VII, Part B)
data from multiple genes and the addition of missing taxa to the analysis.
Problems also arise from a difference of opinion among authors. The term
fungi has assumed an ecological meaning for all organisms with a similar nutri-
tional mode, and therefore, Eumycota, rather than Fungi, is less confusing for the
members of the phylum that encompasses a monophyletic group of these organ-
isms. Pseudofungi (Cavalier-Smith, Chap. 1, Vol. VII, Part A) implies that organ-
isms that lie outside the Eumycota but possess the fungal lifestyle are not fungi,
but in an ecological sense they are fungi. Pseudomycota is therefore used in this
series for these fungal organisms that lie outside the Eumycota.
Volume Preface to the First Edition xvii
Table 2 Taxonomic outline at the kingdom, phylum, and class levels as used in the rest of this
volume compared with that of Cavalier-Smith, Chap. 1, Vol. VII, Part A
Mycota, Vol. VII Chapter 1, Vol. VII, Part A

a
PSEUDOMYCOTA CHROMISTA
Oomycota Bigyra
Peronosporomycetes Oomycetes
Hyphochytriomycota
Hyphochytriomycetes Hyphochytrea
PROTOZOA
Plasmodiophoromycota Cercozoa
Plasmodiophoromycetes Phytomyxea
EUMYCOTA FUNGI
Chytridiomycota Archemycota
Chytridiomycetes Chytridiomycetes
Allomycetes
Zygomycota
Zygomycetes Zygomycetes
Bolomycetes
Glomomycetesb
Trichomycetes Enteromycetes
Zoomycetesc
Microsporidia
Minisporea
Microsporea
Ascomycota Ascomycota
Saccharomycetes Taphrinomycetes
Geomycetes
Endomycetes
Plectomycetes Plectomycetes
Hymenoascomycetes Discomycetes
Pyrenomycetes
Loculoascomycetes Loculomycetes
Basidiomycota Basidiomycota
Urediniomycetes Septomycetes
Ustilaginomycetes Ustomycetes
Heterobasidiomycetes Gelimycetesb
Homobasidiomycetes Homobasidiomycetes
a
Artificial taxon
b
Probably paraphyletic
c
Includes Zygomycetes, Ascomycetes, and Trichomycetes
The Mycota, Vol. VII, includes treatments of the systematics and related
topics of the Eumycota and Pseudomycota as well as specialized chapters on
nomenclature, techniques, and evolution. Certain groups are not treated in this
volume: the Labyrinthulomycetes (Pseudomycota) and the slime molds. The
evolutionary position of the slime molds has been controversial. Recent evidence
suggests that most slime molds are more closely related to the Eumycota than
previously believed (S.L. Baldauf and W.F. Doolittle, 1997, Proc Natl Acad Sci
USA 94:12007), and they should continue to be of interest to those who study
fungi for both ecological and phylogenetic reasons.
Chapters 2 to 4, Vol. VII, Part A, cover the Pseudomycota, and Chaps. 5–14,
Vol. VII A, and Chaps. 1–5, Vol. VII, Part B, the Eumycota. The Pseudomycota
contains distantly related groups of fungi (Table 1). The Chytridiomycota and
xviii Volume Preface to the First Edition
Zygomycota are treated in one and two chapters, respectively, while the Ascomy-
cota and Basidiomycota are treated in five or six chapters each, with separate
chapters for yeasts in each phylum, although the yeasts are not monophyletic
groups. Chapter 14, Vol. VII, Part A, discusses the special problems of anamor-
phic genera and their relationships to the teleomorphic genera and describes the
attempts being made to incorporate anamorphs into modern phylogenetic sys-
tematics. In Chap. 6, Vol. VII, Part B, Hawksworth discusses the development of a
unified system of biological nomenclature. Chapters 7 and 8, Vol. VII, Part B, deal
with techniques for cultivation and data analysis, respectively. The final two
chapters in Vol. VII, Part B, consider speciation and molecular evolution.
The Mycota, Vol. VII, was originally intended to have been Vol. I in the series.
Several changes in editors and the unfortunate death of Paul Lemke delayed its
production. Added to these difficulties was the fact that these are tumultuous
times in systematics because of the rapid development of molecular and phylo-
genetic analysis techniques and the explosive accumulation of data. As these
techniques and new data are more broadly incorporated into systematics, a
more stable and useful classification of the fungi will result.
We thank Heather J. Olson for her substantial efforts in compiling the indices.
St. Paul, MN DAVID J. MCLAUGHLIN

Minneapolis, MN ESTHER G. MCLAUGHLIN
April 2000 Volume Editors
Contents
Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1 Saccharomycotina and Taphrinomycotina: The Yeasts and Yeastlike

Fungi of the Ascomycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
CLETUS P. KURTZMAN, JUNTA SUGIYAMA
2 Pezizomycotina: Pezizomycetes, Orbiliomycetes . . . . . . . . . . . . . . . . . . . . . . 35

DONALD H. PFISTER
3 Pezizomycotina: Sordariomycetes and Leotiomycetes . . . . . . . . . . . . . . . . 57

NING ZHANG, ZHENG WANG
4 Pezizomycotina: Lecanoromycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
CÉCILE GUEIDAN, DAVID J. HILL, JOLANTA MIADLIKOWSKA, FRANCOIS LUTZONI
5 Pezizomycotina: Eurotiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

DAVID M. GEISER, KATHERINE F. LOBUGLIO, CÉCILE GUEIDAN
6 Pezizomycotina: Dothideomycetes and Arthoniomycetes . . . . . . . . . . . . 143

CONRAD SCHOCH, MARTIN GRUBE
Nomenclature and Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
7 The Shifting Sands of Fungal Naming Under the ICN

and the One Name Era for Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
ANDREW M. MINNIS
8 The Role of Herbaria and Culture Collections . . . . . . . . . . . . . . . . . . . . . . . . 205

GERARD J.M. VERKLEY, AMY ROSSMAN, JO ANNE CROUCH
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
9 Subcellular Structure and Biochemical Characters

in Fungal Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
DAVID J. MCLAUGHLIN, T.K. ARUN KUMAR, MEREDITH BLACKWELL,
PETER M. LETCHER, ROBERT W. ROBERSON
xx Contents
10 Fungal Diversity in the Fossil Record . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

THOMAS N. TAYLOR, MICHAEL KRINGS, EDITH L. TAYLOR
11 Phylogenomics Enabling Genome-Based Mycology . . . . . . . . . . . . . . . . 279

JASON E. STAJICH
Biosystematic Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

List of Contributors
MEREDITH BLACKWELL
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA
70803, USA
Department of Biological Sciences, University of South Carolina, Columbia, SC
29208, USA
JO ANNE CROUCH
Systematic Mycology & Microbiology Laboratory, USDA-ARS, Rm. 246, B010A
10300 Baltimore Ave., Beltsville, MD 20705, USA
DAVID M. GEISER
Department of Plant Pathology and Environmental Microbiology, 121 Buckhout
Laboratory, The Pennsylvania State University, University Park, PA 16802, USA
MARTIN GRUBE
Institute of Plant Sciences, Karl-Franzens-University, Holteigasse 6, 8010 Graz,
Austria
CÉCILE GUEIDAN
Department of Life Sciences, The Natural History Museum, Cromwell Road,
London SW7 5BD, UK
CSIRO – National Research Collections Australia, Australia National Herbarium,
Clunies Ross Street, Canberra, ACT 2601, Australia
DAVID J. HILL
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8
1UG, UK
MICHAEL KRINGS
Department of Ecology and Evolutionary Biology, University of Kansas,
Lawrence, KS 66045-7534, USA
Natural History Museum and Biodiversity Institute, University of Kansas,
Department für Geo- und Umweltwissenschaften, Paläontologie und
Geobiologie, Ludwig-Maximilians-Universität, Richard-Wagner-Straße 10,
Munich 80333, Germany
Bayerische Staatssammlung für Paläontologie und Geologie, Richard-Wagner-
Straße 10, Munich 80333, Germany
xxii List of Contributors
T.K. ARUN KUMAR

Department of Botany, The Zamorin’s Guruvayurappan College, Calicut, Kerala
673 014, India
CLETUS P. KURTZMAN
Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for
Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural
Research Service, 1815 North University Street, Peoria, IL 61604, USA
PETER M. LETCHER
Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487,
USA
KATHERINE F. LOBUGLIO
Farlow Herbarium, Harvard University, 22 Divinity Avenue, Cambridge, MA
02138, USA
FRANCOIS LUTZONI
Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA
DAVID J. MCLAUGHLIN
Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA
JOLANTA MIADLIKOWSKA
Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA
ANDREW M. MINNIS
USDA-US Forest Service, Center for Forest Mycology Research, One Gifford
Pinchot Dr., Madison, WI 53726, USA
DONALD H. PFISTER
Farlow Herbarium and Library of Cryptogamic Botany, Department of
Organismic and Evolutionary Biology, Harvard University, Cambridge, MA
02138, USA
ROBERT W. ROBERSON
School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA
AMY ROSSMAN
Systematic Mycology & Microbiology Laboratory, USDA-ARS, Rm. 246, B010A
10300 Baltimore Ave., Beltsville, MD 20705, USA
CONRAD SCHOCH
NCBI/NLM/NIH, 45 Center Drive, Bethesda, MD 20892, USA
JASON E. STAJICH
Department of Plant Pathology & Microbiology and Institute for Integrative
Genome Biology, University of California-Riverside, Riverside, CA 92521, USA
List of Contributors xxiii
JUNTA SUGIYAMA
Chiba Branch Office & Laboratory, TechnoSuruga Laboratory Co., Ltd., 3-1532-13
Hasama-cho, Funabashi-shi, Chiba 274-0822, Japan
THOMAS N. TAYLOR
EDITH L. TAYLOR
GERARD J.M. VERKLEY

CBS Biodiversity Center, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
ZHENG WANG
Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect
Street, New Haven, CT 06520, USA
NING ZHANG
Department of Plant Biology & Pathology, Rutgers University, 59 Dudley Road,
New Brunswick, NJ 08901, USA
Fungi
1 Saccharomycotina and Taphrinomycotina: The Yeasts
and Yeastlike Fungi of the Ascomycota
CLETUS P. KURTZMAN1, JUNTA SUGIYAMA2
CONTENTS I. Introduction
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
II. Occurrence, Distribution, and Ecology . . . . 3 Yeasts are found in nearly all regions of the
III. Importance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
A. Food, Beverage, and Industrial Uses . . . . 4 Earth, including hot deserts, polar areas, in
B. Agriculturally Important Yeasts . . . . . . . . . 5 freshwater, in salt water, and in the atmosphere,
1. Plant Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . 5 where they are commonly transported by pre-
2. Biocontrol Yeasts . . . . . . . . . . . . . . . . . . . . . . . 5 vailing winds. Though yeast growth is mainly
C. Food and Beverage Spoilage . . . . . . . . . . . . . 6 saprotrophic, some yeasts are important patho-
D. Human and Animal Pathogens . . . . . . . . . . 6
IV. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 gens of animals and plants. The term yeast has
A. Asexual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 come to mean those fungi that divide by bud-
1. Budding, Fission, Endospores, ding or fission and that have sexual states unen-
Chlamydospores . . . . . . . . . . . . . . . . . . . . . . . 7 closed in a fruiting body. Consequently, yeasts
2. Pseudohyphae and True (Septate) occur among the Ascomycota and the Basidio-
Hyphae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
B. Sexual Reproduction . . . . . . . . . . . . . . . . . . . . . 9 mycota. The focus of this chapter are those taxa
V. Taxonomic Methods. . . . . . . . . . . . . . . . . . . . . . . . 11 assigned to Saccharomycotina and Taphrino-
A. Phenotypic Characterization . . . . . . . . . . . . 11 mycotina of Ascomycota. As we will discuss,
B. Genotypic Characterization . . . . . . . . . . . . . 12 some members of these subphyla are among
VI. Phylogeny and Classification . . . . . . . . . . . . . . 13 the economically most important fungi known.
A. Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
B. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1. Saccharomycotina. . . . . . . . . . . . . . . . . . . . . 20
2. Taphrinomycotina . . . . . . . . . . . . . . . . . . . . 20 II. Occurrence, Distribution, and
VII. Isolation, Maintenance, and Culture
Availability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Ecology
A. Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
B. Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Although yeasts occur worldwide, some have
C. Culture Availability and Distribution. . . 26
VIII. Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . 26 restricted habitats, whereas others are found
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 in many different environments. The key to
understanding yeast ecology and the extent of
habitat specificity is the accurate identification
of species, which is now possible through DNA-
based methods. Prior to the application of
1
Bacterial Foodborne Pathogens and Mycology Research Unit, molecular methods, the identification of species
National Center for Agricultural Utilization Research, U.S. from phenotype often resulted in misclassifica-
Department of Agriculture, Agricultural Research Service, tion, which rendered results from many ecolog-
1815 North University Street, Peoria, IL 61604, USA; e-mail:
ical studies uncertain or misleading.
cletus.kurtzman@ars.usda.gov
2
Chiba Branch Office & Laboratory, TechnoSuruga Laboratory
Yeasts are often associated with insects, and
Co., Ltd., 3-1532-13 Hasama-cho, Funabashi-shi, Chiba 274- numerous studies have detailed these interac-
0822, Japan; e-mail: jsugiyam@tecsrg.co.jp tions (Phaff et al. 1956; van der Walt and Scott
Systematics and Evolution, 2nd Edition

The Mycota VII Part B
D.J. McLaughlin and J.W. Spatafora (Eds.)
© Springer-Verlag Berlin Heidelberg 2015
4 C.P. Kurtzman and J. Sugiyama
1971; Vega and Blackwell 2005; Wickerham areas of the world (Legras et al. 2007; McGovern
1969). Contemporary studies have expanded et al. 2004), and the production of fermented
on this earlier work and refined it through beverages and foods seems to have paralleled
molecular-based species identification. Notable the beginning of agriculture. Louis Pasteur
has been the work of H. J. Phaff and W. T. provided the insight that fermentation was the
Starmer and colleagues (Phaff et al. 1987; Star- result of microorganisms and noted that yeasts
mer et al. 1992), who examined yeast–Drosoph- occurred on grapes, thereby providing a ready
ila interactions among various species of cacti. source of inoculum for wine (Dubos 1960;
In one of these studies, Pichia kluyveri was Mortimer and Polsinelli 1999). The common-
recognized to be comprised of three closely place processes of baking, brewing, and wine
related and partially interfertile species that making are often taken for granted but repre-
were found to show significant habitat differ- sent major industries with a combined world-
ences. wide annual value that may exceed US$1 trillion
Other yeasts are strongly associated with (Hansen 2004; Verstrepen et al. 2006). Other
plants, including the well-known interaction food-related yeast processes include the natural
of Saccharomyces cerevisiae with grapes (e.g., fermentation of cocoa beans, coffee beans,
Mortimer and Polsinelli 1999). Ripe apples pickles, olives, and similar products.
have significant surface populations of Saccha- The industrial importance of yeasts has
romyces, Torulaspora, Zygosaccharomyces, and vastly expanded over the past few decades to
other yeasts that are attributed to the transfer of include much more than bread making and
soluble sugars onto the surface of the fruit, and brewing. Yarrowia lipolytica was initially used
many species of the genus Ogataea are found for the production of single-cell protein from
on leaves and decaying wood. Species of Oga- hydrocarbons, but the species is now recog-
taea utilize methanol as their sole source of nized as a major producer of citric acid, an
carbon, which is present in the environment important acidulant for industrial and food
as a degradation product of lignin (de Koning and beverage uses. Other industrially signifi-
and Harder 1992) and is formed in leaf respira- cant metabolites from ascomycetous yeasts
tory processes (Fall and Benson 1996). include riboflavin from Eremothecium gossypii
Some yeasts, such as Debaryomyces hanse- (Ashbya gossypii) (Wickerham et al. 1946),
nii and Meyerozyma guilliermondii (anamorph: lactase from Kluyveromyces marxianus
Candida guilliermondii), occur widely in (Rubio-Texeira 2006), biosurfactants, such as
nature and are common in water, plant debris, sophorolipids, from members of the Starmer-
and soil. Both of the aforementioned species ella clade (Kurtzman et al. 2010), and lipases
also represent opportunistic human pathogens. from Y. lipolytica and Candida cylindracea
Although yeasts are commonly isolated from (Gellissen et al. 2005). S. cerevisiae is widely
soil, few are believed to have soil as a primary used in the production of recombinant pro-
habitat. Many Lipomyces species are an excep- teins for medical and other uses, but the
tion and have been isolated only from soil. methanol-assimilating yeasts, such as Komaga-
taella pastoris (Pichia pastoris) and Ogataea
polymorpha (Hansenula polymorpha), are
III. Importance also important in this role (Cregg and Madden
1988; Veenhuis et al. 1983).
A. Food, Beverage, and Industrial Uses The conversion of plant biomass to
biofuels is of major interest, and the yeasts
Since ancient times, human societies world- Pachysolen tannophilus, Scheffersomyces stipitis
wide have used beverages and foods fermented (Pichia stipitis), and Candida shehatae, which
by yeasts (Legras et al. 2007). Archaeologists were discovered to ferment the D-xylose of bio-
have found evidence that fermented beverages mass to ethanol, have been the mainstays in this
were consumed in Neolithic times (8500– effort (Slininger et al. 1987, and references
4000 B.C.) in China, Iran, Egypt, and other therein). More recently, Spathaspora passali-
Saccharomycotina and Taphrinomycotina: The Yeasts and Yeastlike Fungi of the Ascomycota 5
darum and Candida jeffriesii were found to ing cotton boll rot in various species of Gossy-
ferment D-xylose to ethanol (Nguyen et al. pium and cankers on citrus fruit (Batra 1973).
2006). Similarly, E. gossypii causes staining and rot of
cotton bolls and is pathogenic to coffee (Coffea
spp.), soybean (Glycine max), and other crops.
B. Agriculturally Important Yeasts E. coryli can infect cotton, but it is also a
pathogen of hazelnuts, tomatoes, and beans.
1. Plant Pathogens Symptoms are generally disfigurement and dis-
ruption of the infected plant tissue. E. sinecau-
In this section, we discuss two important agri- dum was discovered by Holley et al. (1984) to
cultural aspects of yeasts, those that are plant cause seed infection in oriental and yellow mus-
pathogens and those that are antagonists of tard in Saskatchewan, Canada. The remaining
pathogens. Taphrina and Protomyces, both known species of Eremothecium, E. cymbalar-
members of Taphrinomycotina, are perhaps iae, appears uncommon but has been isolated
the best known of the yeastlike taxa that cause as a pathogen of flax and other plants (Arnaud
plant diseases. T. deformans, the cause of peach 1913). Other known pathogenic yeasts are
leaf curl, is worldwide in its distribution and Galactomyces candidus (anamorph Geotrichum
the most economically devastating of the dis- candidum) and Galactomyces citri-aurantii
eases caused by species of Taphrina (Fonseca (anamorph: Geotrichum citri-aurantii), which
and Rodrigues 2011; Mix 1949). Young leaves, commonly cause sour rot of citrus, tomatoes,
stems, and fruit are often severely distorted cantaloupes, peaches, lychee, and carrots
when infected by T. deformans. Early applica- (Butler et al. 1965; Wells 1977). Losses are
tion of fungicides usually controls peach leaf sufficiently great to require treatment of the
curl (Daughtrey et al. 2003), but failure to do produce by a fungicide.
so can result in significant crop losses. Other
tree crops, such as almonds and pears, may be
severely affected by Taphrina infections. Trees, 2. Biocontrol Yeasts
such as alders and poplars, are also susceptible
to various Taphrina species (Fonseca and Yeasts assigned to Saccharomycotina as well to
Rodrigues 2011; Mix 1949). The effect of these Basidiomycota are used in the biocontrol of
infections on tree health is generally limited, plant diseases (Andrews 1992; Chalutz et al.
but the appearance of infected leaves and flow- 1991). Our focus will be on the ascomycetous
ers can be distressing when the trees are used in yeasts used for this purpose. Yeasts are com-
ornamental plantings. mon on leaf surfaces and on fruits, the latter
Species of Protomyces cause symptoms containing abundant, easily utilizable carbon
similar to those seen from Taphrina infections. sources. The finding that naturally occurring
All known Protomyces species are plant para- yeasts on apples can protect fruit against
sitic and cause galls on stems, leaves, and fruits postharvest diseases (Janisiewicz 1987) mark-
of Compositiae, Umbelliferae, and certain other edly stimulated work on the utilization of
plants (Tubaki 1957). Economic losses are yeasts as an alternative to chemical pesticides
seldom great, but P. macrosporus infection of for the protection of fruits against storage
coriander (Coriandrum sativum) was reported diseases.
to have damaged up to 11 % of the crop during M. guilliermondii (anamorph: Candida
one growing season (Tripathi et al. 2003). guilliermondii) has been successfully used in
Several genera of Saccharomycotina also a number of studies to control fruit rots.
cause plant diseases. Most notable are species Guetsky et al. (2002) reported a 50 % reduction
of Eremothecium, some of which were previ- in Botrytis cinerea rot of strawberries following
ously classified in the genera Ashbya, Nematos- application of M. guilliermondii and suggested
pora, and Holleya, all of which are plant that the control mechanism is competition for
pathogens. E. ashbyi has a long history of caus- nutrients. Similarly, M. guilliermondii was
effective at controlling fungi that cause rot of spoilage. Spoilage of fresh fruits may be caused
citrus (Droby et al. 1993). M. guilliermondii is by Candida stellata, P. kluyveri, Pichia fermen-
widespread in nature, but it is also an opportu- tans, M. pulcherrima, and species of Hansenias-
nistic human pathogen, raising concerns about pora and its anamorph Kloeckera. Species of
its safety as a biocontrol agent on fruit and Brettanomyces and its teleomorph Dekkera are
other produce that will be consumed without often responsible for turbidity and off-flavors
cooking. Candida oleophila, which is not a clin- in wine, beer, and soft drinks.
ical yeast, shows good control of fruit storage
rots and has been commercialized under the
trade name Aspire for the protection of citrus
D. Human and Animal Pathogens
from rots caused by species of Penicillium
(Droby et al. 1998). Metschnikowia fructicola Candida albicans is the most common cause of
and Metschnikowia pulcherrima have also candidiasis and is the species most often
been tested extensively for the biocontrol of isolated from cases of oral infections and vagi-
fruit rots (Janisiewicz et al. 2001; Karabulut nitis (Bialkova and Subik 2006; Kaur et al.
et al. 2004). Additionally, Wickerhamomyces 2005). DNA sequence analysis of C. albicans
anomalus (Pichia anomala) has been effec- strains resulted in the discovery of the closely
tive in the biocontrol of mold-induced spoilage related and phenotypically nearly identical spe-
of ensiled maize (Passoth et al. 2006). A con- cies Candida dubliniensis (Sullivan et al. 2005).
cern is that W. anomalus has been implicated in C. dubliniensis is mainly isolated from oral
some human infections, and its suitability as a candidiasis in HIV-infected patients, but it is
biocontrol agent is uncertain. also isolated from healthy individuals, as is the
case for C. albicans. Following C. albicans, the
second most common cause of blood stream
C. Food and Beverage Spoilage infections is Candida glabrata, and the
increased frequency of infections by this spe-
The spoilage of foods and beverages by con- cies has been attributed to the larger population
taminating yeasts results in major economic of immunocompromised individuals and the
losses worldwide (e.g., Fleet 1990). Yeasts widespread use of antimycotics. DNA sequence
responsible for food spoilage are not known to analysis resulted in the discovery of the clini-
cause infection or food poisoning in humans, as cally important species Candida bracarensis
do certain bacteria. The composition of foods and Candida nivariensis, both of which are
and beverages often determines the species of phenotypically similar to C. glabrata (Alcoba-
yeasts that are likely to be found. Products with Flórez et al. 2005; Correia et al. 2006).
high sugar content (40–70 %) are commonly Numerous other ascomycetous yeasts are
spoiled by Zygosaccharomyces spp., Torulas- obtained as clinical isolates, but most are wide-
pora delbrueckii, Schizosaccharomyces octos- spread in the environment and regarded as
porus, and Wickerhamomyces subpelliculosus opportunistic pathogens. Among them are
(Pichia subpelliculosa). If salt (NaCl) is used M. guilliermondii, Pichia kudriavzevii (ana-
as a preservative, other species can predomi- morph: Candida krusei), Candida parapsilosis,
nate. During the fermentation of cucumbers Candida tropicalis, and even S. cerevisiae. Both
(10–16 % NaCl) and other products, such as animals and humans can develop yeast infec-
soy sauce, Candida etchellsii and Candida ver- tions. Kazachstania pintolopesii has devastated
satilis are common. Species of Debaryomyces, colonies of laboratory mice (Kurtzman et al.
especially D. hansenii, often overgrow aged 2005), and Macrorhabdus ornithogaster
cheeses, salami, and other salted meat pro- appears to be a cause of decline in birds such
ducts, imparting flavor and the potential for as budgerigars (Tomaszewski et al. 2003).
IV. Reproduction leads to the formation of multiple scars or

annellations at the poles of the cell (Streiblová
A. Asexual 1971). Bipolar budding is characteristic of apic-
ulate yeasts. Budding from various sites on a
1. Budding, Fission, Endospores, cell is termed multilateral or multipolar and is
Chlamydospores the most common type of budding among asco-
mycetous yeasts (Fig. 1.1). Budding is also
Asexual reproduction, sometimes termed veg- described in terms of the way successive buds
etative reproduction, occurs in ascomycetous are produced. Sympodial budding occurs on a
yeasts by budding or by fission. The formation conidiophore that extends in growth by a suc-
of pseudohyphae and septate (true) hyphae cession of apices. A blastoconidium is pro-
constitutes other forms of asexual reproduction duced at each apex, and the growth continues
that are discussed subsequently. Buds may arise to the side of the apex; the result is a zigzag
either on yeast cells or on hyphal cells, and appearance, for example, Blastobotrys. Acrope-
budding is initiated by the formation of a tal budding entails the formation of successive
small evagination or outgrowth at some point buds in a chain with the youngest at the apex. In
on the surface of the cell. The parent (mother) basipetal budding, successive buds are formed,
cell remains more or less constant in size as the with the oldest at the apex.
bud (blastoconidium) increases in size to form Reproduction by fission is the division of
a new cell and then usually separates from the an asexual cell by means of a septum growing
parent cell. Budding is termed holoblastic or inward from the cell wall to bisect the long axis
enteroblastic, depending on how the bud is of the cell. The newly formed fission cells,
formed. All layers of the wall of the parent cell which are termed arthroconidia (arthros-
are involved in holoblastic budding and the bud pores), elongate, and the process is repeated.
separates, usually on a narrow base, leaving a Recurrent fission by a cell may give rise to
scar through which no further budding occurs. transverse multiple scars or annellations (Strei-
Von Arx and Weijman (1979) considered holo- blová 1971). This manner of reproduction is
blastic budding to be characteristic of Sacchar- characteristic of Schizosaccharomyces and
omycotina. When budding is enteroblastic, the Dipodascus (Fig. 1.1).
first bud arises through a rupture in the wall of Asexual cells may be globose, subglobose,
the parent cell through which the innermost ellipsoid, ovoid, obovoid, cylindrical, botuli-
layer evaginates and ultimately grows out to form, bacilliform, elongate, apiculate, ogival,
form the outermost layer of the bud. The site lunate, or triangular. Definitions and illustra-
of budding is eventually surrounded by a col- tions of the various possibilities can be found in
larette owing to the recurrent formation and Ainsworth & Bisby’s Dictionary of the Fungi
abscission of a succession of buds arising (Kirk et al. 2008). The shape of the cell may
from the inner layer of the wall of the cell. reflect the mode of reproduction, and in some
Enteroblastic budding is characteristic of cases, it is characteristic of particular genera or
basidiomycetous yeasts and some members of species. Some examples include the lemon-
Taphrinomycotina (Kurtzman and Sugiyama shaped cells of the apiculate yeasts Hansenias-
2001; Sugiyama and Nishida 1995). pora and Wickerhamia, the lunate cells of
Budding is also classified on the basis of the Metschnikowia lunata, and the triangular cells
position of the site where it occurs. Budding of Trigonopsis variabilis (Kurtzman and Rob-
that is restricted to one pole of a cell is termed nett 2007).
monopolar, and budding that occurs at both Endospores are asexual cells that are
poles of a cell is termed bipolar. When the formed within single cells and in hyphal cells
buds are abstricted on a rather broad base by and seem to arise by budding. Endospores are
the formation of a cross wall, the process is not commonly formed, but they have been
referred to as “budding on a broad base” and observed in strains of Candida and a few
“bud fission” (Fig. 1.1). Recurrent budding other genera. No special media have been
Fig. 1.1 Various forms of asexual reproduction. hypha with side branches bearing blastoconidia
(a) Multilateral budding (Pichia nakasei). (b) Bipolar (Candida ontarioensis). (Panels a–d, T. van Beest and
budding on a wide base (Hanseniaspora osmophila). T. Boekhout, CBS Web site; panel e, C.P. Kurtzman).
(c) Fission (Schizosaccharomyces pombe). (d) Pseudo- Bars¼5 mm
hyphae (Metschnikowia gruessii). (e) True (septate)
devised to stimulate the development of endo- parent cell and give rise to chains or clusters
spores (do Carmo-Sousa 1969). of cells. The tendency of some yeasts to form
Chlamydospores have been defined as chains of cells results in the formation of pseu-
thick-walled, nondeciduous, intercalary or ter- dohyphae. A pseudohypha is defined as a fila-
minal asexual spores formed by the rounding of ment composed of a chain of cells that has been
a cell or cells (Ainsworth 1971; Hughes 1985; formed by budding (Fig. 1.1). Pseudohyphae
Stalpers 1987). The asexual nature of the chla- may be either rudimentary, in which case they
mydospore distinguishes it from the teliospore consist of cells of similar size and shape, or they
of basidiomycetous yeasts from which the may be differentiated into elongated cells, each
basidium is produced. Chlamydospores are of which may produce blastoconidia. The form
generally rich in lipids and well adapted to of a pseudohypha can be markedly affected by
maintain viability through periods of dor- cultural conditions (van der Walt 1970). Some
mancy. In older cultures, chlamydospores species of Dekkera form an unusual type of
shed their outer layers just before or during pseudohypha called a blastese, which is a slen-
germination. Chlamydospores are characteris- der, aseptate hypha that develops from germi-
tic of C. albicans, C. dubliniensis, and Metsch- nating blastospores (Langeron and Guerra
nikowia species. Chlamydospores fulfill a dual 1940).
function in Metschnikowia and either germi- Some yeasts produce true septate branch-
nate by budding or are transformed into asci. ing hyphae, which elongate by continuous
growth of the hyphal tip followed by the forma-
2. Pseudohyphae and True (Septate) Hyphae tion of septa (Fig. 1.1). The fine structure of
hyphal septa varies among taxa, but light
Mature buds can either become detached as microscopy does not reveal much detail except
individual cells or remain attached to the for the presence of large septal pore bodies in
Ambrosiozyma. Hyphae may proliferate by which usually one to four ascospores are
simple branching, or they may produce blasto- formed. Asci bearing such vestigial buds are
conidia on differentiated conidiogenous cells. found in Debaryomyces, Torulaspora, Pichia,
The presence of blastoconidia on denticles is a and certain other genera. A process comparable
characteristic of species of Trichomonascus to cell–bud conjugation appears to operate in
(anamorph Blastobotrys), Hyphopichia, and the genus Nadsonia, where karyogamy is
certain other genera. Hyphae are sometimes initiated by the fusion of the nuclei of a bud
joined by a process in which there is the fusion and its parent. The contents of the zygote then
of branches of the same or different hyphae, move into a bud at the opposite pole, which is
and this is called anastomosis. The media abstricted by a septum and becomes the ascus.
most commonly used for detecting pseudohy- When diploidization occurs by the fusion of
phae and true hyphae are corn meal (maize) two independent haploid cells, the cells may
agar, morphology agar, 5 % malt extract agar, form elongated conjugation tubes, which fuse
and potato-dextrose agar, but some clinical to give a dumbbell shape, as is characteristic for
laboratories use rice agar, which will also pro- Zygosaccharomyces, Kodamaea and certain
mote the formation of chlamydospores by other taxa (Fig. 1.2).
C. albicans. Heterothallic species may occur as haploid
mating types or as diploid strains, which are
normally heterozygous for the mating-type
B. Sexual Reproduction genes and are sometimes termed bisexual. Uni-
sexual diploid strains are known (Wickerham
Many yeasts reproduce sexually, resulting in an 1958); asci from these strains are unconjugated,
alternation of generations with the formation of and unisexual haploid ascospores of both mat-
characteristic cells in which reduction division ing types are formed. Ascospores of opposite
takes place. In ascogenous yeasts, the site of mating types either conjugate within the ascus,
meiosis is the ascus where the haploid genera- giving rise to the diplophase, as in Saccharomy-
tion of ascospores is formed by so-called free- codes, or the ascospores are released and ger-
cell formation, i.e., the process by which the minate to give haploid asexual cells of opposite
cytoplasm surrounding the meiotic nuclei mating types. Normally, the diplophase can
becomes enveloped by a wall. Ascogenous only be restored if conjugation of haploid cul-
yeasts may be homothallic or heterothallic, tures of opposite mating types occurs. Active
and the asexual phase may be diploid or hap- cultures of mating types are not invariably sta-
loid, but sometimes both haploid and diploid ble and may revert to sporulating cultures as a
cells are present in the same culture. Higher result of mutation of the mating-type alleles
degrees of ploidy have also been reported (Takano and Oshima 1970). Some species,
for some species, such as S. cerevisiae and such as Pichia membranifaciens, seem to have
Lachancea kluyveri (Saccharomyces kluyveri) both heterothallic and homothallic strains.
(Wickerham 1958). Heterothallic strains of some species may
For haploid homothallic yeasts, plasmog- show sexual agglutination, as in Wickerhamo-
amy, karyogamy, and meiosis occur within myces canadensis (¼Hansenula wingei) and
the zygote, which is often formed by the conju- Lachancea kluyveri (Saccharomyces kluyveri)
gation of two separate budding cells or by (Wickerham 1958), in which cells of opposite
conjugation between a cell and its bud mating types agglutinate when mixed. Aggluti-
(mother–daughter cell conjugation or bud mei- nation in W. canadensis is mediated by comple-
osis). The diplophase is usually restricted to the mentary glycoproteins present on the surface of
diploid zygote within which the ascospores are cells of the opposite mating types (Crandall and
formed. In the case of conjugation between a Brock 1968).
cell and its bud, the bud remains attached to the Ascospores vary in the number present in
parent cell, which is converted into an ascus in asci. Asci with either one or two ascospores
Fig. 1.2 Various forms of ascospore formation. siamensis). (g) Deliquescent asci with bean-shaped
(a) Persistent, unconjugated asci with globose ascos- ascospores (K. marxianus). (h) Hat-shaped ascospores
pores (Saccharomyces paradoxus). (b) Deliquescent, released from deliquescent asci (Cyberlindnera
unconjugated asci with globose and hat-shaped ascos- veronae). (i) Saturn-shaped ascospores formed in deli-
pores (Pichia membranifaciens). (c) Asci with globose quescent asci (Saturnispora ahearnii). (j) Elongated,
ascospores formed from conjugation of complemen- needle-shaped ascospores in a persistent ascus
tary mating types (Kodamaea ohmeri). (d) Persistent (Metschnikowia hawaiiensis). (k) Elongated ascospores
asci with globose ascospores formed by conjugation with a whiplike tail released from a deliquescent ascus
between cells and their buds (Schwanniomyces pseudo- (Eremothecium coryli). (l) Hat-shaped ascospores
polymorphus). (e) Persistent asci with tapered released from an ascus formed at the tip of an asco-
bud-conjugants and globose ascospores (Torulaspora phore (Pachysolen tannophilus). (Panels a, c, d, g, j, T.
delbrueckii). (f) Persistent, unconjugated asci, each van Beest and T. Boekhout, CBS Web site; panels b, e, f,
with a roughened, spherical ascospore (Citeromyces h, i, k, l, C.P. Kurtzman). Bars¼5 mm
are typical for Lodderomyces, Metschnikowia, genera. In contrast, species of Ascoidea and
and some species of Debaryomyces, whereas Vanderwaltozyma may form in excess of 100
four spores are characteristic of some species ascospores in each ascus. The shape of ascos-
of Pichia, Saccharomyces, and certain other pores varies widely and includes globose,
ellipsoidal, cylindrical, reniform, crescentic, V. Taxonomic Methods

clavate, hat-shaped (galeate), cap-shaped,
saturnoid, walnut-shaped, falcate, needle- A. Phenotypic Characterization
shaped, and spindle-shaped with a whiplike
appendage (Fig. 1.2). The surface may be In addition to differentiation by cellular mor-
smooth or rough, but surface ornamentation, phology, responses on fermentation and
including brims and ledges, may be reduced to growth (assimilation) tests are still used in
such an extent that this morphology cannot be some laboratories to identify taxa. The physio-
detected by light microscopy. Phylogenetic logical tests commonly used are fermentation
analyses have shown that ascospore shape is of seven to eight carbohydrates, growth on
of uncertain value for assessing genus assign- various carbon and nitrogen sources, require-
ments and sometimes even varies within strains ments for vitamins, growth at various tempera-
of a species (Kurtzman et al. 2008). An example tures, growth on media with a high content of
of the latter is Kodamaea ohmeri, where both sugar or sodium chloride, hydrolysis of urea,
hat-shaped and globose ascospores have been and resistance to antibiotics.
found depending on the mating types paired There is no known exception to the rule
(Wickerham and Burton 1954). that when a yeast strain ferments a carbohy-
Ascosporulation may be induced under drate, it is also able to grow on it. However, the
conditions that restrict asexual growth, but reverse does not hold true; many yeasts grow
many strains sporulate without any special aerobically on sugars they cannot ferment.
preparation. Some genera sporulate best Yeasts vary in their ability to ferment sugars
on a particular medium. Acetate agar has as measured by the production of carbon
been recommended for Saccharomyces (e.g., dioxide.
McClary et al. 1959) and dilute V8 agar for
Metschnikowia (Pitt and Miller 1968). Many Various tests have been devised to detect the produc-
strains of Pichia and related genera sporulate tion of carbon dioxide from carbohydrates, but
on YM or malt agars. What follows represents Durham tubes are the most useful method for the
routine detection of carbohydrate fermentation (Kurtz-
a suggested start for ascospore detection. man et al. 2011; Wickerham 1951). Durham tubes are
Strains placed on slant cultures of YM, 5 % test tubes with a small inverted tube inserted to collect
malt extract, dilute V8, and RG agars should any gas that may be produced. The fermentation of
be incubated at 15 and 25 C and examined D-glucose, D-galactose, sucrose, maltose, lactose,
weekly for 2 months [see Kurtzman et al. raffinose, and trehalose is generally tested for routine
identification; other compounds, such as inulin, starch,
(2011) for the composition of culture media melibiose, cellobiose, and D-xylose, are sometimes
given in this chapter]. Some yeasts sporulate used. The sugars are tested as 2 % (w/v) solutions,
rapidly, i.e., within 24–48 h, especially when except for raffinose, where 4 % is usually used because
first isolated; others may require much longer, some strains cleave and ferment only part of the mole-
up to 6 weeks or more. If ascosporulation is cule of this trisaccharide.
not detected, then other media, such as Gor-
Assimilation tests determine the ability of a
odkowa and acetate agars, can be tried. Strains
yeast to grow aerobically on a particular carbon
that do not form ascospores may represent
compound supplied as the sole source of
mating types and should be mixed to deter-
energy. The tests can be done either on solid
mine whether conjugation and ascosporula-
tion occur. Conjugation often occurs within media or in liquid media, but liquid media are
24–48 h, but occasionally 1–2 weeks may be believed by some taxonomists to give more
required. Ascospores can be stained with mal- reproducible results.
achite green (Wickerham 1951), but visualiza-
The size of growth tubes and the amount of medium
tion of unstained cells with either bright field used can vary widely among laboratories, but the
or phase contrast microscopy is favored by method employing tubes of liquid media as described
many observers over staining. by Wickerham (1951) is commonly used. The results
are improved by gently shaking the tubes during incu- related species, as well as more distantly related
bation. Some laboratories incubate the tests for a taxa, and a database of sequences can be devel-
period of 3 weeks, others for 4 weeks. These long incu-
bations allow the yeasts to adapt to utilize some com-
oped and expanded for further use.
pounds. Tests on solid media can be done in two ways. The variable domain 2 (D2) from nuclear
The first is the auxanographic method of Beijerinck large subunit ribosomal RNA (LSU rRNA) was
(1889), in which the yeast is suspended in agar in initially examined and found to resolve closely
pour plates and the test sugars are spotted at intervals related species (Peterson and Kurtzman 1991).
around the circumference. The second method is to
incorporate the test compound into a nutrient agar
This work was expanded to include domains
basal medium in petri dishes and inoculate the test 1 and 2 (D1/D2) and applied to all described
yeast as either a streak or a point on the surface. species of ascomycetous yeasts, resulting in a
diagnostic database (barcode) for rapid species
B. Genotypic Characterization identification (Kurtzman and Robnett 1998). A
comparison of nuclear DNA reassociation
During the past 10–15 years, gene sequence values suggested that conspecific strains dif-
analyses have been used for yeast identification, fered by no more than 3 nucleotides among
usually replacing the phenotypic methods the 500–600 nucleotides of the D1/D2 domains,
described earlier. Nonetheless, the collection whereas differences of 6 or more nucleotides
of phenotypic data provides important infor- (1 %) indicated that the strains were different
mation about the biology of the strains under species. The preceding estimates of divergence
study and their potential biotechnological were treated as a prediction (Kurtzman and
applications. DNA comparisons of yeasts have Robnett 1998) because exceptions were
paralleled the increasing sophistication of known. Among them are hybrid species
methods for nucleic acid characterization. (Groth et al. 1999; Peterson and Kurtzman
Initial studies were restricted to determining 1991; Vaughan-Martini and Kurtzman 1985)
the mol% guanine+cytosine (G+C) content of and certain DNA polymorphisms (Lachance
DNA. From this work it was seen that ascomy- et al. 2003).
cetous yeasts had a nuclear DNA content of ca. A significant advantage to using rRNA gene
28–50 mol%, whereas basidiomycetous yeasts sequences is that ribosomes have a common
had a noticeably higher range of 50–70 mol% evolutionary history, and within the sequences
(Nakase and Komagata 1968; Price et al. 1978). are highly conserved regions between the vari-
These studies suggested that strains differing by able regions that serve for pan-specific primer
1–2 mol% were likely to represent different attachment for PCR amplification and sequenc-
species, thereby providing a means for excluding. In contrast, protein coding genes tend to be
ing strains incorrectly assigned to a particular variable across the entire gene, often making
species. Quantitation of gene sequence similar- primer design difficult. Nonetheless, the gene
ity between strains became possible with the sequences encoding several proteins have been
development of DNA reassociation techniques examined for phylogenetically divergent
that measure the extent of pairing of nucleotide groups of species. Daniel et al. (2001) compared
sequences when DNA is made single-stranded Candida spp. from several clades and showed
and allowed to re-pair as a double strand. An that phylogenetic trees generated from actin
interpretation of DNA reassociation data was sequences were congruent with rRNA gene
provided by Martini and Phaff (1973) and Price trees. Daniel and Meyer (2003) compared the
et al. (1978), who suggested that, on the basis of resolution of closely related species from actin
shared phenotype, strains that showed 80 % or sequences and from D1/D2 LSU. As with
greater nuclear DNA relatedness are members D1/D2, actin did not always provide a clear
of the same species. A limitation of DNA reas- separation of species, but in general, actin
sociation experiments has been that genetic sequences had a greater number of substitu-
resolution extends no further than to closely tions, providing easier recognition of closely
related species. In contrast, gene sequence com- related species. Similar resolution was reported
parisons offer the opportunity to resolve closely for the translation elongation factor-1a gene
(Kurtzman et al. 2008) and the cytochrome Ascomycota (e.g., Guilliermond 1912) or that
oxidase II (COX II) gene (Belloch et al. 2000; they represent morphologically reduced forms
Kurtzman and Robnett 2003). of more evolved taxa (Cain 1972; Redhead and
In the examples presented, determination Malloch 1977; von Arx and van der Walt 1987).
of whether strains are conspecific or members The issue of relationships within the Asco-
of separate species can be confused by hybridi- mycota remained uncertain until the use of
zation events, by unexplained sequence poly- gene sequence analysis to estimate phylogeny.
morphisms, and by differences in nucleotide Walker (1985) sequenced 5S rRNA for selected
substitution rates. Multigene analyses offer a ascomycetes, and the analysis of this data set
means for detecting these changes, which divided the Ascomycota into three groups: (1)
would be signalled by lack of congruence for a Schizosaccharomyces and Protomyces, (2) bud-
particular gene tree. ding yeasts, and (3) so-called filamentous fungi.
Berbee and Taylor (1993) analyzed a larger
The multigene approach was recommended by Good- group of species from nuclear SSU rRNA gene
man (1976) for vertebrates, for bacteria by Dykhuizen sequences, which showed the same three major
and Green (1991), and for fungi by Taylor et al. (2000). ascomycete lineages and that yeasts and fila-
The paper by Taylor et al. (2000) provides an inclusive
review of species concepts, and the term genealogical
mentous fungi are sister taxa, whereas Schizo-
concordance phylogenetic species recognition (GCPSR) saccharomyces and relatives diverged prior to
was introduced to describe the concept of multigene these two clades. Kurtzman and Robnett (1994)
analysis for species recognition (see Taylor and Berbee, showed from partial LSU and SSU rRNA
Chap. 1, Vol. VII, Part A). sequences that all currently accepted ascomy-
cetous yeast genera were members of a single
Comparison of strains from single gene clade, which was separate from Schizosacchar-
sequences, such as D1/D2 LSU rRNA and the omyces and members of the filamentous fungi.
sequences of ITS, has provided a rapid means The finding from single-gene analyses that
for species identification. However, the preced- Ascomycota is comprised of three separate
ing discussion makes clear that some closely lineages was supported by multigene sequence
related species are not resolved by these analyses (Fitzpatrick et al. 2006; James et al.
sequences and that the occurrence of hybrids 2006; Kuramae et al. 2006). The phylogenetic
further complicates identification. Conse- trees generated from sequence analyses have
quently, critical species identification requires significantly changed classification within the
a comparison of multiple genes, hence the fungi, and these changes will be discussed.
increasingly widespread application of multi- The outline of ascomycetous yeast classification
locus sequence typing (MLST) in strain identi- is given in Table 1.1 and is based on the classi-
fication. fication of the kingdom Fungi presented by
Hibbett et al. (2007) and from multigene ana-
lyses reported by Kurtzman (2003), Kurtzman
VI. Phylogeny and Classification and Robnett (2003, 2007, 2010, 2013), Kurtz-
man and Suzuki (2010), and Kurtzman et al.
(2007, 2008). Figure 1.3 shows the phylogenetic
A. Phylogeny
relationship among taxa of Saccharomycotina,
The relationship of ascomycetous yeasts with Taphrinomycotina, and Pezizomycotina and
other members of the Ascomycota has been was determined from multigene sequence anal-
controversial for over 100 years. Because yeasts ysis (Sugiyama et al. 2006). Support for early
are morphologically simple, it was proposed diverging lineages in many multigene trees is
that either they represent primitive forms of often weak, and assignment of genera to
Table 1.1 Subphyla, classes, orders, families, and genera of yeasts and yeastlike taxa of phylum Ascomycotaa,b,c
14
Class Order Family Genus

Subphylum Taphrinomycotina
Archaeorhizomycetes Archaeorhizomycetales Rosling & T. Archaeorhizomycetaceae Rosling & Archaeorhizomyces Rosling & T. James (A)
James T. James
Neolectomycetes Neolectales Landvik, O.E. Eriksson, Neolectaceae Redhead Neolecta Spegazzini (T)
Gargas & P. Gustafsson
Pneumocystidomycetes Pneumocystidales O.E. Eriksson Pneumocystidaceae O.E. Eriksson Pneumocystis P. Delanoë & Delanoë (T)
Schizosaccharomycetes Schizosaccharomycetales O.E. Schizosaccharomycetaceae Schizosaccharomyces Lindner (T)
Eriksson, Svedskog & Landvik Beijerinck ex Klöcker
Taphrinomycetes Taphrinales Gäumann & C.W. Dodge Protomycetaceae Gray Burenia M.S. Reddy & C.L. Kramer (T)
Protomyces Unger (T)
Protomycopsis Magnus (T)
Taphridium Lagerheim & Juel ex Juel (T)
Volkartia Maire (T)
Taphrinaceae Gäumann & Lalaria R.T. Moore emend. Á. Fonseca (A)
C.W. Dodge Saitoella S. Goto, Sugiyama, Hamamoto & Komagata (A)
Taphrina Fries (T)
Subphylum Saccharomycotina
Saccharomycetes Saccharomycetales Kudryavtsev Ascoideaceae J. Schröter Ascoidea Brefeld & Lindau (T)
Cephaloascaceae L.R. Batra Cephaloascus Hanawa (T)
Debaryomycetaceae Kurtzman & Debaryomyces Lodder & Kreger-van Rij (T)
M. Suzuki Hyphopichia von Arx & van der Waltd (T)
Kurtzmaniella Lachance & Starmerd (T)
Lodderomyces van der Waltd (T)
C.P. Kurtzman and J. Sugiyama
Meyerozyma Kurtzman & M. Suzukid (T)

Millerozyma Kurtzman & M. Suzukid (T)
Priceomyces M. Suzuki & Kurtzmand (T)
Scheffersomyces Kurtzman & M. Suzukid (T)
Schwanniomyces Klöcker emend. M. Suzuki & Kurtzman (T)
Spathaspora Nguyen, Suh & Blackwelld (T)
Wickerhamia Sonedad (T)
Yamadazyma Billon-Grandd (T)
Dipodascaceae Engler & E. Gilg Dipodascus Lagerheim (T)
Galactomyces Redhead & Malloch (T)
Geotrichum Link:Fries (A)
Magnusiomyces Zender (T)
Saprochaete Coker & Shanor ex D.T.S. Wagner & Dawes (A)
Endomycetaceae J. Schröter Endomyces Reess (T)
Helicogonium W.L. White (T)
Phialoascus Redhead & Malloch (T)
Lipomycetaceae E. K. Novak & Zsolt Dipodascopsis Batra & P. Millner emend. Kurtzman, Albertyn
& Basehoar-Powers (T)
Lipomyces Lodder & Kreger-van Rij (T)
Myxozyma van der Walt, Weijman & von Arx (A)
Metschnikowiaceae T. Kamienski Aciculoconidium King & Jong (A)
Clavispora Rodrigues de Miranda (T)
Kodamaea Y. Yamada, T. Suzuki, Matsuda & Mikata emend.
Rosa, Lachance, Starmer, Barker, Bowles & Schlag-Edler (T)
Metschnikowia Kamienski (T)
Phaffomycetaceae Y. Yamada, Barnettozyma Kurtzman, Robnett & Basehoar-Powers (T)
Kawasaki, Nagatsuka, Mikata & Cyberlindnera Minter (T)
Seki Phaffomyces Y. Yamada, (T)
Starmera Y. Yamada, Higashi, S. Ando & Mikata (T)
Wickerhamomyces Kurtzman, Robnett & Basehoar-Powers (T)
Pichiaceae Zender Ambrosiozyma van der Walt (T)
Brettanomyces Kufferath & van Laere (A)
Dekkera van der Walte (T)
Kregervanrija Kurtzman (T)
Ogataea Y. Yamada, Maeda & Mikata (T)
Pichia E.C. Hansen (T)
Saturnispora Liu & Kurtzman (T)
Saccharomycetaceae G. Winter Cyniclomyces van der Walt & D.B. Scott (T)
Eremothecium Borzi emend. Kurtzman (T)
Kazachstania Zubkova (T)
Kluyveromyces van der Walt (T)
Lachancea Kurtzman (T)
Nakaseomyces Kurtzman (T)
Naumovozyma Kurtzman (T)
Saccharomyces Meyen ex Reess (T)
Tetrapisispora Ueda-Nishimura & Mikata emend. Kurtzman (T)
Torulaspora Lindner (T)
Vanderwaltozyma Kurtzman (T)
Zygosaccharomyces Barker (T)
Zygotorulaspora Kurtzman (T)
Saccharomycodaceae Kudryavtsev Hanseniaspora Zikes (T)
Kloeckera Janke (A)
Saccharomycodes E.C. Hansen (T)
Saccharomycotina and Taphrinomycotina: The Yeasts and Yeastlike Fungi of the Ascomycota
(continued)
15
Table 1.1 (continued)
16
Class Order Family Genus

Saccharomycopsidaceae von Arx & Saccharomycopsis Schiönning (T)
van der Walt
Trichomonascaceae Kurtzman & Blastobotrys von Klopotek (A)
Robnett Diddensiella Péter, Dlauchy & Kurtzman (T)
Spencermartinsiella Péter, Dlauchy, Tornai-Lehoczki,
M. Suzuki & Kurtzman (T)
Starmerella Rosa & Lachance (T)
Sugiyamaella Kurtzman & Robnett (T)
Trichomonascus H.S. Jackson emend. Kurtzman & Robnett (T)
Wickerhamiella van der Walt (T)
Yarrowia van der Walt & von Arx (T)
Zygoascus M.Th. Smith (T)
Saccharomycetales incertae sedis Ascobotryozyma J. Kerrigan, M.Th. Smith & J.D. Rogers (T)
Babjeviella Kurtzman & M. Suzuki (T)
Botryozyma Shann & M.Th. Smith (A)
Candida Berkhout (A) pro parte
Citeromyces Santa Marı́a (T)
Coccidiascus Chatton emend. Lushbaugh, Rowton & McGhee (T)
Komagataella Y. Yamada, Matsuda, Maeda & Mikata (T)
Kuraishia Y. Yamada, Maeda & Mikata (T)
Macrorhabdus Tomaszewski, Logan, Snowden, Kurtzman &
Phalen (A)
C.P. Kurtzman and J. Sugiyama
Nadsonia Sydow (T)

Nakazawaea Y. Yamada, Maeda & Mikata (T)
Pachysolen Boidin & Adzet (T)
Peterozyma Kurtzman & Robnett (T)
Schizoblastosporion Ciferri (A)
Sporopachydermia Rodrigues de Miranda (T)
Trigonopsis Schachner emend. Kurtzman & Robnett (A)
a
(A)¼anamorphic genus, (T)¼teleomorphic genus
b
Anamorphic and teleomorphic genera are placed together in the same family when relationships are known. For many anamorphic and teleomorphic genera, phylogenetic relationships
are unclear and the genera are placed in Saccharomycetales incertae sedis until family relationships become known. The polyphyletic anamorphic family Candidaceae is not listed in this
table
c
Classification is based on the following publications: Hibbett et al. (2007), Kurtzman (2003), Kurtzman and Robnett (2003, 2007, 2010, 2013), Kurtzman and Suzuki (2010), and Kurtzman
et al. (2007, 2008)
d
Placement in the family Debaryomycetaceae is tentative
e
Placement of the genera Brettanomyces and Dekkera in the family Pichiaceae is tentative
Fig. 1.3 Phylogenetic resolution of members of the included vs. excluded) are indicated by thick lines.
Ascomycota into three major clades (Pezizomycotina, Dotted arrows indicate alternative groupings found
Saccharomycotina, Taphrinomycotina) from a multi- with 70 % bootstrap support: I¼parsimony analysis
gene analysis based on the 50 % majority rule consen- with third codon position; II¼both parsimony and NJ
sus of 18,000 Bayesian MCMCMC generated trees. In analyses with third codon position; III¼NJ analysis
this analysis, the alternate positions for Saitoella com- without third codon position, which demonstrates the
plicata provide an example of the effect that weak basal impact of data set composition on the outcome of the
lineages have on taxon placement. Numbers on analysis. For details and GenBank accession numbers
branches are Bayesian posterior probability. Numbers of the four genes (SSU rRNA, D1/D2 LSU rRNA, RNA
separated by a slash (/) indicate bootstrap values based polymerase 2, and beta-tubulin) used in this analysis,
on parsimony analysis with third codon position/parsi- see Sugiyama et al. (2006). Additionally, see Assem-
mony analysis without third codon position/neighbor bling the Fungal Tree of Life Web site (http://aftol.
joining (NJ) analysis with third codon position/NJ anal- org/data.php) and the GenBank homepage (http://
ysis without third codon position. An asterisk (*) indi- www.ncbi.nih.gov/Genbank/index.html). Modified
cates lack of bootstrap support. Branches supported by from Sugiyama et al. (2006); reproduced with permis-
95 % posterior probability and 70 % bootstrap sion from Mycologia. @The Mycological Society of
value in all analyses (parsimony vs. NJ, third codon America
families is tentative for many of the taxa. The B. Classification

analyses presented in Figs. 1.3 and 1.4a, b are
examples of this issue, and the uncertain place- Two events are having a profound impact
ment of the anamorphic genus Saitoella has on the classification of fungi. The first of
been indicated in Fig. 1.3, whereas Fig. 1.4b these, as discussed earlier, is gene sequence
suggests that Taphrinomycotina may consist analyses, which have changed our understand-
of two clades. However, other multigene ana- ing of relationships among members of
lyses have supported the monophyly of Taph- Ascomycota. The genera accepted in Saccharo-
rinomycotina (Liu et al. 2009; Schoch et al. mycotina and Taphrinomycotina are listed
2009). in Table 1.1, and most have now been
Fig. 1.4 (continued)
circumscribed from multigene phylogenetic may now include species with known sexual
analyses. Phylogenetic relationships among states (teleomorphs) as well as species for
the genera are shown in Fig. 1.4a, b. This analy- which sexual states have not been discovered
sis is based on five gene sequences, but boot- (anamorphs). With this change, genera will be
strap support for placement of some genera in phylogenetically circumscribed to include
the phylogenetic tree is weak and their assign- related species whether or not sexual states are
ment to families is tentative. known, and the terms anamorph and teleo-
The second event that is having a major morph will have no status in genus descrip-
impact on the classification of fungi is the result tions. Because this change in rules for
of recent changes in the International Code of classification is so new, few yeast genera have
Nomenclature for algae, fungi and plants (Mel- been recircumscribed to include both sexual
bourne Code, e.g., Hawksworth 2012; Knapp and asexual species. For this reason, the terms
et al. 2011; Norvell 2011). The genera of fungi anamorph and teleomorph are being used in
Fig. 1.4 (a) Phylogenetic relationships among type spe- indicate the coenzyme Q value for each species. Y and
cies of ascomycetous yeast genera and reference taxa YB prefixes are NRRL strain numbers. Type species of
determined from maximum-likelihood analysis using the genera Ascobotryozyma (anamorph, Botryozyma),
concatenated gene sequences for LSU rRNA, SSU Coccidiascus, Endomyces, Helicogonium, Phialoascus,
rRNA, EF-1a, RPB1, and RPB2. Filobasidiella neofor- Macrorhabdus, and Schizoblastosporon (teleomorph,
mans was the designated outgroup species in the anal- Nadsonia), several of which are not known from cul-
ysis. Names in bold font are type species of currently ture, were not included in the analysis (Kurtzman and
recognized genera, whereas names in standard font are Robnett 2013). Basal lineages of some taxa are not well
not type species. Pneumocystis is represented by the resolved, which demonstrates the need to include addi-
type species, but not the type strain. Protomyces and tional gene sequences in analyses. Because of this,
Taphrina are not represented by type species. Boot- many family assignments are tentative. Clade 1, Sac-
strap values (1,000 replicates) >50 % are given at charomycetaceae and Saccharomycodaceae (Saccharo-
branch nodes. Strain accession numbers are NRRL mycodes and Hanseniaspora, which appear more
unless otherwise indicated. Designations in brackets closely related in some analyses); Clade 2, Phaffomyce-
this chapter to provide a reference to the classi- gies is unknown, but the recent description of
fication of yeasts presented in the recently pub- Archaeorhizomyces (the new class Archaeorhi-
lished fifth edition of The Yeasts, A Taxonomic zomycetes) (Rosling et al. 2011), an early
Study (Kurtzman et al. 2011), which was diverging and widely distributed genus, sug-
prepared before the new code was adopted. gests that more effective isolation methods
will reveal Taphrinomycotina to be a larger
1. Saccharomycotina group of species than previously thought.
Taphrinomycotina are characterized by
Many of the teleomorphic clades of Saccchar- ascosporic states that lack ascogenous hyphae.
omycotina include species of Candida. Under Asexual reproduction is by budding or fission.
the old code, the genus Candida was a “dump- With the exception of Neolecta, an apothecial
ing ground” for budding yeasts that do not ascomycete genus (Landvik 1996; Landvik et al.
form ascospores, and it represents a polyphy- 1993), neither ascomata nor conidiomata are
letic group of species. With the introduction of formed. Saccharomycotina have cell walls that
sequence analysis, many species of Candida are show two layers and undergo holoblastic con-
seen to be members of teleomorphic clades and, idiogenesis, in contrast to basidiomycetous
because of the aforementioned changes in the yeasts, which characteristically have multilay-
code, will be transferred to those genera. How- ered cell walls and enteroblastic, repetitive per-
ever, other Candida species are in clades with current conidiogenesis (Moore 1987). However,
no known ascosporic state and will be classified Saitoella complicata and Taphrina wiesneri are
in new genera. What has become apparent from exceptional because, although they have two-
recent research is that much of the future clas- layer cell walls, they show enteroblastic bud-
sification of yeasts will rest on the phylogenetic ding (Goto et al. 1987; Sjamsuridzal et al. 1997).
analysis of gene sequences rather than on the
phenotypic characters that we observe on a Originally, it was thought that Taphrina and Schizosac-
petri dish or under the microscope. charomyces lacked chitin in their cell walls (e.g.,
Cavalier-Smith 1987), thereby separating them from
other fungi, but Sietsma and Wessels (1990) reported
2. Taphrinomycotina glucosaminoglycan in Schizosaccharomyces pombe.
Bowen et al. (1992) determined the DNA sequence of
On the basis of phylogenetic analyses of various a chitin synthase fragment from S. pombe, and Nishida
gene sequences, members of the subphylum and Sugiyama (1994a) demonstrated DNA sequences
Taphrinomycotina are composed of the early for chitin synthase fragments in Taphrina wiesneri,
diverging and biologically diverse members of Protomyces inouyii, and Saitoella complicata. Further-
more, Garner et al. (1991) reported that chitin is an
Ascomycota (e.g., Kurtzman and Robnett 1998; integral part of the cell wall of Pneumocystis carinii
Kurtzman and Sugiyama 2001; Sugiyama et al. trophozoites and cysts. The carbohydrate composition
2006). The number of known taxa assigned to of cell walls from Taphrina, Protomyces, and Saitoella is
Taphrinomycotina is surprisingly small consid- characterized by rhamnose, glucose, and mannose
ering that the more recently evolved Saccharo- (Prillinger et al. 1990; Sugiyama et al. 1985), but Schi-
zosaccharomyces does not contain rhamnose (Prillinger
mycotina and Pezizomycotina are much more et al. 1990). Members of Taphrinomycotina show a
species rich. Whether this reflects an absence of negative diazonium blue B (DBB) reaction, as is typical
species diversity or ineffective isolation strate-
Fig. 1.4 (continued) taceae; Clade 3, Komagataella; Debaryomycetaceae, Metschnikowiaceae, and other
Clade 4, Saccharomycopsis (Saccharomycopsidaceae) related taxa; Clade 7, Alloascoidea, an Ascoidea-like
and Ascoidea (Ascoideaceae); Clade 5, Pichiaceae and new genus; Clade 8, Sporopachydermia; Clade 9, Dipo-
closely related genera. (b) Phylogenetic relationships dascaceae and Trichomonascaceae; Clade 10, Trigonop-
among type species of ascomycetous yeast genera and sis and Botryozyma; Clade 11, Lipomycetaceae; Clade
reference taxa determined from maximum likelihood 12, Taphrinomycotina, which in this analysis shows a
analysis using concatenated gene sequences for LSU dichotomy but in some other analyses all genera share a
rRNA, SSU rRNA, EF-1a, RPB1, and RPB2. Clade 6, common lineage
of Saccharomycotina (Kurtzman and Sugiyama 2001; analyses (Kurtzman and Robnett 1991, 1998;
Kurtzman et al. 2011; Sugiyama and Nishida 1995). Naehring et al. 1995). Strains of Schizosacchar-
omyces are isolated from high sugar substrates,
Order Pneumocystidales. Pneumocystis such as fruit juices, honey, dried fruits, and tree
carinii is a principal causal agent of pneumonia fluxes. Schizosaccharomyces is widely used in
in patients with HIV/AIDS, and for many years studies of molecular biology.
this organism was considered to be a proto- Order Neolectales. The genus Neolecta is
zoan. Edman et al. (1988) showed P. carinii to characterized by clavate, stalked apothecia and
be a fungus based on 18S sequence compari- cylindrical aparaphysate eight-spored asci.
sons. Watanabe et al. (1989) suggested from 5S Korf (1973) placed the genus in Helotiales of
rRNA analysis that Pneumocystis is closely the Discomycetes, whereas Redhead (1977) cre-
related to Zygomycotina, but ultrastructural ated the new family Neolectaceae and tenta-
studies showed cell division by fission (Yoshida tively placed it in Lecanorales. Landvik et al.
1989). Taylor et al. (1994) and Sugiyama and (1993) and Landvik (1996) proposed the new
Nishida (1995) suggested that P. carinii and order Neolectales because their SSU rRNA gene
S. pombe have a similar life cycle, and Nishida sequence analyses placed Neolecta outside
and Sugiyama (1994b) placed Pneumocystis in of Pezizomycotina. Because of these and other
Taphrinomycotina. Subsequently, Hibbett et al. comparisons, Eriksson and Hawksworth (1995)
(2007) accommodated the genus in the class and Sjamsuridzal et al. (1997) placed the genus
Pneumocystidiomycetes in the subphylum Neolecta in what is now Taphrinomycotina.
Taphrinomycotina. According to Redhead’s (1977) description
and illustrations, asci of Neolecta vitellina occa-
Pneumocystis was included for the first time in The sionally are filled with numerous conidia,
Yeasts, A Taxonomic Study, 5th Edition, in which Cush- which has also been observed in Taphrina
ion and Keely (2011) fully redescribed and accepted five
species, i.e., the type species P. carinii and four other
(cf. Sugiyama 1998).
species. Order Taphrinales. Included in this order
are two families, Protomycetaceae and Taphri-
According to the genus diagnosis (Cushion naceae (Table 1.1). Protomycetaceae includes
and Keely 2011), “no species of Pneumocystis six genera, Burenia, Protomyces, Protomycopsis,
has been continuously cultivated outside the Saitoella, Taphridium, and Volkartia. Some of
mammalian lung,” asexual reproduction is by these genera are well studied, others are not,
binary fission, and eight ascospores are pro- and we will discuss three of the better studied
duced within an ascus as a result of meiosis. genera of the order. Protomyces is parasitic on
The correct name for the taxon called Pneumo- plants mainly of Apiaceae (Umbelliferae) and
cystis carinii from human lungs is Pneumocystis Asteraceae (Compositae). Protomyces inouyei,
jirovecii, whereas the name P. carinii is only for example, attacks the composite Youngia
found in the lungs of immunosuppressed rats japonica in Japan, causing a gall on stems
(Cushion and Keely 2011). (Tubaki 1957). The thick-walled resting spores
Order Schizosaccharomycetales. Schizosac- that are formed in infected plant tissue germi-
charomyces is saprotrophic and undergoes nate, giving an ascuslike tube in which spores
asexual reproduction by fission. The genus is develop (Fig. 8 in Kurtzman and Sugiyama
comprised of four species: S. japonicus, S. octos- 2001). Protomyces species generally grow at
porus, S. pombe, and S. cryophilus (Vaughan- lower temperatures (15–25 C) and produce
Martini and Martini 2011). Yamada and Banno pigmented colonies, much as is seen for Taph-
(1987) reassigned S. octosporus and S. japonicus rina and Saitoella, which also tend to grow at
to other genera on the basis of differences in lower temperatures.
ascospore morphology, ubiquinone type, and
cellular linoleic acid content, but this separa- The SSU rRNA gene sequences from most species of
Protomyces contain one or two group I introns (Nishida
tion was not supported by rRNA gene sequence and Sugiyama 1995; Nishida et al. 1993, 1998, 2000),
whereas introns have not been detected in the rRNA (Kurtzman 1993), Protomycetales appear to be synony-
genes of Taphrina species. mous with Taphrinales, hence placement of the preced-
ing genera in a single order, Taphrinales. This
treatment has been supported by the multigene phylo-
The genus Taphrina, which may include as genetic analysis of Hibbett et al. (2007).
many as 95 species, is parasitic on a wide vari-
ety of vascular plants, primarily ferns, the
The history of studies on the anamorphic
Rosales, and the Fagales (Kramer 1973; Mix
genus Saitoella was fully described by
1949). The morphology and life cycles of Taph-
Sugiyama et al. (1993). The first known species
rina spp. are unique. Species are dimorphic
of this genus, S. complicata, was initially iden-
with a saprotrophic haploid, uninucleate state,
tified as Rhodotorula glutinis (Goto and
and a parasitic ascogenous binucleate mycelial
Sugiyama 1970), but later Goto et al. (1987)
state that develops in the host tissue. Moore
proposed that S. complicata was closely related
(1990) described the genus Lalaria for the
to Taphrinales. This supposition, based on phe-
anamorphic states of Taphrina, some of which
notypic comparisons, was verified from ana-
are occasionally isolated from the environment
lyses of SSU rRNA gene sequences (Nishida
as a yeast state. Taphrina deformans, which
and Sugiyama 1993, 1994b; Nishida et al. 1993;
causes peach leaf curl, is especially well studied
Sugiyama et al. 1993). On the basis of multigene
(Kramer 1960; Martin 1940; Syrop and Beckett
phylogenetic analysis, Saitoella appears to be
1976). Another well-studied species is T. wies-
a member of Taphrinomycotina (Fig. 2 in
neri (¼T. cerasi), which attacks the Japanese
Sugiyama et al. 2006). A second species of
cherry tree (Cerasus yedoensis), causing
Saitoella was reported and described as S. color-
witches’ broom (Fig. 9 in Kurtzman and
adoensis (Kurtzman and Robnett 2012). This
Sugiyama 2001; Tubaki 1978). Ascospores of
new species was isolated from insect frass
T. wiesneri bud within asci on the leaves of
occurring in an Engelmann spruce (Picea engel-
the host plant (Fig. 10 in Kurtzman and
mannii) growing in the state of Colorado in the
Sugiyama 2001). Characteristic of most other
USA, and multigene analysis showed that
Taphrina spp., the budding haploid phase of
S. complicata and S. coloradoensis are closely
T. wiesneri appears as pigmented yeastlike
related. The teleomorph of Saitoella is
colonies on agar media. The color is due to
unknown, and the evolutionary relationships
the formation of carotenoid pigments.
between Saitoella and other members within
The yeast states of Taphrina (Fonseca and
Taphrinomycotina remain uncertain. At pres-
Rodrigues 2011), Protomyces (Kurtzman 2011),
ent, the genus Saitoella has two assigned spe-
and Saitoella (Sugiyama and Hamamoto 2011)
cies described from three strains (Kurtzman
are similar culturally, biochemically, and
and Robnett 2012; Sugiyama and Hamamoto
chemotaxonomically. Especially noteworthy is
2011), but the genus may be larger. Allison
the fact that the cell walls of the three genera
et al. (2010) cloned DNA from soil sampled in
are two-layered and typical of ascomycetous
an Alaskan (USA) boreal forest. Of the 433
yeasts, whereas conidiogenesis (budding) is
fungal sequences obtained, 3 (GU212336,
enteroblastic and typical of basidiomycetous
GQ892426, GQ892425) were identical to the
yeasts (Goto et al. 1987; Sjamsuridzal et al.
D1/D2 LSU rRNA gene sequence of S. compli-
1997).
cata.
New major lineages within Ascomycota.
Phylogenetic analysis of SSU rRNA gene sequences Several studies on fungal diversity in soils
from 14 species of Taphrina and 4 species of
Protomyces verified the presence of two genera and
using a total environmental DNA sampling
demonstrated that they are monophyletic (Sjamsurid- have revealed potentially new early diverging
zal et al. 1997). On the basis of rRNA sequence analyses ascomycete lineages independent of Taphrino-
mycotina (Blackwell 2011; Jumpponen and plate techniques can be used for quantitative
Johnson 2005; Schadt et al. 2003; Sugiyama studies.
et al. 2006; Vandenkoornhuyse et al. 2002). Temperatures for isolation and growth.
Subsequently, Porter et al. (2008) suggested a Cultures are usually incubated at 20–25 C
major new clade as Soil Clone Group I (SCGI) because most yeasts are mesophilic; however,
of Ascomycota equivalent to a subphylum. Cur- temperatures between 4 and 15 C are essential
rently the SCGI clade is known only from for psychrophilic taxa. Higher temperatures, in
sequence data. As discussed earlier, the genus the range of 30–37 C, are often required for
Archaeorhizomyces (Rosling et al. 2011), which yeasts that are strictly associated with warm-
was assigned to Archeorhizomycetes, appears blooded sources. Among these species are
to be a member of the Taphrinomycotina. Both human and animal pathogens assigned to
molecular- and cultivation-based methods for Candida, Kazachstania, Macrorhabdus, and
isolation of novel ascomycete taxa from envir- Cyniclomyces. The latter two genera have
onments are needed to elucidate the whole exceptional nutritional requirements (Kurtz-
phylogenetic picture of Ascomycota. man et al. 2011). Incubation temperatures can
also serve to selectively isolate particular
groups of species.
Acidified media (pH 3.5–5). Acidified
VII. Isolation, Maintenance, and media provide selective isolation, and hydro-
Culture Availability chloric and phosphoric acids are often used to
acidify the media. Organic acids, such as acetic
A. Isolation acid, are not recommended because they are
only slightly dissociated at pH 3.5–5, and high
Yeasts are recovered from a wide range of concentrations of undissociated acids have an
aquatic, marine, and terrestrial habitats, as inhibitory effect on many yeasts. Exceptions
well as from the atmosphere. Many yeasts are include Zygosaccharomyces bailii, Z. bisporus,
widely distributed, whereas others appear to be and some strains of Pichia membranifaciens
confined to specific habitats. Yeasts seldom (Yarrow 1998).
occur in the absence of either molds or bacteria. Agar in media with a low pH is hydrolyzed
Consequently, selective techniques are often when autoclaved. To resolve this problem, ster-
used for the recovery of yeasts. The composi- ilized molten agar is cooled to approximately
tion of selective media is determined by the fact 45 C, and a predetermined volume of acid is
that yeasts are generally capable of developing added. The medium and acid are quickly but
at pH levels and water activities that reduce or gently mixed to avoid air bubbles and immedi-
inhibit the growth of bacteria. Antibiotics may ately poured into petri dishes. The addition of
also be used to suppress bacteria. Fungistatic approximately 0.7 % (v/v) 1 N hydrochloric
agents for the suppression of molds can be acid to YM agar or glucose–peptone–yeast
used, but these compounds may also inhibit extract agar usually gives the desired pH of
yeasts. In addition to the methods discussed 3.7–3.8. Many yeasts can be recovered at
in this chapter, the publications of Beech and pH 3.7, but some species, such as those of the
Davenport (1971), Deak (2003), and Kurtzman genus Schizosaccharomyces, are inhibited by
et al. (2011) discuss the isolation of yeasts from high acid media, and moderately acidic media
natural habitats, and the publications of Buck- with a pH in the range 4.5–5.0 will give a higher
ley (1971) and Staib et al. (1989) provide meth- recovery.
ods for isolating clinical yeasts. When yeasts When yeasts are present in low numbers,
are present in high numbers, they may be population size can be increased by incubating
isolated by directly plating the material, or sus- the sample in a liquid medium at a pH of
pensions of the material, on acidified agar 3.7–3.8. The use of antibiotics or high or low
media that may also contain antibiotics or temperatures can provide further selection. The
have other selective formulations. Dilution development of molds can be restricted by
excluding air from the culture by pouring ster- cycloheximide (100–500 mg/L), cyclosporin A
ile paraffin oil over the surface of the medium (4–10 mg/L), and pimaricin (5–100 mg/L).
to form a layer about 1 cm deep. This procedure Selective media can be used to isolate
favors the development of fermentative strains genera, species, or groups of species with a
but may fail to recover aerobic strains. Another particular property. Van der Walt and van Ker-
method for restricting mold development ken (1961) isolated species of Dekkera using
involves incubating flasks of isolation media media containing cycloheximide and sorbic
on a rotary shaker (Wickerham 1951). Molds acid at pH 4.8. Van Dijken and Harder (1974)
in shaken flasks often do not conidiate and tend used a medium containing methanol, cycloser-
to grow as pellets that are outgrown by yeasts. ine, and penicillin G for the isolation of
The yeasts may be separated from the molds methanol-assimilating yeasts of biotechnologi-
either by allowing the pellets of mold to settle cal importance. Various species of the Lipomy-
for a few minutes and then streaking the sus- cetaceae can be recovered from soil and insect
pension of yeasts onto agar in petri dishes or frass by a procedure that depends on the utili-
by removing suspended pellets by filtering zation of thymine as a nitrogen source and
through sterile glass wool. These pregrowth resistance to cycloheximide.
methods cannot be used for the quantitation Isolation using membrane filters. Yeasts
of the initial population size in the sample can be recovered from liquid substrates by
tested. passing the liquid through a 0.45 mm membrane
Osmotic media. Yeasts can often grow on filter (e.g., Mulvany 1969). Solid substrates,
media with concentrations of sugar that are such as soils, can be washed to suspend the
high enough to inhibit the development of yeast cells prior to passing the wash solution
many bacteria. A medium such as glucose–pep- through the membrane. The filters are placed
tone–yeast extract agar or YM agar containing face up on the surface of a selective agar
glucose at a concentration of 30–50 % is medium, followed by incubation at a tempera-
suitable for recovering osmophilic and osmo- ture appropriate for the target organisms.
tolerant yeasts from foodstuffs and juice con- Plates should be inspected daily. This technique
centrates of low water activity. The selective is particularly useful for recovering yeasts when
action of these media can be enhanced by low- they are present in low concentrations, and the
ering the pH to around 4.5. Osmotolerant method can serve as a means to quantitate the
yeasts recovered in this way can usually be densities of yeast communities.
successfully subcultured on media containing Purification of cultures. Isolates are
successively decreasing amounts of sugar, for obtained in pure culture from natural materials
example, 30, 10, 4, and 2 %. or from enriched cultures by streaking on a
Antibiotics and other selective com- suitable medium, such as glucose–peptone–
pounds. Several media containing antibiotics yeast extract agar or YM agar (Kurtzman et al.
have been described (Bills and Foster 2004) 2011). Persistent bacterial contamination can
that can be used to suppress co-occurring often be eliminated by acidifying the media or
microorganisms. Antibacterial antibiotics, by adding antibiotics. Single, well-separated
which are often used, include tetracycline at colonies of each form are selected and streaked
50 mg/L or a combination of penicillin G and again. Twice is generally sufficient to obtain
streptomycin sulfate, each at a concentration of pure cultures. When two or more morphologi-
150–500 mg/L. Many of these antibiotics are cally distinct colonies persistently appear after
heat labile and must be added after the medium replating of a single colony, these may repre-
has been autoclaved and is cool to the touch. In sent morphological or sexual variants of a
contrast, chloramphenicol (actidione) is heat single species. Because of this, it is useful to
stable and can be added to the medium prior initially save multiple colonies from isolations
to sterilization. Antifungal antibiotics, which because some may represent mating types.
suppress filamentous fungi, may also suppress
yeasts. Commonly used antifungals include
B. Maintenance bility. Lyophilized cultures are usually stored in

a refrigerator at 4–5 C.
The maintenance of yeast cultures on a medium
that contains glucose as the only carbon source In the ARS process, ca. 0.1 mL of suspension containing
reduces the risk of changes in growth and cells and bovine serum is added aseptically to the ster-
fermentative patterns due to the selection of ile, labeled lyophil tubes, which have a small cotton
mutants (Scheda 1966). The majority of yeasts plug (Kurtzman et al. 2011). After filling, tubes are
may be stored at temperatures between 4 and inserted into rubber connectors on the manifold of
the lyophil machine. The manifold is lowered so that
12 C and subcultured at intervals of 6–8 the ends of the tubes are immersed in a 50 % ethylene
months. Some yeasts, such as certain Kazach- glycol bath cooled to ca. 30 C with dry ice. Once the
stania spp., need to be subcultured every contents of the tubes are frozen, the bath temperature is
month. Strains of Dekkera and Brettanomyces raised to ca. 20 to 25 C with fresh ethylene glycol,
produce excessive amounts of acetic acid, and and the vacuum pump is started. Drying takes ca. 2 h
and is finalized by raising the manifold and allowing
inclusion of 1–2 % calcium carbonate in the further drying at room temperature. Ampoules are
medium prolongs viability. Nevertheless, these sealed with a gas-oxygen torch. Some lyophil machines
yeasts still need to be subcultured every use larger ampoules that may contain 1 mL of cell
2 months, or sometimes more frequently. suspension. These larger tubes may be initially frozen
Some yeasts lose their ability to produce but do not need immersion in a freezing bath because
evaporation of this larger volume is adequate to keep
ascospores when maintained by serial cultiva- the preparations frozen.
tion on laboratory media, whereas other iso-
lates still sporulate after 50 or more years in A process termed L-drying has been
cultivation. However, for many strains, the abil- successfully used by several large culture collec-
ity to sporulate is either impaired or lost within tions in Japan (Mikata and Banno 1989). The
a period that varies from a few weeks to several process is similar to lyophilization, with the
years. For ascosporogenous heterothallic spe- exception that cells and cryoprotectant are
cies, one mating type may selectively predomi- dried more slowly under vacuum and do not
nate in laboratory culture, which results in the freeze. L-dried ampoules are sealed in the same
loss of the sexual state. For these reasons, it is manner as lyophilized preparations.
important to preserve nomenclatural types and Frozen storage at low temperatures is a
reference strains with one of the more perma- common alternative for preserving microor-
nent conservation techniques as soon as possi- ganisms that do not survive lyophilization or
ble after acquisition. Suitable techniques are L-drying. Storage in 80 and 125 C mechan-
lyophilization (Kirsop and Kurtzman 1988), ical freezers is often satisfactory, but storage at
L-drying (Mikata and Banno 1989), and freez- still lower temperatures is preferable. The
ing in either liquid nitrogen vapor or a mechan- choice is the vapor phase of liquid nitrogen,
ical freezer at temperatures between 60 and which is ca. 180 C. Liquid nitrogen at atmo-
135 C, although liquid nitrogen is preferred. spheric pressure has a slightly lower tempera-
The procedure of lyophilization freeze- ture of 196 C (320 F), but a concern with
dries cultures to an inactive, low-moisture immersion storage is that ampoules occasion-
state. The process starts with suspending ally leak, and when thawed, the trapped liquid
actively growing cultures in a cryoprotectant, nitrogen will expand to gas so rapidly that the
such as sterile bovine serum or skim milk. The ampoules explode.
volume of cryoprotectant depends on the size
of the ampoule used, and ampoules used in the
The general procedure for liquid nitrogen storage of
ARS Culture Collection (NRRL) are made from microorganisms is to suspend cells from an actively
6 mm outside diameter pyrex glass tubing. growing culture in a cryoprotectant such as 10–15 %
Lyophilized strains generally survive for many glycerol. Usually 1 mL of suspension is placed in a 2 mL
decades, though there are exceptions, and the cryoampoule. Ordinarily, the ampoules are transferred
cultures should be checked periodically for via- to a storage box in the liquid nitrogen freezer and
Table 1.2 Some large international culture collections ulations, and some of these regulations have
that maintain a diversity of yeast culturesa developed in recent years because of the threat
Agricultural Research Service Culture Collection of global bioterrorism. Within the USA, ship-
(NRRL) ment of plant and animal pathogens requires a
Peoria, Illinois, USA permit from the USDA Animal and Plant
http://nrrl.ncaur.usda.gov Health Inspection Service (APHIS) (APHIS
All-Russian Culture Collection (VKM) 526 for plant pathogens, APHIS VS 16-3 for
Moscow Region, Pushchino, Russia animal pathogens). A permit is required from
http://www.vkm.ru
the U.S. Public Health Service when a pathogen
American Type Culture Collection (ATCC)
Manassas, Virginia, USA is imported into the USA or when a foreign
http://www.atcc.org pathogen is redistributed within the USA.
Belgian Coordinated Collections of Micro-Organisms Other countries often have their own specific
(BCCM) regulations. Failure to comply with require-
Various cities, Belgium ments can result in the closure of laboratories,
http://bccm.belspo.be/index/php large fines, or even incarceration. The shipment
Centraalbureau voor Schimmelcultures (CBS) of cultures may be governed by the Convention
Utrecht, The Netherlands
http://www.cbs.knaw.nl on Biodiversity, also known as the Rio Treaty.
Japan Collection of Microorganisms (JCM)b Here the concern is that part of a country’s
Tsukuba, Japan national heritage, i.e., unique microbial germ-
http://www.jcm.riken.jp plasm, will be exploited by outside parties with-
National Collection of Yeast Cultures (NCYC) out due compensation. Consequently, before
Norwich, United Kingdom novel or other strains are shipped between
http://www.NCYC.co.uk countries, the requirements of the Rio Treaty
National Institute of Technology Evaluation – must be known. Culture collections that main-
Biological Resource Center (NBRC)c
Chiba, Japan tain a wide diversity of yeasts are listed in
http://www.nbrc.nite.go.jp Table 1.2.
Phaff Yeast Culture Collection, University of California,
Davis, California, USA
http://www.phaffcollection.org
Portuguese Yeast Culture Collection VIII. Future Directions
Caparica, Portugal
http://www.sec-biot@fct.unl.pt Yeasts are central to many present-day agricul-
a
Other culture collections that maintain yeasts can be located tural, medical, and industrial processes, and
at the Web site of the World Federation for Culture Collections with the development of recombinant DNA
(WFCC) (http://www.wfcc.info/datacenters.html) technologies, as well as new societal needs, the
b
Cultures maintained at IAM Culture Collection (IAM) were
role of yeasts in human advancement can be
transferred to JCM in 2007
c
Cultures maintained at Institute for Fermentation, Osaka expected to increase. Rapid DNA sequencing
(IFO), were transferred to NITE-NBRC in July 2002 technologies have made whole genome
sequencing relatively inexpensive, thereby
permitting large-scale comparative genomics
allowed to undergo uncontrolled freezing. Occasion-
ally, the freeze rate needs to be controlled to ensure for yeasts, which will lead to enormous
high cell viability, which can be done using commer- advances in understanding the genetics of
cially available devices that have a programmable freez- metabolic pathways, reproductive processes,
ing rate (see Verkley et al., Chap. 8, this volume). and bioengineering. Molecular systematics will
play a key role in future uses. The application of
molecular methods for strain identification will
markedly assist medical diagnostics as well as
C. Culture Availability and Distribution define industrially and agriculturally important
species, including those important in food
The shipment of cultures is governed by a vari- spoilage and the biocontrol of pests and patho-
ety of national and international laws and reg- gens. Molecular identification methods will
lead to the discovery of numerous new species, chitin synthases. Proc Natl Acad Sci U S A 89:519–
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2 Pezizomycotina: Pezizomycetes, Orbiliomycetes
DONALD H. PFISTER1
CONTENTS 5. Discinaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6. Glaziellaceae. . . . . . . . . . . . . . . . . . . . . . . . . . . 47
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 7. Helvellaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
II. Orbiliomycetes: An Overview. . . . . . . . . . . . . . 37 8. Karstenellaceae . . . . . . . . . . . . . . . . . . . . . . . . 47
III. Occurrence and Distribution . . . . . . . . . . . . . . 37 9. Morchellaceae . . . . . . . . . . . . . . . . . . . . . . . . . 47
A. Species Trapping Nematodes 10. Pezizaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
and Other Invertebrates . . . . . . . . . . . . . . . . . 38 11. Pyronemataceae. . . . . . . . . . . . . . . . . . . . . . 48
B. Saprobic Species . . . . . . . . . . . . . . . . . . . . . . . . . 38 12. Rhizinaceae . . . . . . . . . . . . . . . . . . . . . . . . . . 49
IV. Morphological Features . . . . . . . . . . . . . . . . . . . . 38 13. Sarcoscyphaceae . . . . . . . . . . . . . . . . . . . . . 49
A. Ascomata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 14. Sarcosomataceae . . . . . . . . . . . . . . . . . . . . . 49
B. Asci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 15. Tuberaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
C. Ascospores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 XIII. Growth in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 50
D. Paraphyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 XIV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
E. Septal Structures . . . . . . . . . . . . . . . . . . . . . . . . . 40 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
F. Nuclear Division . . . . . . . . . . . . . . . . . . . . . . . . . 40
G. Anamorphic States . . . . . . . . . . . . . . . . . . . . . . 40
V. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
VI. History of Classification and Current I. Introduction
Hypotheses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
VII. Growth in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 41
VIII. Pezizomycetes: An Overview. . . . . . . . . . . . . . . 41 Members of two classes, Orbiliomycetes and
IX. Occurrence and Distribution . . . . . . . . . . . . . . 41 Pezizomycetes, of Pezizomycotina are consis-
A. Parasitic Species . . . . . . . . . . . . . . . . . . . . . . . . . 42 tently shown in molecular phylogenetic studies
B. Mycorrhizal Species . . . . . . . . . . . . . . . . . . . . . 42 to diverge at the base of the Pezizomycotina
C. Saprobic Species . . . . . . . . . . . . . . . . . . . . . . . . . 42
X. Morphological Features . . . . . . . . . . . . . . . . . . . . 42 phylogeny (Gernandt et al. 2001; Hansen and
A. Ascomata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Pfister 2006; Spatafora et al. 2006). Kumar et al.
B. Asci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 (2012) give ultrastructural data on the septal
C. Ascospores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 structure, ascus wall construction, and nuclear
D. Paraphyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 division that suggest the Orbiliomycetes repre-
E. Septal Structures . . . . . . . . . . . . . . . . . . . . . . . . . 44
F. Anamorphic States. . . . . . . . . . . . . . . . . . . . . . . 44 sent the earliest diverging lineage of the Pezi-
XI. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 zomycotina. The relationship of these two
XII. History of Classification and Current classes and the family diversity within the Pezi-
Hypotheses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 zomycetes are shown in Fig. 2.1. These two
A. Families of the Pezizomycetes . . . . . . . . . . 46 groups show very few shared characteristics
1. Ascobolaceae . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2. Ascodesmidiaceae. . . . . . . . . . . . . . . . . . . . . 46 other than the formation, for the most part, of
3. Caloscyphaceae. . . . . . . . . . . . . . . . . . . . . . . . 46 well-developed apothecial ascomata in which
4. Chorioactidaceae . . . . . . . . . . . . . . . . . . . . . . 46 asci are generally arranged in a hymenium
with paraphyses. Orbiliomycetes was recog-
1 nized as a class only recently (Baral 2003).
Farlow Herbarium and Library of Cryptogamic Botany,
Department of Organismic and Evolutionary
There is a single order, Orbiliales, and a single
Biology, Harvard University, Cambridge, MA, 02138, USA; family, Orbiliaceae, with perhaps 300 species
e-mail: dpfister@oeb.harvard.edu currently arranged in three teleomorphic

36 D.H. Pfister
Fig. 2.1 A diagrammatic representation of the relation- received maximum parsimony, posterior probability,
ships of the Pezizomycetes and Orbiliomycetes, includ- and maximum-likelihood support 75 %, 95 %, and
ing the families of the Pezizomycetes, based on Hansen 75 %, respectively, are in bold
et al. (2013) and Pfister et al. (2013). Branches that
Pezizomycotina: Pezizomycetes Orbiliomycetes 37
genera. An early diverging position of the class In this chapter, the two classes are treated
has been confirmed by morphological studies independently in separate sections.
and molecular phylogenetic analyses (Kumar
et al. 2012; Spatafora 2006; Wang et al. 2006).
Previously, the Orbiliaceae was placed among II. Orbiliomycetes: An Overview
the Helotiales as a family based on Nannfelt’s
(1932) classic work on the nonlichenized inop-
The family Orbiliaceae was proposed by Nann-
erculate discomycetes. The Pezizomycetes
feldt (1932) and was restricted to those fungi
have long been recognized to include a single
with small so-called inoperculate asci, minute
order, the Pezizales, and include 16 families
ascospores, and a waxy texture. The asci and
and approximately 1,200 species. The group,
paraphyses are often agglutinated. Nannfeldt
in whole or in part, has been the subject of
illustrated several species showing details of
recent surveys (Hansen and Pfister 2006; Har-
the thin-walled globose to angular cells of the
rington et al. 1999; Perry et al. 2007; Pfister
excipulum. A monograph on the genus Orbilia
et al. 2008).
by Svrcek (1954) included 16 species that were
In regard to morphological characteristics
distinguished based on ascospore size and
and ecology, the two classes also are distinct.
shape, the color of the ascomata, and the form
The Orbiliomycetes are saprobic and in some
of the paraphyses. The groundbreaking work of
cases supplement their nutrition through the
Hans-Otto Baral and his collaborator, Guy Mar-
capture and consumption of small inverte-
son, has led to a refinement of the family and
brates. Their spores are small, nonseptate
recognition of many more species. Kirk et al.
(save for a single example), uninucleate, and
(2008) proposed 288 species, but the extent of
contain a membrane-bound structure, the
the diversity to be described is in flux. A mono-
spore body (Baral 1994; Benny et al. 1978;
graph has been in preparation by Baral for
Kumar et al. 2012). Spores are discharged
many years. Modern methods employ morpho-
actively. Asci are small and generally lack a
logical and histochemical characters previously
well-developed apical apparatus. Anamorphic
not used and incorporate ecological characters
states are frequently formed, and their number
as well. Of particular note is the reliance on
has increased steadily in the literature in recent
fresh, living material (Baral 1992). There has
years. Species are found on dung, on wet wood
been a burst of activity focusing on the descrip-
on the forest floor, in water-soaked areas where
tion of teleomorphic taxa and associated ana-
they are somewhat ephemeral, or on dead wood
morphs (e.g., Kohlmeyer et al. 1998; Liu et al.
hanging in trees where, because of their
2005a, b, 2006; Mo et al. 2005; Pfister 1994;
drought resistance, they are persistent (Baral
Pfister and Liftik 1995; Qiao et al. 2011; Qin
2011). No species are plant parasitic.
et al. 2010; Su et al. 2011; Tanabe et al. 1999;
The Pezizomycetes are saprobic, plant par-
Webster et al. 1998; Wu et al. 2007; Yang and
asitic, or associated with plants as ectotrophic
Liu 2005; Yu et al. 2007a, b, c, 2011; Zhang et al.
mycorrhizae (Hansen and Pfister 2006; Teder-
2007), and in this work there has been an
soo et al. 2006; Wei et al. 2010). The ascomata
intense interest in the diversity and functional
are always to some degree ephemeral. Spores
morphology of the anamorphic states. This is
are large, generally more than 10 mm and may
particularly the case regarding those that
reach a length of 60 mm, and single-celled.
capture nematodes, i.e., species referred to
Spores generally are actively discharged
Arthrobotrys and allied form genera.
through a well-developed opening created by
the rupture of the ascus wall forming an oper-
culum that is either terminal or eccentric in
position. The trufflelike habit has developed III. Occurrence and Distribution
independently in several lineages across the
class (Læssøe and Hansen 2007). These hypo- Members of the Orbiliomycetes are found
geous taxa generally do not have active spore around the world in boreal to tropical habitats.
release. Furthermore, several species complexes, such
38 D.H. Pfister
as O. luteorubella and O. xanthostigma, that addition to trapping nematodes, some hypho-

represent morphologically very similar collec- mycetes in the orbiliaceous lineage trap other
tions seem to be found broadly distributed small invertebrates such as collembolans and
around the world and across latitudes. There rhizopods (e.g., Barron 1981; Drechsler 1936,
are two major ecological groups—those that 1937b). Even though these fungi may occur on
occur in moist habitats such as on dung, old wood or cellulose-rich substrates, there is evi-
polypores, and moist wood on the forest floor dence that few of the nematode-trapping fungi
or near streams and species that are drought are able to break down cellulose (Park et al.
tolerant. These drought-tolerant species are 2002).
found on dead twigs and small woody branches
in the canopy of living trees. Often species of
both ecological types are found in association B. Saprobic Species
with unicellular green algae, but there is no
evidence that the fungi are lichenized. Until the work of Baral (2011) and Marson, most
studies of diversity within the Orbiliomycetes
were focused on those species on moist sub-
A. Species Trapping Nematodes strates. The discovery that these fungi are
and Other Invertebrates highly diverse in the dry habitats of hanging
branches in living trees has greatly expanded
Since the classic work of Drechsler (1937a) on the number of species that have been described
the nematode-trapping hyphomycetes, these through intense collecting in such habitats.
fungi have attracted many researchers to this Although these species are treated here as
fascinating system. The work of Barron (1977, saprobic, there is little clear evidence of their
1981) further set the stage for ecological and mode of nutrition. Drechsler (1938) discussed
descriptive work on these fungi. Pfister (1994, at length hyphomycetes that are parasitic on
1997) and Pfister and Liftik (1995) documented oospores of Oomycota; these also seem now to
the life history connection between these be clearly referable to the Orbiliaceae. These
nematode-trapping hyphomycetes and teleo- fungi were “considered from a morphological
morphic members of the Orbiliomycetes. The viewpoint. . . [to] fit acceptably in the preda-
several anamorphic form genera involved are cious series” (Drechsler 1938). Whether they
discussed in what follows. fall in the main clade of nematode trapping
Trapping of nematodes and other inverte- Orbiliaceae or outside it remains to be studied,
brates is accomplished by the formation of but that a variety of life styles and biotic modes
constricting rings, hyphal networks, or sticky are found within the family is unquestionable.
knobs. These trapping structures are often, but It is important to note as well that no plant
not exclusively, initiated in the presence of parasitic species have been discovered so far
prey. When nematodes become ensnarled in in this lineage.
the trap, the fungus then grows and penetrates
the body of its prey and proliferates within it.
Conidia are formed in the presence or absence IV. Morphological Features
of nematodes and are generally dispersed
across the medium, that is, they are not con- A. Ascomata
centrated around trapped nematodes (Barron
1977). Earlier classification schemes for these Apothecial ascomata range in size from 0.5 to
fungi relied primarily on conidial morphology, 5 mm and are saucer-shaped to pulvinate.
particularly spore shape and septation. Phylo- Macroscopically they have been described as
genetic studies have indicated that the form of appearing waxy at sight or to touch. This is
the trapping devices gives important informa- a reference at least in part to their shiny, trans-
tion for classification (Hagedorn and Scholler lucent appearance. The sterile tissues of the
1999; Rubner 1996; Scholler et al. 1999). In ascomata are composed of an outer layer, or
ectal excipulum, of globose or angular cells that enhanced magnification. Spores are globose,
in some species give rise to superficial hairs. ellipsoidal, ovoid, reniform, acicular, or filiform
These cells may contain one or more cytoplas- and mostly smooth, although spore wall
mic bodies that may take on several different ornamentations are known, for example, in
shapes but disappear when material is mounted O. xanthostigma. Ascospores of the Orbiliomy-
in KOH. Cells in the outer excipulum may be cetes contain an inclusion, the spore body. This
encrusted with granules or produce glassy pro- membrane-bound body was suggested to derive
jections or the cells may be embedded in an from the plasma membrane by Baral (2003),
amorphous matrix. The inner layers of the but Kumar et al. (2012) and Benny et al.
ascomata are of interwoven hyphae or com- (1978) showed that the spore body is derived
pressed angular cells (Nannfeldt 1932). from mitochondria. The spore body is attached
to the cell membrane and is surrounded by a
rough endoplasmic reticulum (Benny et al.
B. Asci 1978). The contents are electron dense (Benny
et al. 1978). Under a light microscope it is seen
The asci of members of this class are small cylin- in unstained living spores as a refractive body.
drical to broad cylindrical with or without cro- Its appearance can be enhanced by application
ziers; in some cases they have elongate, forked of aqueous brilliant cresyl blue or other vital
bases. Asci contain eight to many ascospores per stains. It disappears in dead spores and can be
ascus. Asci are inamyloid. The apex ranges from destroyed by application of KOH. The spore
hemispherical conical to truncate. Walls are thin, body is a synapomorphy for the class and can
composed of two layers in transmission electron take on many different forms, from globose to
microscopy (Benny et al. 1978; Kumar et al. tear-shaped to elongate. Its shape and orienta-
2012). The apex is generally little developed. tion is consistent within the ascospores of a
Benny et al. (1978) reported no pores; Kumar particular species. The shape and position of
et al. (2012) found an electron-light zone at the the spore body is considered a critical taxo-
apex. In some taxa, an opening develops on the nomic character. Generally spores have a single
shoulder of the truncate asci (Zhang et al. 2007), spore body, but an example of a spore with two
and this was confirmed by Kumar et al. (2012). In spore bodies is known (Wu et al. 2007). As
some species, the apical walls may be thickened mentioned previously, its contents are electron
to form a cap (Kohlmeyer et al. 1998), as in dense; it does not appear to be composed of
Orbilia subgenus Hemiorbilia, in which the lipids but may be proteinaceous (Kumar et al.
apex of the ascus is thickened (Baral 2011). In 2012). No function has been attributed to the
all cases, asci appear to dehisce by irregular tear- spore body, but Benny et al. (1978) offered
ing. Kumar et al. (2012) found similarities three possible functions: for flotation and bal-
between the asci of the Orbilia species they stud- ance in a liquid environment before and during
ied and members of the Taphrinomycotina. Asci germination, storage of waste products, and
and paraphyses may be embedded in a gelati- storage of materials for use during germination.
nous matrix that causes them to stick together None of these functions has been documented,
and not fan out when crushed for microscopic and ultimately the function of the spore body
observation. This is particularly the case in mem- may have to do with special metabolic activities
bers of the genus Hyalorbilia. within the spore.
C. Ascospores D. Paraphyses
The initial striking feature of the ascospores is Paraphyses are sparingly septate, broad, some-
their small size. They range in size from 3 to times with a prominent capitate terminal cell.
8 mm. Observation of them is difficult in some They may be encrusted or contain refractive
situations and requires oil immersion and materials. In some species, the paraphyses and
40 D.H. Pfister
asci are closely adherent and are difficult to and the other adhesive traps, but, importantly,
separate. these fungi form a single clade. Yang et al. (2007)
proposed a scheme for the evolution of trapping
structures.
E. Septal Structures Other form genera are those that produce
staurosporous or helicosporous conidia or are
Ascus, ascogenous, and nonascogenous hyphae associated with aquatic habitats. These include
have simple septa, with septal pores plugged by Angulospora (Pfister 1997; Webster 1992;
unelaborated electron-dense, nonmembranous Webster and Descals 1979), Dicranidion (reports
occlusions. Globose Woronin bodies were summarized by Pfister 1997), Dwayaangam
located on both sides of the septum (Kumar (Kohlmeyer et al. 1998), Helicoon (Pfister 1997),
et al. 2012). Trinacrium (Matsushima 1995), and Triden-
taria. Others are found on a variety of plant
materials in terrestrial habitats, and these
F. Nuclear Division include Brachyphoris (Chen et al. 2007a, c), Dac-
tylellina (Li et al. 2005; Qin et al. 2010), Dactylella
Division is characterized by the retention of an (Chen et al. 2007a, b, c; Pfister 1997; Qin et al.
intact nuclear envelope and a two-layered disk- 2010), and Pseudotripoconidium (Yu et al. 2011).
shaped spindle pole body (Kumar et al. 2012). Staurosporous conidia and those reported
from other aquatic anamorphs are detected in
stem flow, through-fall, and treeholes (Gönczöl
G. Anamorphic States and Révay 2003, 2004, 2006; Révay and Gönczöl
Many members of the Orbiliomycetes have 2011). Among these are conidia referred to
been shown to produce mitospores in culture; form genera of anamorphs associated with
they are hyphomycetous or synnematous. All species of Orbiliomycetes. These include spe-
conidia are formed by holoblastic wall exten- cies of Angulospora, Dactylaria, Dicranidion,
sion. A variety of named form genera have been Dwayaangam, Trinacrium, and Tridentaria.
applied to these conidial states. Anamorphs can Of these Gönczöl and Révay (2004) found
provide some useful information on the biology that Trinacrium in particular was prevalent
and ecology of these fungi. Those that have and that Dwayaangam and Trinacrium species
been demonstrated to trap nematodes and were particularly diverse and geographically
other invertebrates fall into the following widespread (Gönczöl and Révay 2006). Such
genera: Arthrobotrys (Li et al. 2005; Mo et al. observations suggest that canopies are impor-
2005; Pfister 1994, 1997; Pfister and Liftik 1995; tant habitats for these fungi. We expect that
Qiao et al. 2011), Drechslerella (Li et al. 2005), these are likely Orbilia anamorphs that are
Dwayaangam (Barron 1991), Gamsylella, associated with the drought-tolerant, canopy-
known only in its anamorphic state (Scholler inhabiting Orbiliomycetes, which are fungi on
et al. 1999), Lecophagus (Tanabe et al. 1999), dead branches in many woody plants. Gönczöl
and Monacrosporium (Li et al. 2005; Pfister and Révay (2006) say, regarding their findings
1997). There remains debate about the applica- of aquatic hyphomycetes in these terrestrial
tion of some of these names. For general habitats, that “this reinforces our belief that
overviews the interested reader may consult an ecological group of hyphomycetes distinct
Hagedorn and Scholler (1999), Liou and Tzean from those in lotic habitats exists and functions
(1997), and Scholler et al. (1999). Through a in canopies.” By extension, we might assume
multigene analysis Yang et al. (2007) postulated that branches and twigs inhabited by Orbi-
that two lineages evolved trapping mechanisms, liomycetes are a major part of this canopy
with one lineage giving rise to constricting rings ecosystem.
V. Reproduction ascus, the ectal excipulum, spore bodies, the

presence or absence of gelatinous material in
Although many of the Orbiliomycetes have the hymenium, and pigmentation [see Zhang
been grown in culture, few reports have been et al. (2007) for a summary of the genera].
published on apothecial production in the lab.
Spontaneous production of ascomata has been
noted (Drechsler 1937a; Rubner 1994). Zachar- VII. Growth in Culture
iah (1983) was able to induce ascomatal
primorida. Breeding systems have not been One of the most remarkable features of the
studied in part because of the unpredictability Orbiliomycetes is the ease with which most of
of producing ascomata in culture. the species can be cultivated on standard
media. Spores deposited on the surface of agar
media germinate readily, with the exception of
VI. History of Classification and the species of Hyalorbilia for which there are
only a few reports of successful cultivation.
Current Hypotheses Spores germinate quickly, and growth is often
luxuriant on substrates containing glucose.
The family Orbiliaceae was created in 1932 Sporulation is common under room condi-
in Nannfeldt’s (1932) classic treatment of the tions.
inoperculate discomycetes. Recognizing the
special features of this group, he removed the
genera Orbilia and Hyalina from the core group
of inoperculate discomycetes. Primary treat-
VIII. Pezizomycetes: An Overview
ments of the family were those by Svrcek
(1954) and Spooner (1987). Nannfeldt’s cir- The Pezizomycetes comprise a single order,
cumscription of the family was used with few Pezizales, with 16 families currently recognized.
changes until the 1990s. The placement of the The full diversity of the order has not yet been
family outside the Leotiomycetes lineage initi- completely recognized in the classification, and
ally began to be questioned by Pfister (1997), no doubt other lineages will of necessity be
and subsequent work has confirmed the place- named. Ascomata are both epigeous and hypo-
ment as an early diverging lineage within the geous. The epigeous types are apothecial, cleis-
Pezizomycotina (Gernandt et al. 2001; Spata- tothecial, or highly reduced, being composed of
fora et al. 2006). The position of the Orbiliomy- only a few asci in clusters on vegetative hyphae
cetes as an early diverging lineage in the with little or no excipular tissue. In epigeous
filamentous Ascomycota along with the Pezizo- lineages, spores are generally forcibly dis-
mycetes was one of the major surprises in the charged with the asci rupturing by the forma-
era of molecular phylogenetic studies. tion of an operculum. Hypogeous members
Within the Orbiliomycetes there is a single occur in most families containing mycorrhizal
order, and only three teleomorphic genera are members. To date, more than 15 independent
recognized. Other genera may well be proposed origins of trufflelike members are known
as more of these fungi come to be known within a majority of the families (Læssøe and
through wider collection of them and collec- Hansen 2007).
tions come under deeper scrutiny. The
described genera are Orbilia, the largest and
most diverse genus with two subgenera and IX. Occurrence and Distribution
nine sections in Baral’s treatment (2011), Hya-
lorbilia with six to eight species (Baral and Pezizomycetes can be found around the
Marson 2000), and Pseudorbilia, with a single world, but representatives are unevenly
species (Zhang et al. 2007). These genera are distributed. Members of certain families, such
distinguished based on characteristics of the as the Pezizaceae, Morchellaceae, Helvellaceae,
42 D.H. Pfister
Rhizinaceae, and many of the Pyronemataceae, generally do not form rhizomorphs (Tedersoo
show a particularly high diversity in tempera- et al. 2006; Wei et al. 2010). Mycorrhizal species
ture regions. Others are more abundant in seem to predominate in areas that experience
tropical areas, as exemplified by the Sarcoso- xeric conditions (Smith et al. 2006). Mycorrhi-
mataceae and Sarcoscyphaceae. Recent studies zal lineages are often also those that contain
have made clear that, although collecting in this truffle or trufflelike taxa. There are no truffle-
group is robust in many areas of the Northern like species in the families Sarcoscyphaceae and
Hemisphere, the Southern Hemisphere is Sarcosomataceae, and likewise there are no
poorly documented. Nutritionally, Pezizomy- known mycorrhizal species.
cetes are saprobic or mutualistic (Tedersoo
et al. 2006) or are parasitic on bryophytes and
vascular plants (Hansen and Pfister 2006). C. Saprobic Species
Many of the species collected on soil occur in
disturbed areas or in soil that has a high pH and Pezizomycetes occur on organic material of
low content of organic matter. Petersen (1985) various types—decaying wood, dung, leaf litter,
provides a review of the edaphic factors and twigs. The diversity of Pezizales on dung is
involved in growth and reproduction. well documented, with many studies and keys
available (Bell 1983; Doveri 2004; Richardson
and Watling 1997). Some clades are nearly
A. Parasitic Species exclusively found on dung, most notably spe-
cies of Ascobolaceae (Brummelen 1967). Dung-
The plant parasitic species are scattered in var- inhabiting species are found in several clades of
ious lineages. Rhizina undulata is a serious the Pyronemataceae and Pezizaceae (Hansen
root parasite of conifers (Ginns 1968). Pithya and Pfister 2006; Hansen et al. 2001; Perry
species may cause dieback on conifers. Calos- et al. 2007). In most cases, little is known of
cypha fulgens is implicated as a seed pathogen the biology of saprobic species. Sarcoscypha-
of conifers (Paden et al. 1978; Salt 1974). Urnula ceae, Sarcosomataceae, and Chorioactidaceae
craterium has been implicated as the causal are found exclusively on wood, leaves, and
agent of strumella canker in oak (Davidson plant debris.
1950). Phymatothrichopsis ominivora is a root
pathogen of cotton and other dicotyledonous
plants and is known only in its anamorphic X. Morphological Features
state (Uppalapati et al. 2010). A clade in the
Pyronemataceae, including Octospora and A. Ascomata
Lamprospora, is parasitic on bryophytes (Han-
sen and Pfister 2006). Apothecia occur on the Apothecia are the basic type of ascomata. These
thalli or surrounding soil if they are rhizoidal range in size from several millimeters to several
parasites (Döbbler 1979). centimeters and vary from sessile cups of small
to large size to stalked cups to the stipitate
piliate structures found in the Helvellaceae,
B. Mycorrhizal Species Discinaceae, and Morchellaceae. Some highly
reduced members are little more than small
With the advent of molecular approaches to fascicules of asci such as in Ascodesmis. Further
studying mycorrhizal root tips a number of reductions include highly reduced forms found
examples of mycorrhizal Pezizomycetes have in Eleutherascus and Monascella (Guarro and
been documented, and it is now known that Arx 1986; Stchigel et al. 2001). Here the asci are
mycorrhizal lineages are found throughout the formed in clusters on unspecialized hyphae.
class (Tedersoo et al. 2006). Morphologically, Ascomata are often highly pigmented, particu-
the mycorrhizae have a mantle that is often larly the hymenium. Carotenoid pigments have
thin, have a well-developed Hartig net, and been characterized (Arpin 1969).
Variations are found in typical apothecial in the form of an operculum, and its position,
ascomata. On the one hand, there are highly the reaction of the wall layers in various stain-
reduced types that are merely asci scattered ing agents in light microscopy, and the ultra-
on hyphae as mentioned previously, but small structure of the ascus wall are all important
cleistothecial ascomata have also been found in characters. Amyloid asci are found in Peziza-
a few cases (Hansen et al. 2005b). In the case of ceae and Ascobolaceae (Hansen et al. 2001), but
Orbicula parietina, active discharge is lost and this character has been lost several times, most
spores are presented in a powdery mass. Hey- notably in the hypogeous members of the Pezi-
denia species form stalked cleistothecial fruit zaceae. Amyloid asci are not found outside
bodies closely related to O. parietina (Lecht- these families.
mann and Clémençon 2011). The ascomata of The layering of the wall as seen in transmis-
Heydenia, which was recently placed in the sion electron microscopy helps delimit groups,
Pyronemataceae (Lechtmann and Clémençon particularly the taxa with thick lateral walls in
2011), deserve special comment. H. alpina is a the Sarcoscyphaceae and Sarcosomataceae. The
small fungus that occurs on plant debris and asci of these fungi were termed suboperculate
mosses. It is stipitate with a dark stipe that at by Le Gal (1946a, b, 1953). Subsequently,
the top expands to hold a pale mass of hyaline Ecblad (1968) and Samuelson (1975) dis-
spores and radiating hyphae. No asci are pres- counted the term.
ent, but molecular evidence confirms that this Not all members of the class have opercula.
species belongs in the Pyronemataceae as a Hypogeous (Læssøe and Hansen 2007) and
close neighbor of Orbicula, another fungus some cleistothecial taxa (Hansen et al. 2005a,
that produces dry powdery masses of spores b) have lost the operculum and spores remain
at maturity. The two genera share a cleistothe- either within asci until eaten and the fruit body
cial fruit body and asci that seem to disintegrate is broken down, or the asci disintegrate and a
early in the development of the ascomata but powdery mass is formed.
diverge on important morphological features
(Lechtmann and Clémençon 2011). These
genera, often placed outside of the Pezizales, C. Ascospores
have been demonstrated through molecular
systematic studies to be members of the order. No member of the Pezizomycetes has septate
Truffleoid fruit bodies are found in several spores at maturity. Septa may form in germinat-
lineages. Following the terminology of Weber ing spores, but only when a germ tube has
et al. (1997), these are stereothecia, exothecia, already been established. The spores may take
or ptycothecia, depending on the arrangement on a variety of shapes from globose to naviculate,
of the asci and the presence or absence of with smooth or ornamented surfaces. Generally,
well-developed hymenial layers. Truffle-type the ornamentations are derived from secondary
ascomata are derived from apothecial forms. wall material, although a few examples are
Læssøe and Hansen (2007) summarize much known of spore ornamentation without a contri-
of the literature on these taxa. bution of secondary walls. The spore walls are
Some highly reduced forms are found multilayered and may be thin or thick (Merkus
among bryophyte parasites. The fruit bodies 1976). The number of nuclei per spore varies
are partly closed and have only a few asci. from one, two, or four to many, and such varia-
They give the appearance of perithecia. tion has been shown to have some taxonomic
value (Berthet 1964; Korf 1972, 1973).
B. Asci
D. Paraphyses
Brummelen (1978, 1994) summarized the
structural characters of the ascus in the Pezizo- The pigmentation of the hymenium is attribu-
mycetes. The apical apparatus, when present, is ted to the pigments found in the paraphyses.
44 D.H. Pfister
Yellow, red, and orange carotenoid pigments Attempts to germinate mitospores from these
are distinctive features of some of these fungi. mats have failed, and the function of these
These pigments are dissolved in oil droplets spores remains unknown. Several anamorphic
in the paraphyses. A variety of carotenoid pig- states have been discovered through molecular
ments are known to occur in these fungi, as phylogenetic studies to belong within the
summarized by Arpin (1969). Paraphyses are Pezizomycetes, but they have no known
generally septate and may be either uninucleate teleomorphs. Most notable among these is Phy-
or multinucleate, this latter character being matotrichopsis omnivora, the cotton root path-
used in the classification (Berthet 1964; Pfister ogen. This falls within the Rhizinaceae based on
et al. 2008). In some cases, paraphyses are inter- molecular studies, but despite intensive study
spersed with hyaline or darkly pigmented, often of this important pathogen, no evidence of a
thick-walled elements or setae. Examples of teleomorph has been discovered. Another
such setae are found in members of the Rhizi- example is Cephaliophora, a dung-inhabiting
naceae, Sarcosomataceae, Chorioactidaceae, fungus and one that has been implicated in
and Sarcoscyphaceae. causing keratitis (Hoog 2000).
E. Septal Structures XI. Reproduction

Ultrastructural characters of septal pore plugs Extensive studies have been carried out on sev-
and Woronin bodies correlate to some degree eral members of the class. Ascobolus was early
with families and lineages in the order. Kim- incorporated as a genetic tool. Pyronema has
brough (1994) summarizes much of the infor- also been extensively studied. Early cytological
mation. Septal pores in vegetative hyphae studies on members of the Pyronemataceae
generally have lamellae embedded in a matrix. were used to explore mitotic events in asci.
These septa generally have associated Woronin
bodies. The Woronin bodies take on several
forms—globular, hexagonal, or cylindrical,
sometimes considerably elongate. The form of XII. History of Classification and
the Woronin bodies is an important taxonomic Current Hypotheses
and phylogenetic character. The septa at the
base of the asci also provide important charac- The current classification has its origins in the
ters. These septa are occluded by dome-shaped, work of Boudier (1885, 1907), who was the first
pyramidal, or dumbbell-shaped structures. to use the presence of an operculum to define
These plugging structures show electron-dense this group. Hypogeous, truffloid members pre-
bands, lamellae, and raylike extensions. viously treated in the order Tuberales have now
been placed in the Pezizales based on morphol-
ogy, cytology (Trappe 1975), and molecular
F. Anamorphic States sequence analyses (Læssøe and Hansen 2007).
Wide-ranging phylogenetic work within the
Anamorphic states have been reported across order led to the recognition of three primary
many families. Conidia are blastic in develop- lineages (Landvik et al. 1997). Subsequent work
ment and are generally hyphomycetous. has expanded this framework and has sug-
Table 2.1 provides a summary of the known gested groupings of families that in part reflect
anamorphs. In many of these anamorphs, the those previously based on morphological, cyto-
germination of conidia has not been observed, logical, and pigment data and are noted in
suggesting that they may rather act as sperma- earlier work (Korf 1972, 1973; Rifai 1968). The
tia. The investigations by Healy et al. (2013) most recent comprehensive overview of the
identify anamorphic spore mats in several class is that of Hansen and Pfister (2006). Cur-
clades in the Pezizaceae and Tuberaceae. rently, 15 families are recognized (Fig. 2.1),
Table 2.1 Summary of known anamorphs of Pezizomycetes
Family Anamorphic form genus Teleomorphic genus References

Ascobolaceae Rhizostilbella Ascobolus Seifert (1985)
Papulospora Ascobolus Brummelen (1967)
An oidial state Ascobolus Brummelen (1967)
Caloscyphaceae Geniculodendron Caloscypha Paden et al. (1978)
Chorioactidaceae Kumanasamuha geaster Chorioactis geaster Nagao et al. (2009)
Verticicladium Desmazierella acicola Paden (1972)
Morchellaceae Costantinella Morchella Paden (1972)
Pezizaceae Glischroderma Pezizaceae Norman and Egger (1999), Hansen et al. (2001), Healy et al. (2013)
Chromelosporium Peziza, Plicaria, Muciturbo Hennebert (1973), Paden (1972), Warcup and Talbot (1989), Healy
et al. (2013)
Oedocephalum, including highly Peziza, Pachyella, Iodophanus, Hennebert (1973), Hennebert and Bellemère (1979), Paden (1972),
reduced forms Cleistoiodophanus Bezerra and Kimbrough (1976)
Pyronemataceae Actinosporella megalspora Miladina lechithina Descals et al. (1998)
Alciphila vulgaria Byssonectria Harmaja (2002a, b)
Ascorhizoctonia Tricharina Yang and Korf (1985), Barrera and Romero (2001)
Cephaliophora None known Tanabe et al. (1999)
Complexipes Wilcoxina Yang and Korf (1985)
Dichobotrys Trichophaea Hennebert (1973)
Micronematobotrys None known Sun and Guo (2010)
“Nodulosporium”-like Geopyxis majalis Paden (1972)
Pezizomycotina: Pezizomycetes Orbiliomycetes
Rhizinaceae Phymatotrichopsis None known Uppalapati et al. (2010)

Sarcosomataceae Conoplea Urnula craterium, Plectania, Hughes (1958), Paden (1972)
Sarcosoma latahensis
Sarcoscyphaceae Molliardiomyces Phillipsia, Sarcoscypha, Nanoscypha Paden (1984), Pfister (1973b)
Tuberaceae Unnamed sympodulosporous Tuber Urban et al. (2004), Healy et al. (2013)
type
45
46 D.H. Pfister
though the relationship among those families in are formed on a much-reduced excipulum
many instances is not well resolved. Although (Brummelen 1981). These species are saprobic
this group is reasonably well known, future on dung and often are isolated from soil. Asci
sampling will certainly change our understand- are inamyloid and are saccate or pyriform.
ing of the relationships and of these lineages. A Ascospores are brownish and have reticulate
particular focus is on the large and heteroge- spore ornamentations. Septa in ascogenous
neous family Pyronemataceae, in which several cells and the ascus bases are dome-shaped
lineages are recognizable based on both with arrays of radiating tubular elements and
sequence analyses and expanded knowledge of a striate structure associated with the septal
nutritional modes, morphology, and anamor- pore rim in vegetative cells (Brummelen 1989;
phic states. Kimbrough 1994). Four genera are placed in the
General overviews of the families can be family (Hansen et al. 2005b). A recent study by
found in Cannon and Kirk (2007). The follow- Hansen et al. (2013) places this family within
ing augmented descriptions include available the larger and highly diverse sister group to the
structural and cytological information that has Pyronemataceae sensu stricto.
been used in refining the classification.
3. Caloscyphaceae
A. Families of the Pezizomycetes The brightly colored cupulate ascomata stain
blue green when bruised. No hypogeous mem-
1. Ascobolaceae
bers are known. Caloscypha fulgens is a parasite
Only epigeous members are known, and these of conifer seeds and seedlings. The ascomata
occur primarily on dung, but a few species are have a well-developed inner layer of interwoven
found on plant material; Ascobolus carbonarius hyphae and an outer layer of globose or angular
is found on burned material (Brummelen cells. Ascospores are globose, and their walls
1967). All species are saprobic and ascomata are smooth (Harmaja 2002a). The genus Calos-
have multiple cell layers. Developmental pat- cypha was previously placed in the Pyronema-
terns have been characterized by Brummelen taceae, in part because of its carotenoid
(1967). Cleistothecial developmental types are pigments and because of the globose spores
found. Asci blue diffusely along the walls in (Arpin 1969; Korf 1972, 1973). For details on
iodine solutions. Ascospores are uninucleate septal construction see Kimbrough and Curry
(Berthet 1964); in Ascobolus and Saccobolus (1986b). Recently a second genus was added to
the spore walls are ornamented with purple the family, Kallistoskypha, with a single species,
brown pigments that become fissured at matu- K. incarnata, which is associated with Eucalyp-
rity (Brummelen 1967). The placement here of tus found around the Mediterranean region
Thecotheus species, with their hyaline ascos- (Pfister et al. 2013).
pores, has been suggested on morphological
grounds and has been confirmed in molecular
phylogentic studies (Landvik et al. 1997). Septa 4. Chorioactidaceae
associated with the ascogenous system are The ascomata are cupulate or deep urnulate
dome-shaped (Kimbrough 1994; Kimbrough and dark on the outer surface; the hymenium
and Curry 1985). Three genera are generally is beige, brownish, or orange. No hypogeous
recognized. members are known in this family, and all the
species are considered to be saprobic. The asco-
2. Ascodesmidiaceae mata have a well-developed inner layer and an
outer layer of globose or angular cells that give
Ascomata are much reduced in most genera; for rise to thick, brown hairs encrusted with gran-
example, in Eleutherascus species the asci are ular material. The asci are inamyloid and thick-
scattered on hyphae. In Ascodesmis, several asci walled with a terminal operculum. Ascospore
ornamention is in the form of warts or ribs. et al. 2006). The ascomata are characteristically
Four genera have been included (Pfister et al. constructed with an inner layer of interwoven
2008). hyphae and an outer layer of elongate cells
oriented perpendicularly to the outer surface.
Asci are inamyloid. Ascospores are tetranucle-
5. Discinaceae ate (Berthet 1964), smooth, or ornamented with
The ascomata of this family are discoid or gyro- irregular warts, and Woronin bodies are elon-
mitroid. Both epigeous and hypogeous mem- gate, hexagonal, or globose (Kimbrough 1994).
bers are known. Many species in the family are Septa at the base of the asci are electron-
mycorrhizal (Tedersoo et al. 2006, 2010). The opaque. There are hemispherical structures
ascomata are characteristically constructed that become cone- to dumbbell-shaped with
with an inner layer of interwoven hyphae and V-shaped striations; in vegetative cells, an
an outer layer of elongate cells oriented perpen- electron-translucent torus separates the pore
dicularly to the outer surface. Asci are inamy- plug from the septal pore border. Vegetative
loid, and ascospores are tetranucleate (Berthet cells possess globular or slightly angled Woro-
1964), smooth, or elaborately ornamented with nin bodies (Kimbrough 1994; Kimbrough and
warts, often with apiculae. Woronin bodies are Gibson 1989; Kimbrough et al. 1996).
elongate and often surround the septal pore.
The septa at the base of asci are accompanied
by an electron-opaque, hemispherical structure 8. Karstenellaceae
that becomes cone- to dumbbell-shaped with In this family, the ascomata are little more than
V-shaped structures; an electron-translucent a very thin, resupinate crust situated on leaf
torus separates the pore plug from the septal litter and wood surfaces. Asci are scattered on
pore border (Kimbrough 1991, 1994). The this mycelial mat. Nothing is known of its mode
septal ultrastructure is similar to that of the of nutrition. Asci are inamyloid and operculate.
Helvellaceae. Generally, five or six genera are Ascospores are binucleate or multiguttulate
recognized. (Hansen et al. 2008; Harmaja 1969). No ultra-
structural details are known for the vegetative
septal construction or for the septa of the asci.
6. Glaziellaceae Molecular data place it in an unresolved posi-
The ascomata in this family are large and hol- tion near Helvellaceae and Tuberaceae. There is
low with a basal opening. The unisporic, clavate a single genus with a single species recognized,
to globose asci are embedded in the rindlike Karstenella vernalis.
wall. Ascus walls break down, but the spores
remain embedded in this peridiumlike wall.
The single species, Glaziella aurantiaca, is pre- 9. Morchellaceae
sumed to be mycorrhizal. See Gibson et al. The morels are characterized by stipitate
(1986) for ultrastructural details that initially apothecia, with an interrupted hymenial layer
supported the placement of Glaziella in the giving a honeycomb appearance, or by cupulate
Ascomycota. forms such as Disciotus. Hypogeous members
include Leucoangium (Li 1997) and Kalapuya
7. Helvellaceae (Trappe et al. 2010). Species are saprobic and
have been implicated in mycorrhizal relation-
Ascomata are sessile cupulate or stipitate cupu- ships (Buscot 1994; Dahlstrom et al. 2000). Asci
late, saddle-shaped, or columnar (Abbott and are inamyloid. Ascospores are multinucleate
Currah 1997; Dissing 1966). Both epigeous and (Berthet 1964) and lack both prominent wall
hypogeous species are found in the family ornamentation and internal oil droplets. Tradi-
(Hansen and Pfister 2006; Kimbrough et al. tional classifications of the species accept six to
1996). Many or most are mycorrhizal (Tedersoo eight species, but phylogenetic studies indicate
48 D.H. Pfister
the presence of many species (Du et al. 2012; phyletic and will need to be broken down into
Kuo et al. 2012; Taskin et al. 2010) that seem several additional genera (Hansen et al. 2001,
restricted to specific continents (O’Donnell 2005a, b).
et al. 2011). Some of these species are morpho-
logically distinct; others are cryptic (O’Donnell
et al. 1997, 2011). The septal construction is 11. Pyronemataceae
similar to that found in the Helvellaceae. At
the base of the asci, dome-shaped structures Pyronemataceae is the largest of the families of
with V-shaped striations are found at the septal the class and the most diverse ecologically and
pore. Lamellate structures are found within morphologically. As recognized here, the family
septal pores of most vegetative cells. Elongate includes Aleuriaceae, Humariaceae, Otideaceae,
Woronin bodies have been found along with and Geneaceae. These fungi are saprobic,
hexagonal ones (Kimbrough 1994). Five or six mycorrhizal vascular plants or parasitic on
genera are recognized. bryophytes. Epigeous members are cupulate,
often possessing hairs on the outer surface.
Hypogeous members are scattered in the family.
10. Pezizaceae The ascomata show considerable variation in
construction and development. Asci are inamy-
Both epigeous and hypogeous taxa are found; loid, and ascospores vary in shape and orna-
there are multiple origins of the hypogeous taxa mentation from smooth to variously
within several clades (Hansen et al. 2001, 2002, ornamented (Perry et al. 2007). Several different
2005a; Healy et al. 2009). Saprobic members septal types are present; at least four types of
include those on dung, wood, and other plant septa plugging have been associated with asci
debris. Mycorrhizal taxa are found in several (Kimbrough 1994). These are the aleurioid type,
lineages. Some taxa are found exclusively on which have a granular, opaque matrix that bor-
burned areas. The ascomatal structure varies, ders both the ascal and ascogenous hyphal sides
but multiple layers composed of large, thin- of pores; later this becomes fan-shaped with a
walled globose cells are often present. Asci gen- lamellate electron-translucent torus adjacent to
erally become blue in iodine solutions. This the pore rim. The otideoid type, is characterized
bluing can take several forms. The reaction by double translucent bands in a granular,
may be diffuse along the entire wall or concen- opaque pore matrix. Later the pore plug is dif-
trated in the upper portion of the ascus but ferentiated into two zones, an inner dense zone
most intensely at the tip, or in the upper por- and an outer less opaque zone. The pulvinuloid
tion with a more intense ring surrounding the type, is similar to the Helvellaceae at maturity
opercular region (Hansen et al. 2001). The blu- with V-shaped striations, but these are fewer
ing reaction has been lost in several taxa, and less prominent than in Helvellaceae. The
including some that are hypogeous. Ascospores scutellinioid type has a large hemispherical sep-
are uninucleate (Berthet 1964), with or without tal pore plug that becomes zonate; the inner
secondary wall ornamentations (Hansen et al. zone adjacent to the pore lumen is electron-
2001, 2002). Oil droplets may or may not be opaque, and an outer, thicker zone is composed
present in the ascospores. Septa in vegetative of less dense material. In general, in this family,
cells are lamellate with large globose Woronin vegetative cells have a lamellate structure in the
bodies. At the ascus bases, septa are occluded septal pores. Woronin bodies are small and
by convex or biconvex bands that become cov- largely globular, but often hexagonal.
ered with electron-opaque amorphous material The pyronema type has asci and ascogen-
or by an additional secondary wall (Curry and ous hyphae that are similar to those of the
Kimbrough 1983; Kimbrough 1994; Kimbrough Ascobolaceae but with a different electron den-
et al. 1991). Approximately 30 genera are cur- sity core and small, radiating tubular bands
rently recognized in the family, but it is well similar to those of Ascodesmis (Kimbrough
established that the genus Peziza is nonmono- 1994; Kimbrough and Curry 1986a, b).
The family includes approximately 80 Li and Kimbrough 1995b). Twelve genera are
genera. It is perhaps highly heterogeneous and commonly recognized within this family.
one that will see future division and refinement. Romero et al. (2012) review the classification
The family was recently studied by Hansen et al. within the family.
(2013), and this work provides the most accu-
rate overview of the family.
14. Sarcosomataceae
12. Rhizinaceae In this family, ascomata are cupulate or urnu-

late, tough and leathery of moderate to large
The ascomata in members of this family are flat, size, and black or dark brown on the outside,
recurved, or pulvinate. Rhizina undulata is a often with a paler hymenium. No hypogeous
pathogen of conifer roots. Also included here is members are known. Most species are pre-
Psilopezia, whose species are found on water- sumed to be saprobic on plant debris, but in
soaked wood and which is presumed to be a the case of Urnula, the species is said to be
saprobe (Pfister 1973a). The construction of the parasitic. The ascomata have a well-developed
ascomata follows a pattern of interwoven inner layer, with gelatinous material, and an
hyphae forming the medulla, with more densely outer layer of globose or angular cells that give
interwoven hyphae contributing to the cortical rise to dark hyphoid hairs. Asci are inamyloid
layer. These fungi are characterized by indeter- and thick-walled with a terminal operculum.
minate marginal growth. The asci are inamy- The ultrastructure of the asci and ascospores
loid, and ascospores are smooth or ornamented have been studied by Bellemère et al. (1990).
with warts and apiculae. The spores are pre- The ascospores are smooth or with wrinkled or
sumed to be tetranucleate. Studies are needed folded walls or with low warts. In addition, they
on the septal structure. The family encom- are multinucleate. Septal structures are com-
passes the two genera mentioned earlier as posed of an electron-dense matrix and globose
well as Phymatotrichopsis omninvora, which is Woronin bodies that are often accompanied by
known only as an anamorph. short or long, cylindrical, hexagonal Woronin
bodies. Septa at the ascus bases are composed of
a dumbbell-shaped matrix with V-shaped
13. Sarcoscyphaceae bands. The septal structure was studied by Li
and Kimbrough (1995b). Five or six genera are
Ascomata are generally cupulate, bright col-
commonly accepted.
ored, stipitate, or sessile. No hypogeous mem-
bers are known; all are considered saprobic.
The ascomata have a well-developed inner 15. Tuberaceae
layer and an outer layer of globose or angular
cells or of tightly interwoven hyphoid cells ori- Until recently, all members of this family were
ented parallel to the outer surface. Cells on the considered to be hypogeous. The recent place-
outer surface sometimes give rise to single or ment of Nothojafnea thaxteri among these
fasciculate hairs. Ascospores are smooth or fungi is notable as the first epigeous member
ornamented with ridges and ribs. In vegetative of the family (Bonito et al. 2013). The truffles
cells, lamellate structures are poorly differen- of commerce (Tuber melanosporum and
tiated or missing. Septa in vegetative cells are T. magnatum) belong here. Ascomata are
imperforate, plugged by an electron-opaque, highly convoluted and folded, often with cham-
fan-shaped matrix surrounded by a number of bers or pockets of asci. The large, primarily
globose electron-dense Woronin bodies. The Northern Hemisphere genus Tuber has been
Woronin bodies are globose or occasionally investigated by Bonito et al. (2010). All mem-
hexagonal and electron-opaque. At maturity bers are mycorrhizal on a range of plant
obscure V-shaped striations may be found families (Tedersoo et al. 2006). The asci are
(Benny and Samuelson 1980; Kimbrough 1994; inamyloid, globose, or pyriform with one to
50 D.H. Pfister
eight spores per ascus. The ascospores, in addi- behavior, whereas some members of the Orbi-
tion to being often multinucleate, are often liomycetes trap and consume invertebrates.
elaborately ornamented with reticula, ridges, Recent studies have placed many hypogeous
and warts. Septal structure was studied by Li Pezizomycetes within families. These studies
and Kimbrough (1995b), who determined that highlight the evolutionary patterns within the
there was some heterogeneity among those spe- class in which suites of changes, including loss
cies examined. In that study, the researchers of active spore discharge mechanisms and
found long, cylindrical Woronin bodies in extended ascomata developmental times, take
some of the species, similar to those of the place in distantly related clades. Most hypoge-
Morchellaceae, Discinaceae, and Helvellaceae, ous Pezizomycetes are found within clades with
and rectangular ones in another, similar to other mycorrhizal members. The diversity in
those of the Pyronemataceae. Septa associated both Orbiliomycetes and the Pezizomycetes
with ascogenous hyphae and ascus bases also has proven to be greater than previously antici-
differed among the sampled taxa. O’Donnell pated, and much researches remains to be done
et al. (1997) included a series of genera in addi- regarding the biogeography and evolutionary
tion to Tuber. Seven genera are commonly histories of the group.
recognized. Bonito et al. (2013) estimate a late
Jurassic origin of the genus Tuber and discuss
the Southern Hemisphere diversity. References
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3 Pezizomycotina: Sordariomycetes and Leotiomycetes
NING ZHANG1, ZHENG WANG2
CONTENTS VII. Culture and Maintenance . . . . . . . . . . . . . . . . . . 80

VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
II. Sordariomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
A. Ecology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
B. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
1. Ascomata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 I. Introduction
2. Centrum Development. . . . . . . . . . . . . . . . 60
3. Asci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4. Ascospores . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 A close evolutionary relationship between the
5. Anamorphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Sordariomycetes and Leotiomycetes was initi-
6. Ultrastructure . . . . . . . . . . . . . . . . . . . . . . . . . 61 ally supported through phylogenetic analyses of
C. Molecular Phylogeny . . . . . . . . . . . . . . . . . . . . 61 DNA sequence data and subcellular characters.
D. Classification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
1. Hypocreomycetidae . . . . . . . . . . . . . . . . . . . 62
Using various data sets and tree reconstruction
2. Sordariomycetidae . . . . . . . . . . . . . . . . . . . . 65 methods, several recent studies corroborated
3. Xylariomycetidae. . . . . . . . . . . . . . . . . . . . . . 67 that the two classes are sister clades in Ascomy-
4. Orders Incertae Sedis . . . . . . . . . . . . . . . . . 68 cota (Celio et al. 2006; Fitzpatrick et al. 2006;
III. Leotiomycetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Lumbsch et al. 2005; Robbertse et al. 2006;
A. Classification History. . . . . . . . . . . . . . . . . . . . 69
1. History of Leotiomycetes . . . . . . . . . . . . . 69
Schoch et al. 2009a; Spatafora et al. 2006; Wang
2. Current Classification of et al. 2009; Zhang et al. 2006). A rankless taxon,
Leotiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . 70 “Sordariomyceta,” was proposed by Schoch
B. Molecular Phylogeny Update . . . . . . . . . . . 72 et al. (2009a) for the clade that includes the
1. Higher-Level Relationships of classes Sordariomycetes, Laboulbeniomycetes,
Leotiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . 72
2. Phylogeny Within Leotiomycetes . . . . 73
and Leotiomycetes. Although Sordariomycetes
C. Characters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 and Laboulbeniomycetes are characterized
1. Apothecium (Ascoma) Morphology, by the production of perithecial ascoma
Anatomy, and Ultrastructure. . . . . . . . . 75 (Figs. 3.1–3.13) and Leotiomycetes by apothe-
2. Ecology and Biogeography . . . . . . . . . . . 76 cial ascoma (Figs. 3.14–3.18), and members in
IV. Geoglossomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . 77
V. Laboulbeniomycetes. . . . . . . . . . . . . . . . . . . . . . . . 78
these classes have diverse ecology and nutri-
A. Laboulbeniales . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 tional modes, they likely evolved from a com-
B. Pyxidiophorales. . . . . . . . . . . . . . . . . . . . . . . . . . 78 mon ancestor that was terrestrial, saprotrophic,
VI. Problems and Perspectives . . . . . . . . . . . . . . . . 78 and nonlichenized. A putative synapomorphy
A. Genome Project. . . . . . . . . . . . . . . . . . . . . . . . . . 78 of “Sordariomyceta” is their inoperculate, uni-
B. Environmental Study . . . . . . . . . . . . . . . . . . . . 79
tunicate, and poricidal asci.
1
Department of Plant Biology & Pathology, Rutgers University,
59 Dudley Road, New Brunswick, NJ 08901, USA; e-mail: II. Sordariomycetes
zhang@aesop.rutgers.edu
2
Department of Ecology and Evolutionary Biology, Yale Uni-
versity, 165 Prospect Street, New Haven, CT 06520, USA; Based on the 10th edition of the Dictionary of
e-mail: wang.zheng@yale.edu the Fungi (Kirk et al. 2008), Sordariomycetes

58 N. Zhang and Z. Wang
50 μm 10 μm
10 μm
1 2 3 2 cm 4 0.5 mm
6 10 μm
8 9 10
11 12
10 mm
10 mm 10 mm
13 14 15
10 mm 10 mm 10 mm
16 17 18
Figs. 3.1–3.18 Morphology of species in Sordariomy- Figs. 3.5 and 3.6 Asci and ascospores of Anisogramma
cetes (1–13) and Leotiomycetes (14–18). Fig. 3.1 Peri- anomala. Figs. 3.7–3.12 Xylaria globosa (Spreng. ex Fr.)
theicia of Melanospora sp. Fig. 3.2 Ascospores of Mont. Courtesy of Yu-Ming Ju. Fig. 3.7 Stromata.
Melanspora sp. Fig. 3.3 Stromata of Anisogramma Fig. 3.8 Reticulately cracked stromatal surface showing
anomala (Peck) E. Müll. on hazelnut stem. Courtesy ostioles. Fig. 3.9 Colony on 9-cm petri dish containing
of Guohong Cai. Fig. 3.4 Anisogramma anomala Difco oatmeal agar. Fig. 3.10 Stromata produced on OA
perithecia in a stroma. Courtesy of Guohong Cai. with red exudates. Fig. 3.11 Ascospores showing an
Pezizomycotina: Sordariomycetes and Leotiomycetes 59
represents a large clade in Ascomycota, with fungal systematic framework using multilocus
1,119 genera and over 10,000 known species. It phylogenies and other traits, such as subcellu-
contains most nonlichenized ascomycetes with lar and other morphological characters.
perithecial (flask-shaped) or, less frequently, In the current classification sensu Lumbsch
cleistothecial (nonostiolate) ascomata and and Huhndorf (2010) (outline of Ascomycota,
inoperculate unitunicate or prototunicate asci initiated by Eriksson and Winka in 1997, now
(Alexopoulos et al. 1996). hosted at the Field Museum: http://fieldmuseum.
org/explore/myconet), Sordariomycetes com-
The term “Pyrenomycetes” was formerly used to unite prises 18 orders with core taxa in 3 subclasses,
fungi with perithecial ascomata and unitunicate asci i.e., Hypocreomycetidae, Sordariomycetidae,
(Luttrell 1951), and its use was discontinued based on and Xylariomycetidae. The subclasses were first
the placement of perithecial species outside of the clade
and the inclusion of species with prototunicate asci
proposed by Eriksson and Winka (1997) based
(e.g., Corollospora and Ophiostoma) in order to avoid on morphological characters and SSU phyloge-
confusion (Eriksson and Winka 1997). nies. This classification is further supported by a
number of multilocus phylogenies, such as the
Sordariomycetes share some features with four-gene phylogeny by Zhang et al. (2006).
Dothideomycetes and Leotiomycetes but are
unique in having a true perithecium and inop- Classes and orders in Sordariomycetes have been clas-
erculate, unitunicate asci. In the 10 years since sified based on ascoma shape and developmental types.
However, convergent evolution often results in similar
the first edition of the Mycota VII, 115 articles ascoma morphology and even ontogenesis, as shown in
with the keywords “Sordariomycet* OR pyre- recent reviews by Liu and Hall (2004) and Lumbsch and
nomycet*” and “phylogen*” have been pub- Huhndorf (2007a).
lished, when a search in the Web of Science
was conducted. A number of new taxa were
discovered from nature and placed in the Sor- A. Ecology
dariomycetes, from both terrestrial and aquatic
habitats (Campbell et al. 2009; Raja and Shearer Members of Sordariomycetes are ubiquitous
2008; Samuels et al. 2009). and cosmopolitan. They function in virtually
Since the early 1990s, molecular sequence all ecosystems as saprotrophs involved in
data have been used to test fungal systematics decomposition and nutrient cycling, as endo-
and evolutionary hypotheses. Until recently, phytes and pathogens of plants, arthropods,
most molecular phylogenetic studies had and mammals, and even as mycoparasites
comprised single-gene phylogenies, usually of attacking other fungi. Most plant pathogens
rRNA genes. In general, nuclear small subunit in Sordariomycetes are distributed in Dia-
(SSU) rRNA genes were used to infer relation- porthales, Glomerellales, Hypocreales, Micro-
ships at family and higher taxonomic levels, ascales, Ophiostomatales, Phyllachorales, and
large subunit (LSU) rRNA genes at the genus Xylariales. These include the best-known plant
and family levels, and the internal transcribed pathogens, for example, Cryphonectria parasi-
spacer (ITS) region at species or infrageneric tica (the causal agent of chestnut blight), Mag-
levels. In the past decade, through initiatives naporthe oryzae (the cause of rice blast),
such as Assembling the Fungal Tree of Life Ophiostoma ulmi and O. novo-ulmi (the Dutch
(AFTOL), Deep Hypha, and Myconet, mycolo- elm disease causal agents), Fusarium species,
gists and bioinformatists have pursued collab- and Rosellinia species (Alexopoulos et al. 1996;
orative efforts to generate a relatively stable Samuels and Blackwell 2001).
⁄

Figs. 3.1–3.18 (Continued) oblique germ slit. Fig. 3.12 G. W. Hudler) Fig. 3.15 Leotia lubrica Fr. (Courtesy
Asci with an apical apparatus staining blue in an of M. Wood) Fig. 3.16 Chlorencoelia versiformis
iodine solution and ascospores. Fig. 3.13 Cyttaria (Pers.) J. R. Dixon. (Courtesy of E. Bosman) Fig. 3.17
espinosae Lloyd. (Courtesy of G. Palfner) Fig. 3.14 Spathularia flavida Pers. Fig. 3.18 Trichoglossum
Rhytisma americanum Hudler & Banik. (Courtesy of hirsutum (Fr.) Boudier
In addition to pathogens of plants, Sordar- of Nectriaceae and the Stachybotrys clade

iomycetes includes endophytes that live inside and the ergot and other alkaloids produced by
apparently healthy plants (Alexopoulos et al. Claviceps and Epichloë. Marine fungi are also a
1996; Carroll 1988). Numerous endophytic good source of novel bioactive compounds
fungal survey studies have demonstrated that (Bugni and Ireland 2004).
Sordariomycetes is one of the most frequently
isolated fungal classes in land plant foliage
(Arnold and Lutzoni 2007; Carroll 1988). The B. Morphology
best-studied endophytes belong to Hypocreales
(e.g., Balansia and Epichloë), Xylariales (e.g., 1. Ascomata
Nemania and Xylaria), and Glomerellales (e.g., The majority of Sordariomycetes produce peri-
Colletotrichum). Their infected host plants often thecial ascomata (Fig. 3.1). The shape, size,
benefit from increased drought resistance, pigmentation, texture, and position of asco-
reduced feeding by insects, and limited pathogen mata are examined in traditional taxonomy.
infections (Alexopoulos et al. 1996). For example, the position of ascomata in
Some members of Hypocreales, Ophiosto- relation to substrates was used in the family
matales, and Microascales are associated with delimitation of Diaporthales by Barr (1978),
opportunistic infections of humans and other but this classification was not supported by
animals (e.g., Sporothrix schenkii, Fusarium phylogenetic analyses using molecular charac-
solani species complex, and Trichoderma spp.) ters (Castlebury et al. 2002; Zhang and Black-
(Gugnani et al. 1976; O’Donnell et al. 2010; Sum- well 2001). A phylogenetic study by Miller and
merbell 2003). As symbionts of arthropods, Sor- Huhndorf (2005) suggested that ascomal wall
dariomycetes comprise a diverse assemblage of morphology is a better character than asco-
species that range from antagonistic to mutual- spore morphology in defining genera in
istic (Samuels and Blackwell 2001). Sordariales. Leal et al. (2010) assessed fungal
wall heteromannans as a phylogenetically
Members of Microascales and Ophiostomatales (e.g.,
Ceratocystis, Ophiostoma, and Ambrosiella) are asso-
informative character in ascomycetes, includ-
ciated with bark beetles in fungal spore dispersal. Spe- ing Sordariomycetes.
cies of Hypocreales (e.g., Cordyceps and Torrubiella)
directly parasitize a broad range of arthropods. The
Hypocreales is also known to be rich in mycoparasites. 2. Centrum Development
Most species of Hypomyces are parasitic on fleshy fun-
gal fruit bodies such as mushrooms and large apothecia Nannfeldt (1932) and Luttrell (1951) first
(Rogerson and Samuels 1989). Some species of Tricho- applied ontogenetic characters in filamentous
derma, used alone or combined with other biocontrol ascomycete classification. Although ontoge-
agents, have been applied in agriculture for plant netic characters do not always correspond well
disease management as an alternative to pesticides
(Harman et al. 2004). with molecular phylogenies, they are still infor-
mative characters in ordinal level classification
Saprobic Sordariomycetes function in the within Sordariomycetes (Hibbett et al. 2007;
decomposition and nutrient cycling of plant Samuels and Blackwell 2001). For a detailed
litter, including wood, herbaceous stems, and description and illustration of perithecium
dung. Important taxa include Neurospora ontogeny and centrum types in Sordariomy-
crassa, the model organism widely used in cetes, see Samuels and Blackwell (2001). For a
molecular and genetic studies (Alexopoulos review and recent progress on ontogenic stud-
et al. 1996), and Chaetomium, an important ies of Sordariomycetes, see Lord and Read
cellulolytic organism responsible for the (2011). In Samuels and Blackwell (2001), nine
destruction of paper and fabrics. Sordariomy- centrum types were outlined and illustrated:
cetes also contains species known as producers Melanospora type, Nectria type, Ophiostoma
of some of the most important fungal second- type, Xylaria type, Epichloë type, Sordaria
ary metabolites. These include the trichothe- type, Eutypa type, Meliola type, and Phaeotri-
cene mycotoxins produced by many members chum type.
3. Asci anamorphs also occur, most notably in the Glo-

merellaceae and Diaporthales. In common with
Asci usually arise from ascogenous hyphae teleomorph characters, many characters used to
through the mediation of a crozier, a structure delimit anamorph genera (e.g., variations in
that functions to ensure that only two compati- conidiomata, pigmentation, conidiophore
ble nuclei are sequestered in a developing ascus. branching, and conidial septation) are homo-
The arrangement of asci in Sordariomycetes is plastic in Sordariomycetes. Despite this, recog-
basal or peripheral in a hymenium (Fig. 3.1). nizable patterns of anamorph morphological
Asci of Sordariomycetes either are capable of characters often allow recognition of phyloge-
forceful ejection and wind dissemination of netic groups (Seifert and Gams 2001).
ascospores or lack any apical discharge mecha-
nism. A sphincterlike ring at the ascal apex is
likely to effect forceful ejection (Read and Beck- 6. Ultrastructure
ett 1996; Samuels and Blackwell 2001). The pres-
ence or absence of an apical ring (amyloid or Subcellular or ultrastructural and biochemical
chitinoid) is an important feature in the classi- characters also play a major role in understand-
fication of Sordariomycetes (Figs. 3.5 and 3.12). ing fungal evolution, but reports on these char-
Typically, the asci of Sordariomycetes are octos- acters are scattered in the literature. As part of
porous (Figs. 3.2, 3.5, and 3.6). the AFTOL project, a searchable structural and
biochemical database for selected fungal taxa is
now available at http://aftol.umn.edu. This
4. Ascospores database includes descriptions and illustrations
of characters such as nuclear division, septum/
Traditionally, mycologists have put consider- pore cap, sterol data, hyphal tip organization,
able weight on ascospore morphology (e.g., and meisporangium.
ascospore shape, size, pigmentation, wall According to Celio et al. (2006), Sordariomy-
ornamentation) in delimiting ascomycete cetes was strongly supported by the ascogenous
genera. Sordariomycetes is no exception; how- hypha/ascus pore occlusions in immature and
ever, many molecular phylogenetic studies sug- mature hyphae. Unique characters of the Sordar-
gest that these generic deliminations do not iomycetes include the following: (1) for imma-
always accurately reflect evolutionary history. ture ascogenous hypha/ascus: endoplasmic
reticulum associated with toroid occlusion
For instance, in Melanosporales, genera delimited (donutlike with central pore), torus with radiat-
based on ascospore shape and ornamentation are not ing tubular cisternae; (2) for mature ascoenous
monophyletic, likely owing to convergent evolution
(Zhang and Blackwell 2002). In Sordariales, ascomal
hypha/ascus: subspherical pore cap membrane,
wall morphology was found to be a better character simple membrane enclosing cytoplasm. Leotio-
than ascospore morphology in defining genera (Miller mycetes and Sordariomycetes appear to have the
and Huhndorf 2005). In Hypocreales, asexual states same spindle pole body form and nuclear enve-
seem to be more informative in defining monophyletic lope organization during nuclear division, i.e.,
taxa because the sexual stages tend to be more con-
served (Chaverri et al. 2003).
unlayered disc with intact nuclear envelope and
internal microtubule organizing center.
5. Anamorphs C. Molecular Phylogeny
Sordariomycetes is an anamorph-rich class, Recent comprehensive taxonomic and phyloge-

with significant diversity represented by hypho- netic studies of families and higher taxa of
mycete and coelomycete species. Many species Sordariomycetes were published by Samuels
of Hypocreales, Ophiostomatales, and Chaeto- and Blackwell (2001), Eriksson (2006), and
sphaeriales have two or more distinguishable Zhang et al. (2006); they were based on both
anamorphs (synanamorphs). Hyphomycetes morphological characters and molecular
occur throughout the class, but coelomycete sequence data and reviewed in Hibbett et al.
Coronophorales
Glomerellales
Hypocreales Hypocreomycetidae
Melanosporales
Microascales
Calosphaeriales
Diaporthales
Main Body of
SORDARIOMYCETES Magnaporthales
Ophiostomatales
Sordariomycetidae
Boliniales
Chaetosphaeriales
Coniochaetales
Sordariales
Xylariales Xylariomycetidae
Koralionastetales
Lulworthiales
SORDARIOMYCETES
Meliolales incertae sedis
Phyllachorales
Trichosphaeriales
Pyxidiophorales
LABOULBENIOMYCETES
Laboulbeniales
LEOTIOMYCETES
LICHINOMYCETES
LECANOROMYCETES
EUROTIOMYCETES
DOTHIDEOMYCETES
ARTHONIOMYCETES
GEOGLOSSOMYCETES
ORBILIOMYCETES
PEZIZOMYCETES
Fig. 3.19 Phylogeny and classification of Sordariomycetes. Branch lengths are not proportional to genetic
distances
(2007) and Lumbsch and Huhndorf (2010). We Erysiphales is now placed in Leotiomycetes,
provide here a compiled classification to reflect which was supported by rRNA gene sequences
the well-supported phylogenetic relationship of (Wang et al. 2006b), and Coryneliales is con-
Sordariomycetes and allied taxa (Fig. 3.19). firmed as a member of Eurotiomycetes (Geiser
There are three major clades in Sordario- et al. 2006; Schoch et al. 2009a). The extralimital
mycetes, corresponding to the three subclasses pyrenomycete Laboulbeniales sensu Samuels
of Eriksson (2005) (Fig. 20). Xylariomycetidae and Blackwell (2001) is now recognized as a
(one order) is placed as the earliest diverging separate class close to Sordariomycetes
group of Sordariomycetes, followed by the (Lumbsch and Huhndorf 2010; Schoch et al.
monophyletic clade comprising Hypocreomy- 2009a; Weir and Blackwell 2001).
cetidae (five orders) and Sordariomycetidae
(eight orders), which are supported by several
multilocus phylogenetic studies (Schoch et al.
D. Classification
2009a; Tang et al. 2007; Zhang et al. 2006).
These three well-delimited subclasses consti- 1. Hypocreomycetidae
tute the main body of Sordariomycetes. The
position of five sordariomycetous orders, Hypocreomycetidae is recognized as a mono-
including Lulworthiales, Koralionastetales, phyletic subclass based on recent molecular
Meliolales, Phyllachorales, and Trichosphaer- phylogenetic studies (Lumbsch and Huhndorf
iales, is uncertain. Erysiphales and Coryneliales 2010; Réblov et al. 2011; Schoch et al. 2009a;
sensu Barr (1990) are excluded from the Sor- Tang et al. 2007; Zhang et al. 2006). It includes
dariomycetes (Samuels and Blackwell 2001). five orders: Coronophorales, Glomerellales,
Hypocreales, Melanosporales, and Microas- distinctions, and Wanderlei-Silva et al. (2003)

cales (Fig. 3.19). Members of Hypocreomyceti- recognized Glomerella as a family in Hypocreo-
dae typically possess light-colored perithecia, mycetidae. Further investigation is required to
but with many exceptions. Asci are amyloid or test whether Verticillium dahliae, a sister taxon
chitinoid or lack apical rings, and true para- to G. cingulata according to Zhang et al. (2006),
physes are lacking in most members. Melanos- is actually a member of Glomerellales.
pora and allied species were excluded from
Hypocreales and recognized as a distinct The family Glomerellaceae was invalidly published by
order by Zhang and Blackwell (Hibbett et al. Locquin (1984) and was validated by Seifert and Gams
2007). Réblov et al. (2011) validated and delim- (Zhang et al. 2006). Réblov et al. (2011) validated and
delimited order Glomerellales.
ited the order Glomerellales based on morphol-
ogy and a three-gene phylogeny.
a) Coronophorales c) Hypocreales
Coronophorales is composed of primarily Hypocreales is a monophyletic order that
wood-inhabiting taxa and defined by species includes virulent plant and insect pathogens
with erumpent to superficial ascomata that as well as useful mycoparasitic, endophytic,
often collapse upon drying and that sometimes and saprobic species (Rossman et al. 1999).
possess an extensive hyphal subiculum or basal This order is defined by species with soft-
stroma. Many taxa contain Munk pores (small textured, generally light- to bright-colored peri-
pores each of which is surrounded by a ring or thecial fruiting bodies, and unitunicate asci that
thickening) in their ascomatal cell walls and a develop within apical (and centripetal in some
Quellkörper (a gelatinous mass of cells in the species) paraphyses that often dissolve at
apical region of an ascoma, believed to function maturity. The colorless ascospores vary from
in rupturing the ascoma) in their centrum nonseptate, globose to one- to multiseptate,
(Huhndorf et al. 2004a). While Munk pores ellipsoidal to filiform. Hypocreales includes
are found in a few taxa outside the order, the over 200 genera in seven families: Bionectria-
Quellkörper is a character unique to Corono- ceae, Clavicipitaceae, Cordycipitaceae, Hypo-
phorales. Filiform paraphyses are absent in the creaceae, Nectriaceae, Niessliaceae, and
group, and in most species the asci are thin- Ophiocordycipitaceae [see details in Kirk et al.
walled, clavate, and stipitate and lack an apical (2008), Lumbsch and Huhndorf (2010), and
ring. Ascospores usually are hyaline, small, and Rossman et al. (1999)].
allantoid. Anamorphs are hyphomycetous Bionectriaceae and Nectriaceae consist
when known. primarily of species previously placed in the
very large and artificial genus Nectria, which is
b) Glomerellales characterized by its nonstromatic perithecia.
Glomerellales is a monotypic order character- The genus Nectria (anamorph: Tubercularia) is
ized by dark perithecia, well-developed now restricted to a related group of 23 species.
periphysate ostioles, abundant thin-walled Nectriaceae (e.g., Haematonectria, Nectria, and
paraphyses, ascal apex thickened without visi- Pseudonectria) is defined by the orange to red,
ble discharge mechanism or with a distinct KOH+ perithecia. Bionectriaceae (e.g., Bionec-
apical annulus, and hyaline or pigmented tria, Hydropisphaera, and Roumegueriella)
ascospores. Known anamorphs include Colleto- consists primarily of pallid, Nectria-like species
trichum, Monilochaetes, Cylindrotrichum, and such as Bionectria (anamorph: Clonostachys),
Sporoschismopsis. Glomerella had been placed some of which are used to control greenhouse
in Phyllachorales, but some of its features are pathogens. Hypocreaceae includes Hypocrea
clearly distinct from those of other Phyllachor- (anamorph: Trichoderma), increasingly useful
ales, such as its lack of stromatic tissue and its in the biological control of plant pathogens
exclusively Colletotrichum anamorphs. Molec- (Samuels 2006), and Hypomyces with diverse
ular phylogenetic analyses confirmed these anamorphic states. Clavicipitaceae produces a
wide range of secondary metabolites and plant- e) Microascales

associated (e.g., Claviceps, Epichloe) and insect- Microscales is an order of primarily saprobic
associated genera including Metacordyceps and fungi in soil, rotting vegetation, dung, and
Hypocrella. Cordycipitaceae and Ophiocordyci- marine environments. A few species of this
pitaceae comprise the majority of arthropod order cause plant diseases such as Ceratocystis
pathogenic species of Hypocreales (e.g., Cordy- fimbriata, transmitted by beetles to living trees
ceps and Ophiocordyceps) as well as several and causing cacao wilt and many other eco-
lineages of mycoparasites (e.g., Elaphocordy- nomically important diseases. Other members,
ceps of Ophiocordycipitaceae). The anamorphs such as species of Pseudallescheria, cause
of Hypocreales are generally phialidic, produc- incurable diseases in humans. The order is
ing powdery to slimy conidia; chlamydospore- characterized by nonstromatic black perithe-
like anamorphs are also produced by many cial ascomata with very long necks or, rarely,
species. The anamorphs include the well- with cleistothecial ascomata that lack para-
known phialidic genera Gliocladium, Tricho- physes, and by globose and evanescent asci,
derma (Hypocreaceae), Clonostachys, Stilbella developing singly or in chains. The nonseptate,
(Bionectriaceae), Cylindrocarpon, Cylindrocla- colorless ascospores often bear ornamenting
dium, Fusarium, Tubercularia (Nectriaceae) ridges or wings. The anamorphs of Microasca-
(Rossman et al. 1999) and Beauveria, Hirsutella, ceae produce percurrently proliferating coni-
Metarhizium, and Tolypocladium (Cordycipita- diogenous cells (annellides) and sometimes
ceae, Clavicipitaceae, Ophiocordycipitaceae) chlamydosporelike or aleurioconidial synana-
(Hodge 2003; Sung et al. 2007). The “toxic morphs that are mostly classified in the hypho-
mold” fungus, Stachybotrys chartarum, repre- mycete genera Scopulariopsis, Graphium, and
sents part of a lineage separate from any of the Scedosporium (Abbott et al. 1998; Okada et al.
defined families within Hypocreales (Castlebury 1998).
et al. 2004). Halosphaeriaceae, a marine clade within
Microascales, is characterized by usually
submerged perithecial ascomata; an interascal
d) Melanosporales
tissue is absent, but the centrum is filled by a
The typical characteristics of Melanosporales
thin-walled pseudoparenchyma that dissolves
include translucent perithecial or cleistothecial
or breaks up to form filamentous catenophyses.
ascomata, pseudoparenchymatous centra,
A small number of species in this family are
absence of paraphyses in development, clavate
found in freshwater, but the majority consists
evanescent asci, and dark-colored ascospores.
of marine species. The ascus wall in most spe-
Previous phylogenetic studies based on rDNA
cies deliquesces, releasing the ascospores,
sequences suggested that Melanospora was
which are forced into the neck of the ascocarp
within or near Hypocreales (Spatafora and
by the production of additional asci and ascos-
Blackwell 1994; Zhang and Blackwell 2002).
pores. Several anamorphs have been attributed
When analyzed with more taxa of the Sordar-
to Halosphaeriaceae, including chlamydos-
iomycetes, Melanospora formed a distinct clade
porelike anamorphs and several genera of
outside Hypocreales (Castlebury et al. 2004). A
so-called Ingoldian hyphomycetes. The hypho-
four-gene phylogeny (Zhang et al. 2006) sup-
mycetous anamorph Varicosporina ramulosa
ported the exclusion of Melanospora from the
produces conidia and sclerocarps, sclerotium-
Hypocreales. Furthermore, a close relationship
like fruiting bodies that lack sexual capacity. It
between Coronophorales and Melanosporales
was postulated that V. ramulosa was an asco-
was recognized. Similar morphological and
mycete that had lost its ability to reproduce
ecological features of the two orders include a
sexually (Kohlmeyer and Charles 1981).
pseudoparenchymatous ascomal wall, clavate,
Sclerocarps are morphologically similar to
deliquescent asci, lack of paraphyses (with a
ascomata of Corollospora species and well
few exceptions in Coronophorales), and often
adapted to extreme conditions of sandy bea-
a mycoparasitic habit.
ches, where they may be exposed to long and often have conspicuous refractive apical
periods of drying and extreme high or low rings. The ascospores vary from colorless, allan-
temperatures. toid (Valsa), one-septate, ellipsoidal (Diaporthe),
or elongated (Cryptosporella) to large, multisep-
tate, and black (Melanconis). Commonly
2. Sordariomycetidae encountered anamorphs are coelomycetes, such
The members of Sordariomycetidae possess as Cytospora (Valsa), Phomopsis (Diaporthe),
light- to dark-colored perithecia. Asci are non- and Melanconium (Melanconis), producing pyc-
amyloid, amyloid, or lack apical rings, and true nidia with phialidic conidiogenous cells, often on
paraphyses are present in some members. The the same stroma as the sexual state.
subclass consists of eight orders divided into
two clades. Calosphaeriales, Diaporthales, c) Magnaporthales
Magnaporthales, and Ophiostomatales belong Magnaporthales is characterized by nonstro-
to one clade, and Boliniales, Chaetosphaer- matic black perithecia, usually with long hairy
iales, Coniochaetales, and Sordariales belong necks, tapering paraphyses, persistent asci, and
to the other clade. Representative taxa of the elongate fusiform or filiform ascospores. The
Annulatascaceae and Papulosaceae were found anamorphs are hyphomycetous and variable
to be close to the Ophiostomatales, according but can be categorized as two types: Pyricu-
to recent multilocus phylogenetic analyses laria-like or Phialophora-like. This order con-
(Schoch et al. 2009a; Zhang et al. 2006). tains devastating fungal cereal and grass
pathogens, such as Magnaporthe oryzae (rice
blast fungus, formerly known as M. grisea),
a) Calosphaeriales M. poae (summer patch pathogen of turf
Species in Calosphaeriales are saprobes grasses), and Gaeumannomyces graminis (take-
associated with woody plants. They produce all fungus of various cereals and grasses). His-
perithecial ascomata, which are often gregari- torically, these species were placed in various
ous, with separate or convergent periphysate orders in Ascomycota because of a lack of
ostioles. The peridium is two-layered, exter- convincing morphological and developmental
nally dark, and internally pallid. Asci are characters, such as Diaporthales (Krause and
formed in fascicles or spicate clusters, usually Webster 1972), Phyllachorales (Barr 1977), and
with a refractive nonamyloid apical ring. Para- Xylariales (Barr 1977). A new single-family
physes are lacking or few when present. Ascos- order, Magnaporthales, was recently established
pores are light colored, allantoid or ellipsoid, for these fungi (Thongkantha et al. 2009). Molec-
and biseriate or crowded in the ascus. Ana- ular phylogenetic studies indicated that this
morphs are hyphomycetous where known. group of fungi belongs to the subclass Sordario-
The six-gene Ascomycota phylogeny by Schoch mycetidae (Huhndorf et al. 2008; Thongkantha
et al. (2009a) grouped Calosphaeriales with Dia- et al. 2009; Zhang and Blackwell 2001; Zhang
porthales, Magnaporthales, and Ophiostoma- et al. 2006). A recent study by Zhang et al.
tales, which is followed here. (2011) indicated that anamorphic and ecological
features are more informative than the teleo-
b) Diaporthales morphic characters in defining monophyletic
Diaporthales is a strongly supported monophy- groups among taxa in Magnaporthales.
letic order and includes primarily plant-asso-
ciated fungi, such as the agent of chestnut d) Ophiostomatales
blight (Cryphonectria parasitica) and dogwood Ophiostomatales includes fungi associated with
anthracnose (Discula destructiva). The order is wood and bark such as the agents of Dutch elm
characterized by black perithecial fruiting bodies disease (Ophiostoma ulmi and O. novo-ulmi)
that may or may not be aggregated and and blue stain of hardwood and softwood tim-
immersed in a stroma, evanescent interthecial ber (O. piliferum). Most species of the order,
elements, and asci that float free at maturity however, are saprobic and usually are asso-
ciated with beetle dispersers, while related ana- f) Chaetosphaeriales

morphs, i.e., Sporothrix schenckii, are reported Chaetosphaeriales includes saprobic, often
to cause diseases in humans (Summerbell wood-inhabiting fungi. Small, dark perithecial
2003). The order is characterized by solitary, ascomata that are often setose are found in a
black perithecia, and most species have long number of genera in the group. Ascomata
necks from which sticky ascospores ooze and are typically associated with dematiaceous,
are transported by insects to new substrates. hyphomycetous anamorphs. Members of the
The asci are globose and dissolve early in devel- Chaetosphaeriales have filiform paraphyses
opment, while the ascospores often have orna- and cylindrical asci, usually with a pronounced
menting sheaths, ridges, or wings. Many of the apical refractive ring. Ascospores are often hya-
hyphomycete genera once associated with line and can range from ellipsoidal, nonseptate
Ophiostomatales have been simplified by the to elongate, almost filiform, and septate in spe-
recognition that the apparent variety in coni- cies of Chaetosphaeria, with fusiform, one- to
dium ontogeny represents a minor variation of three-septate ascospores being typical of most
a common pattern. In genera such as Leptogra- members of the order (Fernndez and Huhndorf
phium and Pesotum, there is percurrent prolif- 2005).
eration of the conidiogenous cells and delayed
secession of conidia, giving a false impression
g) Coniochaetales
of sympodial proliferation. In addition, some
Members of Coniochaetales typically possess
species have exclusively sympodially proliferat-
small, setose perithecial ascomata and occur
ing, denticulate conidiogenous cells that are
on wood, dung, or soil. The asci are more or
classified in Sporothrix (Wingfield et al. 1993).
less clavate without an apical ring. Ascospores
Ophiostoma was once considered synonymous
are brown, ellipsoidal, fusoid, or discoid, and
with Ceratocystis in Microascales, and their
most possess a germ slit. The relatively simple
long-necked, fleshy perithecia with sticky
ascomatal morphology can cause members of
ascospores associated with insects in wood rep-
the group to be confused with taxa in Chaeto-
resent a morphological homoplasy (Cain and
sphaeriales or Sordariales. The known ana-
Weresub 1957; Samuels and Müller 1978;
morphs of this order are classified as species
Spatafora and Blackwell 1994).
of Lecythophora. These are phialidic hyphomy-
cetes with the phialide often reduced to a col-
e) Boliniales larette lateral on a hyphal cell (Gams 2000).
Members of Boliniales occur primarily on
wood. It is the only order in the Sordariomyce-
h) Sordariales
tidae that contains taxa with large, stromatic,
Sordariales consists of mostly wood- and dung-
carbonaceous ascomata. Other taxa in the
inhabiting species with relatively large, erum-
group may possess soft-textured stromata that
pent, or superficial ascomata with large-celled,
may be brightly colored. Perithecia may be
membraneous, or coriaceous ascomatal walls.
polystichous or monostichous, wherein they
Filiform papraphyses are present in some taxa,
often will be distinctively vertically elongate.
as are subapical globules within the asci. Ascos-
Members of this order possess small asci and
pores in this group show variation on a distinct
brown, ellipsoidal, frequently flattened ascos-
developmental theme and range from cylindri-
pores that often have polar germ pores. No
cal, hyaline ascospores to ellipsoid, brown
anamorphs are known in this order. Boliniales
ascospores, often with appendages or sheaths.
in the four-gene phylogeny is represented by
Many of the species in this order lack ana-
four species of Camarops encompassing dispa-
morphs, but some have lightly pigmented,
rate morphologies and is more strongly allied
phialidic, Phialophora-like anamorphs, or the
with Lasiosphaeriella nitida and Linocarpon
ascospores germinate by directly producing
appendiculatum (taxa that currently are placed
phialides (Gams 2000; Réblov and Winka
in Sordariomycetidae inc. sed.).
2000). The most important discovery in these
four-gene analyses is that the internode repre- netics within the family (e.g., Snchez-
senting Sordariales is strongly supported by Ballesteros et al. 2000; Smith et al. 2003; Triebel
both bootstrap and posterior probability, et al. 2005; Hsieh et al. 2005, 2010; Pelaez et al.
which was not shown in previous studies 2008; Ju et al. 2011). Anamorphs of Xylariaceae
(Huhndorf et al. 2004b; Miller and Huhndorf have sympodially proliferating conidiogenous
2005). The present taxon sampling encom- cells with characteristic scars and holoblastic,
passes some of the great morphological varia- single-celled conidia with detachment scars
tion within the group, but the use of four genes corresponding with those on the conidiogenous
does not distinguish any well-supported new cells. Variation in conidiophore branching and
lineages. In previous studies, well-supported the nature of the conidiogenous cell prolifera-
clades correlated with distinct ascomal wall tion (ampulliform, irregularly swollen, genicu-
characters were uncovered [see details in Miller late) allows the distinction of anamorph genera,
and Huhndorf (2005)]. Sordariaceae (e.g., Neu- such as Nodulisporium and Geniculosporium,
rospora and Sordaria) remains a well- and several others, which often delimit mono-
supported monophyletic group, and this is the phyletic groups (Ju and Rogers 1996). Amphi-
only traditional family recognized in the order. sphaeriaceae in the sense of Müller and von Arx
While Chaetomium is monophyletic with (1973) included taxa that greatly resemble
strong support, the genera Podospora and Cer- members of Xylariaceae, but with ascospores
cophora are polyphyletic. that (mostly) lack defined germination sites.
Barr (1990) presented a much more restricted
circumscription of the family, and Kang et al.
3. Xylariomycetidae (2002) published a molecular-based phylogeny,
including the restricted group. The versico-
This well-supported monophyletic subclass lored, multiappendaged conidia of the Pestalo-
contains a single order, Xylariales, which is tia complex seem to be a synapomorphic
characterized by well-developed stromata, character for these coelomycetous anamorphs
dark-colored perithecia, persistent asci often of Amphisphaeriaceae (Kang et al. 2002).
with amyloid apical rings, and true paraphyses Cainiaceae was established for a small
(Figs. 3.7–3.12). Most species of Xylariales are assemblage of monocot inhabitants with two-
saprotrophs or plant parasites in terrestrial celled ascospores ornamented with striations or
habitats. While most marine species are classi- multiple germination slits and asci with amy-
fied in Halosphaeriaceae and Lulworthiales loid apical rings (Kang et al. 1999). The two-
(Schoch et al. 2007; Spatafora et al. 1998), locus phylogeny of Lutzoni et al. (2004) places
some species, for example, Anthostomella tor- Cainia among Xylariales. Clypeosphaeriaceae
osa, represent aquatic lineages in Xylariales sensu Barr (1990) includes genera with mostly
(Kohlmeyer and Volkmann-Kohlmeyer 2002; iodine-negative ascal apices and germination
Zhang et al. 2006). Xylariales is one of the larg- sites, when present, that are pores. Important
est orders in Sordariomycetes, which includes genera include Clypeosphaeria and Endoxyla.
over 2,400 species in 209 genera and 9 families Graphostromatace contains Graphostroma,
according to the Dictionary of the Fungi (Kirk and although species have a stroma reminiscent
et al. 2008). Some of its families are larger and of Diatrypaceae and ascospores somewhat sug-
more diverse than small orders in Sordariomy- gestive of that family, its affinities are clearly
cetes. Therefore, we will discuss each family so with Xylariaceae. Diatrypaceae has long been
as not to oversimplify the picture. recognized as a natural assemblage allied with
Xylariaceae is a large assemblage and the Xylariaceae. A cardinal feature is the pale
type family of Xylariales. Perithecia are embed- brown allantoid ascospore. The anamorphs of
ded in stromata, asci usually possess amyloid Diatrypaceae also have sympodially proliferat-
apical rings, and mature ascospores are usually ing, as well as percurrently proliferating,
unicellular with a prominent germ slit. A num- conidiogenous cells (sometimes on the same
ber of recent reports have dealt with phyloge- conidiophore) but lack conspicuous scars, and
often they have tiny conidiogenous cells and dages. The members of Lulworthiales degrade
conidia densely packed into rudimentary coe- wood and marsh plants in marine and estua-
lomycetous conidiomata, sometimes little more rine environments. Recently, two species of an
than cavities in stroma (Glawe and Rogers enigmatic genus, Spathulospora, obligate para-
1982). Although circumscription of the family sites on red algae, were placed in this order
is clear and widely accepted (Barr 1990), the based on nuclear ribosomal data (Inderbitzin
delimitation of genera is, in part, artificial et al. 2004). It was noted by Inderbitzin et al.
(Acero et al. 2004). Hyponectriaceae as circum- (2004) that Spathulospora shares the apical
scribed by Barr (1990) contains 13 genera. The mucus-filled chambers of the ascospores with
type genus Hyponectria falls within Xylariales several lulworthialean species. However, major
in the two-locus phylogeny of Lutzoni et al. features of Spathulospora species, absent in
(2004). Phylogenetic analyses based on a single other members of Lulworthiales, include the
locus, ITS, found Hyponectriaceae to be mono- presence of sterile ascomatal hairs, antheridia,
phyletic (Triebel et al. 2005). Myelostromata- and trichogynes. Haloguignardia, another
ceae is monotypic; it is generally assigned to genus with algae-inhabiting species and ascos-
Xylariales, but its true affinities remain uncer- pores characterized by mucus-filled apical
tain. Iodosphaeriaceae is a recently established chambers, also is nested within Lulworthiales
small family containing only two species (Campbell et al. 2005). The only reported ana-
(Hilber and Hilber 2002). morphs in this order are the hyphomycetes,
Zalerion maritimum (teleomorph Lulworthia
uniseptata) (Nakagiri 1984), with helically
4. Orders Incertae Sedis coiled conidia, and Anguillospora marina (tele-
omorph Lindra obtusa) (Nakagiri and Tubaki
a) Koralionastelales
1983), with filiform conidia. The placement of
Based on rDNA sequences, Campbell et al.
Lulworthiales in Sordariomycetes is not settled.
(2009) assigned two marine genera, Koralio-
According to the four-gene phylogeny of Zhang
nastes and Pontogeneia, to a new order, Kora-
et al. (2006), this order is an early diverging
lionastelales, a sister group to Lulworthiales.
clade of Sordariomycetes, suggesting a new
Species in Koralionastelales have distinct para-
subclass-level lineage. Other phylogenetic stud-
physes and periphyses, while species of
ies placed it either within or close to Hypocreo-
Lulworthiales lack hamathecia in mature asco-
mycetidae, but with no statistical support
mata. Furthermore, unlike most Lulworthiales,
(Schoch et al. 2007, 2009a; Tang et al. 2007).
species in the Koralionastelales do not have
apical ascospore structures, such as apical
mucus-containing chambers or gelatinous c) Meliolales
sheaths. Koralionastelales differs from other Species in Meliolales are biotrophic and have
Sordariomycetes by the characteristic forma- never been grown in pure culture. They are all
tion of antheridia on germinating ascospores. leaf parasites in tropical regions. Perithecia are
superficial on host leaves, nonstromatic, but
associated with coarse hyphae that bear
b) Lulworthiales
“hyphopodia” that themselves are modified
Lulworthiales was recently segregated from
conidiophores. Pigmentation is black. Asci
Halosphaeriaceae based on molecular data
form a basal hymenium and tend to be broadly
(Kohlmeyer et al. 2000). Lulworthiales is char-
clavate, and they lack an apical discharge mech-
acterized by dark ostiolate ascomata, with or
anism. Ascospores are ellipsoidal to cylindrical,
without a subiculum, and the absence of inter-
dark brown, and multiseptate. Conidiogenesis
ascal tissue, although the centrum is initially
is enteroblastic. They have been suggested as
filled with a thin-walled pseudoparenchyma.
being Dothideomycetes, but developmental and
Asci are thin-walled and deliquesce early, and
phylogenetic studies place the order among
ascospores are usually filamentous, mostly with
Sordariomycetes (Luttrell 1989; Vijaykrishna
mucus-containing apical chambers or appen-
et al. 2004). Hansford (1961, 1963) has mono- transmission electron microscopy (e.g., Baral
graphed this group. 1987; Bellemère 1994; Mengoni 1986; Minter
and Cannon 1984; Verkley 1994). Although
d) Phyllachorales ultrastructural characters like the ascus dehis-
The ordinal limits of Phyllachorales are ambig- cence mechanism, along with other morpholog-
uous because of a lack of reliable morphological ical characters, and ecological or biochemical
characters that clearly delimit this group (Alex- features can be informative for classification at
opoulos et al. 1996; Samuels and Blackwell different levels for some ascomycetous groups,
2001). Recent molecular data strongly support the difficulties in gathering and interpreting
the segregation of the fleshy stroma Glomerella such data across the highly diverse Leotiomy-
(anamorph Colletotrichum) from the nonfleshy cetes cause these traditional methods to fail to
black stroma Phyllachora (Réblov et al. 2011; keep up with molecular approaches in resolving
Wanderlei-Silva et al. 2003). Glomerellales is the phylogeny of the class, especially at higher
now a member of Hypocreomycetidae, but the taxonomic ranks.
phylogenetic position of the remaining Phylla- The Assembling the Fungal Tree of Life
chorales senso lato still needs further investiga- (AFTOL) project represents a peak of effort
tion. Here we follow the Myconet (Lumbsch and achievement in studying molecular phy-
and Huhndorf 2010) and treat this order as logeny in fungi (Blackwell et al. 2006; Hibbett
Sordariomycetes incertae sedis. et al. 2007), and the traditional classification of
Leotiomycetes at high levels has experienced
considerable challenges from hypotheses
e) Trichosphaeriales
based on molecular systematics (Schoch et al.
Trichosphaeriales is a small single-family order
2009a, b; Wang et al. 2006a, b). However, sug-
whose members have carbonized, relatively
gestions such as the inclusion of Erysiphales,
thin perithecial walls and tend to be lignicolous,
Myxotrichum, and Pseudogymnoascus and the
less frequently fungicolous. Perithecia are
exclusion of Geoglossaceae were solely or
superficial, nonstromatic, often with hairs, or
mainly based on molecular data with little evi-
setose conidiophores arising from the surface
dence from ecology and morphology, and this
of the peridium. Asci have a reflective, inamy-
makes it very difficult to teach or to study
loid apical ring. Ascospores are variable.
Leotiomycetes in a taxonomic context (Weber
Phylogenetic analysis of LSU sequences of
2009). The traditional classification of Leotio-
Cryptadelphia, a constituent genus, suggested
mycetes was reviewed by Pfister and Kim-
a close relationship with Sordariomycetidae
brough (2001) within the morphological group
(Réblov and Seifert 2004).
Discomycetes for The Mycota VII (McLaughlin
et al. 2001). Additional studies with a wide
taxon sampling in Leotiomycetes at different
III. Leotiomycetes levels have been published since then, and
reconciliation is thus indispensable for Leotio-
Following Eriksson (2005), Leotiomycetes, mycetes and Geoglossomycetes between the
including Geoglossaceae, was referred to as traditional classification and recent develop-
the “inoperculate discomycetes,” a group of ments in molecular phylogenies.
nonlichenized ascomycetes characterized by
the production of open ascomata (apothecia)
and asci opening with an apical perforation or A. Classification History
pore (inoperculate) for releasing ascospores. 1. History of Leotiomycetes
The types of dehiscence in asci have been one
of the central issues in discomycetes classifica- Leotiomycetes was based on Leotiales
tion, and sophisticated terms have been devel- (Helotiales) for nonlichenized inoperculate
oped to describe and to classify structural discomycetes, and the inclusion of the other
characters revealed by light microscopy and three orders, Cyttariales, Erysiphales, and
Rhytismatales, was primarily supported with phology and ecology, their interordinal
SSU rDNA data (Eriksson and Winka 1997). relationships within Leotiomycetes have not
However, some members of Helotiales were yet been well established regarding the relation-
positioned paraphyletically among other asco- ships among them and other members of the
mycetes and within Leotiomycetes in published class (Schoch et al. 2009a; Wang et al. 2006a, b).
phylogenies, and Leotiomycetes was not
accepted by some classifications (e.g., Pfister Cyttariales hosts a group of weak parasites in southern
and Kimbrough 2001). As discussed subse- South America and southeastern Australasia that pro-
quently, the monophyly of Leotiomycetes in its duce the largest fruiting bodies (apothecia) in Leotio-
mycetes on Nothofagus trees, and the biogeography of
current concept has not yet been verified with a Cyttaria species has been of special interest to mycolo-
proper taxon sampling and a robust phylogeny, gists and evolutionary biologists since Charles Darwin.
and the use of Leotiomycetes should be consid-
ered more as a practicable solution for manag- Close relationships between Cyttariales and
ing the extreme diversity within these fungi than members of Helotiales were hypothesized based
a solid rank for a fully matured classification. on morphological characters, and molecular
The contents of the order Helotiales and Leotio- phylogenies supported the placement of Cyttar-
mycetes have changed dramatically during the iales in Leotiomycetes [reviewed in Peterson
history of major classifications in Ascomycetes, and Pfister (2010) and Peterson et al. (2010)].
and a summary of these changes is updated In contrast, the smallest but delicately
from Spooner (1987) in Table 3.1. structured fruiting bodies (cleistothecia) are
found in Erysiphales (Takamasu 2004). Given
the dramatic differences in morphology, connec-
2. Current Classification of Leotiomycetes
tions between powdery mildew fungi and other
Because of the important and diverse ecological Leotiomycetes are difficult to interpret directly at
roles of these fungi, morphological classifica- the morphological level, and the inclusion of
tion of Leotiomycetes has been extensively Erysiphales in Leotiomycetes has been only
studied, and their classification has experienced inferred with molecular phylogeny (LoBuglio
many changes over a century, especially and Pfister 2010; Wang et al. 2006a, b). Although
recently, as molecular characters have become Erysiphales has a high diversity at the species
available. Many of these revisions have been level, monophyly of the order is strongly sup-
controversial. The classification of Leotiomy- ported with morphology, ecology, and molecular
cetes published recently by Lumbsch and phylogeny, and the single family Erysiphaceae
Huhndorf (2007b) on Myconet and by Kirk consists of 16 genera and approximately 650
et al. (2008) in the Dictionary of the Fungi species (Takamasu et al. 2010). Similar to the
are widely accessible and the most inclusive case of Erysiphales, the placement of Thelebo-
ones beyond the family level. Kirk et al. (2008) lales in Leotiomycetes is solely suggested with
accepted five orders, Cyttariales, Erysiphales, molecular characters from one or two Thelebolus
Helotiales, Leotiales, and Rhytismatales, and species (Gernandt et al. 2001; Landvik et al. 1998;
included Thelebolales with uncertainty, and LoBuglio and Pfister 2010; Peterson and Pfister
these orders, except Leotiales, were recognized 2010). Thelebolaceae had been included in the
in Lumbsch and Huhndorf (2007b) for Leotio- Pezizales for a long time because the species
mycetes as well. Accommodating the SSU rRNA produce small, globose to disk-shaped ascomata
phylogeny in Hambleton and Sigler (2005), with sometimes polysporus asci, and the devel-
Eriksson (2006) treated Myxotrichaceae as fam- opment of the ascomata varies from a cleistohy-
ily incertae sedis for the class, and this place- menial type to a eugymnohymenial type (de
ment was followed in Lumbsch and Huhndorf Hoog et al. 2005; Wang et al. 2006b). Some spe-
(2007b) (Table 3.1). cies of Thelebolales might be true members of
Although the orders Cyttariales, Erysi- Pezizales with respect to their ascus dehiscence
phales, and Thelebolales are distinct in mor- mechanisms (Hansen and Pfister 2006).
Table 3.1 Family structure of Helotiales (left six columns) and the development of the Leotiomycetes (right four columns) in major classifications (updated from Spooner
1987)
Helotiales (Leotiales) Leotiomycetes (Leotiomycetidae)

Eriksson (2006),
Lumbsch and
Dennis Hawksworth et al. Eriksson Kirk et al. Huhndorf Kirk et al.
Nannfeldt (1932) Korf (1973) Barr (1976) (1978) (1983) (1983) Eriksson (1999) (2001) (2007) (2008)
Dermateaceae + + + + + + + + +
Geoglossaceae + + + + + + + +
Hyaloscyphaceae + N/A + + + + + + +
Orbiliaceae + + + + +
Helotiaceae Leotiaceae N/A Helotiaceae Helotiaceae Leotiaceae + + + +
Phacidiaceae + + + + + +
Sclerotiniaceae + + + + + + + +
Ascocorticiaceae + + + ? ? + ? +
Hemiphacidiaceae + + + + + + +
Mycocaliciaceae Caliciaceae
Gelatinodiscaceae +
Medeolariaceae +
(Medeolariales)
Baeomycetaceae +
Leotiaceae + + + (Leotiales)
Loramycetaceae + + +
Rutstroemiaceae + + +
Vibrisseaceae + + +
Cyttariaceae + (Helotiales) + +
(Cyttariales)
Erysiphaceae + +
(Erysiphales)
Ascodichaenaceae + + +
(Rhytismatales)
Cryptomycetaceae +
(Rhytismatales)
Rhytismataceae + + +
(Rhytismatales)
Bulgariaceae + + (Leotiales)
Cudoniaceae + +
(Rhytismatales) (Rhytismatales)
Thelebolaceae ?
(Thelebolales)
Myxotrichaceae?
The name Helotiales, instead of Leotiales adaptive morphology and parasitic lifestyle
suggested by Carpenter (1988), is used here (Hawksworth 1995; Johnston 1997; Livsey and
following Korf (1973), but the content of the Monter 1994; Minter and Cannon 1984).
order is modified from Eriksson (2006) and However, a recent study showed a parasitic or
Lumbsch and Huhndorf (2007b) with the endophytic lifestyle playing critical roles via
exclusion of the family Geoglossaceae. The divergent, convergent, and parallel evolution
order Helotiales, one of the largest nonlichen- in shaping the evolution of morphology in the
forming ascomycetous groups, is composed of Ascomycota (Schoch et al. 2009b; Wang et al.
fungi of diverse morphology and ecology, and 2009). Molecular data of Ascodichaenaceae and
helotialean fungi have been reported from a Cryptomycetaceae were recently available for
broad range of niches. To make the classifica- phylogenetic analysis, and significant changes
tion of this order more complicated, correla- in the classification of Rhytismatales are
tions between teleomophs and anamorphs in expected in the near future (Lantz et al. 2010;
the families Dermateaceae, Hyaloscyphaceae, LoBuglio and Pfister 2010).
and Leotioaceae (as Helotiaceae) were regarded
as heterogeneous (Huhtinen 1989; Sutton and
Hennebert 1995), and the monophyletic status B. Molecular Phylogeny Update
of Helotiales is also rejected based on ultra- 1. Higher-Level Relationships of Leotiomycetes
structural study and molecular phylogeny (Pfis-
ter and Kimbrough 2001; Schoch et al. 2009a; The evolutionary history of Leotiomycetes was
Wang et al. 2006b). Twelve families, including investigated haphazardly in the AFTOL-1 proj-
Ascocorticiaceae, Bulgariaceae, Dermateaceae, ect. Only two out of six or seven orders were
Helotiaceae, Hemiphacidiaceae, Hyaloscypha- sampled using multilocus sequence data,
ceae, Leotiaceae, Loramycetaceae, Phacidia- resulting in an unresolved phylogeny with poly-
ceae, Rutstroemiaceae, Sclerotiniaceae, and tomies and long branches at diverse levels
Vibrisseaceae, are accepted in Helotiales by (James et al. 2006; Lutzoni et al. 2004; Schoch
Lumbsch and Huhndorf (2007b) and Kirk et al. 2009a; Wang et al. 2006a, b). Close rela-
et al. (2008). Hosoya et al. (2010) accepted the tionships between members of Leotiomycetes
Lachnaceae as well for lachnoid fungi based on and species of Sordariomycetes, rather than
an rDNA phylogeny. Except for monotypic or operculate discomycetes, are suggested by a
small families such as Ascocorticiaceae, Bulgar- study of the mechanisms of fertilization,
iaceae, Leotiaceae, and Loramycetaceae, which ascomatal development, and ascal structures
reflect our lack of knowledge about relation- (Kimbrough 1981; Pfister and Kimbrough
ships among these fungi and other helotialean 2001; Verkley 1994, 1996). Recent molecular
species, monophyly of other families in Helo- phylogenies have consistently recognized a sib-
tiales has been questioned with morphological ling relationship between these two classes, but
and molecular characters from limited taxon significant support for a monophyletic Leotio-
samplings (Pfister and Kimbrough 2001; mycetes has only been achieved with multilocus
Schoch et al. 2009a), and retaining these ranks sequence data from a very limited taxon sam-
is primarily for practical purposes. pling (e.g., James et al. 2006; LoBuglio and
The order Rhytismatales hosted only leaf Pfister 2010; Lutzoni et al. 2004; Schoch et al.
endophytic fungi and pathogens causing seri- 2009a, b; Spatafora et al. 2006). Sibling relation-
ous plant diseases, before the saprotrophic ships between Leotiomycetes and Sordariomy-
Cudoniaceae was added to Ascodichaenaceae, cetes have not yet been established with an
Cryptomycetaceae, and Rhytismataceae in this inclusive taxon sampling of Leotiomycetes
order (Pfister and Kimbrough 2001; Kirk et al. that includes Cyttariales, Theleborales, and
2008; Wang et al. 2006a, b). Although Erysiphales, as well as traditional inoperculate
challenged with ultrastructural observations, discomycetes (e.g., Gernandt et al. 2001;
the traditional classification of Rhytismatales LoBuglio and Pfister 2010; Peterson and Pfister
has been supported with the reduced but highly 2010; Saenz et al. 1994; Schoch et al. 2009a, b;
Fig. 3.20 Outline of previous classifications of Sordariomycetes
Spatafora et al. 2006; Wang et al. 2006a, b). To restricted separately to South American and
delimit Leotiomycetes and to resolve relation- eastern Asia (Takamasu et al. 2005). Species
ships among major clades of Leotiomycetes and of the genus Thelebolus (Thelebolales) have
Sordariomycetes, multilocus data for some been frequently isolated from the Arctic and
lichenized lineages, for species of Cyttariales, Antarctic, and the position of Thelebolales in
Erysiphales, and Thelebolales, and for species Leotiomycetes is only weakly supported by
of Bulgaria, Chlorociboria, and Bisporella in rDNA data (de Hoog et al. 2005). Species of
Helotiales are critical. Helotiales, the largest order of nonlichenized
ascomycetes and apparently not monophyletic,
2. Phylogeny Within Leotiomycetes are reported from all over the world, but some
major clades in this order may not historically
In summary, phylogeny within Leotiomycetes reside in the Southern Hemisphere. Problems
is not well resolved with molecular data in resolving lower-level clades (family and
(Fig. 3.20), and polytomies and long branches genus) in Leotiomycetes may be caused by
encountered in Leotiomycetes phylogeny recent adaptation to novel ecological roles in
reflect the complicated natural history of these new environments, with the result that almost
fungi. Three recent publications, by LoBuglio all morpho families in Helotiales collapse in
and Pfister (2010), Peterson and Pfister (2010), recently published gene trees. Evidence for
and Lantz et al. (2011), are among the most this lack of resolution includes unresolved rela-
important developments toward resolving the tionships among clades composed of taxa that
phylogeny of Leotiomycetes. are morphologically very different yet share
Biogeographic isolation and disjunction similar ecological characters [e.g., the aquatic-
may be one of the major reasons for the diffi- saprobe clade in Wang et al. (2005)], and the
culties in resolving phylogenetic relationships evidence also includes lineages with very long
within orders of Leotiomycetes. Cyttariales, a branch lengths (e.g., worldwide distributed
single-genus order with a dozen species, so far small or monotypic genera Bisporella, Neobul-
has been found only in the Southern Hemi- garia, Medeolaria, Chlorociboria, or Cordierites
sphere (Peterson and Pfister 2010; Peterson (LoBuglio and Pfister 2010; Peterson and Pfister
et al. 2010). Erysiphales has two basal lineages 2010; Wang et al. 2006a, b).
Sampled genera Families Orders

Ciboria Sclerotiniaceae
Sclerotinia Sclerotiniaceae
Monilinia Sclerotiniaceae
Scleromitrula Sclerotiniaceae HELOTIALES
Rutstroemia Rutstroemiaceae
Piceomphale
Heyderia Hemiphacidiaceae
Chlorencoelia Hemiphacidiaceae
Hemiphacidium Hemiphacidiaceae HELOTIALES
Meria Hemiphacidiaceae
Fabrella Hemiphacidiaceae
Bryoglossum Helotiaceae HELOTIALES
Lachnum Hyaloscyphaceae HELOTIALES
Hydrocina Hyaloscyphaceae HELOTIALES
Hyaloscypha Hyaloscyphaceae HELOTIALES
Mitrula Helotiaceae HELOTIALES
Gremmeniella Helotiaceae HELOTIALES
Asocoryne
Chloroscypha Helotiaceae HELOTIALES
Cudoniella Helotiaceae
Ombrophila HELOTIALES
Neobulgaria Helotiaceae HELOTIALES
Bisporella Helotiaceae HELOTIALES
Chlorocibora Helotiaceae HELOTIALES
Chlorovibrissea Helotiaceae HELOTIALES
Cordierites Helotiaceae HELOTIALES
Hymenoscyphus Helotiaceae HELOTIALES
LEOTIOMYCETES Dermea
Neofabraea Dermateaceae HELOTIALES
Pezicula
Loramyces Loramycetaceae
Mollisia Dermateaceae HELOTIALES
Vibrissea Vibrisseaceae
Phialocephala
Microglossum Leotiaceae HELOTIALES
Leotia
Bulgaria Bulgariaceae HELOTIALES
Phacidiopycnis
Phacidium Phacidiaceae HELOTIALES
Pseudophacidium Ascodichaenaceae RHYTISMATALES
Potebniamyces Cryptomycetaceae RHYTISMATALES
Holwaya Bulgariaceae HELOTIALES
Ascodichaena Ascodichaenaceae RHYTISMATALES
Cudonia Cudoniaceae
Rhytisma Rhytismataceae RHYTISMATALES
Cryptomyces Cryptomycetaceae
Medeolaria Medeolariaceae MEDEOLARIALES
Thelebolus Thelebolaceae THELEBOLALES
Myxotrichum Myxotrichaceae
Pseudeurotium Pseudeurotiaceae
Erysiphe
Golovinomyces Erysiphaceae ERYSIPHALES
Blumeria
Encoelia Encoelioideae HELOTIALES
Cyttaria Cyttariaceae CYTTARIALES
LABOULBENIOMYCETES
SORDARIOMYCETES
LICHINOMYCETES
LECANOROMYCETES
EUROTIOMYCETES
DOTHIDEOMYCETES
ARTHONIOMYCETES
Thuemenidium
GEOGLOSSOMYCETES Sarcoleotia
Trichoglossum Geoglossaceae GEOGLOSSALES
Geoglossum
ORBILIOMYCETES
PEZIZOMYCETES
Fig. 3.21 Phylogeny of Leotiomycetes. Branch lengths are not proportional to genetic distances
Besides the order Medeolariales with as monophyletc with at least rRNA sequence
only one species, the orders Cyttariales, Erysi- data; however, taxon sampling is not
phales, and Thelebolales have been supported considered as sufficient for the species-rich
Erysiphales and morphologically diverse The- lematic group in Leotiomycetes, the order
lebolales (LoBuglio and Pfister 2010; Peterson Helotiales, and this is likely due to the difficul-
and Pfister 2010; Schoch et al. 2009a). Although ties in identifying and generating data from
Medeolaria farlowii was reported almost a cen- proper genetic markers for evolutionary events
tury ago, reliable rRNA sequence data were not in this epoch, when there seems to be a quick
available until LoBuglio and Pfister (2010), and radiation of helotialean diversity. Consistent
its position in Leotiomycetes was confirmed. A with Wang et al. (2006a, b), Schoch et al.
close relationship between M. farlowii and the (2009a, b) proposed eight lineages to be recog-
always problematic Chlorociboria was sug- nized in Helotiales for future research; they are
gested without support in consensus trees in (1) Cyclaneusma and Naemacyclus clade;
the study, and top hits resulting from a BLAST (2) Leotiaceae (including Leotia and Microglos-
search with LSU-rRNA of M. farlowii are vari- sum); (3) Bulgariaceae (including Bulgaria and
ous leotiomycetous fungi but did not include Holwaya); (4) Dermeataceae sensus stricto
any Chlorociboria species. Multilocus data, (including Dermea, Neofabraea, and Pezicula);
including the protein-coding gene EF1-alpha, (5) Sclerotineaceae, Rutstroemiaceae, and
were generated from the same lab for Cyttaria Hemiphacidiaceae; (6) gelatinous species such
species (Peterson and Pfister 2010). In the as Ascocoryne and Chloroscypha; (7) Hymenos-
study, the inclusion of Cyttariales in Leotiomy- cyphus and Cudoniella related species; and (8)
cetes was further verified with multilocus data, aquatic Vibrissea, Loramyces, root endophytes
and Encoelioideae of Helotiales, including spe- Phialocephala, and Mollisia. Among these
cies of Ionomidotis, Encoelia, and Cordierites, lineages, the Sclerotineaceae, Rutstroemiaceae,
together with Chlorociboria species, were iden- and Hemiphacidiaceae clade should be justified
tified as sister groups to Cyttariales with mod- as an order with morphology (including bio-
erate support (Peterson and Pfister 2010). The chemistry), ecology, and molecular systematics.
apothecia of Encoelioideae fungi excrete violet
pigmentation in aqueous KOH solution, a so-
called ionomidotic reaction, and Encoelioideae
is likely polyphyletic based on morphology and C. Characters
molecular characters (Peterson and Pfister 1. Apothecium (Ascoma) Morphology,
2010; Zhuang 1988; Zhuang et al. 2000). It Anatomy, and Ultrastructure
would also be desirable to have data of
M. farlowii included in the Cyttaria studies. Apothecial morphology has been well studied
Shortly after LoBuglio and Pfister (2010), for major groups traditionally classified
who pointed out that there were no data from within Leotiomycetes, and a detailed review is
Ascodichaenaceae and Cryptomycetaceae for provided in Pfister and Kimbrough (2001).
resolving the phylogeny of the order Rhytisma- Considerable differences in morphology
tales, Lantz et al. (2011) reported a core clade of among species in Leotiomycetes make it one
Rhytismatales using mitochondrial SSU rRNA of the most difficult groups for taxonomy; how-
and nuclear LSU rRNA data from representa- ever, an ascoma morphology definition for the
tives of all four families in the order. Not orders Cyttariales, Erysiphales, Medeolariales,
unanticipated, Rhytismatales and its families and Thelebolales is well established. For most
Ascodichaenaceae, Rhytismataceae, and prob- genera in Helotiales, macromorphological
ably Cryptomycetaceae did not form a mono- characters are reliable, but exceptions do exist,
phyletic group, but data from a wider taxon for example, in Ascoryne and Vibrissea (Bun-
sampling for genes that are more informative yard et al. 2008; Wang et al. 2006b). At the
are required to further accept or reject these family level, current molecular phylogenies
suggestions, which were based on a poorly do not support traditional classification using
resolved phylogeny. ascoma morphology for the families Helotia-
Little progress has been made in molecular ceae, Leotioaceae, Hyaloscyphaceae, and Der-
phylogeny for the polyphyletic and most prob- mateaceae in Helotiales and the families
Ascodichaenaceae and Cryptomycetaceae in tions, hosts, and distribution, they are poten-
Rhytismatales. Anatomical characters, such as tially good indicators for historical and ongoing
the structure of the excipulum, epithecium, events in global climate changes.
gelatinous layer, paraphyses, and hymenium, Species of Leotiomycetes also contribute a
are informative for classification at the genus significant diversity to fungal endophytes of
or lower levels, as are histochemical characters leaves (U’Ren et al. 2010; Wang et al. 2009),
of asci and ascospores. The ultrastructure of including conditional pathogens. Leaf endo-
asci has been used in classifications in some phytes are concentrated in the families
groups in both Leotiomycetes and Geoglosso- Rhytismataceae, Cryptomycetaceae, and Asco-
mycetes (Pfister and Kimbrough 2001), but a dichaenaceae in the Rhytismatales and Derma-
lack of widespread use of these characters in teaceae, Hemiphacidiaceae, Rutstroemiaceae,
many other groups makes it hard to evaluate and Sclerotiniaceae in the Helotiales. Appar-
the usefulness of these techniques, which usu- ently many leaf endophytes are adapted to the
ally require special equipment and training. leaf environment by producing highly reduced,
Difficulties encountered in recognizing homo- dark-colored, and often covered ascomata, and
logies between ultrastructural characters are their close saprotrophic relatives, in contrast,
further complicated by the complex terminol- produce large and bright-colored fruiting bod-
ogy used by different researchers. ies (Lantz et al. 2011; Wang et al. 2009). Thus,
molecular data are critical in classifying Leotio-
mycetes that produce a small and highly
2. Ecology and Biogeography reduced reproductive structure, because mor-
phological characters in these fungi are gener-
More and more evidence, especially inferred
ally limited and not very informative. A highly
from molecular data, suggest ecology and bio-
reduced morphology and life history are also
geography have been critical for understand-
found in Leotiomycetes associated with roots
ing the evolution of Leotiomycetes. Within
of certain groups of plants, such as the Phialo-
the incredible variety of ecology observed for
cephala fortinii s.l.—Acephala applanata spe-
Leotiomycetes, three groups that are highly
cies complex, also known as dark-septate root
adapted to unique niches require more atten-
endophytes, which are found in conifer roots
tion, i.e., leaf epiphytic powdery mildews, leaf
(Queloz et al. 2011). Although the mating-type
endophytes—plant pathogens, and aeroaquatic
locus has been partially characterized, sexual
fungi—root endophytes.
reproduction has not yet been observed for
this species complex (Zaffarano et al. 2010).
The powdery mildews are a group of obligate bio-
trophic fungi attacking about 10,000 angiosperm
Molecular phylogenies identified fungi in
species, and the host range is strictly confined to the mostly aeroaquatic Loramyces-Vibrissea-
angiosperms for this fungal group that originated in Mollisia clade as closely related to these root
the late Cretaceous (Amano 1986). endophytes (Grünig et al. 2008; Upson et al.
2009; Wang et al. 2006a, b), and a shared evolu-
Molecular phylogenetic analyses suggested tionary history between root endophytes and
that Erysiphaceae is a monophyletic group aquatic hyphomycetes has been hypothesized
(Wang et al. 2006b). The molecular phylogenies based on molecular and morphological data
of powdery mildews also suggested that tree- (Sati and Belwall 2005; Selosse et al. 2008).
parasitic species usually take basal positions Independent origins of ectomycorrhizae are
and herb-parasitic species have derived posi- observed in the Helotiales (Tedersoo et al.
tions, but multiple host shifts from trees to 2009), and this includes species in Hymenoscy-
herbs may have then occurred during the phus and related genera often isolated from the
Tertiary [a detailed review is provided in Taka- roots of Ericaceae (Hambleton and Sigler 2005).
masu et al. (2010)]. Because powdery mildews Some species in Leotiomycetes are found to
have been well documented as pathogens for be associated with mosses, with the highest
various flowering plants for their identifica- diversity of bryosymbiotic fungi being in
Leotiomycetes among all the major ascomyce- long-distance gene flow seems also to be
teous lineages, as revealed by a five-gene restricted because of the geographic isolation
phylogeny (Stenroos et al. 2009). Some Leotio- of freshwater bodies. It is worthy of mention
mycetes associated with mosses were found to that an isolate related to Ascocoryne, Helotiales,
be capable of colonizing lichens, and a high was found to produce hydrocarbon derivatives
similarity of moss endophytes and endolichenic of potential interest for the biofuel industry
fungal communities was observed (U’Ren et al. (Strobel et al. 2010).
2010).
Leotiomycetes also show biogeographic
isolation and disjunction within major clades. IV. Geoglossomycetes
The order Cyttariales is found only in the
Southern Hemisphere, and a recent study The class Geoglossomycetes with a single fam-
based on the cophylogeny and biogeography ily, Geoglossaceae, was created for Geoglossum,
of these fungal parasites and its southern Trichoglossum, and Sarcoleotia to accommo-
beech hosts accepted both a vicariance hypoth- date recent molecular phylogenies and a new
esis and long-distance dispersal model for the interpretation of previously observed develop-
distribution of Cyttaria species (Peterson et al. mental morphology (Schoch et al. 2009a, b;
2010). The basal lineages of the Erysiphales are Schumacher and Sivertsen 1987; Spooner
restricted to South America and eastern Asia 1987; Wang et al. 2006a, b). Geoglossaceae was
(Takamasu et al. 2005). The distribution of initially proposed to include club-shaped,
Thelebolus (Thelebolales) is largely in the Arc- unitunicate, inoperculate discomycetes (earth
tic and Antarctic (de Hoog et al. 2005). Species tongues), but the content of Geoglossaceae has
of Helotiales are reported from all over the experienced significant changes, especially with
world, but some major clades, such as lineages the development of molecular systematics
in Sclerotiniaceae, may not historically reside (Korf 1973; Spooner 1987; Wang et al. 2002,
in some areas of the Southern Hemisphere. It is 2006a, b).
also worth mentioning that Southern Hemi-
sphere species of Chlorovibrissea and Vibrissea Species of Geoglossum, Trichoglossum, and Sarcoleotia
albofusca evolved a morphology that is remark- possess a hymenium that freely develops toward the
ably similar to that of the also aquatic Vibrissea base of the stalk. Other earth tongues, such as species
species found in the Northern Hemisphere, of Leotia, Microglossum, Bryoglossum of Helotiales,
although the two biogeographic groups are or Cudonia of Rhytismatales, show a distinct bond
between the developing hymenium and infertile stalk,
not closely related in Helotiales (Wang et al. implying an ancestral state of covered (cleistohyme-
2006b). nial) hymenium that has not been lost in Cyttaria,
Many helotialean fungi are now known to Erysiphales, Rhytismatales, and probably some species
be associated with ectomycorrhizas, and such in Thelebolales in Leotiomycetes.
relationships probably evolved independently
among biogeographic groups, as inferred from The relationships between Geoglossomy-
molecular phylogenies (Tedersoo et al. 2009). cetes and other basal lineages and lichenized
Interestingly, no biogeographical pattern has ascomycetes in the Pezizomycotina require fur-
been identified in the root-associated Phialoce- ther investigation (Lutzoni et al. 2004; Schoch
phala fortinii s.l.—Acephala applanata species et al. 2009a, b; Spatafora et al. 2006).
complex after a worldwide sampling, and long- Conflicting placements of Geoglossomy-
distance distribution mechanism, e.g., by con- cetes within Pezizomycotina were inferred
idia, has been shown to be rare, if it even from different genes in different analyses, and
occurs, for this root endophyte complex (Que- the relationships among Geoglossomycetes,
loz et al. 2011). Furthermore, Leotiomycetes Orbiliomycetes, Pezizomycetes, and two lichen
closely related to these root endophytes include lineages, Lecanoromycetes and Lichinomy-
some aeroaquatic species in Vibrissea and cetes, are not resolved given the so far poorly
Loramyces (Grünig et al. 2008, 2009), whose supported backbone in the phylogeny of
Pezizomycotina (Schoch et al. 2009a, b). In Mallach 1989). Species in Pyxidiophorales

addition to the genera Geoglossum, Trichoglos- occur in diverse habitats, such as dung, bark
sum, and Sarcoleotia, Ohenoja et al. (2010) beetle galleries, and plant debris. Unusual
suggested that one of the three species of Thue- features of Pyxidiophora include perithecia
menidium, T. arenarium, is also a species in consisting of a single layer of wall cells, and
this class, while the other Thuemenidium spe- three-ascospore asci. Ascospores of Pyxidio-
cies are probably related to Microglossum. phora are carried by mites (Alexopoulos et al.
1996).
V. Laboulbeniomycetes
VI. Problems and Perspectives
This class contains over 2,000 species in two
orders, most of which are minute obligate eco-
parasites of living beetles or other arthropods A. Genome Project
(Tavares 1985; Weir and Hammond 1997). Genomics has a significant impact on biology as
These fungi had been placed in pyrenomycetes a whole, and fungal genome projects provide
or ascomycetes incertae sedis (Alexopoulos new insights into reproduction, gene gain and
et al. 1996). Recent molecular studies of Laboul- loss, horizontal gene transfer, gene structure,
beniomycetes supported a distinct but close and regulation for a better understanding of
relationship with the main body of Sordario- the evolution of fungal diversity. In systematic
mycetes (Schoch et al. 2007; Weir and Blackwell biology, phylogenetic analysis of DNA or pro-
2001). tein sequence data currently is considered the
most powerful tool for the elucidation of evolu-
tionary relationships. However, the analysis of
A. Laboulbeniales single or few loci often yields conflicting phy-
logenies. A robust systematic framework that
More than 2,000 species in 146 genera have
stands the test of time should be based on
been described in Laboulbeniales, which are
genetic information at the genomic scale,
cosmopolitan. These fungal species are often
which includes various independently evolving
attached to specific exoskeleton areas of a single
regions. It was estimated that at least 20
host insect. Typical characteristics include
unlinked genes or 8,000 randomly selected
hyaline to darkly pigmented thalli, perithecial
orthologous nucleotides are required to reach
ascomata, often surrounded by complex appen-
this goal (Rokas et al. 2003). Advancements in
dages, an absence of hamathecial structures,
high-throughput genome sequencing technol-
evanescent asci maturing sequentially, and
ogies have made large-scale phylogenomic
two-celled ascospores. The species are usually
studies possible.
haustorial parasites of arthropods. Four
families are recognized: Ceratomycetaceae,
Neurospora crassa (Sordariales, Sordariomycetes), the
Euceratomycetaceae, Herpomycetaceae, and
red bread mold, is a classic model organism in genetic
Laboulbeniaceae (Kirk et al. 2008; Lumbsch studies. The genome of N. crassa, which is approxi-
and Huhndorf 2010). Monographs of Laboulbe- mately 43 megabases long and includes approximately
niomycetes were written by Thaxter (1896– 10,000 genes, was reported as completely sequenced in
1918) and Tavares (1985). 2003 (Galagan et al. 2003).
Soon thereafter, tremendous progress was

B. Pyxidiophorales made in the genomics of a number of other
model organisms in Sordariomycetes and Leo-
This single-family order contains 22 species in tiomycetes (Couch et al. 2005; Aguileta et al.
five genera. Molecular phylogenetic analyses 2009; Fillinger et al. 2007; Xu et al. 2007).
supported their sister group relationship with According to the Genome Online Database
Laboulbeniales (Blackwell 1994; Blackwell and (www.genomesonline.org), on May 25, 2012,
there were 105 Sordariomycetes and 15 Leotio- described species, 100 genomes are far from
mycetes complete and ongoing genome pro- enough for investigating their biological and
jects, which constitue 22 % of the 544 genome genetic diversity. With the high-throughput
projects for all ascomyceteous fungi. next-generation sequencers, exponential
Fungal species being sequenced for gen- growth of the genome data for these fungi in
omes are mostly economically important, and the coming decades is expected. However,
many of them are pathogens. For instance, the downstream bioinformatic analyses, including
genome project for Magnaporthe oryzae (for- genome assembly and gene annotation, will
mer name: M. grisea), the rice blast fungus, was likely be a bottleneck and a challenge.
completed by the International Rice Blast
Genome Consortium and the BROAD Institute
(http://www.broadinstitute.org). The genomes B. Environmental Study
of its closely related species, M. poae (summer
patch pathogen of turfgrass) and Gaeumanno- Traditional approaches to the systematics and
myces graminis var. tritici (take-all pathgen of taxonomy of fungi to a large extent rely on the
cereals), have been sequenced and are currently presence of distinctive morphological charac-
in the process of gene annotation. The BROAD ters of reproductive structures or on culture
Institute and Department of Energy Joint studies in the laboratory. However, many
Genome Institute have sequenced several fungi do not produce detectable reproductive
plant pathogenic Fusarium genomes (teleo- morphological structures, and the majority of
morphs: Nectria and Gibberella), which enables fungal lineages defy attempts at keeping them
comparative genomic studies (Ma et al. 2010). in cultures. Significant methodological
In 2005, the international consortium Botrytis advancements in DNA extraction and next-
& Sclerotinia genome project, led by M.H. Leb- generation sequencing have become a useful
run (INRA-Bioger), was initiated to support the tool in the characterization of the diversity of
sequencing and genome annotation of these environmental or otherwise cryptic fungi.
two very closely related necrotrophic and poly- Conversely, so-called invisible fungal lineages
phageous fungi (Fillinger et al. 2007). These are identified from environmental samples provide
the first two Leotiomycetes to be fully important insight into the ecological and
sequenced. Botrytis cinerea was sequenced at biological diversity of so-called visible fungi,
the French National Sequencing Center, and which often are herbarium collections lacking
Sclerotinia sclerotiorum was sequenced at the ecological characters (Wang et al. 2011). Fungi
BROAD Institute. Genome sequences for these in both Sordariomycetes and Leotiomycetes
two species were completed and released for have been shown in many environmental stud-
public access, and publications of these two ies with samples from soil and from roots or
genomes will be available soon. In addition, leaves of different plants (e.g., Arnold et al.
the genomes of three powdery mildew fungi 2007; Arnold and Lutzoni 2007; Herrera et al.
were sequenced; they have a genome size of 2010; Higgins et al. 2011; Napoli et al. 2010;
more than 120 Mb—more than four times Seephueak et al. 2010; Soca-Chafre et al. 2011;
larger than the median of other ascomycetes— U’Ren et al. 2010). Interestingly, in many cases
and experienced many gene losses during the of environmental study, more species of
evolution of obligate biotrophy (Spanu et al. Sordaromycetes than Leotiomycetes were
2010). Two other Leotiomycetes, Leotia lubrica recovered from samples when culturing was
(Leotioaceae, Helotiales) and species of Rhy- used to isolate fungi from the samples (e.g.,
tisma (Rhytismataceae, Rhytismatales), have Gallery et al. 2007; Higgins et al. 2011), but
been nominated as candidates to be sequenced with direct PCR and cloning approaches or
for genomes in the near future (http://www. high-throughput sequencing techniques,
broadinstitute.org/). Genome data for Laboul- more species of Leotiomycetes were discovered
beniomycetes are still lacking. compared with the culturing isolation method,
For highly diverse Sordariomycetes and especially from plant materials (e.g., Hartmann
Leotiomycetes that include over 15,000 et al. 2009; Morris et al. 2008; O’Brien et al.
2005). A possible explanation is that many leo- ascomata and conidia (sporulation) is some-
tiomycetous fungi are likely strict endophytes times enhanced by adding natural substrates
and cannot be cultured easily in laboratory to synthetic agar, such as sterilized host plant
conditions. Considering endophytic species, tissue. For example, carnation leaf is often used
sordariomycetous endophytes are more often to enhance conidium production of Fusarium.
found in tropical forests, and leotiomycetous However, biotrophic species usually require
endophytes can be detected in temperate and more elaborate nutritional formulations. No
boreal forests with comparatively less fre- member of Laboulbeniales or Meliolales has
quency (Arnold and Lutzoni 2007). Sordario- been cultured. Furthermore, spores including
mycetes also show a higher endophytic conidia and ascospores of many fungi in Leotio-
diversity than Leotiomycetes in flowering mycetes do not germinate and grow in culture
plants and lichens, while Leotiomycetes are using standard techniques and media, and this
more common in conifers and dead plant tis- may be due to strict parasitic/endophytic stages
sues (U’Ren et al. 2010). in their life histories (e.g., Higgins et al. 2011).
Formulations of a number of general and
Within the class Sordariomycetes, the orders Sordar- selective media for phytopathogenic fungi are
iales, Xylariales, and Hypocreales are often reported available in Singleton et al. (1992). For methods
from environmental samples, while species from the used with insect-associated groups see Benja-
orders Helotiales and Rhytismatales are the most
often encountered Leotiomycetes.
min et al. (2000). The preservation of fungal
cultures in a metabolically inactive state in
liquid nitrogen has been used to minimize
But the availability of reference sequences in
mutation. With the increased availability of
GenBank or International Nucleotide Sequence
ultra-low-temperature freezers, more labora-
Database (INSD) databases for annotated spe-
tories store their cultures in glycerol solution
cies in different groups of fungi is also a restric-
under 80 C. Other methods include lyophili-
tive factor for identifying unknown fungi from
zation or storage in sterile soil, in sterile dis-
environmental samples (Brock et al. 2009), espe-
tilled water, or on oil-covered agar slants.
cially when pragmatic attempts to characterize
Protocols of these techniques are described in
the diversity of these so-called invisible fungi
Smith and Onions (1983) and Singleton et al.
from environmental samples have relied on
(1992).
operational taxonomic units (OTUs) that have
been delimited as discrete units of sequences
that share, for example, 95–97 % pairwise simi-
larity (Buée et al. 2009; Jumpponen and Jones VIII. Conclusion
2009; O’Brien et al. 2005). As for almost all envi-
ronmental studies targeting fungal diversity, Recent phylogenetic studies using DNA
the ITS regions of rDNA are standard genetic sequences and ultrastructural data support a
markers. Recently, an automatic alignment close evolutionary relationship between the
program called SATé was developed for dealing perithecial Sordariomycetes and apothecial
with large data sets (Liu et al. 2009), and phylo- Leotiomycetes. Extant members of these two
genetic analysis with data from environmental highly diverse classes likely evolved from a
samples and annotated specimens may provide common ancestor that was a nonlichenized
profound insights about fungal ecology and saprotroph with inoperculate, unitunicate asci.
diversity (Wang et al. 2011). During the past two decades, rapid devel-
opment in molecular phylogeny and genome
sequencing has had a great impact on fungal
VII. Culture and Maintenance classification. However, progress in these inno-
vations is not well balanced across the fungal
Most saprotrophic and necrotropic species of kingdom owing to some historic and technical
Sordariomycetes and Leotiomycetes are easily issues. Sordariomycetes and Leotiomycetes
cultured on synthetic media. The production of present good examples showing how such
imbalances would affect current classification from Hypocreomycetidae, and Sordariomyceti-

of higher ascomycetes. While ordinal-level clas- dae is rich in coprophilous taxa.
sification of Sordariomycetes is well supported Ascospore wall ornamentation has been
by multilocus sequence data, the classification used to delimit many fungal genera, but
and molecular phylogeny at the higher taxo- phylogenetic studies on Melanosporales and
nomic levels for Leotiomycetes have not yet Sordariales show that this character is prone
reached a common ground. Nevertheless, to convergence. Ecological features, such as
molecular data suggest that ecology played the root vs. shoot/leaf association, have been
important roles in the recent evolution of Leo- recognized as informative characters at the
tiomycetes. generic level for Sordariomycetes and Leotio-
A synapomorphy of Sordariomycetes is the mycetes (Zhang et al. 2011).
perithecial ascoma, which is postulated to be
evolved from the apothecium of ancestral Acknowledgements We are grateful to Dr. Yu-Ming Ju
for photographs of Xylaria globosa and Dr. Guohong
Pezizomycotina. However, taxa in a number of Cai for photographs of Anisogramma anomala.
unrelated lineages of Sordariomycetes have lost
ostioles, which is usually associated with the
loss of forcible discharge of ascospores (Mal-
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4 Pezizomycotina: Lecanoromycetes
CÉCILE GUEIDAN1,2, DAVID J. HILL3, JOLANTA MIADLIKOWSKA4, FRANCOIS LUTZONI4
CONTENTS L. Teloschistales . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

M. Trapeliales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 N. Umbilicariales . . . . . . . . . . . . . . . . . . . . . . . . . . 110
II. Occurrence and Distribution . . . . . . . . . . . . . . 90 VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
III. Substrate Range and Ecology . . . . . . . . . . . . . . 91 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
A. Substrate Range . . . . . . . . . . . . . . . . . . . . . . . . . 91
B. Lifestyles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
C. Mycobiont–Photobiont Associations . . . 91
IV. Mycobiont–Photobiont Cellular Contacts 92
V. Morphological and Chemical Features . . . . 93 I. Introduction
A. Thalli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
B. Ascomata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Lecanoromycetes (formally named by Eriksson
1. Ascoma Morphology. . . . . . . . . . . . . . . . . . 93 and Winka in 1997) constitutes one of the larg-
2. Ascoma Development. . . . . . . . . . . . . . . . . 95
C. Asci. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 est classes of Ascomycota with 14,199 known
1. Ascus Walls . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 species (Kirk et al. 2008). The class mainly
2. Ascus Apical Structure. . . . . . . . . . . . . . . . 96 includes ascomycetes characterized by apothe-
3. Dehiscence Mechanisms . . . . . . . . . . . . . . 97 cial ascomata, amyloid asci with a two-layered
D. Ascospores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 wall and an apical thickening, and a hamathe-
E. Interascal Filaments . . . . . . . . . . . . . . . . . . . . . 98
F. Pycnidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 cium (interascal hyphae) formed with para-
G. Asexual Propagules. . . . . . . . . . . . . . . . . . . . . . 98 physes or pseudoparaphyses. This group
H. Secondary Compounds . . . . . . . . . . . . . . . . . 99 comprises the largest number of lichen-
VI. Origin and Diversification . . . . . . . . . . . . . . . . . 99 forming ascomycetes, with approximately
VII. Orders and Classification . . . . . . . . . . . . . . . . . 100 90 % of all known lichen-forming fungi (Mia-
A. Acarosporales. . . . . . . . . . . . . . . . . . . . . . . . . . . 100
B. Baeomycetales . . . . . . . . . . . . . . . . . . . . . . . . . . 102 dlikowska et al. 2006). However, not all mem-
C. Caliciales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 bers of this class are lichenized; several
D. Candelariales . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 nonlichenized species are nested within this
E. Lecanorales. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 lineage (Lutzoni et al. 2001, 2004; Schoch et al.
F. Lecideales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 2009). Lecanoromycetes are well known for the
G. Ostropales. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
H. Peltigerales. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 broad range of secondary compounds, such as
I. Pertusariales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 depsidones, terpenoids, and xanthones (Huneck
J. Rhizocarpales . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 and Yoshimura 1996), that they exclusively pro-
K. Sarrameanales . . . . . . . . . . . . . . . . . . . . . . . . . . 108 duce, in some cases in large quantities out of
proportion to the dry weight of their thalli.
1
Department of Life Sciences, The Natural History Museum, In the past, ascoma structure or develop-
Cromwell Road, London SW7 5BD, UK ment and ascus types were used as the main
2
CSIRO, NRCA, Australian National Herbarium, GPO box 1600, characters in the classification of ascomycete
Canberra, ACT 2601, Australia; e-mail: Cecile.Gueidan@csiro.au fungi. Nannfeldt (1932) classified the euasco-
3
School of Biological Sciences, University of Bristol, Woodland
mycetes according to their type of ascomatal
Road, Bristol BS8 1UG, UK; e-mail: D.J.Hill@bristol.ac.uk
4
Department of Biology, Duke University, Box 90338, Durham, development: Plectascales, Ascoloculares, and
NC 27708, USA; e-mail: jolantam@duke.edu; flutzoni@duke.edu Ascohymeniales, where most Lecanoromycetes

90 C. Gueidan et al.
belonged. Luttrell (1951, 1955) in his classifica- 2011; Lumbsch and Huhndorf 2010; Schmull
tion system gave more importance to the ascus et al. 2011).
types and classified the euascomycetes as Bitu-
nicatae (the Ascoloculares) and Unitunicatae II. Occurrence and Distribution
(the Ascohymeniales and Plectascales). Unitu-
nicatae were then subdivided according to their
Lecanoromycetes have a broad distribution
ascomata morphology: Plectomycetes, Pyreno-
and can be found from the tropics to the
mycetes, and Discomycetes, where most Leca-
poles. Their biomass is substantial in boreal to
noromycetes belonged (Luttrell 1955).
arctic climates, especially in the tundra, where
However, subsequent ultrastructural and onto-
genetical studies showed that the complexity of species such as Cladonia rangiferina can con-
these traits had been underestimated and that stitute the main vegetation cover and are the
main primary producer and food source for
many taxa did not fit into these broad cate-
large herbivorous mammals (Brodo et al.
gories because of intermediate types (e.g., Bel-
2001). In Antarctica, lichens (mostly from the
lemère and Letrouit-Galinou 1987; Henssen and
class Lecanoromycetes) form the most species-
Jahns 1974; Honegger 1982a). The reliability of
diverse group within the vegetation cover, and
these characters for higher-level classification
some of them are found in the coldest and
was therefore doubted even before the begin-
ning of the molecular era (e.g., Bellemère 1994; driest habitats of continental Antarctica
Poelt 1973; Reynolds 1989). (Green et al. 1999; Øvstedal and Lewis Smith
2001), where they are the basis of terrestrial life
Molecular studies confirmed that classifica-
closest to a pole on Earth.
tions based on ascomatal characters often failed
Lecanoromycetes are also very common
to capture monophyletic lineages within the
and diverse in temperate regions, where they
ascomycetes (Berbee 1996; Lindemuth et al.
have been extensively studied (e.g., Brodo et al.
2001; Lumbsch and Huhndorf 2007a; Lutzoni
2001; Clauzade and Roux 1985; McCarthy 2003;
et al. 2001; Liu and Hall 2004; Spatafora et al.
2006; Schoch et al. 2009). In particular, some Smith et al. 2009). In the tropics, they are
groups within Lecanoromycetes (e.g., Ostro- thought to be as species diverse as in temperate
regions, if not more so (Aptroot and Sipman
pales, Caliciales) were shown to include a
1997; Coppins and Wolseley 2002; Lücking
much larger diversity of ascoma and asci types
2008). A recent study in Papua New Guinea
than expected (Grube et al. 2004; Schmitt et al.
showed that a single Elaeocarpus tree could
2005; Wedin and Tibell 1997; Wedin et al.
harbor up to 173 species of lichens, among
2000a). Molecular phylogenetic studies also
which approximately 130 species were Lecanor-
cast doubts on the morphology-based delimi-
tation of orders and families in Lecanoromy- omycetes (Aptroot 2001). Many tropical
cetes. As a result, many families and genera regions of the world are still mainly understu-
died, and many new species remain to be dis-
were segregated from the large order Lecanor-
covered (Sipman and Aptroot 2001).
ales, while several additional families were
Lecanoromycetes are found in a large num-
included in Ostropales, the second largest
ber of natural terrestrial habitats, such as
order in Lecanoromycetes (Grube et al. 2004;
woodlands, heathlands, lowland and alpine
Hofstetter et al. 2007; Kauff and Lutzoni 2002;
grasslands, deserts, and arid shrublands. They
Lumbsch et al. 2004; Miadlikowska and Lutzoni
2004; Miadlikowska et al. 2006; Wedin et al. are also commonly found in urban areas, waste-
2005). Lecanoromycetes currently includes 14 lands, and other anthropogenous habitats
(Gilbert 1990; Smith et al. 2010). Although
orders and 3 subclasses: Acarosporomycetidae,
most species occur in terrestrial environments,
Lecanoromycetidae, and Ostropomycetidae
some species can grow on temporarily
(Gaya et al. 2012; Hodkinson and Lendemer
submerged substrates, in freshwater (Gilbert
Pezizomycotina: Lecanoromycetes 91
1996; Gilbert and Giavarini 1997; Thüs and nonlichenized saprotrophs, depending on the
Schultz 2009), or in saltwater in the supralittoral substrate (Wedin et al. 2004). Some lichenized
zone (Brodo and Sloan 2004; Fletcher 1980). fungi can also be parasitic on other lichens, either
throughout their life (Lawrey and Diederich 2003)
or only in the first stage of their development
III. Substrate Range and Ecology (e.g., Diploschistes muscorum) (Friedl 1987).
Finally, several lineages within Lecanoromycetes
A. Substrate Range are always nonlichenized, most likely because of
secondary losses of lichenization (Baloch et al.
Lecanoromycetes grow predominantly on tree 2010; Gueidan et al. 2008; Lutzoni et al. 2001;
bark (corticolous species) and rocks (saxico- Schoch et al. 2009). These nonlichenized lineages
lous species) but are also found on leaves (folii- can have diverse lifestyles, ranging from sapro-
colous species), soil (terricolous species), wood phytism to parasitism (Lawrey and Diederich
(lignicolous species), mosses (muscicolous spe- 2003; Sherwood 1977a, b; Sherwood-Pike 1987).
cies), and other lichens (lichenicolous species). They mostly occur on lichens (Lawrey and Die-
Most species tend to be specific to a particular derich 2003) but are also found on diverse sub-
type of substrate, although some can colonize strates such as bark or wood (e.g., Stictidaceae,
several (e.g., Parmelia sulcata). In addition to a Odontotremataceae).
natural substrate, some Lecanoromycetes can
grow on human-made materials, such as
brick, concrete, asphalt, metal, plastic, glass, C. Mycobiont–Photobiont Associations
and rubber (Brodo et al. 2001; Gilbert 1990).
Most Lecanoromycetes species associate with a
A few species even occur on the bones or shells of land single photobiont, a green alga (chlorobiont) or
tortoises (Brodo et al. 2001) and indeed on any material a cyanobacterium (cyanobiont). Some lichen-
with a stable surface exposed for long enough. A few forming fungi are associated with both types of
others, mainly from the genera Aspicilia s.l. and Xantho- photobionts, forming tripartite thalli where the
parmelia, do not attach to a substrate and occur as an
erratic or vagrant (e.g., Pérez 1997; Rosentreter 1993).
cyanobacterial photobiont is an accessory part-
ner. The cyanobiont is then restricted to special
organs of the thallus called cephalodia [although
see exceptions in Henskens et al. (2012)], which
can be internal or external outgrowths. Cyano-
B. Lifestyles
bionts fix atmospheric nitrogen, transferring
In Lecanoromycetes, most lichen-forming (myco- ammonia to the other partners. These tripartite
bionts) fungi form mutualistic associations with associations are common in Lecanorales (e.g.,
one or two photosynthetic partners, either a green Stereocaulon) and Peltigerales (e.g., Lobaria,
alga or a cyanobacterium (photobiont). Myco- Nephroma, Peltigera).
bionts obtain carbon from their photobiont, in The majority of photobionts belong to
the form of glucose when associated with cyano- Chlorophyta [approximately 90 % according
bacteria or in the form of polyhydric sugar alco- to Tschermak-Woess (1988a); see also Ahmad-
hols (polyols) when associated with green algae jian (1967, 1993)]. The green algal genera Tre-
(Palmqvist et al. 2008). Cyanobacteria found in bouxia, Asterochloris (Trebouxiophyceae), and
lichens, such as Nostoc, can fix nitrogen and, Trentepohlia (Ulvophyceae) are the most com-
therefore, can be a source of nitrogen for the mon photobionts for Lecanoromycetes.
mycobiont. The mycobiont protects the photo-
biont from UV light, temperature extremes, and, Other genera of photobionts associated with Lecanor-
to some extent, dessication (Nash 2008). The omycetes include Chlorella (e.g., in Pseudocyphellaria)
(Tschermak-Woess 1988b), Chlorosarcinopsis (in Leci-
great majority of lichenized fungi in Lecanoromy- dea) (Plessl 1963), Coccomyxa (e.g., in Icmadophila,
cetes are obligate mutualists, but a few species of Peltigera, Solorina) (Jaag 1933), Dictyochloropsis (e.g.,
Stictis can occur either as lichen symbionts or as in Lobaria, Pseudocyphellaria) (Tschermak-Woess 1984),
Elliptochloris (e.g., in Baeomyces) (Tschermak-Woess 70 % of lichen thalli across worldwide popula-

1985), Leptosira (in Vezdaea and Thrombium) tions of Lobaria pulmonaria were derived from
(Tschermak-Woess 1953; Tschermak-Woess and Poelt
1976), Myrmecia (e.g., in Psora) (Geitler 1963), and
asexual propagules. No recombination was
Phycopeltis (e.g., in Porinaceae) (Santesson 1952). detected for the photobiont, while it was
detected in only 7.7 % of the mycobiont indivi-
Cyanobacterial photobionts are associated duals. Therefore, vertical transmission of the
with a relatively small number of lichen-forming photobiont through thallus fragments or differ-
fungal species (10 %, Tschermak-Woess 1988a; entiated asexual propagules might be more
approximately 1,700 species, Rikkinen 2002) prominent than expected in shaping lichen
and, in Lecanoromycetes, are restricted to Arc- populations and could explain in part the pat-
tomiaceae, Stereocaulaceae, Trapeliaceae, and tern of association observed at a high taxo-
Peltigerales. The most common cyanobacterial nomic level within Lecanoromycetes.
(primary or accessory) lichen photobionts are
Nostoc and Rhizonema (Lücking et al. 2009b; A population ecology study of the geographically wide-
spread lichen Cetraria aculeata revealed that climate
Rambold et al. 1998; Tschermak-Woess 1988a). and codispersal are the most relevant factors shaping
The cyanobacterial genus Scytonema was also the genetic structure of the photobiont and that rare
listed in the past as one of the most common photobiont switches enabled the mycobiont to achieve
lichen photobionts, but a recent molecular study a broad geographical distribution crossing bioclimatic
showed that all lichenized strains of Scytonema zones (Fernandez-Mendoza et al. 2011).
studied (including those from the Lecanoromy-
cetes genera Coccocarpia and Stereocaulon) In Lecanoromycetes, the following patterns
belonged to Rhizonema, a new strictly lichenized of mycobiont–photobiont associations have
cyanobacterial lineage genetically distinct from been observed (Miadlikowska et al. 2006; Ram-
Scytonema (Lücking et al. 2009b). The current bold et al. 1998). Certain lineages, such as Par-
status of the genus Scytonema as a lichen photo- meliaceae and Teloschistales, are mainly
biont is therefore in need of determination using associated with Trebouxia, whereas Cladonia-
molecular data. Stigonema and Gloeocapsa are ceae, Stereocaulaceae, and Icmadophilaceae are
also often found in lichens but more often as mostly associated with Asterochloris. In Peltiger-
accessory photobionts (Rambold et al. 1998). ales, Nostoc is the main photobiont, and in
Ostropales, Trentepohlia is the most common
Other cyanobacteria occasionally found as photobionts photobiont. Although some photobiont associa-
are Anabaena (in Stereocaulon) (Duvigneaud 1955), tions could potentially be used to classify higher
Calothrix (in Coccotrema, Stereocaulon) (Brodo 1973; taxa within Lecanoromycetes, current knowl-
Lamb 1977), Dichothrix (in Placynthium nigrum) (Gei- edge on the identity of lichen photobionts is
tler 1934), Hyphomorpha (in Spilonema) (Henssen
1981), and Tolypothrix (in Hertella) (Henssen 1985).
still too sparse. They are notoriously difficult to
identify when lichenized, or even when cultured,
based on morphology only (Friedl and Büdel
Interestingly, patterns of mycobiont–
2008), and molecular data are currently only
photobiont associations have been observed at
available for a limited number of taxa.
a high taxonomic level (Miadlikowska et al.
2006; Rambold et al. 1998). This is somewhat
surprising for a symbiotic system that is
believed to transmit its photobiont mostly hor- IV. Mycobiont–Photobiont Cellular
izonally across generations. However, the con- Contacts
tribution of fungal sexual (horizontal
transmission) versus asexual (vertical trans- The type of cellular contacts between sym-
mission) reproduction to the genetic composi- bionts ranges from appressoria to haustoria
tion of the mycobiont and photobiont in Lecanoromycetes (Honegger 2008), and
populations has rarely been quantified. Dal their structure depends on various factors.
Grande et al. (2012) estimated that more than The taxonomic affiliation of photobionts plays
a large role, especially its morphology and cell superficial layer of the substrate, either in rock
wall composition (Honegger 2008). A correla- (endolithic species such as Clauzadea immersa)
tion has been observed between thallus growth or in bark (endophloeic species such as Calo-
forms and types of cellular contact between placa cerinella or Lecanora persimilis).
symbionts, with simple crustose species tend- The largest thalli are generally found in
ing to have less complex contact structures than squamulose, foliose, and fruticose lichens
do foliose or fruticose species (Honegger 2008). (macrolichens). Squamulose lichens are com-
Environmental conditions can also be respon- posed of scattered or imbricated scalelike lobes.
sible for variation in the extent of penetration Foliose lichens have dorsiventral thalli formed
by the fungus (Ben-Shaul et al. 1969; Galun by lobes mostly with a lower cortex, which are
et al. 1970), as well as thallus age (Collins and often only loosely attached to the substrate,
Farrar 1978; Galun 1988; Geitler 1963). As a most often by attachment structures such as
result, this character has not been used in the rhizines (e.g., Peltigera) (Fig. 4.1c). Foliose
classification of lichen-forming fungi (Poelt thalli attached to the substrate with a single
1973). holdfast are called umbilicate and can be
formed either by a single lobe or by several
lobes (e.g., Umbilicaria) (Hestmark 1997).
Lichens with long striplike or cordlike branches
V. Morphological and Chemical that are hanging or standing upward from their
Features substrates are called fruticose (e.g., Usnea,
Ramalina) (Fig. 4.1d). Some fruticose lichens
A. Thalli (e.g., Cladonia) have a mixed thallus formed by
a basal squamulose primary thallus and trum-
The lichen thallus is a vegetative structure petlike or spikelike structures (podetia) grow-
formed by the fungal hyphae and algal cells. ing upward from the primary thallus and
In Lecanoromycetes, thallus structures range bearing fruiting bodies (secondary thallus).
from simple to more complex organizations.
The simplest thalli are found in leprose (pow-
Thallus growth forms were of prime importance in
derlike, Fig. 4.1a) and byssoid (cottonlike) spe- shaping the first classifications of lichens (Zahlbruck-
cies, which lack differentiation into strata, and ner 1926). With improved microscopical techniques,
are formed of loosely intermingled fungal anatomical and ultrastructural characters became
hyphae and algal cells (Ekman and Tønsberg available, and ascomatal characters replaced thallus
2002; Kantvilas 1996; Poelt 1987). The complex morphology as the main characters used in classifica-
tion (e.g., Hafellner 1984; Henssen and Jahns 1974;
thalli (heteromerous) are differentiated into Luttrell 1951, 1955; Nannfeldt 1932; Poelt 1973). Molec-
layers (Fig. 4.2): upper cortex, photobiont ular data confirmed that thallus morphology could not
layer, medulla, and lower cortex. be strictly used for classification purposes because most
Two main types of lichen thalli are recog- growth forms were shown to have evolved several times
nized, mainly for convenience: microlichens independently in Lecanoromycetes and in lichens in
general [e.g., Grube and Arup 2001; Schmitt et al.
and macrolichens. Microlichens can consist of 2001; Stenroos and DePriest 1998; see also the review
powderlike or cottonlike thalli (leprose and in Grube and Hawksworth (2007)].
byssoid thalli) or corticated granules (granu-
lose thalli) or appear as crusts tightly appressed
to the substrate and generally lacking rhizines
and a lower cortex (crustose thalli) (Fig. 4.1b).
Crustose thalli are quite diverse, ranging from B. Ascomata
continuous or slightly cracked crusts to crusts
1. Ascoma Morphology
formed by areoles or small lobes often on a
fungal hyphal mat (hypothallus). Crustose spe- Ascomata (Fig. 4.3) are structures producing
cies generally grow above the substrate, but spore-bearing cells called asci. They generally
some have a thallus developing within the consist of a hymenium enveloped by a pro-
Fig. 4.1 Different types of thallus in Lecanoromycetes. gera rufescens, with white rhizines visible on lower
(a) Powderlike leprose thallus of Lepraria membrana- surface (white arrows). (d) Cordlike branches of fruti-
cea. (b) Crustose areolate thallus of saxicolous species cose thallus of Usnea subfloridana. Bars¼5 mm
Rhizocarpon macrosporum. (c) Foliose thallus of Pelti-
in Lecanoromycetes. The most common type is

the disc-shaped apothecium, where the upper
portion of the hymenium is mostly exposed
(Fig. 4.3a). When laterally elongated they are
referred to as lirellate apothecia (Fig. 4.3b). In
Lecanoromycetes, lirellate apothecia are
restricted to Ostropales but can also be found
in Arthoniomycetes, a distinct class within Leo-
tiomyceta (Pezizomycotina), which also
includes many lichen-forming fungi. The sec-
ond type of fruiting body in Lecanoromycetes is
the flask-shaped perithecium (Fig. 4.3c), where
the hymenium is exposed only through an
ostiole. The term perithecium was restricted
in the past to species with an ascohymenial
development (ascostromatic flask-shaped asco-
mata were called pseudothecia), but it is now
used in a broad sense for all flask-shaped asco-
Fig. 4.2 Schematic representation of a cross section in
mata, regardless of their development type
thallus of Xanthoria parietina. The thallus is composed (Kirk et al. 2008). Perithecia are only found in
of four layers: upper cortex, photobiont layer, medulla, a few lineages in Lecanoromycetes (e.g., Pori-
and lower cortex. Bar¼20 mm naceae, Protothelenellaceae, and Thelenella-
ceae). Some species have apothecia that can be
confused with perithecia because they are
tective structure called an excipulum. The immersed in the thallus and the hymenium
hymenium comprises asci and sterile interascal is exposed only through a small aperture.
hyphae. There are two main types of ascomata Called perithecioid apothecia (Fig. 4.3d), these
Fig. 4.3 Examples of fruiting bodies in Lecanoromy- fruiting bodies (perithecia) in Porina guentheri. (d)
cetes. (a) Disc-shaped fruiting bodies (apothecia) in Enclosed disc-shaped fruiting bodies (perithecioid
Xanthoparmelia tinctina. (b) Elongated fruiting bodies apothecia) in Thelotrema lepadinum. Bars¼2.5 mm
(lirellate apothecia) in Graphis scripta. (c) Flask-shaped
ascomata are frequently found in the subclass have evolved. These perithecioid fruiting ascomata
Ostropomycetidae. may therefore have a neotenic origin (Grube et al.
2004; Schmitt et al. 2009).
In early classifications, ascoma morphology
was used together with ascus characters to clas- 2. Ascoma Development
sify ascomycetes (e.g., Luttrell 1955; Nannfeldt
1932). However, molecular phylogenies demon- Types of ascoma development have been used
strated that some types of ascomata evolved in the past to define higher divisions in filamen-
several times independently in Lecanoromy- tous ascomycetes. Originally, two main types of
cetes [e.g., Grube et al. 2004; Schmitt et al. development were distinguished, ascolocular
2005, 2009; Wedin and Tibell 1997; see also and ascohymenial (Nannfeldt 1932). In ascolo-
the review in Grube and Hawksworth (2007)]. cular ascomata, the asci develop in the cavity in
In particular, some groups with perithecioid a preformed stroma, whereas in ascohymenial
ascomata (e.g., Porinaceae, Protothelenellaceae, ascomata, the asci develop in a hymenium not
and Thelenellaceae) were shown to be nested located in a preformed stroma (Kirk et al.
within the predominantly apothecial subclass 2008). In Lecanoromycetes, ontogenetic studies
Ostropomycetidae (Grube et al. 2004; Schmitt have helped describe in more detail the devel-
et al. 2005, 2009). In Lecanoromycetes, the con- opment of ascomata (e.g., Henssen 1976; Hens-
vergent evolution of ascoma types also prevents sen and Jahns 1974; Letrouit-Galinou and
the use of this character for defining orders and Bellemère 1989). Henssen and Jahns (1974)
families, although some broad trends can be recognized two main types of development in
observed (e.g., predominantly lirellate apothe- ascohymenial lichen-forming fungi, depending
cia in Graphidaceae). on the origin of the structure enveloping the
ascogonia (or primordium), as well as other
In Ostropomycetidae, a correlation between ascoma
more specific types. These development types
type and ascoma development (Schmitt et al. 2009) can lead to apothecia (angiocarps), perithecia
suggested that angiocarpous development was a pre- (gymnocarps), or perithecioid apothecia
requisite adaptation in lineages in which perithecia (hemiangiocarps).
Letrouit-Galinou and Bellemère (1989) recognized two Among unitunicate ascus types are the Lecanora, Per-
types of development depending on the differentiation tusaria, and Teloschistes types from Honegger (1982a,
of the excipulum (or parathecium, proper margin). b). Bellemère and Letrouit-Galinou (1987) grouped
Among the types without any differentiated excipulum these three ascus types within the “archeacés” and
are the Pertusaria and Thelotrema types for perithe- distinguished the further Lecidella, Catillaria, Psora,
cioid apothecia and the Graphis and Baeomyces types Lecidea, Cladonia, and Collema types. Unitunicate
for apothecia or lirellate apothecia. Among the types asci are also found in Anzina (Scheidegger 1985), Baeo-
with a differentiated excipulum are the Aspicilia and myces (Bellemère 1977; Honegger 1983), Dactylospora
Gyalecta types (excipulum reduced to a crown), the (Bellemère and Hafellner 1982), Gyalectaceae (Kauff
Peltigera, Diploicia, and Xanthoria types (typical exci- and Büdel 2005), and Trapeliopsis (Bellemère and
pulum), and Cladonia and Parmelia types (atypical Letrouit-Galinou 1987).
excipulum).
Functionally bitunicate asci are less com-
Molecular studies showed that the classifi- mon in the Lecanoromycetes. They are found in
cation in ascolocular and ascohymenial fungi Collemataceae, Peltigeraceae, and Rhizocarpa-
did not reflect monophyletic groupings (e.g., ceae (Bellemère and Letrouit-Galinou 1987;
Berbee 1996; Lindemuth et al. 2001; Lumbsch Honegger 1982a). Prototunicate asci are rather
and Huhndorf 2007a; Lutzoni et al. 2004). As a rare in this fungal class and are only found in
result, these characters are now rarely men- the families Caliciaceae and Sphaerophoraceae
tioned in classification work (e.g., Hibbett et al. (Wedin and Tibell 1997; Wedin et al. 2000b), in
2007). Although the use of ascoma developmen- which the released spores form a loose mass.
tal types is inadequate for delimiting higher taxa
of Ascomycota, their use at the ordinal and fam-
ily levels may have some value since they can be 2. Ascus Apical Structure
characteristic of some groups of Lecanoromy- With improving microscopy technologies, the
cetes (e.g., Parmeliaceae and Agyriaceae) (Hens- apical structure of asci was shown to be partic-
sen et al. 1981; Lumbsch et al. 2001a). ularly variable within Lecanoromycetes (e.g.,
Chadefaud 1973; Chadefaud et al. 1963; Honeg-
ger 1978, 1980; Letrouit-Galinou 1973a).
C. Asci Among the variations were the presence or
1. Ascus Walls absence of differentiated apical structures (api-
cal thickening, ocular chamber, apical ring, api-
The first studies of asci with light microscopy cal nasse, subapical bourrelet) and their
(e.g., Chadefaud 1942; Luttrell 1951; Nannfeldt reaction to various stains such as iodine (e.g.,
1932) and electron microscopy resulted in the Chadefaud 1973; Hafellner 1984; Letrouit-
use of ascus characters in combination with Galinou 1973a). Hafellner (1984) was the first
ascomatal characters to establish higher-rank to use these characters to systematically revise
classification systems among filamentous asco- the classification within Lecanorales s.l. at the
mycetes (Barr 1983; Eriksson 1982; Luttrell 1951, family and genus levels. He recircumscribed
1955; Nannfeldt 1932). Thus, the structure of the some species-rich families (e.g., Lecanoraceae
ascus wall was one of the main ascus features and Lecideacae) and described new families
used in higher classification. Currently, ascus (e.g., Catillariaceae and Dactylosporaceae)
walls are classified in three main types: a thin based on the ascus apical structure (Bellemère
ascus wall formed of a single layer (prototuni- and Hafellner 1982; Hafellner 1984). His classi-
cate ascus), an ascus wall formed of two layers fication system was broadly accepted (e.g.,
functioning as a single layer (unitunicate ascus), Eriksson and Hawksworth 1993; Hafellner
and an ascus wall formed of two layers function- 1994; Rambold and Triebel 1992), until molec-
ing as two layers (bitunicate ascus). Most mem- ular data showed that these characters were
bers of Lecanoromycetes have unitunicate asci not as conserved as initially thought and that
(Honegger 1982a; Letrouit-Galinou 1973a). similar apical structures have evolved several
times independently in unrelated lineages released after deliquescence of prototunicate

[Ekman and Wedin 2000; Ekman et al. 2008; asci (evanescent type of dehiscence) (Wedin
Lumbsch et al. 2001b, 2007c; Wedin et al. 2005, and Tibell 1997). Although ascus dehiscence
2009; see also the review in Grube and Hawks- has been used in high-rank classifications (Lut-
worth (2007) and Printzen (2010)]. trell 1955), the systematic value of this character
has been questioned because dehiscence types
more likely correspond to adaptations to various
3. Dehiscence Mechanisms environmental conditions (Bellemère 1994).
The type of dehiscence (or the process leading
to the liberation of ascospores from mature
asci) varies greatly in Lecanoromycetes (Belle- D. Ascospores
mère and Letrouit-Galinou 1987; Honegger
1982a). In most members of this class, dehis- Ascospore characters, such as septation, pig-
cence occurs by apical rupture of the ascus, mentation, size, shape, and number per ascus,
combined with the ejection of the internal part were originally used to delimit families or
of the ascus wall. When the ejection is not genera in many groups of Lecanoromycetes
coupled with a sliding between the two layers [e.g., in Graphidaceae (Müller 1880, 1882) or
of the ascus wall, this type of dehiscence is in Acarosporaceae (Zahlbruckner 1907)]. How-
called a rostrum type or Lecanora type (Belle- ever, many authors have recognized subse-
mère and Letrouit-Galinou 1987; Honegger quently that classification based on these
1978). It is the most common type of dehis- characters might not reflect evolutionary his-
cence in Lecanoromycetes (e.g., Lecanoraceae, tory because of convergent evolution in ascos-
Physciaceae, Parmeliaceae), and it has been pores of lichenized and nonlichenized fungi
suggested that it might be the ancestral dehis- (e.g., Poelt 1973; Vainio 1890). Morphologically
cence type in this class based on both anatomi- similar ascospores are indeed often found in
cal data (“type archaeascé”) (Chadefaud et al. distantly related groups, as confirmed by
1967) and molecular data (Miadlikowska et al. molecular data.
2006; Wedin et al. 2005). Molecular phylogenetic results have further
thrown doubts on the use of ascospores as a
In Rhizocarpon, the internal layer of the ascus wall main source of characters for classification at
slides slightly along the external layer (Rhizocarpon the generic level. Several Lecanoromycetes
type or hemifissitunicate type) (Bellemère 1994; Hon-
egger 1980). In Peltigera, the sliding of the internal
genera that were primarily circumscribed on
layer along the external layer is more important (Pelti- ascospore characters were shown to be poly-
gera type, fissitunicate type or “Jack in the box”) (Bel- phyletic, and characters such as ascospore sep-
lemère and Letrouit-Galinou 1987; Honegger 1978). tation and color were frequently shown to be
homoplasious (e.g., Ihlen and Ekman 2002;
Dehiscence can also occur by apical rupture Rivas Plata and Lumbsch 2011; Staiger et al.
of the ascus without ejection of the internal part 2006). Despite this conclusion, when combined
of the ascus wall (e.g., Trapelia and Coccocarpia) with other features, ascospore characters pro-
(Bellemère 1994). In Teloschistes, the apical pore vide important taxonomic information for
forms after predehiscent elongation of the ascus most groups of lichenized fungi at the species
wall (Teloschistes type or chimney type of dehis- and generic levels (e.g., within Thelotremata-
cence) (Bellemère and Letrouit-Galinou 1987; ceae) (Frisch et al. 2006). Ascospore characters
Honegger 1978). In Pertusaria, a predehiscent can also, in some cases, be useful at higher
elongation also occurs, but the ascospores are taxonomic levels since some broad trends can
released after bursting or splitting of the ascus be observed in some groups. For example,
tip (Honegger 1982b). Finally, in Caliciaceae and polarilocular ascopores are characteristic of
Sphaerophoraceae, ascospores are passively the order Teloschistales and ascospores with
lens-shaped lumina, of the family Graphidaceae (Ahmadjian 1969; Bailey 1976; Vobis 1977).
s.s. (Poelt 1973). Microscopical observations revealed that con-
idiospores most probably act as spermatia since
they have been found attached to trichogynes in
E. Interascal Filaments several species (e.g., Honegger 1984a, b; Jahns
1970).
Sterile filaments are usually present in fruiting
The systematic value of pycnidial charac-
bodies alongside asci. These interascal fila-
ters was recognized early on (Choisy 1954; Stei-
ments constitute the hamathecium and are
ner 1901; Zahlbruckner 1903–1907), but their
thought to protect the asci or promote their
use in systematic studies remains rather lim-
function (Poelt 1973). Historically, two broad
ited, mostly because they are difficult to
categories of interascal filaments were
observe. In the 1970s and 1980s, cytological
described in ascomycetes depending on their
and ontogenetical studies triggered a renewed
origin in the development of the ascoma. Asco-
interest in pycnidial characters, and several
hymenial development leads to the formation
types of pycnidia and conidiophores were
of paraphyses, whereas ascolocular develop-
described in the Lecanoromycetes (e.g., Honeg-
ment forms paraphysoids or pseudopara-
ger 1984b; Janex-Favre 1977, 1982; Letrouit-
physes. These two types of filament may look
Galinou 1972, 1973b; Letrouit-Galinou and Lal-
very similar in mature fruiting bodies and can
lement 1977; Vobis 1980). However, data avail-
be confused, so they have been applied only in
able on pycnidia remain sparse (Roux et al.
more recent classification systems of Ascomy-
1986), and only relatively few studies have
cota (e.g., Barr 1983). At lower taxonomic
tried to assess their use for classification within
levels, hamathecial characters (septation, ana-
Lecanoromycetes (e.g., Krog 1982; Matsumoto
stomoses and branching, color, chemistry, and
and Deguchi 1999; Thell et al. 2002).
shape of the upper cell) have been used together
with other characters to delimitate genera or
families in Lecanoromycetes (e.g., Kärnefelt
and Thell 1992; Staiger and Kalb 1999; Timdal G. Asexual Propagules
1992). The importance of some of these char-
Lecanoromycetes can disperse asexually by
acters has been discussed in light of molecular
thallus fragmentation aided by undifferentiated
data (e.g., Rivas Plata and Lumbsch 2011; Stai-
or specialized structures. The two most com-
ger et al. 2006). However, a comprehensive
mon types of specialized dispersal structures
reevaluation of the evolution of hamathecial
are isidia (corticated and more or less cylindri-
characters and their taxonomic value is still
cal thallus outgrowths) (Fig. 4.4a) and soredia
needed.
(ecorticated thallus granules produced via
openings in the cortex called soralia)
F. Pycnidia (Fig. 4.4b). Such propagules facilitate successful
codispersal of both lichen partners, in contrast
In lichenized fungi, pycnidia (¼conidiomata) to fungal dispersal through sexual ascospores,
are minute flask-shaped structures located on which requires finding appropriate photobionts
the surface of, or embedded within, lichen to reestablish lichen thalli de novo. The taxo-
thalli and producing small asexual spores nomic value of asexual propagules was first
called pycnidiospores (also called conidios- recognized by Du Rietz (1924). He described
pores or conidia). The role of pycnidia has and classified them and introduced the concept
been insufficiently studied in lichens. Culture of species pairs for species that differ only by
studies first suggested that conidiospores might their mode of reproduction, either primarily
act as asexual dispersal units (Möller 1888), but sexual or primarily vegetative. Poelt (1970,
this possibility was questioned because more 1972) later developed this concept, and since
recent studies showed that, in Lecanoromy- then, it has been debated whether they corre-
cetes, conidiospores germinate only rarely spond to conspecific individuals or separate
lichens (Elix and Stocker-Wörgötter 2008).

Most of these compounds are found exclusively
in lichens. They are derived from three main
biosynthetic pathways (acetyl-polymalonyl,
shikimic, and mevalonic acid pathways) and
belong to different chemical groups such as
anthraquinones, depsidones, triterpenes, pulvi-
nic acids, and xanthones (Asahina and Shibata
1954; Culberson and Elix 1989; Huneck 2001).
Mostly present in the cortex and the medulla,
they can help repel herbivores and microorgan-
isms (Lawrey 1986, 1989) but in the cortex also
act as light and UV screens (Armaleo et al. 2008;
Millot et al. 2012; Solhaug et al. 2003). Armaleo
et al. (2011) were the first to identify a gene
cluster involved in the biosynthesis of a depside
and a depsidone.
Approximately 5,000 lichen species have
been investigated for their secondary com-
pounds (Elix and Stocker-Wörgötter 2008).
Methods of detection vary from simple spot
tests to analysis of extracts with thin-layer chro-
matography and high-performance liquid chro-
Fig. 4.4 Asexual propagules in Lecanoromycetes. (a) matography (Huneck and Yoshimura 1996). The
Upper surface of thallus of Parmelina tiliacea covered presence of secondary compounds has been
with small and easily detached, corticated outgrowths
called isidia. In this species, isidia are simple to used at all taxonomic levels, for both classifica-
branched and brown at the tip (inset: detail of a long tion and species identification. Most secondary
branched isidium). (b) Round openings (soralia) in the compounds are found widely in Lecanoromy-
upper cortex of Pertusaria flavicans releasing ecorti- cetes and only rarely form synapomorphies for
cated granules called soredia. Bars¼0.5 mm single lineages. However, the presence of some
secondary compounds was shown to be infor-
sister species (e.g., Mattsson and Lumbsch mative at the genus and family levels (Lumbsch
1989; Tehler 1982). Molecular studies on sev- 1998a; Schmitt and Lumbsch 2004). The use of
eral species pairs have so far not confirmed chemical characters for species delimitation has
them as separate species (e.g., Buschbom and been controversial (Lumbsch 1998b). Recent
Mueller 2006; Kroken and Taylor 2001; Myllys molecular studies show that, depending on the
et al. 2001). The contribution of asexual repro- group studied, chemotypes may correspond to
duction to the ecology and evolution of lichens separate species (e.g., Tehler and Källersjö 2001)
has rarely been addressed (e.g., Buschbom and or can represent infraspecific variability (e.g.,
Barker 2006; Dal Grande et al. 2012; Fedrowitz Leavitt et al. 2011a, b; Nelsen and Gargas 2009;
et al. 2011; Hestmark et al. 2011). Velmala et al. 2009).
H. Secondary Compounds VI. Origin and Diversification
Secondary compounds are insoluble metabo- Dating the divergence time of fungi is not an
lites that are often deposited extracellularly by easy task because of the relatively poor fossil
the fungal partner on the surface of hyphae. record for these organisms and the variable
These compounds are very diverse and numer- rates of nucleotide substitution across this
ous, with more than 700 described so far from kingdom (Lumbsch et al. 2008a; Lutzoni and
Pagel 1997; Woolfit and Bromham 2003; Zoller well-preserved lichen thalli fragments in silt-
and Lutzoni 2003). As a result, estimates of stone of the lower Devonian (415 mya)
divergence times in fungi once varied consider- provided the oldest record of modern lichens.
ably depending on the methods and calibra-
tions used (Berbee and Taylor 1993, 2001; One of the Parmelia fossils and the Alectoria fossil were
Heckman et al. 2001; Padovan et al. 2005; Tay- used in a recent study to investigate the origin of Par-
lor and Berbee 2006). With the reinterpretation meliaceae, one of the largest families within Lecanor-
omycetes (Amo de Paz et al. 2011). Results show that
of fossil data and the development of new phy- this family radiated around the Cretaceous–Tertiary
logenetic methods allowing for rates to vary boundary (65 mya), just before a climatic period char-
across lineages, divergence estimates for the acterized by temperature and atmospheric CO2 max-
main fungal lineages have now reached a con- ima. Most major parmelioid genera originated during
sensus (Lücking et al. 2009a; Taylor and Berbee the Eocene and early Oligocene and diversified during
the cooler periods of late Oligocene to early Pliocene.
2010), and divergences of more recent lineages
have started to be investigated (Amo de Paz
et al. 2011; Gueidan et al. 2011). VII. Orders and Classification
Soon after the divergence of Pezizomyco-
tina, Leotiomyceta underwent a radiation dur-
ing which the Lecanoromycetes lineage A. Acarosporales
originated (Gazis et al. 2012; Schoch et al. Acarosporales (Acarosporomycetidae)
2009; Spatafora et al. 2006). So far, all ancestral (Fig. 4.5) is a small order with a single family,
state reconstruction studies agree that licheni- Acarosporaceae, 11 genera, and 183 species
zation in Lecanoromycetes evolved at or prior (Kirk et al. 2008). They are mostly crustose
to the onset of the evolution of this fungal class. species with apothecial ascomata, poorly to
This acquisition of lichenization has been moderately branched and anastomosed para-
placed at the base of a lineage including Leca- physes, unitunicate polysporous asci, and
noromycetes, Eurotiomycetes, and Lichinomy- small hyaline simple ascospores (Hibbett et al.
cetes (James et al. 2006; Lutzoni et al. 2001), at 2007). They occur worldwide and mostly colo-
the base of a lineage including Lecanoromy- nize rocks. Traditionally, Acarosporaceae had
cetes and Lichinomycetes (Gueidan et al. been placed in the order Lecanorales based on
2008), or at the base of Lecanoromycetes ascus characters (Kirk et al. 2001). However,
(Schoch et al. 2009). According to Gueidan molecular studies showed that they did not
et al. (2011), Lecanoromycetes diverged from belong to this order but represent instead the
Eurotiomycetes during the late Devonian, earliest diverging lineage in Lecanoromycetes
around 371 million years ago (mya) (between (Lumbsch et al. 2007b; Miadlikowska et al.
322 and 424 mya), and the diversification of 2006; Reeb et al. 2004; but see Hofstetter et al.
extant Lecanoromycetes species (crown 2007). The order Acarosporales was therefore
group) originated during the Carboniferous, formally described for this family (Hibbett et al.
approximately 322 mya (between 269 and 2007).
380 mya). Although polyspory is not uncommon in
Within Lecanoromycetes, several well- nonlichenized and lichenized ascomycetes, the
interpreted fossils from amber are available to family Acarosporaceae was originally charac-
calibrate the molecular clock. A species of terized by its true polyspory (polyspory result-
Anzia was described from European amber ing from a meiosis followed by several mitoses
(35–40 mya) (Mägdefrau 1957), two species of generating more than 100 ascospores, as
Parmelia from Dominican amber (15–45 mya) opposed to polyspory resulting from budding
(Poinar et al. 2000), and a species of Alectoria or fragmenting ascospores). However, in Leca-
from Baltic amber (35–40 mya) (Rikkinen noromycetes, true polyspory is not restricted to
and Poinar 2002). A more recent discovery the Acarosporaceae. As a result, the circum-
by Honegger et al. (2013) of exceptionally scription of this family has been variable, with
Fig. 4.5 Schematic representation of phylogeny and et al. 2007; Baloch et al. 2010; Bylin et al. 2007; Ekman
classification of Lecanoromycetes based on selected et al. 2008; Gaya et al. 2012; Hodkinson and Lendemer
published sources (Andersen and Ekman 2005; Arup 2011; Hofstetter et al. 2007; Kirk et al. 2008; Lumbsch
many polysporous genera (e.g., Maronea, sally septate hyaline ascospores (Hibbett et al.
Pleopsidium, and Thelocarpon) being succes- 2007; Johnston 2001). The species grow on
sively excluded from, or included in, the family soils, rocks, and bryophytes in moist areas
depending on the system adopted for their clas- and often are primary colonizers of disturbed
sification [Golubkova 1988; Hafellner 1995; substrates (Johnston 2001). First placed in
Magnusson 1936; Zahlbruckner 1907; see the Lecanorales close to Cladoniaceae (Henssen
detailed review in Reeb et al. (2004)]. and Jahns 1974; Poelt 1973), this family (includ-
Molecular data have helped resolve the cir- ing at the time the genera Baeomyces and Icma-
cumscription of Acarosporaceae (Reeb et al. dophila) was then transferred to the
2004; Wedin et al. 2005), and the genera Acar- predominantly nonlichenized order Helotiales
ospora, Glypholecia, Pleopsidium, Polysporina, (now Leotiales) based on its Leotia-type ascus
Sarcogyne, Thelocarpella, and Timdalia were (Chadefaud 1960, 1973; Hafellner 1988; Honeg-
confirmed as part of this family, whereas the ger 1983; Rambold et al. 1993; Tehler 1996).
genera Biatoridium, Maronea, Sporastatia, Early molecular studies suggested that
Strangospora, and Thelocarpon were excluded. Baeomyces might not be related to Leotiales
Molecular studies also confirmed the multiple (Platt and Spatafora 1999; Stenroos and DePr-
independent origins of true polyspory in Leca- iest 1998). Later, this genus was shown to form
noromycetes (Reeb et al. 2004), i.e., Acarospor- a sister group to the Ostropales s.l., and the
aceae (with a loss in the two species Acarospora ordinal name Baeomycetales was then sug-
macrospora and Glypholecia scabra, which have gested for this lineage (Kauff and Lutzoni
only 30–100 ascospores), Maronea, Sporastatia, 2002). Additional molecular data confirmed
Strangospora, and a lineage including Thelocar- its phylogenetic placement within Ostropomy-
pon and Biatoridium. cetidae (Lumbsch et al. 2007b; Miadlikowska
et al. 2006), and the order Baeomycetales was
therefore formally erected for the family Baeo-
B. Baeomycetales mycetaceae (Hibbett et al. 2007).
The order Baeomycetales (Ostropomycetidae) The delimitation of Baeomycetaceae also underwent

(Fig. 4.5) currently includes two families, Baeo- some important changes during the last two decades.
mycetaceae and the monotypic Anamylopsora- Some genera previously placed in this family (e.g.,
Dibaeis, Icmadophila, Siphulella) were segregated into
ceae (Hodkinson and Lendemer 2011; Lumbsch the new family Icmadophilaceae based on morphologi-
et al. 1995). The family Baeomycetaceae com- cal evidence (Rambold et al. 1993), and subsequent
prises the three genera Ainoa, Baeomyces, and molecular studies confirmed that these genera are not
Phyllobaeis (Hibbett et al. 2007; Lumbsch and related to Baeomycetaceae (Platt and Spatafora 1999;
Huhndorf 2010) and a total of approximately 15 Stenroos and DePriest 1998). A new genus (Phyllobaeis)
was also described for Baeomyces species with squamu-
species with crustose to squamulose or foliose lose primary thalli and brown apothecia (Gierl and Kalb
thalli, sessile to stipitate apothecia, branched 1993). Finally, molecular data showed that the genus
paraphyses, nonamyloid asci or asci with a Ainoa also belongs to Baeomycetales (Lumbsch et al.
slightly amyloid apex, and simple to transver- 2007b, c). This genus had previously been described to
⁄

Fig. 4.5 (continued) and Huhndorf 2010; Lumbsch at the same level of support. The number of recognized
et al. 2004, 2007b, 2008b; Lutzoni et al. 2004; Miadli- families in each order is provided in parentheses after
kowska et al. 2006; Muggia et al. 2011; Reeb et al. 2004; the order name. Shaded boxes represent subclasses and
Rivas Plata et al. 2012; Schmitt et al. 2005, 2012; Schoch suborders. Oblique bars across branches indicate
et al. 2009; Wedin et al. 2009; Widhelm and Lumbsch families with unknown placement in the Lecanoromy-
2011; Zhou and Wei 2007). Phylogenetic relationships cetidae or Ostropomycetidae. Stars indicate families for
among taxa (families, orders, and subclasses) were which no DNA sequence is currently available in Gen-
compiled using a “super tree” approach and are Bank. Question mark indicates that Dactylosporaceae
shown as resolved if reported with posterior probability may be placed outside of Lecanoromycetes (in Euro-
95 % or maximum likelihood bootstrap 70 % in tiomycetes) (Schoch et al. 2009)
multiple studies (in most cases) and are not in conflict
accommodate two morphologically different species of Lecanorales (Wedin et al. 2000a, 2005). Both
Trapelia that did not cluster with Trapelia s.s. in a Physciaceae and Caliciaceae were later tenta-
molecular phylogenetic study (Lumbsch et al. 2001b).
tively placed in Teloschistales as part of the
suborder Physciineae (Miadlikowska et al.
The circumscription of the order Baeomy- 2006). In a more recent molecular study focus-
cetales is likely to undergo more changes in ing on Teloschistales s.l., Gaya et al. (2012)
the future. A morphological study of the family demonstrated phylogenetic instability for rela-
Anamylopsoraceae shows that this monotypic tionships among Physciineae, Teloschistineae,
family shares several characters with Baeomy- and Lecanorales, where the two suborders did
cetaceae, such as the ascocarp ontogeny, stipi- not always form a monophyletic group. To
tate ascogonia, annular exciple, and ensure the classification was resilient to the
conidiophore type (Lumbsch et al. 1995). How- various resolution of these three clades, Gaya
ever, these two families also differ in other et al. (2012) elevated Physciineae to the ordinal
characters, such as the ascus type. Anamylop- level by resurrecting the order Caliciales and
soraceae had been placed tentatively in Agyrii- restricted Teloschistales to Brigantiaeaceae,
neae and Agyriales (Lumbsch et al. 1995, 2001a, Letrouitiaceae, Megalosporaceae, and Tel-
respectively), but a transfer to Baeomycetales oschistaceae. In this new phylogenetic context,
was recently suggested (Hodkinson and Lende- Caliciales includes two families: Caliciaceae
mer 2011; Lumbsch et al. 2007b). A comprehen- (with two subfamilies Calicioidea and Buellioi-
sive phylogenetic analysis is needed to dea) and Physciaceae.
determine whether this family belongs to Baeo-
mycetales.
D. Candelariales
C. Caliciales The order Candelariales (Candelariomyceti-

dae?) (Fig. 4.5) includes a single family, Cande-
The order Caliciales (Lecanoromycetidae) lariaceae, and four genera: Candelaria,
(Fig. 4.5) was in the past recognized as a phe- Candelariella, Candelina, and Placomaronea
notypically well-delimited group that included (Lumbsch and Huhndorf 2010; Miadlikowska
species with mazaedium-forming ascomata et al. 2006; Westberg et al. 2009). It is a small
and passive ascospore dispersal (e.g., Zahl- group with approximately 66 lichenized species
bruckner 1903–1907; see detailed review in (Kirk et al. 2008) characterized by yellow to
Tibell 1984) and was erected as an order by orange thalli (secondary chemistry based on
Bessey (1907). However, this order underwent pulvinic acid and derivatives), disciform
drastic changes in circumscription after a first apothecia, unitunicate asci (often polysporous),
extensive revision by Tibell (1984). Based on a mostly unbranched paraphyses, and hyaline
careful morphological study, Tibell excluded mostly simple ascospores (Hibbett et al. 2007;
several families and genera from Caliciales s.s. Westberg et al. 2007, 2009). Formerly, the fam-
(e.g., Sphaerophoraceae and Chaenotheca) and ily Candelariaceae had been placed in Lecanor-
restricted the order to the three families Cali- ales s.l. (Henssen and Jahns 1974; Poelt 1973).
ciaceae, Mycocaliciaceae, and Sphinctrinaceae Molecular data showed that this family did not
(Tibell 1984, 1996). More recently, a study cluster within Lecanorales as currently delim-
using molecular data demonstrated that Myco- ited (Hofstetter et al. 2007; Miadlikowska et al.
caliciaceae and Sphinctrinaceae belong in fact 2006; Wedin et al. 2005), and so the order Can-
to Eurotiomycetes and that only the family delariales was erected for this family (Hibbett
Caliciaceae was strongly supported as part of et al. 2007). Candelariales was considered to be
the order Lecanorales (Wedin and Tibell 1997). a sister order to Acarosporales (Wedin et al.
Subsequent studies with broader taxon 2005) or possibly formed the second lineage to
samplings showed that Caliciaceae were in fact diverge within Lecanoromycetes after Acaros-
closely related to Physciaceae, in the order porales (Miadlikowska et al. 2006) or perhaps
the first diverging lineage in Lecanoromycetes species-rich Cladoniaceae, Lecanoraceae, Par-

(Hofstetter et al. 2007). Because of this phylo- meliaceae, and Ramalinaceae (Lumbsch and
genetic uncertainty, this order was only tenta- Huhndorf 2010).
tively recognized as the subclass Parmeliaceae is the largest family within
Candelariomycetidae by Miadlikowska et al. Lecanorales, with approximately 2,500 species
(2006) and Hofstetter et al. (2007). A morpho- and 88 genera (Kirk et al. 2008). Earlier, many
logical and molecular revision of the family groups within Parmeliaceae had been tentatively
showed that the current generic delimitation segregated from this family based on morpho-
does not reflect monophyletic groups, and fur- logical, anatomical, and chemical characters
ther work will be needed to clarify the generic (e.g., Alectoriaceae, Anziaceae, Hypogymnia-
boundaries (Westberg et al. 2007, 2009). ceae, and Usneaceae). But most of these segre-
gate families were not confirmed by molecular
phylogenies (e.g., Arup et al. 2007; Mattsson and
Wedin 1999; Wedin et al. 1999), and a wider
E. Lecanorales
circumscription of Parmeliaceae is currently
Lecanorales (Lecanoromycetidae) (Fig. 4.5) is accepted (Eriksson 2006; Kirk et al. 2008;
the largest order in Lecanoromycetes with 19 Lumbsch and Huhndorf 2010). This family com-
families and approximately 250 genera prises a large variety of growth forms (e.g., par-
(Lumbsch and Huhndorf 2010). The number of meliod, usneoid, or cetrarioid species), which do
species in this order was estimated to be 5,695 not define large monophyletic groups within
(Kirk et al. 2008). It includes mostly lichenized Parmeliaceae but nevertheless were found to be
species with varied thallus types, predominantly useful in characterizing and identifying smaller
apothecial ascomata, usually unbranched para- clades within the family (Crespo et al. 2007).
physes, mostly thick-walled unitunicate asci
(often amyloid), and varied ascospores (Kirk The generic concept is a strongly debated area in lichen
et al. 2008). Traditionally, this apparently heter- research (e.g., Elix 1993; Hale 1984a; Nimis 1998). In
Parmeliaceae, large genera (e.g., Parmelia s.l.) have
ogenous order included most lichenized apothe- been split into multiple smaller genera (e.g., Culberson
cial ascomycetes, with the exception of those and Culberson 1981; Elix and Hale 1987; Elix et al. 1986;
included in Ostropales (e.g., Henssen and Jahns Hale 1974, 1984b). Molecular phylogenetic studies have
1974; Poelt 1973; Rambold and Triebel 1992). A shown that many of these newly segregated genera are
new circumscription of this order based on the not monophyletic (e.g., Blanco et al. 2004, 2005; Crespo
et al. 2010; Nelsen et al. 2011), and the taxonomic
ascus apical structure led to the exclusion of importance of some characters traditionally used to
several taxa from Lecanorales (e.g., Peltigerales classify parmelioid lichens (e.g., chemistry of the cor-
and Teloschistales) (Hafellner 1988). This cir- tex, presence or absence of pores and pseudocyphellae)
cumscription was further narrowed after may have been overestimated (Blanco et al. 2006).
molecular data were used to test the relation-
ships among the main Lecanorales taxa. This Similar problems were found in other
circumscription was confirmed, and the trend families of Lecanorales (e.g, Lecanoraceae,
continued with the advent of molecular phylo- Ramalinaceae). In the predominantly crustose
genetics, resulting in the following orders cur- groups of Lecanorales, which were mainly clas-
rently being recognized outside the Lecanorales: sified based on ascus characters (Hafellner
Acarosporales, Caliciales (with the Physciaceae), 1984), molecular phylogenetic studies reported
Candelariellales, Lecideales, Peltigerales, Pertu- that many of these crustose groups (e.g., Baci-
sariales, Teloschistales, and Umbilicariales (e.g., diaceae, Lecanoraceae, Micareaceae) were not
Gaya et al. 2012; Miadlikowska and Lutzoni monophyletic and that the evolution of the
2004; Miadlikowska et al. 2006; Reeb et al. ascus was more complex than had been antici-
2004; Schmull et al. 2011; Wedin et al. 2005). pated and of limited value for classification at
Among the remaining 19 families in Lecanorales this taxonomic level (Andersen and Ekman
are the broadly distributed and well-known 2004; Ekman 2001; Ekman and Wedin 2000;
Ekman et al. 2008). Because of a similar com- (Henssen and Jahns 1974; Poelt 1973; Zahl-
posite thallus, Cladoniaceae was placed bruckner 1903–1907), was shown to belong to
together with Stereocaulaceae and two other Ramalinaceae, a family classified within Leca-
families within the suborder Cladoniineae norales (Ekman 2001). Molecular phylogenies
(Lecanorales s.l.) (Poelt 1973). Molecular phy- also shed a light on Hafellner’s classification
logenies showed that, although the composite system (1984). Characters of the ascus tip used
growth form evolved several times in Lecanor- by this author to redelimitate genera and
omycetes (Stenroos and DePriest 1998), Clado- families within Lecanorales do not seem to
niaceae and Stereocaulaceae do form a sister characterize monophyletic entities in Lecidea-
group to which the suborder Cladoniineae is ceae and related taxa (e.g., Porpidiaceae)
now restricted (Miadlikowska et al. 2006; (Buschbom and Mueller 2004). Some taxa pre-
Myllys et al. 2005; Wedin et al. 2000b). viously attributed to Lecideaceae were shown to
belong to different lineages within Lecanoro-
mycetes, and genera within this family were
F. Lecideales shown to be poorly delimited (Schmull et al.
2011). The phylogenetic positions of most
Lecideales (Lecanoromycetidae) (Fig. 4.5) is an members of Lecideaceae are still unknown or
order recently resurrected for a single family, unsettled (Miadlikowska et al. 2006; Schmull
Lecideaceae, now restricted to the genus Leci- et al. 2011). However, they were found to form
dea s.s. (sensu Hertel) and some species of five distinct groups within Lecanoromycetidae,
Porpidia (Schmull et al. 2011). In Zahlbruck- one of which included only saxicolous species
ner’s classification system (1903–1907), Leci- belonging to the genera Lecidea and Porpidia,
deaceae was a large artificial family within including the type species Lecidea fuscoatra,
the order Lecanorales that included a hetero- which led to the resurrection of the order Leci-
geneous assemblage of crustose taxa with lecideales s.s. (Schmull et al. 2011). Additional
deine or biatorine apothecia (e.g., Bacidia, molecular data are greatly needed to further
Catillaria, Toninia), among which Lecidea was investigate this species-rich and broadly
one of the largest lichen genera. The delimita- defined lichen group.
tion of this poorly studied family was ques-
tioned in later taxonomic works and
classification systems (Henssen and Jahns G. Ostropales
1974; Hertel and Rambold 1985; Poelt 1973;
Santesson 1952; Timdal 1987). In his classifica- Ostropales (Ostropomycetidae) (Fig. 4.5) is a
tion of Lecanorales, Hafellner (1984) was the large order of mostly crustose lichenized and
first to attempt to recircumscribe the two nonlichenized species, with high species diver-
families Lecideaceae and Lecanoraceae. Based sity in the tropics. It includes approximately
on ascus characters, he segregated several new 2,750 species (Kirk et al. 2008) currently classi-
families from the Lecideaceae, among which fied in ten families: Coenogoniaceae, Graphida-
was Porpidiaceae. His system was broadly ceae (including Gomphillaceae and
accepted (e.g., Eriksson and Hawksworth Thelotremataceae), Gyalectaceae, Myeloconida-
1993; Hafellner 1994; Rambold and Triebel ceae, Odontotremataceae, Phaneromycetaceae,
1992), although also sometimes criticized (e.g., Phlyctidaceae, Porinaceae, Sagiolechiaceae, and
Timdal 1992). Stictidaceae (Baloch et al. 2010; Lumbsch and
Molecular phylogenetic studies confirmed Huhndorf 2010; Rivas Plata et al. 2012). This
the heterogeneity of early circumscriptions of order is characterized by ascomata ranging
Lecideaceae and Lecanoraceae (Andersen and from perithecial to apothecial, with unbranched
Ekman 2004, 2005; Buschbom and Mueller or anastomosate paraphyses, unitunicate non-
2004; Ekman 2001; Ekman et al. 2008; Schmull amyloid asci, and morphologically variable
et al. 2011). For example, the genus Bacidia, ascospores (Kirk et al. 2008; Lücking et al.
included in Lecideaceae in early classifications 2004; Lumbsch et al. 2007b).
The circumscription of the order Ostro- ceae) is now excluded from Ostropales s.l.
pales has undergone many changes in the (Grube et al. 2004; Miadlikowska et al. 2006).
past. It was originally described to accommo- Although still recently largely debated (Grube
date the nonlichenized family Ostropaceae et al. 2004; Lücking et al. 2004; Lumbsch et al.
(Nannfeldt 1932), now known as Stictidaceae. 2004; Miadlikowska et al. 2006), the broader
Gilenstam (1969) was the first to include liche- delimitation of Ostropales (but without Trape-
nized taxa within Ostropales. He recognized the liaceae) has been accepted in current classifica-
close relationship between the lichenized genus tion systems (Hibbett et al. 2007; Lumbsch and
Conotrema and the nonlichenized genus Stictis Huhndorf 2010).
and attributed Conotrema to Ostropales. He The phylogenetic placement of Ostropales
also suggested that the lichenized genera within Lecanoromycetes has also long been
Diploschistes, Graphis, and Thelotrema should unclear due to an unstable backbone of the
be transferred to Ostropales because of their Lecanoromycetes phylogeny (Lumbsch et al.
close relationship with Conotrema (Gilenstam 2007b). Ostropales has been found to be sister
1969). Henssen and Jahns (1974) considered to all other Lecanoromycetes (Grube et al. 2004;
these genera and further lichenized groups Lücking et al. 2004; Lumbsch et al. 2004), sister
(then included in Asterothyriaceae, Graphida- to Trapeliales and Hymeneliaceae (Kauff and
ceae, and Thelotremataceae) as part of Ostro- Lutzoni 2002; Miadlikowska and Lutzoni
pales. Subsequently, in a morphological 2004) or to a lineage including Trapeliales and
revision of Ostropalean fungi, Sherwood Baeomycetales (Miadlikowska et al. 2006), sis-
(1977a, b) restricted Ostropales to Odonto- ter to Fuscideaceae, a family incertae sedis in
trema, Ramonia, most current genera of Sticti- Lecanoromycetes (Reeb et al. 2004), and sister
daceae, and other genera now excluded from to a lineage including Anzina and Arthroraphis
Lecanoromycetes. In this classification, many (Wedin et al. 2005), although none of these
lichenized taxa (e.g., Graphidaceae and Thelo- relationships were strongly supported. Schmitt
tremataceae) were excluded from Ostropales et al. (2005) reported the Thelenellaceae as sis-
based on differences in ascospore type (Sher- ter to the Ostropales s.l. with a high posterior
wood 1977a, b). probability, supporting the resolution shown in
Early molecular phylogenetic studies con- Fig. 4.5. Nevertheless, more loci and broader
firmed the close relationship between Stictis taxon samplings are needed to establish the
and Conotrema, and between Stictidaceae and sister taxa of Ostropales with high phylogenetic
both Graphidaceae and Thelotremataceae confidence (Lumbsch et al. 2007b).
(Winka et al. 1998). The two families Coenogo-
niaceae and Gyalectaceae (Gyalectales) were
then shown to be related to Graphidaceae and H. Peltigerales
Thelotremataceae based on molecular data, and
a broad delimitation was adopted for Ostro- Peltigerales (Lecanoromycetidae) (Fig. 4.5) is
pales (Kauff and Lutzoni 2002): Ostropales s.l. an order of mainly foliose species, with
with Coenogoniaceae, Graphidaceae [including rounded apothecia, unbranched paraphyses,
Thelotremataceae, as shown by Mangold et al. bitunicate asci with fissitunicate dehiscence,
(2008)], Gyalectaceae, Stictidaceae, and Trape- and multiseptate ascospores (Honegger 1978;
liaceae. Other families were subsequently Kirk et al. 2008). They have a worldwide distri-
attributed to Ostropales s.l. based on additional bution and colonize diverse substrates, mostly
molecular data: Asterothyriaceae and Gomphil- in humid habitats. Most species in this order
laceae (Lücking et al. 2004), Phlyctidaceae and are associated with cyanobacteria, either as
Solorinellaceae (Miadlikowska et al. 2006), the primary or secondary photobionts. All Leca-
reinstated family Sagiolechiaceae (Baloch et al. noromycetes with cyanobacteria as their pri-
2010), and, more surprisingly, Porinaceae, a mary photobiont belong to this order [with
family of lichenized perithecioid ascomycetes the only exception being Arctomiaceae, which
(Grube et al. 2004). Trapeliaceae (as Agyria- are classified in Ostropomycetidae (Lumbsch
et al. 2005)]. When cyanobacteria occur only as and Lutzoni (2004) and are now largely adopted
secondary photobionts, the primary photo- (Hibbett et al. 2007; Lumbsch and Huhndorf
bionts are then green algae from the genera 2007b, 2010).
Coccomyxa, Dictyochloropsis, or Myrmecia
(Tschermak-Woess 1988a), and the cyanobac- The number of families within Peltigerales changed
terial secondary photobionts are restricted to greatly over time [see details in Miadlikowska and Lut-
gall-like structures called cephalodia. Peltiger- zoni (2004)]. The two families Lobariaceae and Peltiger-
aceae have always been included in Peltigerales
alean species associated only with a green alga (Hafellner 1988; Poelt 1973), but Coccocarpiaceae, Col-
are rare. The most recent common ancestor of lemataceae, and Pannariaceae have sometimes been
Peltigerales was inferred to be associated with a excluded and transferred to the Lecanorales s.l. or clas-
cyanobacterium as its primary photobiont, sified as incertae sedis within Lecanorales s.l. (Eriksson
which means that the green algal photobionts et al. 2003; Hafellner 1988; Henssen and Jahns 1974; Kirk
et al. 2001; Poelt 1973). Moreover, Nephrotomataceae
were most likely acquired secondarily in this and Solorinaceae were recognized as separate families
order (Miadlikowska and Lutzoni 2004). More- from Peltigeraceae by certain authors (Hafellner 1988;
over, the anatomically nonlayered gelatinous Poelt 1973). Molecular phylogenetic studies have con-
thalli mostly found in some genera of Collema- firmed the placement of Coccocarpiaceae, Collemata-
tineae (e.g., Collema and Leptogium) seem to ceae, Pannariaceae, and Placynthiaceae within the
Collematineae, and Lobariaceae, Nephromataceae, and
have evolved from more complex and anatomi- Peltigeraceae within the Peltigerineae (Miadlikowska
cally layered thalli (Wedin et al. 2009). The and Lutzoni 2004; Miadlikowska et al. 2006; Wedin and
phylogenetic relationships, an overview of phe- Wiklund 2004; Wedin et al. 2009). The families Massa-
notypic characters, and the major types of longiaceae, Vahliellaceae, and Koerberiaceae were more
ascus structures within Peltigerales are recently described and attributed to Peltigerineae (Spri-
bille and Muggia 2013; Wedin et al. 2007, 2011).
reported in Spribille and Muggia (2013).
Peltigerales currently includes two subor-
ders, Collematineae and Peltigerineae (Miadli- I. Pertusariales
kowska and Lutzoni 2004), and ten families
(Spribille and Muggia 2013). Collematineae The order Pertusariales (Ostropomycetidae)
includes four families: Coccocarpiaceae, Colle- (Fig. 4.5) mostly comprises crustose species
mataceae, Pannariaceae, and Placynthiaceae. with disciform to poriform apothecia, thick-
Peltigerinae includes six families: Koerberia- walled asci, branched paraphysoids, and gener-
ceae, Lobariaceae, Massalongiaceae, Nephro- ally large ascospores (Lumbsch et al. 1994;
mataceae, Peltigeraceae, and Vahliellaceae Schmitt et al. 2006). They have a worldwide
(Miadlikowska and Lutzoni 2004; Muggia et al. distribution and colonize a broad range of
2011; Spribille and Muggia 2013; Wedin et al. habitats and substrates. Earlier, these species
2007, 2011). Previously, peltigeralean lichens were classified in the suborder Pertusarineae
had been recognized at either the ordinal level within the order Lecanorales s.l. (Henssen and
(Peltigerales) (Hafellner 1988; Kirk et al. 2001) Jahns 1974; Poelt 1973) and later as the order
or the subordinal level within the order Leca- Pertusariales (Hawksworth and Eriksson 1986).
norales (Peltigerinae) (Eriksson et al. 2003; Molecular phylogenies revealed that Pertusar-
Henssen and Jahns 1974; Poelt 1973; Rambold iales belongs to Ostropomycetidae (Lutzoni
and Triebel 1992; Tehler 1996). Despite this et al. 2004; Miadlikowska et al. 2006; Reeb
ranking inconsistency, all large-scale molecular et al. 2004), and this order was accepted in all
phylogenetic studies confirmed the placement recent classifications of Ascomycota (Hibbett
of this lineage within Lecanoromycetes (e.g., et al. 2007; Lumbsch and Huhndorf 2010).
Kauff and Lutzoni 2002; Lutzoni et al. 2001, A recent molecular study showed that the
2004; Miadlikowska et al. 2006; Wedin and type species of Agyrium did not cluster with
Wiklund 2004). The current recognition of other Agyriaceae but nested within Pertusar-
this clade at the ordinal level and the establish- iales (Schmitt et al. 2010). As a result, these
ment of the two suborders Peltigerineae and authors reduced the order Pertusariales to syn-
Collematineae were proposed by Miadlikowska onymy with Agyriales based on the priority
principle. The name Agyriales would then be J. Rhizocarpales

used for a group including a large majority of
species traditionally classified in Pertusariales The order Rhizocarpales (Lecanoromycetidae?)
and only a few mostly nonlichenized and poorly (Fig. 4.5) includes two families, Rhizocarpa-
known species of Agyrium. Hodkinson and ceae and part of the Catillariaceae (the genus
Lendemer (2011) proposed that the name Per- Sporastatia), and approximately 489 species
tusariales should be retained over Agyriales (Kirk et al. 2008). They are characterized by
because the principle of priority is not manda- crustose areolate thalli, immersed to sessile
tory for taxa above the family rank, and because apothecia, branched and often anastomosed
the name Agyriales was most recently misapplied paraphyses, asci with an amyloid apex, and
to a monophyletic group, including the family simple to muriform ascospores (Hafellner
Trapeliaceae, now recognized as Trapeliales. 1984). The sister relationship between Sporas-
tatia (Catillariaceae) and Rhizocarpon (Rhizo-
Pertusariales currently includes seven families: Agyria- carpaceae) was first demonstrated in the study
ceae (currently only represented by its generic type by Reeb et al. (2004). Further studies confirmed
Agyrium rufum), Coccotremataceae, Icmadophilaceae, this result (Buschbom and Mueller 2004; Lut-
Megasporaceae, Miltideaceae, Ochrolechiaceae, and zoni et al. 2004), and the order Rhizocarpales
Pertusariaceae (Hodkinson and Lendemer 2011; was proposed by Miadlikowska et al. (2006) to
Lumbsch and Huhndorf 2010; Schmitt et al. 2010; Wid-
helm and Lumbsch 2011), but its circumscription has accommodate selected taxa from these two
been problematic. Only species from the families Coc- families (many members were never subjected
cotremataceae, Pertusariaceae, and Ochrolechiaceae to phylogenetic studies). The phylogenetic
had traditionally been included in Pertusariineae/Per- position of Rhizocarpales as the first split
tusariales (Eriksson and Hawksworth 1986; Henssen within Lecanoromycetes has rarely been sup-
1976; Henssen and Jahns 1974; Poelt 1973). Species
from the Coccotremataceae family were segregated ported. If confirmed, this order should be con-
from Pertusariaceae based on differences in ascomata sidered as a member of Lecanoromycetidae.
structure and ontogeny (David and Hawksworth 1991;
Henssen 1976). Because species of Coccotremataceae
also differ from those of Pertusariaceae in other aspects
(e.g., the ascus structure, the presence of cephalodia), K. Sarrameanales
members of Coccotremataceae had previously been
excluded from Pertusariales (Lumbsch et al. 1994), The order Sarrameanales (Ostropomycetidae)
but molecular phylogenetic analyses confirmed their (Fig. 4.5) includes a single family, Sarrameana-
placement within this order (Lumbsch et al. 2002).
The segregation of Ochrolechia from Pertusariaceae
ceae, and the two genera Loxospora and Sarra-
was first suggested by Harris (1990). Schmitt et al. meana. It is a small family of approximately ten
(2006) then formally described and redelimited this species occurring mostly in cool temperate
family to also include the genus Varicellaria. Icmado- regions of both Northern and Southern Hemi-
philaceae had earlier been classified within Baeomyce- spheres (Kantvilas 2004). They are crustose
taceae (Henssen and Jahns 1974; Poelt 1973), but
molecular phylogenies supported the placement of
species with dark lecideine to lecanorine
this family within Pertusariales (Miadlikowska et al. apothecia, simple to sparingly branched para-
2006; Reeb et al. 2004). The family Megasporaceae was physes, asci with an amyloid domelike tholus
erected for Megaspora verrucosa, a species previously lacking an ocular chamber, and simple to trans-
classified as part of the genus Aspicilia (Clauzade and versally septate hyaline ascospores. The genus
Roux 1984) and placed in Pertusariales (Lumbsch et al.
1994). This species was later shown based on molecular
Loxospora was segregated from Haematomma-
data to be sister to Aspicilia and part of Pertusariales taceae and placed in Loxosporaceae (Staiger
(Schmitt et al. 2006). The genera Aspicilia and and Kalb 1995), which was later synonymized
Lobothallia were therefore transferred to Megaspora- with Sarrameanaceae (Kantvilas 2004). Because
ceae, and Aspiciliaceae ined. was regarded as a syno- of a unique combination of morphological
nym of this family (Lumbsch and Huhndorf 2010;
Schmitt et al. 2006).
characters, the systematic position of the
genus Sarrameana has always been problematic, were recognized within Teloschistales (Miadli-
possibly as related to Fuscideaceae (Eriksson and kowska et al. 2006): Physciineae (Physciaceae,
Hawksworth 1986), Haematommataceae (Hafell- including Caliciaceae) and Teloschistineae
ner 1984; Vězda and Kantvilas 1988), Lecideaceae (Letrouitiaceae, Megalosporaceae, and Tel-
(Vězda and James 1973), and Ophioparmaceae oschistaceae). However, the relationship
(Kantvilas and Vězda 1996). Recent molecular between Physciineae and Teloschistineae
phylogenetic analyses that included several spe- never obtained strong support (Miadlikowska
cies of Loxospora showed that Sarrameanaceae is et al. 2006).
not related to any of these families (Lumbsch In a more recent molecular study, Gaya
et al. 2007a, b, 2008b). In several studies, the et al. (2012) detected two competing hypotheses
genus Loxospora forms the earliest diverging line- for the relationships among the three clades
age within the subclass Ostropomycetidae Lecanorales, Physciineae, and Teloschistineae:
(Lumbsch et al. 2007b; Miadlikowska et al. 2006; either Lecanorales is sister to a lineage includ-
Schoch et al. 2009). As a result, Hodkinson and ing Physciineae and Teloschistineae, or Phys-
Lendemer (2011) erected the new order Sarra- ciineae is sister to a lineage including
meanales for Sarrameanaceae. Lecanorales and Teloschistineae. To avoid this
phylogenetic uncertainty contributing to taxo-
nomic instability, Gaya et al. (2012) proposed
L. Teloschistales to restrict the name Teloschistales to the Tel-
oschistiineae and resurrect the order Caciliales
Teloschistales (Lecanoromycetidae) (Fig. 4.5), for the Physciineae. This study also showed
as recently recircumscribed by Gaya et al. that Brigantiaeaceae, a family classified as
(2012), comprises four families classified in incertae sedis in Lecanoromycetidae (Lumbsch
the two suborders Teloschistineae (Megalos- and Huhndorf 2010) or as part of Lecanorales
poraceae and Teloschistaceae) and Letrouiti- (Kirk et al. 2008), belongs to Teloschistales and
neae (Brigantiaeaceae and Letrouitiaceae). It is sister to Letrouitiaceae (Gaya et al. 2012).
includes mostly lichenized species with crus-
tose to foliose or fruticose thalli with a yellow
to orange color (anthraquinone pigments), M. Trapeliales
apothecial ascomata, unbranched paraphyses,
unitunicate asci with an apical thickening, and Trapeliales (Ostropomycetidae) (Fig. 4.5) cur-
mostly hyaline polarilocular ascospores (Kär- rently includes a single family, Trapeliaceae
nefelt 1989; Kirk et al. 2008). They are found (Hodkinson and Lendemer 2011). This family
worldwide and often favor nutrient-rich sub- had been described for the four genera Orceo-
strates. lina, Placopsis, Trapelia, and Trapeliopsis
First recognized as a suborder within Leca- (Hertel 1970). Originally, Trapeliaceae was
norales s.l. (Teloschistineae; Henssen and placed in the Agyriineae, a suborder of Leca-
Jahns 1974) or placed within the suborder Buel- norales with a similar ascus structure (Hafell-
liineae (Poelt 1973), the families Letrouitiaceae, ner 1994). In a comprehensive morphological
Teloschistaceae, and, tentatively, Fuscideaceae revision of Agyriineae, the placement of this
were grouped by Eriksson and Hawksworth suborder within Lecanorales was questioned
(1986) in the order Teloschistales, which they (Lumbsch 1997). Early molecular studies
formally described. With the advent of molecu- showed that Agyriineae were indeed not related
lar phylogenetics, several taxa were added to to Lecanorales, and Agyriales was resurrected
Teloschistales, namely, Megalosporaceae for this group (Lumbsch et al. 2001a). More
(Helms et al. 2003; Lutzoni et al. 2004) and recent molecular studies showed that the type
both Caliciaceae and Physciaceae (Miadli- species of Agyrium (A. rufum) belongs to Per-
kowska et al. 2006), which were shown to tusariales (Lumbsch et al. 2007c; Schmitt et al.
form a monophyletic group (Helms et al. 2003; 2010). Because no ordinal name was then avail-
Wedin et al. 2000a). As a result, two suborders able for the lineage including all genera of
Trapeliaceae and other genera previously unbranched paraphyses, unitunicate asci with a
placed in Agyriaceae, Hodkinson and Lende- small amyloid cap, and simple to muriform
mer (2011) proposed to erect the new order ascospores (Hibbett et al. 2007; Louwhoff
Trapeliales for them. Previous molecular data 2009). They mostly grow on rocks at high alti-
had confirmed the placement within Trapelia- tudes or high latitudes (Davydov 2007). They
ceae of the four genera originally included in were traditionally placed in Lecanorales s.l.
this family (Lumbsch et al. 2007b, c; Miadli- (Henssen and Jahns 1974), sometimes in a sep-
kowska et al. 2006; Poulsen et al. 2001; Schmitt arate suborder, Umbilicariineae (Poelt 1973).
et al. 2003). Early molecular studies showed that Umbilicar-
iaceae was not related to Lecanorales (Kauff
In addition, the genera Aspiciliopsis, Placynthiella, Pty- and Lutzoni 2002; Lutzoni et al. 2001; Stenroos
chographa, Rimularia, and Xylographa had also been and DePriest 1998). They form a well-
shown to belong to this family based on molecular data supported monophyletic group, and although
(Lumbsch et al. 2001b; Schmitt et al. 2003). The genera
Amylora, Coppinsia, Lignoscripta, and Sarea have also
their placement within Lecanoromycetes is not
been suggested as belonging to Trapeliaceae (Hodkin- resolved, they do not seem to belong to any of
son and Lendemer 2011), but their phylogenetic place- the three currently recognized Lecanoromy-
ments still need confirmation from molecular cetes subclasses Acarosporomycetidae, Leca-
phylogenetic studies. noromycetidae, and Ostropomycetidae
(Hofstetter et al. 2007; Lumbsch et al. 2004;
Miadlikowska et al. 2006; Reeb et al. 2004;
Wedin et al. 2005). Miadlikowska et al. (2006)
N. Umbilicariales suggested that this group might be recognized
as a separate subclass in the future.
The order Umbilicariales (Lecanoromycetes
incertae sedis) (Fig. 4.5) is an order of approxi-
mately 191 species classified in four families: VIII. Conclusion
Elixiaceae, Fuscideaceae, Ophioparmaceae
[including Rhizoplacopsidaceae, as it is now Morphological, anatomical, and chemical char-
considered a synonym of Ophioparmaceae acters have traditionally been used to classify
(Lumbsch and Huhndorf 2010)], and Umbili- orders, families, and genera within Lecanoro-
cariaceae (Kirk et al. 2008). The delimitation of mycetes, the class of Ascomycota with the larg-
the order Umbilicariales has changed greatly est number of lichen-forming fungi. In the last
over the last decade. Diverse lichen families two decades, molecular phylogenies showed
have recently been shown to belong to this that traditional classification systems were not
order: Elixiaceae (Lumbsch et al. 2007a), always consistent with the evolutionary history
Ophioparmaceae and Fuscideaceae (Bylin of this fungal class, resulting in changes in the
et al. 2007; Miadlikowska et al. 2006), Rhizopla- delimitation of orders and families. For exam-
copsidaceae (Zhou and Wei 2006), and Ropa- ple, many families were segregated from the
losporaceae, although without support for this large and heterogenous order Lecanorales and
last family (Bylin et al. 2007). Umbilicariales raised to the ordinal level. As a result, the clas-
was formally described in two different publi- sification system of the Lecanoromycetes is
cations during the same year (Hibbett et al. now more on par with the overall supraordinal
2007; Zhou and Wei 2007), but the authorship classification of the Fungi (Hibbett et al. 2007)
was attributed to the earliest one (Zhou and and comprises 14 orders and 3 subclasses.
Wei 2007). Additional changes are expected in the future
Umbilicariaceae, the largest family within when better taxon and gene sampling will be
this order, includes lichenized foliose umbili- available to resolve phylogenetic relationships
cate species, with disciform apothecia, mostly within Lecanoromycetes.
Acknowledgments Thanks to Armando Mendez and in Ostropales (Ascomycota: Lecanoromycetes).

Andrea Hart (Botany Library, Natural History Taxon 59:1483–1494
Museum) for their help in locating and providing the Barr ME (1983) The ascomycete connection. Mycologia
required literature. 75:1–13
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5 Pezizomycotina: Eurotiomycetes
DAVID M. GEISER1, KATHERINE F. LOBUGLIO2, CÉCILE GUEIDAN3
CONTENTS I. Phylogeny and Taxonomy

I. Phylogeny and Taxonomy . . . . . . . . . . . . . . . . . . 121
A. Eurotiomycetidae . . . . . . . . . . . . . . . . . . . . . . . . . 123 The phylogenetically informed taxonomic con-
1. Eurotiales. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2. Onygenales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 cept of Eurotiomycetes has expanded greatly
3. Arachnomycetales, Ascosphaera, and from the one first proposed by Eriksson (1999)
Eremascus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 and summarized as “the monophyletic Plecto-
4. Coryneliales . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 mycetes” by Geiser and LoBuglio (2001), which
B. Chaetothyriomycetidae . . . . . . . . . . . . . . . . . . . 130 comprised the orders Eurotiales, Onygenales,
1. Chaetothyriales. . . . . . . . . . . . . . . . . . . . . . . . . 130
2. Verrucariales . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 and Ascosphaerales. The monophyletic Plecto-
3. Pyrenulales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 mycetes, consisting mostly of saprotrophs and
4. Celotheliales ad int. (Gueidan et al. mammalian pathogens, is strongly associated
2014). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 with the morphological concept of Plectomy-
C. Taxa of Uncertain Placement . . . . . . . . . . . . 131 cetes developed by Nannfeldt (1932) and Luttrell
1. Mycocaliciomycetidae/Mycocaliciales. 131
2. Marine Species of Dactylospora and (1951), although fungi with plectomycetous
Sclerococcum . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 characters are known to fall in several other
3. Cirrosporium novae-zelandiae . . . . . . . . 132 non-Eurotiomycete clades (Berbee and Taylor
II. Ecology and Economic Importance . . . . . . . . 132 1992; Geiser et al. 2006). Key characteristics of
A. Extremophilism. . . . . . . . . . . . . . . . . . . . . . . . . . . 132 monophyletic Plectomycetes include (1) asco-
B. Animal Pathogens . . . . . . . . . . . . . . . . . . . . . . . . 132
1. Onygenalean Pathogens . . . . . . . . . . . . . . . 132 mata that are cleistothecial when present, with
2. Eurotialean Pathogens . . . . . . . . . . . . . . . . . 134 no ostiolar opening, often borne in some kind of
C. Plant Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . 134 stromatic tissue (Figs. 5.1 and 5.4); (2) spherical
D. Mutualistic Interactions. . . . . . . . . . . . . . . . . . 134 asci (Fig. 5.2) with a thin wall that deliquesces at
1. Ectomycorrhizae . . . . . . . . . . . . . . . . . . . . . . . 134 maturity to release oblate to lens-shaped ascos-
2. Lichens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
3. Myrmecophytes . . . . . . . . . . . . . . . . . . . . . . . . 135 pores (Fig. 5.3) free within the ascomata (proto-
E. Food Contamination. . . . . . . . . . . . . . . . . . . . . . 135 tunicate); (3) no distinct hymenial layer within
F. Food Production . . . . . . . . . . . . . . . . . . . . . . . . . . 135 the ascomata; (4) no paraphyses or periphyses;
G. Toxic Secondary Metabolites . . . . . . . . . . . . 135 and (5) undifferentiated, hyphal gametangia
H. Industrial Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 (Fig. 5.5). However, early molecular phyloge-
III. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 netic studies revealed that Capronia pilosella
(Chaetothyriales), a fungus with ascolocular
development and bitunicate asci more typical
1
Department of Plant Pathology and Environmental Microbiol- of modern Dothideomycetes, resolved with
ogy, 121 Buckhout Laboratory, The Pennsylvania State Univer-
Eurotialean and Onygenalean fungi (Berbee
sity, University Park, PA 16802, USA; e-mail: dgeiser@psu.edu
2
Farlow Herbarium, Harvard University, 22 Divinity Avenue, 1996; Spatafora et al. 1995). Subsequent phylo-
Cambridge, MA 02138, USA; e-mail: klobuglio@oeb.harvard.edu genetic results bolstered the connection between
3
CSIRO – National Research Collections Australia, Australia Chaetothyriales and Eurotiomycetes (Liu et al.
National Herbarium, GPO Box 1600, Canberra, ACT 2601, 1999; Lumbsch et al. 2000; Silva-Hanlin and
Australia; e-mail: Cecile.Gueidan@csiro.au

122 D.M. Geiser et al.
1 2 5 6 7
4
3
8 9 10 11
12 13 14 17
16 18 21
22
19 23 24 25
15
20
Figs. 5.1–5.25 Morphological features of Eurotiomy- Elaphomyces sp. (Eurotiales inc. sed.); bar¼1 cm. 10.
cetes. Bars represent approximate scale. Figs. 5.1–5.16 Stromatic ascomata of Pseudotulostoma volvata (Euro-
Eurotiomycetidae. Figs. 5.1–5.10 Eurotiales. Figs. 5.1– tiales inc. sed.): developmental series in cross section
5.8 Aspergillaceae. 1. Cleistothecia (arrow); bar¼ [reproduced from Miller et al. (2001)]; bar¼1 cm.
100 mm. 2. Asci; bar¼5 mm. 3. Ascospore from Euro- Figs. 5.11–5.14 Onygenales. 11. Spore balls within a
tium (Aspergillus section Aspergillus) [reproduced ruptured Ascosphaera spore cyst [reproduced from
from Geiser et al. (2006)]; bar¼5 mm. 4. Emericella Chorbinski and Rypula (2003)]; bar¼10 mm. 12.
sexual stage (Aspergillus subgenus Nidulantes) (arrow: Mazaedial ascomata of Onygena sp. on a sheep’s horn
hülle cell-encrusted cleistothecium); bar¼100 mm. 5. (photographer: Ben Mitchell); bar¼5 mm. 13. Histo-
Gametangial coils typical of Aspergillaceae [repro- plasma capsulata, featuring yeast phase (left arrow)
duced from Benjamin (1955)]. 6. Uniseriate conidio- and tightly coiled peridial hyphae (right arrow) (pho-
phores of Aspergillus fumigatus [reproduced from tograph courtesy David Malloch); bar¼50 mm. 14. Coc-
Samson et al. (2007)]; bar¼10 mm. 7. Terverticillate cidioides arthroconidia (photograph credit: Dr. Hardin,
conidiophores of Penicillium chrysogenum [repro- Centers for Disease Control); bar¼5 mm. Figs. 5.15 and
duced from Frisvad and Samson (2004)]; bar¼10 mm. 5.16 Corynelia uberata (Coryneliales) [reproduced
8–10. Stromatic ascomata: 8. Trichocoma paradoxa, from Geiser et al. (2006)] (photographs courtesy Peter
intact and vertical sections [reproduced from Geiser Johnston). 15. Released unitunicate ascus. Arrow: rem-
et al. (2006)]; bar¼5 mm. 9. Stromatic ascomata of nant of outer ascus wall; bar¼10 mm. 16. Ascostromatic
Pezizomycotina: Eurotiomycetes 123
Hanlin 1999; Winka et al. 1998), supporting the A. Eurotiomycetidae

erection of Chaetothyriomycetes and Chae-
tothyriomycetidae (Eriksson and Winka 1997; Key among the evolutionary trends within Euro-
Kirk et al. 2001). tiomycetes is that plectomycetous characteristics
Subsequently, five-locus analyses of Pezizo- (Sect. I) are derived within Eurotiales and Ony-
mycotina (Spatafora et al. 2006) and Dothideo- genales (Fig. 5.26), which comprised the original
mycetes (Schoch et al. 2006) provided strong concept of Eurotiomycetes as suborders Euroti-
statistical support for the connection between neae and Onygeniniae within Eurotiales (Eriks-
Chaetothyiales and Eurotiomycetes. In addition, son and Winka 1997). Coryneliales, which is
the orders Coryneliales, Pyrenulales, Verrucar- basally derived within Eurotiomycetidae, pos-
iales, and Mycocaliciales resolved within a sesses an ascus morphology that is intermedi-
strongly supported clade within which Euro- ate between the classic plectomycetous
tiales, Onygenales, and Chaetothyriales are characters found in Eurotiales and Onygenales
derived (Geiser et al. 2006). Based on these and the ascolocular characters found in Chae-
results, three strongly supported monophyletic tothyriomycetidae and Mycocaliciomycetidae
subclasses were proposed: Eurotiomycetidae (Geiser et al. 2006). This morphological trend
(Eurotiales, Onygenales, Arachnomycetales, is accompanied by a transition to primarily sap-
Coryneliales), Chaetothyriomycetidae (now con- rophytic ecology in Eurotiales and Onygenales,
sisting of Celotheliales ad int., Chaetothyriales, along with some biotrophic associations with
Verrucariales, Pyrenulales), and Mycocaliciomy- plants and animals.
cetidae (Mycocaliciales) (Geiser et al. 2006;
Gibas et al. 2002; Gueidan et al. 2014; Hibbett 1. Eurotiales
et al. 2007; Spatafora et al. 2006). Mycocalicio-
mycetidae was included with caution since its Eurotiales houses some of the most commonly
inclusion does not receive robust statistical sup- encountered microfungi, including the very
port across all analyses. Importantly, the classic important species with Aspergillus and Penicil-
plectomycetous characters observed in Euro- lium anamorphs. Geiser and LoBuglio (2001)
tiales and Onygenales are rarely observed in recognized three families in Eurotiales: Elapho-
these newly included orders, so this increased mycetaceae, Monascaceae, and Trichocomaceae.
phylogenetic breadth is accompanied by a large This chapter will follow Houbraken and Sam-
increase in morphological as well as ecological son’s (2011) proposed segregation of Trichoco-
diversity (Figs. 5.1–5.25). Figure 5.26 is a sche- maceae into three families, Aspergillaceae,
matic summary phylogeny of Eurotiomycetes Thermoascaceae, and Trichocomaceae. Monas-
that attempts to represent the consensus of caceae is absorbed into Aspergillaceae, and Ela-
molecular phylogenetic studies. phomycetaceae is considered unresolved.
⁄

Figs. 5.1–5.25 (Continued) ascomata on Podocarpus enula concatervans (Pyrenulales) thallus on bark, with
leaf. Figs. 5.17–5.23 Chaetothyriomycetidae. Figs visible perithecia; bar¼5 mm. [Figs. 5.21 and 5.22
5.17–5.20 Chaetothyriales. 17. Dimorphic black yeast reproduced from Geiser et al. (2006); photographs
colony (photograph courtesy Nina Gunde-Cimerman). courtesy Cecile Gueidan.] 23. Perithecium of Celothe-
18. Cross section of domatium from plant Leonardoxa lium longisporum (Celotheliales ad int.), showing
africana ssp. africana, revealing its mutualistic plant– marginal ostiole (arrow); bar¼1 mm. [Fig. 5.23 repro-
ant Petalomyrmex phylax tending a black fungus duced from Gueidan et al. (2014); photograph courtesy
[reproduced from Mayer et al. (2014)]. 19. Phialo- André Aptroot.] 24. Mazaedial ascomata of Mycocali-
phora anamorph; bar¼2 mm. 20. Bitunicate asci from cium (Mycocaliciales/Mycocaliciomycetidae; photo-
Chaetothyrium. [Figs. 5.19 and 5.20 reproduced from graph courtesy Helge G. Gundersen). 25. Pycnidia of
Geiser et al. (2006); photographs courtesy Wendy Cirrosporium novae-zelandiae (Eurotiomycetes inc.
Untereiner]; bars¼2 mm. 21. Placopyrenium canellum sed.) producing chains of meristematic arthroconidia
(Verrucariales), a parasitic species growing on the [reproduced from Réblov and Seifert (2012)]; bar¼
rock lichen Aspicilia calcarea; bar¼100 mm. 22. Pyr- 1 mm
Fig. 5.26 Phylogenetic tree representing current state ies of Berbee and Taylor (1992), Bowman et al. (1996),
of knowledge regarding phylogenetic relationships del Prado et al. (2006), Diederich et al. (2013), Gargas
within Eurotiomycetes. Taxa of uncertain or poorly et al. (1995), Geiser and LoBuglio (2001), Geiser et al.
supported phylogenetic position are represented as (2006), Gibas et al. (2002), Gueidan et al. (2014), Henkel
dashed lines. “Core Onygenales” refers to Onygenaceae, et al. (2001), Houbraken and Samson (2011), Hyde et al.
Gymnoascaceae, Arthrodermataceae, and Nannizziop- (2013), Klinger et al. (2013), Réblov and Seifert (2012),
siaceae (Sect. I.A.2.a). “Unnamed lineages” refer to Stchigel et al. (2013), Tibell and Wedin (2000), and
unnamed lineages 1 and 2 in Gueidan et al. (2014). Untereiner et al. (2002)
Phylogenetic positions are inferred based on the stud-
a) Aspergillaceae biverticillate Penicillium species previously

i. Taxonomy treated as Penicillium subgenus Biverticillium
(Pitt 1979), which were long known to be phy-
Houbraken and Samson’s (2011) Aspergilla-
logenetically distinct (Berbee et al. 1995), were
ceae encompasses a large majority of Aspergil-
segregated into the genus Talaromyces. This
lus and Penicillium species distributed in 14
genus resolves phylogenetically within Tricho-
strongly supported clades. At least 16 teleo-
comaceae sensu Houbraken and Samson (2011)
morph genera are represented, some with
(Sect. I.A.1.c).
clear associations to monophyletic groups
The strongly supported clade corresponding
(e.g., Eupenicillium, Emericella, Neosartorya,
to Penicillium s. str. comprises two subclades,
Eurotium). Houbraken and Samson’s (2011)
proposed as subgenera Aspergilloides and Penicil-
analysis of four protein coding genes resolved
lium (Houbraken and Samson 2011). Subgenus
monophyletic groups proposed as Penicillium
Aspergilloides roughly corresponds to the mono-
s. str. and Aspergillus s. str., although only the
verticillate and biverticillate (with flask-shaped
former received strong statistical support. Most
conidiogenous cells) subgenera Aspergilloides
and Furcatum sensu Pitt (1979) and subgenus asexual states (Figs. 5.6 and 5.7). Aspergillus and
Penicillium with ter- and quaterverticillate spe- Penicillium anamorphs generally produce coni-
cies, mostly representing Pitt’s previous concept dia from phialides on unbranched and branched
of subgenus Penicillium. In support of a unitary conidiophores, respectively. Conidia are borne
generic taxonomy, the International Commis- from the phialides by successive budding, usually
sion on Penicillium and Aspergillus (ICPA; forming long chains (Clutterbuck 1969; Mims
www.aspergilluspenicillium.org) has recom- et al. 1988; Sewall et al. 1990a, b). Conidiophore
mended use of the name Penicillium over the development is tied closely to the expression of
teleomorph names Eupenicillium and other secondary metabolism, with light being a key
genera typified in the Penicillium clade. environmental factor in the regulation of both
The Aspergillus s. str. clade, in contrast, processes (Bayram et al. 2008; Park and Yu 2012).
receives weak support as a whole but comprises There are some interesting exceptions to
ten well-supported clades. Some of these clades the Aspergillus and Penicillium asexual mor-
correspond strongly to teleomorph generic phology that dominates Aspergillaceae, found
concepts, including Emericella, Neosartorya, mostly in 2 of the 14 clades resolved by
and Eurotium. As it did with Penicillium, the Houbraken and Samson (2011). Derived from
ICPA has transferred all species within the within the Aspergillus clade is a strongly
Aspergillus clade into the genus Aspergillus, supported group comprising several canine
reviving the very broad generic concept put pathogenic species with highly reduced coni-
into place by Raper and Fennell (1965). In a diophores, consisting of simple phialides bear-
reinterpretation of the Houbraken and Samson ing chains of conidia. Some of these fungi were
(2011) data set, Pitt and Taylor (2014) resolved previously classified in the genus Sagenomella
a tree that showed the ICPA’s concept of Asper- and later segregated into the new genus Phialo-
gillus to be paraphyletic, with Penicillium simplex (Sigler et al. 2010). To accommodate
derived within it, and recommended generic this paraphyletic morphological concept of
segregation of clades associated mostly with Aspergillus with respect to Phialosimplex, the
teleomorph concepts (Emericella, Neosartorya, ICPA recommends transfer of these species into
Chaetosartorya, Eurotium, Phialosimplex). Pitt Aspergillus despite their reduced asexual mor-
and Taylor (2014) advocated a narrower con- phology; Pitt and Taylor’s (2014) proposal
cept of Aspergillus, provisionally retaining a retains Phialosimplex. In addition, a second
nonmonophyletic core group of species that clade consists of fungi in the genera Monascus,
includes A. flavus, A. niger, A. ochraceus, and Xeromyces, and Leiothecium, all of which pro-
A. terreus. This split is bound to have a signifi- duce Chrysosporium-like thallic anamorphs.
cant impact due to the importance of these Finally, Dichotomomyces produces a Paecilo-
organisms in agriculture, industry, medicine, myces-like anamorph, despite its clear phyloge-
and basic research, but Pitt and Taylor (2014) netic affinity to Aspergillus Section Fumigati
argue that the phenotypic differences between (Houbraken and Samson 2011).
these clades are too great to be accommodated
in a single genus. Indeed, their conclusion is
iii. Sexual Morphology and Self-Fertility
corroborated by genomic comparisons among
Aspergillus species: Emericella nidulans (A. Aspergillaceae produce a wide variety of sexual
nidulans) and Neosartorya fumigata (A. fumi- stages (Geiser 2009). Asci are formed either
gatus) have average protein sequence identities within a cleistothecium or on an unenclosed
similar to those observed between birds and mycelium, which in turn may or may not be
mammals (Fedorova et al. 2008). produced on or within some form of stromatic
tissue. In Aspergillus and Penicillium, simple
cleistothecia represent the most common
ii. Asexual Morphology
form, with walls consisting of flattened cells or
A large majority of species in Aspergillaceae pro- hyphal elements (Fig. 5.1). These include the
duce recognizable Aspergillus and Penicillium teleomorph generic concepts associated with
Neosartorya, Eurotium, Eupenicillium, and A. flavus, A. lentulus, A. oryzae, A. parasiticus,

Fennellia. More elaborate stromatic forms and A. terreus, possess both MAT1-1 and MAT1-
vary from the production of thick-walled sterile 2 individuals (Dyer and O’Gorman 2012; Henk
elements, called hülle cells, surrounding the et al. 2011) enabled the discovery of their hetero-
cleistothecium, as seen in Emericella (Fig. 5.4), thallic sexual stages (Arabatzis and Velegraki
to sclerotiumlike structures that may enclose 2013; Bohm et al. 2013; Horn et al. 2009a, b,
multiple cleistothecia, as seen under the con- 2013; O’Gorman et al. 2009). While sexual stages
cepts of Petromyces and Neopetromyces. for a majority of Aspergillus and Penicillium
One notable feature of Aspergillaceae is the species remain undiscovered, most are probably
high frequency of homothallism, with approxi- heterothallic with cryptic sexual stages. Still,
mately one-third of the species being self-fer- homothallism is unusually common in Aspergil-
tile (Geiser 2009). Relatively few heterothallic laceae, perhaps under positive selection due to
species are known, and the majority of species adaptations associated with ascospore-based
with no known sexual stage were thought of as propagation (Geiser 2009).
largely asexual (Geiser et al. 1996) until fairly
recently. The genetic basis of homothallism in b) Thermoascaceae
Pezizomycotina was first elucidated in a num- i. Taxonomy
ber of Sordariomycetes (Glass and Smith 1994;
Thermoascaceae was first proposed to accom-
Pöggeler et al. 1997; Yun et al. 2000) and Dothi-
modate the thermophilic genera Thermoascus
deomycetes (Yun et al. 1999), but because of a
and Dactylomyces, the latter now recognized as
lack of knowledge about mating type in Asper-
a synonym of the former (Apinis 1967). Based
gillaceae, its basis remained undiscovered until
on its phylogenetic resolution distinct from
complete genome sequences were available. In
Aspergillaceae and Trichocomaceae, Houbra-
the canonical situation outside of Aspergilla-
ken and Samson (2011) defined Thermoasca-
ceae, there is a single MAT1 mating-type
ceae to include Thermoascus and Byssochlamys
locus, harboring either MAT1-1 or MAT1-2 idi-
species.
omorphic genes in heterothallic species. Homo-
thallics, however, typically possess a
combination of genes from both mating-type ii. Asexual and Sexual Morphology
idiomorphs in the same MAT1 locus, providing
Members of Thermoascaceae may produce
compatibility factors in the same haploid
both blastic and thallic conidia. Blastic conidia
genome and enabling self-fertility. Because
are borne from phialides and annellides in
these homothallic MAT1 arrangements tend to
branched, Paecilomyces-like conidiophores.
be unique to species, they are inferred as having
Thallospores are often thick-walled and pro-
been derived uniquely through convergent evo-
duced terminally or intercalary in hyphae. In
lution (Yun et al. 1999). It turns out that at least
Thermoascus, asci are produced in a firm cleis-
some homothallic Aspergillaceae species devi-
tothecium with a peridium composed of pseu-
ate from the canonical situation. The homo-
doparenchymatous cells, while in Byssochlamys
thallic Aspergillus species A. nidulans
no enclosed cleistothecium is produced. These
(Emericella) and A. fischeri (Neosartorya) also
two genera have similar ascospores that, how-
possess both MAT1-1 and MAT1-2 genes in a
ever, possess rough walls and lack a furrow or
single haploid genome, but the two idiomorphs
slit (Houbraken and Samson 2011).
are localized on separate chromosomes (Pao-
letti et al. 2007). In a third homothallic species,
c) Trichocomaceae
A. alliaceus, both idiomorphs are colocalized in
i. Taxonomy
the same locus (Ramirez-Prado et al. 2008), as
typically occurs in other classes of Pezizomyco- Malloch’s (1985) Trichocomaceae approxi-
tina. The discovery that species known previ- mated the sum of Aspergillaceae, Thermoasca-
ously only by their anamorphs, including ceae, and Trichocomaceae as outlined here.
Penicillium chrysogenum, Aspergillus fumigatus, Malloch (1985) recognized two subfamilies
within Trichocomaceae: Dichlaenoideae, which families and typified in Thermoascaceae (P.

corresponded closely to the current concept of variotti), are common in Trichocomaceae. The
Aspergillaceae, except with the inclusion of type genus Trichocoma produces large (up to
Thermoascus, and Trichocomoideae, which 1 cm), stromatic ascomata shaped like a
corresponded closely to the current concept of shaving brush, with hyphae bearing asci
Trichocomaceae, except with the inclusion of extending from a woodlike base (Fig. 5.8), and
Byssochlamys. Malloch defined these two sub- a biverticilliate Penicillium-like anamorph.
families based on morphology, physiology, and Talaromyces species produce enclosed asco-
ecology. Morphologically, Dichlaenoideae were mata with hyphal peridia.
likely to produce true cleistothecia with pseu-
doparenchymatous walls, often borne in a d) Disputed Families: Monascaceae and
pseudoparenchymatous stroma, bearing oblate Elaphomycetaceae
ascospores with a distinct equatorial furrow, Geiser and LoBuglio (2001) recognized Monas-
and usually associated with starchy or oily sub- caceae and Elaphomycetaceae within Euro-
strates. Trichocomoideae produce asci among tiales. We abandon Monascaceae here because
hyphae that may or may not form an enclosed three of the four genera in the family, Monas-
structure, sometimes borne on some kind of cus, Xeromyces, and Leiothecium, resolve
sterile base. Ascospores are spherical to ellipti- together in a well-supported clade in Houbra-
cal, usually roughened, and lack an equatorial ken and Samson’s (2011) Aspergillaceae; a
furrow; these fungi are more likely to be found fourth genus, Ascorhiza, remains unresolved.
on woody or cellulosic substrates. Elaphomycetaceae encompasses two inter-
The Trichocomaceae lineage sensu esting genera, Elaphomyces and Pseudotulos-
Houbraken and Samson (2011) includes five toma, that are both ectomycorrhizal (Henkel
genera: Talaromyces, Thermomyces, Sagen- et al. 2006; Miller and Miller 1984). Elapho-
omella, Rasamsonia, and Trichocoma. Rasam- myces has large, hypogeous ascomata with a
sonia is a new genus in this lineage comprising thick, multilayered peridium/stroma (Hawker
thermotolerant and thermophilic Talaromyces 1954). In what appears to be convergent evolu-
and “Geosmithia” species. Interestingly, it is tion with true truffles, Elaphomyces forms
a sister clade to Trichocoma paradoxa in spherical, heavily ornamented ascospores pro-
Houbraken and Samson’s analyses (Houbraken duced in a pseudocapillitium. In Pseudotulos-
and Samson 2011). toma volvata, a stalked ascoma (mazaedium)
reminiscent of a stalked puffball is produced,
extending upward from a volvalike structure,
ii. Asexual and Sexual Morphology
which itself is very similar to the hypogeous
Many of the fungi within Trichocomaceae pro- ascoma produced by Elaphomyces (Miller
duce biverticillate Penicillium-like anamorphs et al. 2001). An anamorph for Elaphomyces or
bearing tightly packed, spinulose phialides Pseudotulostoma has not been observed, and
(formerly Penicillium subgenus Biverticillium), Elaphomyces grows only very slowly in culture
which distinguishes them from biverticilliate (Miller and Miller 1984).
Penicillium species that are retained in that The lack of resolution in the subordinal
genus, in Aspergillaceae. Most are associated ranking and placement of Elaphomyces has
phylogenetically with Talaromyces (LoBuglio long been a source of frustration. Trappe
et al. 1993; Pitt 1979) and have been transferred (1979) referred to it as “the spurned stepchild
to that genus. Rasamsonia species produce con- of the hypogeous Ascomycota” because of the
idiophores similar to those of Talaromyces but taxonomic confusion resulting from its weak
with roughened stipes and asci borne in chains connections to other orders. Many authors
in ascomata with a superficial covering placed Elaphomyces in Plectomycetes because
(Houbraken and Samson 2011; Stolk and of its irregularly arranged asci and cleistothe-
Samson 1972). Paecilomyces-like anamorphs, cial ascomata, but others placed it in Tuberales
which are found in all three Eurotialean based on its hypogeous mycorrhizal habit and
ascospore size and shape. Finally, Trappe (2013), Arachnomycetaceae did not resolve
(1979) proposed Elaphomycetales as a unique with strong statistical support within Onygen-
monotypic order. Phylogenetic studies by ales, leading those authors to propose ordinal
LoBuglio et al. (1996) and Landvik et al. status as Arachnomycetales. However, no mul-
(1996) demonstrated a strong affinity between tilocus analysis utilizing a full taxon set of Ony-
Elaphomyces and Eurotiales. However, even genales has been performed, leaving the
with the discovery of Pseudotulostoma, the relationships between these families, as well as
phylogenetic status of Elaphomycetaceae their status as monophyletic groups, unclear. In
remains unclear (Fig. 5.26). Based on sequences addition, the genera Ascosphaera and Eremas-
of the small subunit (SSU) ribosomal RNA cus are poorly resolved in this order and recog-
gene, these two genera resolve together basally nized as members of Ascosphaeraceae and
within the order, but with poor statistical sup- Eremascaceae in the NCBI GenBank taxonomy.
port (Miller et al. 2001). Geiser et al. (2006) Stchigel et al. (2013) resolved Ascosphaeraceae
resolved a moderately supported clade that as a family within Onygenales, and Eremascus
included both Trichocoma and Pseudotulos- as an outgroup with respect to Onygenales.
toma, but this analysis included only DNA However, because of the single-locus nature of
sequences from nuclear SSU and large subunit that analysis, caution is advisable about draw-
ribosomal RNA (LSU) genes, and Elaphomyces ing firm taxonomic conclusions. A stable sub-
was not sampled. ordinal taxonomy for this group awaits a
thorough multilocus phylogenetic analysis uti-
lizing a rich taxon sample.
2. Onygenales
Members of Onygenales are also extremely a) Core Families of Onygenales
common, known mostly as saprotrophs, partic- Based on the LSU analysis of Stchigel et al.
ularly in soil and on keratin-rich substrates, (2013), there is a well-supported group of
and parasites of mammals, causing dermato- clades within Onygenales that represent the
phytoses and other infections. Currah (1985) core families of this order: Onygenaceae, Gym-
recognized four families in Onygenales—Ony- noascaceae, Arthrodermataceae, and Nanniz-
genaceae, Arthrodermataceae, Myxotrichaceae, ziopsiaceae. These families share a range of
and Gymnoascaceae—but noted morphological characteristics outlined in Currah’s (1985)
and ecological similarities between Myxotri- treatment of the order. This includes a wide
chaceae and some members of Leotiomycetes; spectrum of cleistothecial peridium types,
this was confirmed by later molecular phyloge- including pseudoparenchymatous peridia,
netic work resulting in its reclassification peridia composed of wefts of hyphae, and
(Sugiyama et al. 2002; Wang et al. 2006). Geiser those with a more netlike peridium with and
and LoBuglio (2001) followed Currah’s (1985) without appendages. Asexual stages are domi-
family-level taxonomy; since then, Arachnomy- nated by thallic Trichophyton, Chrysosporium,
cetaceae/Arachnomycetales [segregated from Malbranchea, and Microsporum anamorphs.
Gymnoascaceae (Gibas et al. 2002)], Ajellomy- Onygena is perhaps the most distinctive mem-
cetaceae [segregated from Onygenaceae ber of the Onygenaceae because it produces
(Untereiner et al. 2004)], and Nannizziopsia- relatively large, stalked mazaedia on keratinous
ceae [segregated from Onygenaceae (Stchigel substrates (Fig. 5.12). Members of Onygenaceae
et al. 2013)] have been proposed based on characteristically produce pitted ascospores
phylogenetics. Much of this segregation reflects and arthroconidia and aleuroconidia, some-
the discovery of a deep clade structure within times both. Arthrodermataceae are distin-
Onygenales identified by Bowman et al. (1996). guished by their smooth ascospores and
In the analysis of the nuclear large ribosomal cleistothecia that bear ossiform (having a bone-
RNA subunit (LSU) gene by Stchigel et al. like appearance) or derived ossiform cells. Nan-
nizziopsaceae comprises reptile pathogens that remains in doubt. Ascosphaera produces a

produce a skunklike odor (Stchigel et al. 2013). unique double-walled cleistothecium within
Members of Gymnoascaceae produce a hyphal which ascospores form, and these aggregate
cleistothecium. into spore balls (Fig. 5.11). Eremascus is a xer-
ophilic fungus that produces prototunicate asci
naked on a thin mycelium. Berbee and Taylor
b) Ajellomycetaceae
(1992) first revealed the connection between
Based on analyses of the nuclear SSU ribosomal
these taxa and Onygenales. Geiser and LoBuglio
RNA gene, Bowman et al. (1996) noted that the
(2001) placed both taxa within order Asco-
major human pathogen Coccidioides immitis
sphaerales in the monophyletic Plectomycetes
was more closely related to the saprobic fun-
clade because the two genera fell out in a clade
gus Uncinocarpus reesii than it was to the
distinct from the remainder of Onygenales. The
pathogens Histoplasma capsulatum and Blas-
multilocus analysis of Geiser et al. (2006)
tomyces dermatitidis. The latter two species,
resolved these genera within Onygenales with
along with Paracoccidioides brasiliensis, have
strong support, but this conclusion remains in
since been segregated into the new family Ajel-
doubt. LSU sequences resolve Ascosphaerales
lomycetaceae (Untereiner et al. 2004), with Coc-
as a basal family within a monophyletic Ony-
cidioides retained in Onygenaceae. The sexual
genales and Eremascus as an outgroup with
stage associated with Histoplasma and Blasto-
respect to Onygenales, Arachnomycetales, and
myces is characterized by distinctive, tight peri-
Eurotiales (Stchigel et al. 2013). Klinger et al.
dial coils that extend radially from the
(2013) performed a multilocus analysis of Asco-
cleistothecium (Fig. 5.13; Kwon-Chung 1973).
sphaera species, showing that the genus repre-
Asci in these taxa are distinguishable from
sents a diverse, monophyletic assemblage. As
those of other members of Onygenales and
stated earlier, hypotheses regarding the supra-
Eurotiales by their pyriform shapes; this may
generic relationships among these genera with
be a derived ancestral character connecting this
respect to Onygenales and its families remain
family with Coryneliales (Sect. I.A.3 below).
largely untested.
3. Arachnomycetales, Ascosphaera, and 4. Coryneliales

Eremascus
Schoch et al. (2009) and Geiser et al. (2006)
The phylogenetic distinctiveness of Arachno- both resolved a clear sister relationship
mycetaceae/Arachnomycetales is well- between Eurotiales+Onygenales and the enig-
established as a unique clade (Gibas et al. matic order Coryneliales. This finding
2002); its position within or outside of Onygen- provided an important insight into the evolu-
ales remains poorly resolved (Geiser et al. tion of one of the most distinctive character-
2006). Stchigel et al.’s (2013) analysis of LSU istics of the Eurotiomycetidae: the globose,
sequences resolved an Arachnomycetales clade evanescent, prototunicate ascus. Coryneliales
sister to all Onygenalean families, but the are biotrophic associates of woody plants, par-
backbone support in this tree was weak. Geiser ticularly Podocarpaceae in the Southern Hemi-
et al. (2006) postulated a possible phylogenetic sphere, as well as some northern temperate
connection between Ascosphaera and Arachno- species. These fungi produce ascolocular asco-
mycetaceae, but Stchigel et al.’s (2013) LSU mata similar to the loculoascomycetes that
phylogeny showed Ascosphaera as sister to the dominate Dothideomycetes (Figs. 5.15 and 5.16).
core Onygenales clade and separate from Ara- In early developmental studies, Coryneliales
chnomyces. While the position of this taxon was considered an order within Pyrenomycetes
remains poorly resolved, it is generally recog- because their long-tailed asci terminate in a
nized at the ordinal rank. globose sac that appeared to be unitunicate or
Ascosphaera and Eremascus are distinctive prototunicate, having a thin wall that deli-
taxa whose inclusion in the Eurotiales+Ony- quesces at maturity to release the single-celled
genales clade is clear, but their exact position ascospores in the ascomata. In his landmark
developmental treatise on the taxonomy of Pyr- ecologically diverse taxa that occupy
enomycetes, Luttrell (1951) expressed frustra- Chaetothyriomycetidae (Figs. 5.17–5.23). They
tion with this taxon, treating it last among the often produce ostiolate perithecial ascomata
11 orders described: with hamathecial elements, and asci that are
bitunicate (Fig. 5.20) and either fissitunicate
The Coryneliales represents a serious obstacle to the sys- or evanescent (Gueidan et al. 2014). They
tem of classification adopted in this review. At this point include diverse mutualists, parasites, and
the correlation between the bitunicate ascus and ascos- saprotrophs. One common ecological trait
tromatic nature of the ascocarp breaks down completely.
Since, however, primary emphasis is placed upon ascus
observed, primarily in the orders Chaetothyr-
structure, the order is tentatively assigned to the Unitu- iales and Verrucariales, is an association with
nicatae and included among the Pyrenomycetes. extreme xeric habitats, such as high salt and
rocky environments. In addition, members of
With its apparently unitunicate asci, Cory- this clade are known to be associated with ants,
neliales defied one of the hallmarks of Luttrell’s bryophytes, and deep marine habitats.
taxonomic system, the connection between
ascostromatic development and the bitunicate,
jack-in-the-box ascus. However, more than 1. Chaetothyriales
three decades later, it was discovered that the Geiser et al. (2006) discussed two families
Corynelialean ascus is not unitunicate. Using within Chaetothyriales; a multilocus phyloge-
light microscopy, Johnston and Minter (1989) netic analysis focusing on Chaetothyriomyce-
documented the bitunicate nature of asci in tidae recognizes five (Chaetothyriaceae,
Coryneliales (Fig. 5.15), with outer walls that Herpotrichiellaceae, Epibryaceae, Cyphello-
break to release thin-walled, young asci. This is phoraceae, and Trichomeriaceae), with an
easy to miss since the bitunicate stage is present unnamed family-level clade known only from
only very early in development, before ascos- sequences derived from rock-inhabiting fungi
pores form. Other than a barely detectable rem- (Gueidan et al. 2014). Chaetothyrialean fungi
nant of the outer wall at the base, the mature are diverse saprotrophs and parasites of plants
ascus appears to be unitunicate. Much like and animals, including humans. Anamorphs
members of the core Eurotiomycetidae, the glo- vary, including black yeasts and dematiaceous
bose apex of the ascus breaks open in an irreg- hyphomycete forms that include genera such as
ular fashion, releasing the ascospores in the Exophiala and Phialophora. Perithecia have
ascomatal cavity. This was interpreted as an thin walls that are often setose and bear fissitu-
intermediate form between the bitunicate and nicate clavate asci. Hamathecial elements
prototunicate ascus (Read and Beckett 1996). include ostiolar periphyses and short apical
Based on phylogenetics, Geiser et al. (2006) pseudoparaphyses.
postulated that Coryneliales represents a tran-
sitional ascomatal form, perhaps between an
ancestral ascostromatic bitunicate form 2. Verrucariales
retained in Chaetothyriales in Chaetothyrio-
mycetidae and the cleistothecial prototunicate Members of Verrucariales are mostly liche-
form seen in core Eurotiomycetidae. This nized. They are notable among lichens for
represents just one of several inferred transi- their ecological diversity, which ranges from
tions to the prototunicate ascus in the Pezizo- xeric terrestrial to freshwater, and even
mycotina (Wedin and Tibell 1997). marine habitats, where they are particularly
common. Much of their diversity occurs on
rock, particularly in temperate regions
B. Chaetothyriomycetidae (Fig. 5.21). Some Verrucarialean fungi are para-
sites of lichens, either as free-living or as liche-
Unlike Eurotiomycetidae, few obvious charac- nized forms. Thalli vary tremendously in form
ters dominate the morphologically and and pigmentation (Muggia et al. 2010), with
melanin being common, a character shared ad interim to the ordinal level, together with
with Chaetothyriales (Geiser et al. 2006). Asex- fungi in the plant pathogenic genera Phaeomo-
ual propagules such as isidia and soredia are niella and Dolabra. No anamorph is known for
rare. Ostiolate perithecia may be produced Celothelium, so its potential morphological
immersed in the thallus or superficially. Asci connection to these nonlichenized anamorph
are bitunicate with either fissitunicate or eva- taxa currently cannot be investigated. Celothe-
nescent release of ascospores. Hamathecial ele- lium produces an elongated, carbonized peri-
ments are similar to those in Chaetothyriales, thecium with a marginal ostiole (Fig. 5.23)
with interascal elements absent at maturity bearing filamentous, multiseptate ascospores
(Gueidan et al. 2014). Although phylogenetic (Gueidan et al. 2014).
studies have started to address generic delimi-
tation within Verrucariaceae, the largest family
of Verrucariales (Gueidan et al. 2007, 2009), no C. Taxa of Uncertain Placement
molecular data are currently available to con-
firm the position of a second family, the Adelo- 1. Mycocaliciomycetidae/Mycocaliciales
coccaceae.
Mycocaliciales, consisting of families Mycoca-
liciaceae and Sphinctrinaceae, was proposed as
3. Pyrenulales an order distinct from Caliciales (Lecanoromy-
cetes) based on morphology and its distinct
Pyrenulales are also mostly lichenized but usu- SSU sequences, which placed it close to Euro-
ally occur on tree bark (Fig. 5.22), and in tropi- tiales and Onygenales (Gargas et al. 1995; Tibell
cal regions (Gueidan et al. 2014). Members of and Wedin 2000). These fungi are nonliche-
this group tend to be associated with algae of nized and occur as parasites or commensals
the genus Trentepohlia (Weerakoon et al. on the thalli of lichens or saprobic fungi.
2012). Thalli are usually thin and immersed or Mycocaliciales received moderate bootstrap
superficial in or on the substrate and lacking in support forming a clade with Eurotiomycetidae
asexual propagules. Unlike most lichen groups, and Chaetothyriomycetidae in the analysis of
secondary metabolites are uncommon. Perithe- Geiser et al. (2006), and it was later given sub-
cia are ostiolar and contain unbranched para- class status in Eurotiomycetes as Mycocalicio-
physes and fissitunicate asci. Many taxa mycetidae (Hibbett et al. 2007). Some
formerly placed in Pyrenulales have been redir- characters seen with varying frequency in Euro-
ected to Dothideomycetes (Geiser et al. 2006; tiomycetidae and Chaetothyriomycetidae
Hyde et al. 2013), and Celotheliaceae was raised [mazaedial ascomata (Fig. 5.24), occasional
to interim ordinal status within Chaetothyrio- evanescent asci, their role as parasites or com-
mycetidae (Gueidan et al. 2014; see below). This mensals on lichens and other fungi] are some-
leaves two families in the order, Requiellaceae times present in Mycocaliciomycetidae. The
and Pyrenulaceae, the latter of which includes group is associated with both coelomycetous
the great majority of species and consists of two and hyphomycetous anamorphs.
strongly supported subgroups (Weerakoon
et al. 2012).
2. Marine Species of Dactylospora and
Sclerococcum
4. Celotheliales ad int. (Gueidan et al. 2014)
Among the taxonomically untreated clades
Del Prado et al. (2006) resolved Celothelium identified in the analysis of Gueidan et al.
close to Pyrenulales, leading to its later family- (2014) is a group that includes marine species
level circumscription (Aptroot et al. 2008). The of Dactylospora and Sclerococcum sphaerale, an
family Celotheliaceae is a strongly supported anamorph that occurs on lichens. The clear
monophyletic group within Chaetothyriomyce- phylogenetic connection between these taxa
tidae, leading Gueidan et al. (2014) to elevate it and their placement in Eurotiomycetes was
first noted by Diederich et al. (2013). However, concentrations that may exceed 30 % (Gunde-
these two studies, which utilized different mar- Cimerman et al. 2000). While saprobic species
kers and taxon selection, resolve them in dominate in Eurotiales, Onygenales, and Chae-
slightly different positions within Eurotiomy- tothyriales, this metabolic adaptability provides
cetes, so their rank status at the ordinal or them with some degree of pathogenic potential
subclass level remains in question. on plants and animals, usually when the host is
weakened by disease or other stress (Gostincar
et al. 2011). Chaetothyriomycetidae includes a
3. Cirrosporium novae-zelandiae very diverse array of inhabitants of rock
Cirrosporium novae-zelandiae is an unusual (Gueidan et al. 2008, 2014), many of which
coelomycete that appears to be a Eurotiomy- remain unclassified. This group also includes
cete, grouping in a poorly supported clade with fungi from deep-sea hydrothermal vents (Le
Mycocaliciomycetidae (Réblov and Seifert Calvez et al. 2009) and other harsh environ-
2012). This fungus is highly unusual in that it ments, even isolates capable of resisting condi-
produces meristem arthroconidia (Hughes tions simulating space and Martian conditions
1953), where arthroconidia form in parallel (Onofri et al. 2008).
chains from meristematic growth of the conid-
iophore apex (Fig. 5.25). While some members
of Mycocaliciomycetidae possess coelomyce- B. Animal Pathogens
tous anamorphs, the cylindrical pycnidium of
Cirrosporium is unique, and there is little else to The Eurotiomycetes include most of the ani-
connect these taxa. While the statistical support mal pathogenic fungi and occur in Onygenales,
for the inclusion of Cirrosporium in Eurotiomy- Eurotiales, and Chaetothyriales. Most of the
cetes was not significant, the analysis was based mammalian pathogens have a wide host range,
on portions of five genes that are useful for and none appear to be obligate pathogens. Dis-
resolving relationships at this level. Firm phy- ease takes a range of forms from superficial
logenetic placement of this taxon within Euro- epidermal infections to gravely serious deep
tiomycetes may require more sequence data or mycoses that may invade the respiratory and
the inclusion of additional taxa. circulatory systems and disseminate to other
parts of the body.
II. Ecology and Economic Importance 1. Onygenalean Pathogens
A. Extremophilism Many of the most important fungal pathogens

of vertebrates are found in Onygenales. These
Eurotiomycetes can degrade a wide variety of fungi are often keratinophilic, adapted to
organic substrates, sometimes under the most degrading keratin, the major protein compo-
challenging osmotic and temperature condi- nent of skin, hair, fingernails, feathers, and
tions. Some, such as Aspergillus species in the hooves. This association probably provides
Eurotium clade, are capable of growing at these fungi with the opportunity for occasional
water activities (aw) as low as those found in pathogenicity. With the exception of the der-
saturated NaCl solutions (Pitt and Hocking matophytes, infections are not believed to be
1997), and species such as A. fumigatus (Neo- transmitted between individuals; rather, they
sartorya clade) grow well at composting tem- are caused by fungi normally present in the
peratures [>50 C (Raper and Fennell 1965)]. environment as saprobes. Some of the most
Dimorphic dematiaceous fungi in Chaetothy- important onygenalean pathogens also tend to
riales (black yeasts) are abundant in hyper- be dimorphic, growing as yeasts at 37 C and as
saline waters found in salterns, in NaCl a mycelium at lower temperatures.
a) Histoplasmosis, Blastomycosis, and of individuals showed a positive serum reaction

Paracoccidioidomycosis to spherulin, an antigen produced by C. immi-
The diseases histoplasmosis, blastomycosis, tis. Valley fever is usually characterized by a
and paracoccidioidomycosis are caused by mild to moderate respiratory infection, but
three dimorphic members of Ajellomycetaceae: dissemination can result in far more serious
Histoplasma capsulatum, Blastomyces dermati- outcomes, including death (Stevens 1995).
tidis, and species of Paracoccidioides. All three Disseminated disease is a risk to both
usually begin in the respiratory cavity but fre- immune-compromised and immune-competent
quently disseminate to other parts of the body. individuals, with differences in dissemination
One of the most serious onygenalean patho- rates associated with ethnicity and gender
gens, Histoplasma capsulatum, is associated (Nguyen et al. 2013). The disease is probably
with bird and bat excreta and shows some contracted by inhaling arthroconidia sent air-
evidence for endemism in the Mississippi and borne by the disturbance of dry soil, particularly
Ohio River valleys of the central United States in association with agriculture, but also arche-
and all of Central and South America. The ological work, disturbance of soil associated
disease it causes, histoplasmosis, is usually with the excreta and decaying remains of small
marked by a mild lung infection but can dis- mammals, earthquakes, and dust storms. Once
seminate to other organs, where it is often fatal inhaled, arthrospores swell in the host to form
(Kauffman 2007). Histoplasmosis is a signifi- multinucleate spherules, which then release
cant problem in immune-compromised popu- uninucleate endospores, causing further
lations, particularly individuals stricken with spread of the infection in the individual
HIV/AIDS, presenting in as much as 26 % of (Pappagianis 1988). No sexual stage has been
AIDS patients in regions where the fungus is observed, but population genetic evidence
endemic (Wheat et al. 1990). Another causal exists for recombination in natural populations
agent of histoplasmosis is H. duboisii (previ- (Burt et al. 1996).
ously H. capsulatum var. duboisii), which is
endemic to Central Africa (Loulergue et al. c) Dermatophytes
2007). Blastomycosis is a disease similar to his- Other Onygenalean fungi tend to exist as
toplasmosis and is caused by another related weaker pathogens of the skin and nails, termed
fungus, Blastomyces dermatitidis. Blastomyco- dermatophytes. Members of Arthrodermata-
sis is often mistaken clinically for cancer or ceae with Microsporum and Trichophyton ana-
tuberculosis. Paracoccidioidomycosis, caused morphs cause tinea infections on the skin,
by Paracoccidioides brasiliensis, is yet another including ringworm and athlete’s foot. Onygen-
similar disease, endemic to South and Central alean dermatophytes live in a range of habitats,
America. This disease also occurs in armadillos from mostly soil (geophilic) to mostly animal
in regions where it is endemic (Bagagli et al. and human associates (zoophilic or anthropo-
1998). philic) (Kwon-Chung and Bennett 1992).
b) Coccidioidomycosis or “Valley Fever” d) Chalkbrood of Bees

Coccidioidomycosis is a disease caused by Coc- Several Ascosphaera spp. cause chalkbrood dis-
cidioides immitis and its cryptic sister species ease of bees but probably exist mainly as sap-
C. posadasii. These fungi show a high degree of robes on their stored pollen and nectar
endemism in the dry valleys extending from the deposits, as well as on their feces (Skou 1972,
southwestern United States through Central 1975). Other Ascosphaera spp. can be found
and South America, with C. immitis dominating infesting the pollen stores and nesting material
in Central and Southern California and C. posa- of bees but are either nonpathogenic or oppor-
dasii extending from Arizona into South Amer- tunistic pathogens (Klinger et al. 2013). The
ica (Fisher et al. 2001). In the southern San name chalkbrood describes the disease symp-
Joaquin Valley of California, more than 95 % toms; dead larvae are swollen, with spore balls
packed underneath the larval integument. The infection have been demonstrated (Pitt et al.
larval cuticle eventually bursts, releasing the 1991). However, postharvest infection by Peni-
spore balls, which have a chalklike appearance cillium and Aspergillus species of a variety of
(Skou 1975). fruits and vegetables is a serious problem. The
greatest agricultural concern regarding Euro-
tiomycetes involves the production of aflatox-
2. Eurotialean Pathogens ins, mostly by A. flavus and A. parasiticus on
A number of Aspergillus species are serious stored grains and nuts (see Sect. II.G), a process
human pathogens. In general, the most com- that is frequently initiated by preharvest pene-
mon species capable of growing at 37 C are tration of the plant by these fungi (Cotty et al.
the most common pathogens, including A. 1994). Members of Coryneliales can be bio-
fumigatus, A. terreus, A. niger, A. ochraceus, trophic parasites of woody hosts, including
and A. nidulans. Disease generally starts in pines in northern temperate regions and Podo-
the lungs after spores are inhaled. Aspergillosis carpaceae in the Southern Hemisphere. Some
can vary from an allergic respiratory infection plant pathogens are also known sporadically in
to an invasive disease that produces a fungus Chaetothyriales and Celotheliales. One of the
ball (aspergilloma) in the lung or disseminate newly erected families of Chaetothyriaceae,
to other parts of the body. Invasive aspergillosis Epibryaceae, consists mostly of species of
is rare in immune-competent individuals, but it Epibryon, a biotrophic parasite of bryophytes.
is a serious killer of transplant patients under-
going cytotoxic chemotherapy. A. sydowii has
been associated with major diebacks of sea fan D. Mutualistic Interactions
corals in the West Indies (Geiser et al. 1998).
Talaromyces marneffei is an important disease 1. Ectomycorrhizae
agent associated with HIV/AIDS in Southeast
Asia. This fungus, which is associated with The Eurotialean genera Elaphomyces and Pseu-
bamboo rats, is unusual for Eurotiales because dotulostoma are known to form ectomycorrhi-
it is dimorphic; at 37 C, vegetative hyphae zal associations with trees (Henkel et al. 2006;
break up into arthroconidia, which then pro- Miller and Miller 1984; Trappe 1962). Both
liferate in the host as a fission yeast (Boyce and fungi form an organized mantle on the host
Andrianopoulos 2013). root surface and form a Hartig net within the
root cortex. Elaphomyces is cosmopolitan in
temperate forests, forming hypogeous fruiting
bodies in association with both conifer and
C. Plant Pathogens
hardwood hosts. Pseudotulostoma’s distribu-
In comparison to Sordariomycetes and Dothi- tion is poorly documented, known only in
deomycetes, relatively few plant pathogens Guyana and Japan (Asai et al. 2004; Miller
reside in Eurotiomycetes. Members of Euro- et al. 2001).
tiales are equipped with the enzymes necessary
to degrade complex plant compounds such as
cellulose and pectin (Dean and Timberlake 2. Lichens
1989) but tend to do so as agents of decay. Many members of Chaetothyriomycetidae and
Some Aspergillus species are considered weak Mycocaliciomycetidae are lichenized, usually
plant pathogens, meaning they are capable of producing small thalli on trees and rocks. Euro-
penetrating living plant hosts and occasionally tiomycete lichens occur worldwide in many ter-
producing rot symptoms. Generally, penetra- restrial, freshwater, and marine habitats and
tion occurs via wounding by insects or other tend to produce relatively few secondary meta-
damaging agents, or in plants that are stressed, bolites and rarely produce asexual propagules.
but penetration of healthy tissues and systemic
3. Myrmecophytes F. Food Production

Myrmecophytes (known as ant plants) form fac- Producing cheese from milk is an effective way
ultative or obligate mutualisms with ants, produc- of preserving its nutritional properties, and
ing internal cavities called domatia (Fig. 5.18) that fungi often play a role in the process. Penicil-
house the insects (Mayer et al. 2014). In addition lium roquefortii and Penicillium camemberti
to mutual benefits of protection and host dis- are used to make Roquefort-type and
persal occurring between the insect and plant camembert-type cheeses, producing unique
partners, this mutualism includes fungi and pos- flavors by oxidizing fatty acids into methyl
sibly bacteria that serve as a food source for the ketone flavor compounds (Girolami and
insects and the plants. The domatial cavities are Knight 1955). While Western cultures have
often lined with a thin black mycelium, which concentrated on using these fungi on milk pro-
are cultivated by and provide nutrients to the tein, in the East they have been used mostly on
ants (Blatrix et al. 2013; Defossez et al. 2009). As soy and rice. Aspergillus oryzae, A. sojae, and A.
is the case in fungus-cultivating ambrosia beetles, tamarii have been used for as long as
more than one fungal symbiont may be present, 4,000 years in a number of food fermentation
but they are always Chaetothyrialean (Blatrix processes, including the production of soy
et al. 2013). sauce, miso, and sake. Some of these fungi
appear to be domesticated forms of wild Asper-
gillus species; A. oryzae is a domesticated ver-
E. Food Contamination sion of A. flavus, and A. sojae is derived from
A. parasiticus (Kurtzman et al. 1986). A. niger
Because they aggressively decay organic mate- is used to produce organic acids, particularly
rial, fungi in Eurotiales are often associated citric acid that is used as a flavoring agent. The
with human and animal foods. Furthermore, red color of ang-khak (Chinese red yeast rice) is
most Eurotiales can tolerate very challenging produced traditionally by culturing Monascus
osmotic conditions, and some are true xero- purpureus on rice. Many mold-ripened sau-
philes, preferring aw<0.85 (Pitt 1975). Some sages in Europe utilize Penicillium and Asper-
genera are capable of growth in aw <0.75, the gillus species, coating them with a white
equivalent of a saturated NaCl solution. This mycelium.
provides these fungi with the ability to grow
in dried and highly concentrated foods, includ-
ing jams, salted fish, fruitcake, and confection- G. Toxic Secondary Metabolites
ary (Pitt 1975). These adaptations have been
exploited, and certain Eurotialean fungi are Members of Eurotiales, particularly Aspergilla-
used to preserve foods, particularly in the pro- ceae, are notable among fungi for making an
duction of cheeses and Asian sauces (see extremely wide variety of toxic secondary meta-
Sect. II.F). On the other hand, this association bolites. Two of the most important are the poly-
permits these fungi to contaminate foods, ketides aflatoxin, produced mostly by A. flavus
making them unpalatable at least if not toxic and A. parasiticus, and ochratoxin, produced
through the production of secondary metabo- by a variety of Aspergillus and Penicillium spe-
lites. In a spoilage mycoflora survey of 294 food cies. Aflatoxins were first discovered after over
samples sent to a British food microbiology 100,000 turkeys died from consuming contami-
laboratory, 68.2 % of the isolates were found nated groundnut meal (Sargeant et al. 1961).
to be Aspergillus or Penicillium (Williams and Concerns about acute exposure have been
Bialkowska 1985). Contamination can be prob- raised in recent years after incidents including
lematic even on foods that undergo heat pro- the discovery of high aflatoxin levels in peanut
cessing because some Eurotiales produce butter consumed by primary school children in
ascospores that are highly heat-resistant. South Africa (South African Medical Research
Council 2001), an outbreak of aflatoxicosis in
Kenya associated with stored maize that killed scale resulted in the discovery of a high-yield
125 people (Centers for Disease Control and strain of Penicillium chrysogenum, still used
Prevention 2004), and a 2005 veterinary out- today, from a moldy cantaloupe. Additional
break in the southeastern United States asso- useful compounds produced by Eurotialean
ciated with contaminated dog food fungi include the echinocandin class of antifun-
(Anonymous 2006). In addition to such acute gal drugs, cyclic peptides that inhibit the syn-
effects following the consumption of high levels thesis of cell wall glucans, and the
of them, aflatoxins are known to be potent anticholesterolemic drug mevinolin, which
carcinogens, particularly of the liver. Typical acts by inhibiting the HMG-CoA reductase
of secondary metabolite biosynthesis, all of enzyme. Mevinolin is also produced by Monas-
the enzymatic activities known to be involved cus purpureus, the Eurotialean fungus used to
directly in aflatoxin biosynthesis are encoded make ang-khak (Endo 1979).
in a large cluster of 25 genes (Brown et al. A. oryzae and A. niger are widely used for
1996). While the aspergilli used in food produc- the industrial production of extracellular
tion, A. oryzae and A. sojae, have clear connec- enzymes, including cellulases, amylases, pro-
tions to aflatoxigenic species, these teases, lipases, and pectinases. These fungi are
domesticated variants lack the ability to pro- well suited to large-scale industrial production
duce aflatoxins (Sato et al. 2011). Ochratoxin is because of their highly efficient secretion sys-
reasonably anticipated to be a human carcino- tems. The enzymes produced include native
gen based on animal studies and shows acute enzymes produced by Aspergillus and heterolo-
effects as well, particularly nephrotoxicity gous proteins from other organisms. The most
(Pfohl-Leszkowicz and Manderville 2007). In widely used industrially produced enzymes are
the poultry industry ochratoxin toxicity can lipases and proteases, as additives to deter-
have a significant economic impact when sus- gents.
ceptible poultry are exposed to ochratoxin-
contaminated feed. In human foods, ochratoxin
is known to occur in coffee and grapes and is a III. Summary
particular concern because it can accumulate in
meats. In addition to these well-studied toxic Since the advent of fungal molecular systemat-
polyketides, most Aspergillus and Penicillium ics, the taxonomy of the fungi discussed here as
species produce a wide variety of diverse sec- Eurotiomycetes has changed drastically. First,
ondary metabolites, including terpenes, ster- the morphological concept of “Plectomycetes”
oids, and amino-acid-derived compounds proved to be nonmonophyletic, leaving a core
(Turner and Aldridge 1983). clade of orders described as monophyletic Plec-
tomycetes (Geiser and LoBuglio 2001). This
large clade of economically important fungi
H. Industrial Uses was somewhat morphologically cohesive. How-
ever, further advances in molecular systematics
The bright side of the secondary metabolic cre- brought morphologically and ecologically dis-
ativity shown by Eurotialean fungi is the variety tinct taxa, particularly fungi with bitunicate/
of useful compounds produced. These include fissitunicate asci, into this phylogenetic realm,
food additives, antibiotics, and other pharma- resulting in three subclasses, Eurotiomycetidae,
ceuticals. Perhaps the most celebrated second- Chaetothyriomycetidae, and, eventually, Myco-
ary metabolite is the beta-lactam antibiotic caliciomycetidae. The inclusion of Coryneliales
penicillin, first discovered by Fleming (1929), in Eurotiomycetes helped to bridge the gap in
who found a contaminant Penicillium that our understanding of the evolutionary relation-
showed antibacterial effects on staphylococci. ship between the original concept of Eurotio-
The successful effort during World War II to mycetes and its newer, much broader
develop and produce penicillin on an industrial definition. The recent inclusion of the unusual
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47:25–35
6 Pezizomycotina: Dothideomycetes and Arthoniomycetes
CONRAD SCHOCH1, MARTIN GRUBE2
CONTENTS mycetes as sister to Dothideomycetes

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 (Lumbsch et al. 2005; Lutzoni et al. 2004; Spa-
II. Character Evolution: Divergence and tafora et al. 2006). Following this, a new infor-
Convergence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 mal superclass taxon name has been proposed
A. Morphology and Development . . . . . . . . . . 144 for this monophyletic group of fungi, dothi-
B. Brief Taxonomic History . . . . . . . . . . . . . . . . . 149
III. Ecology and Distribution . . . . . . . . . . . . . . . . . . . 150 deomyceta, based on combined DNA sequence
A. Associations with Plants and comparisons from protein coding and ribo-
Plant Debris. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 somal structural genes (Schoch et al. 2009b,
B. Aquatic Species . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 c). Dothideomycetes is by far the largest class
C. Associations with Algae and of Ascomycota, with more than 19,000 species
Other Lichens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
D. Rock Environments and Oligotrophism. 153 described on morphological comparisons
IV. Modern Classification and Phylogeny . . . . . 153 alone (Kirk et al. 2008). Its sister class, Artho-
A. Dothideomycetes . . . . . . . . . . . . . . . . . . . . . . . . . 154 niomycetes, with the single order Arthoniales,
1. Pleosporomycetidae. . . . . . . . . . . . . . . . . . . . 155 is the largest taxonomic group of primarily
2. Dothideomycetidae . . . . . . . . . . . . . . . . . . . . 158 lichenized fungi outside of Lecanoromycetes.
3. Incertae Sedis Lineages . . . . . . . . . . . . . . . . 160
B. Arthoniomycetes. . . . . . . . . . . . . . . . . . . . . . . . . . 163 Arthoniomycetes comprises 1,500 species and
V. Maintenance and Culture . . . . . . . . . . . . . . . . . . . 166 74 genera, according to the literature (e.g., Kirk
VI. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 et al. 2008), but, like the number for Dothideo-
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 mycetes, this estimate needs to be updated.
It is clear that the use of DNA and protein
sequence comparisons is crucial in the defini-
I. Introduction tion of this broad and diverse group of Fungi,
and efforts to reconcile morphological and
The classes Dothideomycetes and Arthonio- molecular characters continue. However, it
mycetes were introduced, relying on compar- remains a monumental task. This is illustrated
isons of nuclear ribosomal small subunit DNA by the fact that less than 16 % (more than 3,000)
sequences (Eriksson and Winka 1997), follow- of currently accepted species names of Dothi-
ing work by a number of authors (Berbee 1996; deomycetes were deposited under their bino-
Berbee and Taylor 1992; Gargas et al. 1995; mial names in GenBank and the International
Spatafora et al. 1995). Subsequent phylogenetic Nucleotide Sequence Databases (INSD) by the
analyses based on DNA sequences compared end of 2013. At the same time, for Arthoniomy-
from multiple genes indicated support for a cetes, DNA sequence data are available for less
monophyletic clade with lichenized Arthonio- than 200 species, 13 % of proposed species.
These numbers clearly underrepresent diver-
1 sity estimates as phenotypic diversity remains
NCBI/NLM/NIH, 45 Center Drive, Bethesda, MD 20892, USA;
e-mail: schoch2@ncbi.nlm.nih.gov underrepresented in molecular analyses.
2
Institute of Plant Sciences, Karl-Franzens-University, Holtei- Dothideomyceta contains a diverse set of
gasse 6, 8010 Graz, Austria; e-mail: martin.grube@uni-graz.at species that subsist as general and specialized

144 C. Schoch and M. Grube
saprobes in terrestrial and marine ecosystems, before fusion of opposing mating types occurs. In
plant and animal pathogens, parasites of other contrast, during ascohymenial development the repro-
ductive structures form in conjunction with growth of
fungi, lichens, and mycorrhizal mutualists. A the cells after mating fusion.
small number of Dothideomycetes are liche- Flask-shaped, perithecioid ascomata are referred
nized, while this is the case for the majority of to as pseudothecia when their ascolocular development
Arthoniomycetes. There are also a considerable distinguishes them from the similarly shaped structures
number of lichenicolous, fungi, and only a few with ascohymenial development. A number of lineages
in Dothideomycetes also have open, apothecioid asco-
saprobes in the latter class. Dothideomyceta is mata. These include a smaller subset of navicular or
also notable for several species that produce boat-shaped structures called hysterothecia, which
biologically active compounds, many of which open with a central suture. Flattened ascomata with a
are known to function in plant pathogenicity. scutate or shieldlike wall of outer cells are referred to as
Secondary compound diversity is high in thyriothecia. Cleistothecioid ascomata are enclosed
ascomata that often rupture at maturity.
Arthoniomycetes and characterized by a spec-
trum of compounds apparently correlating with
The presence and morphology of types of
the lichenized lifestyle. Numerous compounds
hamathecial tissues played an important role in
are shared with the unrelated, yet also primarily
delineating orders in Dothideomycetes. Luttrell
lichenized, Lecanoromycetes. Among these are
(1965) used developmental differences to dis-
diverse orcinol derivates, such as despides
tinguish between seemingly similar hamathe-
and depsidones and other typical lichen com-
cial elements. A representative set of ascomata
pounds (Boustie and Grube 2005), which sug-
based on illustrations adapted from von Arx
gests a parallel chemical evolution in unrelated
and Müller (1975) are shown in Fig. 6.1. Lut-
lichenized lineages. A large body of work on
trell’s (1973) centrum concepts consisted of a
numerous plant pathogenic and other eco-
Pleospora type (pseudoparaphyses present), a
nomically important species is available, and
Dothidea type (bundles or fascicles of apara-
molecular-based phylogenetic studies continue
physate asci), and an Elsinoë type (monascous
to expand the resolution of deep nodes in
locules), which now constitute the modern
dothideomyceta while expanding taxon sam-
orders Pleosporales, Dothideales, and Myrian-
pling.
giales and coincide well with DNA-sequence-
based phylogenies (Figs. 6.1a–c and 6.2). The
clearest phylogenetic distinction remains that
II. Character Evolution: Divergence between the presence and absence of pseudo-
and Convergence paraphyses in the centrum, correlating with the
subclasses Pleosporomycetidae and Dothideo-
A. Morphology and Development mycetidae (see section on IV).
A more detailed description of morphological The centrum concept was broadly defined as all tissues
features can be found in Barr and Huhndorf inside ascomata. This was refined with the introduction
of the term hamathecium by Eriksson (1981), which
(2001). We highlight the most basic concepts was used to refer only to sterile hyphae or other tissues
here. between the asci.
Ascomata. The historical concept advo- Pseudoparaphyses are sterile cells extending down
cated by Lutrell for loculoascomycetes centered from the upper portion of ascomata, initially attached
on the designation of a number of morphologi- at both ends, although the upper part may become free,
while paraphysoids, although similar in appearance,
cal types, combined with ascoma structure for result from interascal tissue stretching during ascoma-
five defined orders (Luttrell 1951, 1955). This tal development. Pseudoparaphyses can be anastomos-
still influences current taxonomy. ing and also show regular septation. Other terms for
hamathecial tissues include interascal pseudoparen-
The ascolocular ascomata of Dothideomycetes are chyma (tissue that remains unchanged or becomes
formed by vegetative stromatic plectenchyma (tissue compressed when the asci develop), periphysoids
formed by hyphae becoming fixed together), giving (short hyphae stretching downward, never reaching
rise to one or several locules. The reproductive struc- the bottom of the ascomal cavity), and periphyses
tures developing in the locules are derived from cells (hyphae in the ostiole).
Pezizomycotina: Dothideomycetes and Arthoniomycetes 145
Fig. 6.1 Hamathecial and ascomal variation in dothi- Arthoniomycetes). (e) Zopfia rhizophila (Pleosporales
deomyceta exemplified by selected type species. (a) Pleosporomycetidae). (f) Microthyrium microscopi-
Elsinoë canavaliae (Myriangiales, Dothideomycetidae). cum (Dothideomycetes incertae sedis, Microthyria-
(b) Dothidea sambuci (Dothideales, Dothideomyceti- ceae). Adapted from von Arx and Müller (1975),
dae). (c) Pleospora herbarum (Pleosporales, Pleospor- used with permission
omycetidae). (d) Arthonia dispersa (Arthoniales,
Generally, dothideomycete ascomata are dehiscence of a circular apical cap (Corlett

closed, perithecioid structures that may some- 1967). A number of lineages in Dothideomy-
times be joined in a large stroma. However, cetes have open, apothecioid ascomata (see sec-
several ascomal variations occur; for example, tion on Hysteriales and Mytilinidiales and
Clathrospora heterospora var. simmonsii has Figs. 6.2 and 6.3). The same general morphol-
the Pleospora type of development, but asco- ogy can also be observed in some Arthonio-
genous hyphae arise from pseudoparaphyses, mycetes (Fig. 6.1d). Recent DNA-sequence-
and an operculate ostiole is formed by the based phylogenies have confirmed that these
RAxML analysis (nodes below 50% bootstrap collapsed), 6299 characters, 5 genes, 400 taxa
Key:
P Lineages with economically important plant pathogens
L Lineages with lichenized species
100
Pleosporaceae P
74
Leptosphaeriaceae P
Lineages with aquatic fungi

Pleosporineae 65
Cucurbitariaceae
A 100
73
Phaeosphaeriaceae P
77 87 Didymellaceae P
54
94
Dothidotthiaceae
79 100
Halojulellaceae
100 Massarinaceae
Massarineae 89 100
Montagnulaceae P
100 Lentitheciaceae
100 100
98 Morosphaeriaceae A L
94 87
68
Trematosphaeriaceae E
100
Corynesporascaceae P
O
86 Melanommataceae S
87 100
100
Pleomassariaceae P
72 Roussoellaceae
100
‘Arthopyrenia’ Pleosporales O
78
100
100 Halotthiaceae A R
77
100
Sporormiaceae O
Lophiostomataceae M
96 100 Wicklowia A
Y
100
Amniculicolaceae
Lindgomycetaceae A
C
98
100
Aigialaceae A E
79 Tetraplosphaeriaceae T
89 84
84
Testudinaceae I D
100
Platystomaceae D O
59 89 Lophiotremataceae
97
Anteagloniaceae A T
100
99
Hypsostromataceae E H
91
99 Delitschiaceae I
92 Zopfiaceae D
100
Massariaceae E
94
Hysteriaceae Hysteriales
93
99 Mytilinidiaceae O
100
Gloniaceae Mytilinidiales M
69
99
Cenococcum geophilum Y
100
Acrospermaceae Acrospermales C
100 Farlowiella / Minutisphaera E
84
100
Aliquandostipitaceae A Jahnulales T
100
100 Botryosphaeriaceae P
98 Phyllostictaceae E
100
Aplosporellaceae Botryosphaeriales S
99
Saccharataceae
88
Patellariaceae Patellariales
100
Kirschsteiniotheliaceae
94
Tubeufiaceae Tubeufiales D
100 Trypetheliaceae L
Trypetheliales O
Mycosphaerellaceae P
53
100
Schizothyriaceae P T
57
100 Dissoconiaceae H
82
100
Micropeltidaceae P
I
80 98
Cystocoleus / Devriesia / RIF L
Capnodiales D
98
Teratosphaeriaceae E
Capnodiaceae
99 97
100 Cladosporiaceae P O
58 Zasmidium cellare M
100 100
Racodium L Y
100 Dothideaceae/ Dothioraceae Dothideales C
100
92
Elsinoaceae P
E A
97 63 Myriangiaceae Myriangiales R
RIF T
100
100
Endosporium I T
99
98 Venturiaceae P
Venturiales D H
86 100
Natipusillaceae A O
62
Microthyriaceae E N
100
Phaeotrichaceae I
Asterinaceae
100
Monoblastiaceae L O
100
93 Roccellaceae M
98
100
Opegraphaceae L
Y
76
Roccellographaceae L
C
100
82
Lecanographaceae L Arthoniales E
88
Arthoniaceae L
99
Chrysotrichaceae L T
97 87
RIF E
100
Pyrenula (outgroup) S
Fig. 6.2 A simplified maximum-likelihood tree ob- level clades were collapsed, and provisional or uncer-
tained from 401 taxa and 5 genes with two Pyrenula tain family names are indicated in quotes. RIF denotes
species as outgroup, showing only relationships recov- “rock-inhabiting fungi”
ered in more than 50 % of bootstrap trees. All family-
Fig. 6.3 Variety and convergence in hysteriate asco- chaeliana (Dothideomycetes incertae sedis), Opegrapha
mata. Top left to right: Hysterium angustatum (Hys- filicina (Artoniales, Opegraphaceae). Bars¼0.5 mm.
teriales, Hysteriaceae), Anteaglonium abbreviatum Photo credits: Opegrapha filicina Walter Obermayer,
(Pleosporales, Anteagloniaceae), Farlowiella carmi- remainder Sabine Huhndorf
structures are found in several divergent (following hemiangiocarpous development),

lineages across the two classes. In addition, open apothecialike ascomata, and branched
several unrelated lineages with cleistothecioid and strongly elongate to branched ascomata.
ascomata occur, such as several species in Spor- Moreover, there are types where asci are more
ormiaceae and Zopfiaceae (Fig. 6.1e). The same or less scattered over the entire thallus (Cryp-
situation is also true of species producing thyr- tothecia, Stirtonia) and tightly surrounded by
iothecia in Asterinaceae, Microthyriaceae, and networks of hyaline hyphae. An independent
several other families in Dothideomycetes. An reduction of ascomatal structures has occurred
example of such a scutate ascoma in Microthyr- in Arthonia intexta, a lichenicolous species that
ium microscopicum is shown in Fig. 6.1f. forms individual asci in the ascomata of its
In contrast to the ascolocular ontogeny hosts. An example of an arthoniomycete
found in Dothideomycetes, the ascomal devel- ascoma is shown in Fig. 6.1d.
opment of Arthoniomycetes prompted designa- Tehler (1990) introduced a detailed termi-
tion as an “in-between group” or nology to describe the diversity of ascomatal
“Zwischengruppe” (Henssen and Jahns 1974; structures in Arthoniomycetes. The main char-
Henssen and Thor 1998). This refers to a com- acters to distinguish ascomata were the number
bination of ascolocular and ascohymenial of locules in an ascostroma and the number
characters: the asci are bitunicate, whereas a of asci per locule. An additional type has been
paraphysoid hamathecium develops from gen- described since then, with new ascomatal units
erative plectenchyma. With this combination, a proliferating from existing ascomata (Henssen
plethora of ascomatal types have evolved, and Thor 1998). This latest described mode and
ranging from closed perithecialike ascomata the formation of rather diverse ascomata in
Arthoniales might be correlated with the flexi-

ble architecture and a prolonged growth of
ascogenous hyphae, which also can form ana-
stomoses at their tips with sterile haploid
hyphae of the ascomata (Grube and Lücking
2001). Fluorescence microscopy studies of folii-
colous species also revealed that after a primary
formation of ascogenous hyphae and asci, new
ascogenous hyphae can be developed in the
older, central parts of ascomata. In other spe-
cies, the central parts of the ascomata appar-
ently degrade (Sundin and Tehler 1998), while
ascus formation proliferates at the periphery of
the ascomata.
Asci. The term bitunicate was recognized
by Luttrell (1951) after usage in earlier descrip-
tions (quoted in Reynolds 1989). It was applied
to asci with two distinct walls where the outer
wall separates and ruptures upon expansion of
the inner wall to forcibly eject the ascospores.
Bitunicate asci are noticeably thick-walled
when viewed under a light microscope and
appear to consist of outer and inner mem-
branes, the ectotunica and the endotunica.
Fig. 6.4 Ascus types of bitunicate ascomycetes after
Electron microscopy has revealed that two, sep- Eriksson (1981) and Bellemère (1994). (a) Fissitunicate.
arate, morphologically distinct layers do not (b) Semifissitunicate. (c) Rostrate. (d) Pseudoprototu-
strictly occur. Instead, the ascus wall is com- nicate. Adapted with permission from Hofmann (2009)
prised of several layered zones characterized by
fibrils (Funk and Shoemaker 1967; Furtado and nica separates from the ectotunica, and a part of the
Olive 1971; Reynolds 1971). Although some endoascus is protruded, but the ecto- and endotunica
subsequent authors regarded the use of the do not separate. Semifissitunicate dehiscence is an
term bitunicate as still appropriate despite this intermediate type between fissitunicate and rostrate
dehiscence where the exoascus ruptures by an apical
information (e.g., Müller 1981), others have slit and the endoascal layers extend upward.
pointed out that this also applies to several
types of ascostromatic asci and that a focus on
Members of Arthoniomycetes have dehis-
the variable types of dehiscence may be more
cence that tends to be more closely related to
appropriate (Reynolds 1989). Eriksson (1981)
what Eriksson defined as either semifissituni-
distinguished between seven types of dehis-
cate or rostrate (Eriksson 1981), but rostrate
cence, of which four may apply to the broad
dehiscence is also found in Asterinaceae in
concepts of bitunicate asci. A traditional con-
Dothideomycetes (Hofmann 2009).
cept of bitunicate asci is that they follow a
An additional variation occurs in species
pattern of fissitunicate dehiscence. A schematic
with enclosed ascomata (cleistothecia), where
drawing based on a selection of Eriksson’s con-
the asci dissolve at maturity, and is referred to
cepts is indicated in Fig. 6.4.
as pseudoprototunicate (Eriksson 1981; Rey-
nolds 1989). This occurs in species of the
In fissitunicate dehiscence, the inner wall extends
families Phaeotrichaceae and Zopfiaceae, for
completely and the rigid outer wall is ruptured, similar
to a Jack-in-the-box (Ingold 1933). This occurs in the example, Phaeotrichum and Zopfia spp. Passive
majority of Dothideomycetes, but variations abound. In ascospore release is also known from Arthonio-
rostrate dehiscence, only the apical part of the endotu- mycetes and found in Tylophoron (Lumbsch
et al. 2009), Tylophorella, Sporostigma (Grube one prominent example is the mycorrhizal spe-
2001a), and Wegea. The latter three genera were cies Cenococcum geophilum (LoBuglio et al.
classified as members of Arthoniales based on 1996).
anatomical evidence (e.g., structure of juvenile The way the nomenclature of these asexual
asci), but their position in Arthoniales still forms has been treated to date will change
needs to be confirmed by molecular data. An drastically. The International Code for algae,
apothecioid species with ostensibly unitunicate fungi and plants, which governs fungal nomen-
asci and explosive spore release, Catinella oli- clature, underwent expansive changes after the
vacea, was only recently placed in Dothideomy- 2011 Melbourne congress (Hawksworth 2011).
cetes (Greif et al. 2007) and hints at more One of these changes will be to remove the dual
unaccounted variation in ascus dehiscence. system for sexual and asexual morphs that was
Anamorphs and sterile species. Asexual allowed under previous editions of the code and
morphs (anamorphs) play an important role only allow a single name. Attempts are under
in the life cycle of several Dothideomycetes. It way to use a selection process that will address
is often the dominant form encountered by the stability of commonly used names (Hawks-
pathologists isolating fungi from diseased worth 2012). However, this means that ana-
plant and other tissues, and therefore the spe- morph and teleomorph generic names will
cies with the largest body of genetic and geno- compete to label monophyletic groups irre-
mic work are often encountered in their asexual spective of the presence of a particular morph
states. Although early mycologists included (Hawksworth 2012; Wingfield et al. 2012). For
anamorphic fungi in their classification sys- instance, Cladosporium may likely be an
tems, separate systems of classification were accepted generic name instead of Davidiella,
proposed by Fuckel (1870) and Saccardo and Bipolaris could replace Cochliobolus (Man-
(1882–1931). The use of DNA-based compari- amgoda et al. 2012). Whatever the final choices
sons have now shown that asexual species can of the relevant nomenclatural bodies, this will
be placed within the current classification sys- result in a huge number of changes affecting
tem without requiring that sexual and asexual nomenclature in Dothideomycetes (and, to a
structure be produced in culture. Despite this, lesser extent, Arthoniomycetes).
these artificial groups still facilitate the identifi-
cation of species, and many mycologists con-
tinue to use terms informally to distinguish B. Brief Taxonomic History
between species bearing their asexual spores
(conidia) in structures that are enclosed (coe- Historically, ascomycete taxonomy relied
lomycetes) (Sutton 1980) or unenclosed heavily on morphological characters such as
(hyphomycetes) (Seifert et al. 2011). The devel- the morphology of the ascus, septation of
opment of conidia was studied in great detail in ascospores, the morphology and development
the previous century, initially with light micros- of the ascoma, and the structure and organiza-
copy and later using time-lapse photography tion of the centrum. Luttrell’s proposed class,
and scanning electron microscopy (Cole and Loculoascomycetes (Luttrell 1955), was mainly
Sampson 1979; Mangenot and Reisinger 1976). based on the Ascoloculares of Nannfeldt (1932),
In Dothideomycetes, coelomycetes and hypho- in which the asci develop in cavities in a pre-
mycetes occur interchangeably throughout the formed stroma, and included lichenized fungi
class while only a small number of asexual such as the constituent families of the Artho-
forms have been tied to Arthoniomycetes, for niomycetes. A major difference was that Lut-
example, the coelomycete genus Briancoppinsia trell placed more emphasis on ascus structure
(Diederich et al. 2011) and the hyphomycete than Nannfeldt.
genus Reichlingia (Ertz and Tehler 2011). Asex- The first descriptive taxonomic work in
ual fungi can also exist as sterile mycelia and Dothideomycetes is credited to Fries (1818),
produce specific vegetative structures such as who circumscribed the genus Dothidea. This
thick-walled sclerotia that can survive in soil; was done without designating a type species;
only recently could this genus and the resulting shown for a separate lineage that included
taxa be stabilized by conserving Dothidea sam- Dothideomycetes and Arthoniomycetes
buci as the type species and by designating an (James et al. 2006; Lutzoni et al. 2001).
epitype (Shoemaker 2003; Shoemaker and
Hambleton 2005). The first descriptions of a
distinctive bitunicate ascus were done in the III. Ecology and Distribution
mid-nineteenth century (Currey 1856; Desma-
zières 1843; Pringsheim 1858), and by 1887 the A. Associations with Plants and
asci and the fissitunicate action of ascospore Plant Debris
ejection could be summarized (De Bary 1887).
However, the common occurrence of bitunicate An important set of species in Dothideomycetes
asci and the correlation between this and other subsist as what can loosely be defined as plant
important criteria were not realized until the associates, while none of the members of
early twentieth century. The development of the Arthoniomycetes could so far be detected in a
ascoma as an important distinctive feature was similar role. The vast majority of species in
used by Nitschke and Fuckel when they applied Dothideomycetes appear to be encountered as
the family name Dothideaceae (Fuckel 1870) to saprobes breaking down dead leaves and
fungi with asci in locules within a stroma, woody material, but several Dothideomycete
rather than in a true perithecial wall. This fam- species grow in close proximity to living plants,
ily was raised to ordinal rank by Lindau (1897), as endophytes in leaves, stems, or roots, as
but the first close description to the modern epiphytes growing on leaf surfaces, or as para-
class Dothideomycetes was provided by von sites, which includes acting as pathogens caus-
Höhnel (1909) when he expanded Lindau’s con- ing disease. At least one unique lineage in
cept to include species with monascous and Dothideomycetes, containing the asexual spe-
polyascous locules. Nannfeldt (1932) followed cies Cenococcum geophilum, occurs in a mycor-
these concepts to emphasize the importance of rhizal relationship with a wide range of plant
ascomatal development and its correlation hosts across a broad geographic distribution
with ascus characters. It was one of Nann- (LoBuglio et al. 1996). It has only been reported
feldt’s students, Rolf Santesson, who com- as hyphae, sclerotia, and host-colonized ecto-
pleted the first integration of the separate mycorrhizal root tips (Tedersoo et al. 2010).
classification of lichenized fungi in a com- Surprisingly, recent phylogenetic results indi-
bined classification (Santesson 1952). Santes- cate that this lineage is closely related to mem-
son regarded lichenized pyrenomycetes as bers of the Gloniaceae, saprobic species often
closely related to nonlichenized pyrenomy- found on wood with no indication of a bio-
cetes or loculoascomycetes. This meant that trophic lifestyle to date (Schoch et al. 2009a;
constituents of Arthoniomycetes were classi- Spatafora et al. 2012). This highlights the fact
fied with other nonlichenized bitunicate spe- that much of the biology of these organisms
cies for the first time. In the ensuing decades, remains poorly described. Often species are
several mycologists produced influential only reported from one environmental niche,
works agreeing or disagreeing with these con- while they may in fact have diverse and com-
cepts in varying degrees as they relate to plex life cycles that can only be revealed after
Dothideomycetes and Arthoniomycetes [see intensive sampling and analysis. To date, only a
Barr and Huhndorf (2001) and Hawksworth small number of species in the dothideomyceta
(1985) for a more detailed taxonomic history]. has been studied in this fashion.
With the rise of molecular methods in fungal The most extensive body of biological knowl-
classification, a first integration of lichenized edge on Dothideomycetes exists on parasitic
fungi in a fungal molecular phylogeny was plant pathogens. Numerous economically impor-
corroborated by Gargas et al. (1995). This uni- tant species occur throughout this class, but the
fied view of lichenized and nonlichenized fungi most focus has been on species in suborder
was subsequently refined, and support was Pleosporineae. Species in Alternaria, Cochliobo-
lus, Didymella, Pyrenophora, Leptosphaeria, and groves in aquatic (freshwater, marine, and
several other closely related species have been brackish) environments. These species exhibit
used as models for plant pathogenicity in molec- their main adaptations in the spores they pro-
ular biology, genetics, and, more recently, in duce, such as the gelatinous sheaths and appen-
genomics and related fields. Smaller groups of dages that help them to adhere to the
species in Botryosphaeriaceae, Elsinoaceae, submerged surfaces of plants and wood. Marine
Mycosphaerellaceae, and Venturiaceae continue species have been defined as fungi that grow
to be studied in more detail. exclusively in marine environments (Kohl-
Most plant-parasitic interactions of disease- meyer and Kohlmeyer 1979). Sixty-six genera
causing fungi exist along a spectrum from necro- and 110 species are currently listed under
trophy (killing the host cells in advance to use Dothideomycete species, which are found pre-
them for energy and nutrients) to biotrophy dominantly in marine environments (Jones
(living inside living plant cells). The majority of et al. 2009). These species occur in or on macro-
plant-parasitic fungi in Dothideomycetes are algae, sea grasses, and decaying wood, but they
necrotrophs, with only a few described so far as are most commonly isolated from intertidal
biotrophs (e.g., Simon et al. 2005). However, zones and mangroves, where they play an
several necrotrophs also require periods of bio- important role in breaking down complex car-
trophy. The variability of this hemibiotrophic bohydrates. Several lineages in the Dothideo-
lifestyle is exemplified by the causal agent of mycetes are dominated by marine or freshwater
black leg on Brassica species, Leptosphaeria species, often with a mixture isolated from both
maculans, which recently had its genome environments. The majority of aquatic species
sequence published (Rouxel et al. 2011). This tend to reside in the Pleosporomycetidae,
pathogen sequentially occurs as a saprobe on with only a few species isolated from within
stem residues, a necrotrophic pathogen on the the Dothideomycetidae, but several aquatic
plant host, and an endophyte without causing lineages still remain unresolved in the latest
any symptoms (Rouxel and Balesdent 2005). phylogenetic analyses (Shearer et al. 2009; Sue-
During this process, the fungus needs to employ trong et al. 2009). Shearer et al. (2009) specu-
an array of extracellular enzymes to kill and then lated that the absence of pseudoparaphyses
break down plant material and evade plant outside Pleosporomycetidae may limit survival
defenses. To improve understanding of these in habitats with fluctuating water levels because
interactions, much recent research has focused aquatic species often have abundant pseudo-
on a diverse group of molecules called effectors paraphyses embedded in gel that could poten-
that are employed during these processes. Effec- tially protect them from desiccation during dry
tors suppress plant recognition systems for conditions. Fungi from aquatic environments
pathogens (de Wit et al. 2009). Progress is continue to be of interest for their ability to
being made in understanding how these patho- produce secondary metabolites (Mayer et al.
gens interact with plants by mining new genome 2007; Rateb and Ebel 2011).
data (Ohm et al. 2012; van de Wouw and Howlett Several novel families, predominantly con-
2011). At the same time, molecular ecological taining fungi from aqueous environments, have
sampling is providing a more complete picture only recently been described. These include
of the diversity, host affinities, and geographic Aigialaceae, Morosphaeriaceae, Lentithecia-
dispersal of fungal endophytes in this group ceae, Lindgomycetaceae, and Amniculicola-
(Higgins et al. 2007; U’Ren et al. 2010). ceae, all in Pleosporales (Suetrong et al. 2010).
A new order, Jahnulales, has been described to
contain the predominantly freshwater Aliquan-
B. Aquatic Species dostipitaceae, but indications are that marine
species also form part of this group (Suetrong
In addition to the large group of terrestrial sap- et al. 2010, 2011).
robes, Dothideomycetes includes a number of It is clear that aquatic fungi represent an
saprobic species on woody debris and man- important part of the poorly documented
diversity in dothideomyceta, and several un- the genus Phycopeltis. These planar growing
classified lineages require more in-depth docu- algae are particularly common partners of folii-
mentation. colous species in Arthoniomycetes. According to
cell sizes and branching patterns of algal fila-
ments, there seem to be species-specific relations
C. Associations with Algae and with different species of Trentepohlia and Phy-
Other Lichens copeltis (Lücking 1995; Thor 1990), but the tax-
onomy and phylogenetic relationships of
Lichenization appears to be a primary ecologi- Trentepohliales are still poorly resolved. Chryso-
cal trait in Arthoniomycetes, whereas only a few trichaceae and certain species assigned to Artho-
separate lineages in Dothideomycetes contain niaceae form lichens with coccal green algae.
lichen-forming fungi. This discrepancy is well Several Arthonia species in Mediterranean habi-
reflected by the difference in thallus complexity tats are poorly (or temporarily) lichenized. Their
in both classes. Most lichen-forming species in hyphae permeate the surface of woody twigs.
Dothideomycetes develop only thalli with rather Arthonia muscigena is lichenized and specialized
simple organization (Trypetheliaceae, Arthopyr- to grow on mosses, whereas Dawsophila (Artho-
eniaceae, Monoblastiaceae). In Cystocoleus and niaceae) is a bryosymbiotic, nonlichenized genus
Racodium (lineages in Capnodiales) the shape of Arthoniaceae (Grube 1998). These fruit bodies
of the microfilamentous thalli is determined by of Dawsophila are extremely small and
the algal partner (conglutinate hyphae encage a specialized to growing between the leaf lamellae
central algal thread (Muggia et al. 2008)). of the moss Dawsonia (Polytrichaceae).
In comparison, the thallus structures in Approximately 215 species of Arthoniomy-
Arthoniomycetes are more diverse. cetes (13 %) are known as lichenicolous fungi
“Advanced,” surface-detached thallus forms (Lawrey and Diederich 2003), a number that
are particularly common in Roccellaceae, will very likely increase. Most of these licheni-
which includes crustose, placodioid, shrubby, colous Arthoniales are highly specialized on
and pendant fruticose thalli (foliose thallus their hosts and represent commensals or locally
types, which are ubiquitous in Lecanoromy- destructive parasites. Lichenicolous species are
cetes, are hardly developed in Arthoniomy- often assigned to mainly lichenized genera
cetes). Studies of Tehler and coworkers (such as Arthonia, Enterographa, Mazosia,
suggest that fruticose thallus growth has Opegrapha, or Schismatomma), suggesting
evolved repeatedly in Roccellaceae and Opegra- that lichenized habit frequently and indepen-
phaceae (e.g., Tehler and Irestedt 2007; Ertz and dently gave rise to lichenicolous lifestyles in
Tehler 2011). Roccella and Dendrographa seem unrelated Arthoniomycetes. However, phyloge-
to represent independent evolutions of fruti- netic studies should test relationships more
cose lichens within Roccellaceae, and, similarly, carefully because lichenicolous species do not
Ingaderia and Pentagenella in Opegraphaceae. always form monophyletic groups with seem-
Representatives of Trentepohliales are the ingly congeneric, lichenized relatives (Ertz and
most common photobionts in both Arthoniomy- Tehler 2011). Observations suggest that the
cetes and Dothideomycetes. These algal partners lichenicolous species in Arthonia retained an
usually form three-dimensional filamentous affinity to the algal symbionts by densely
plane and platelike thalli in the free-living stage. entwining the host’s algal cells (Grube and Mat-
In the lichenized stage, the fertile structures of zer 1997; M. Grube personal observation). The
the algae are not developed, and the shape of lichenicolous fungi in Arthoniomycetes appar-
algal filaments can variably be modified. Fila- ently share the strategy for obtaining nutrients
mentous growth of Trentepohlia is often strongly with their lichenized ancestors but gave up on
modified in lichenized stages, and the filaments the formation of their own thallus structure.
are reduced to concatenations of a few algal cells. This may coincide with a switch of the algal
The growth modification is not so distinct in partner: many Arthonia species infect lichens
lichen mycobionts associating with members of with coccal, trebouxioid algae.
Approximately 500 species of Dothideomy- likely represent important environments con-

cetes are lichen-forming, and 266 species taining undescribed Dothideomycete diversity.
represent lichenicolous fungi (Lawrey and
Diederich 2003). In contrast to the likely singu-
lar lichenization transition giving rise to
D. Rock Environments and Oligotrophism
Arthoniomycetes, the Dothideomycetes may
comprise the highest number of transitions Rock-inhabiting fungi (RIF) are nonlichenized
to the lichenized lifestyle in a class (six inde- fungi that are adapted to subsistence in
pendent origins) (Nelsen et al. 2009). The liche- extreme conditions on rock surfaces. These oli-
nicolous life style is found in three orders of gotrophic fungi are slow-growing and asexual
Dothideomycetes, one of which also contains with poor diagnostic features. RIF often show
lichenized members. According to the different meristematic or microcolonial growth, and they
patterns of lichenized and lichenicolous fungi tend to have high concentrations of melanin in
in Dothideomycetes and Arthoniomycetes, the their cell walls. These features generally confer
evolution of lichenicolous fungi has different high levels of stress tolerance in fungi and allow
foundations, and a generalized view of their them to tolerate desiccation, osmotic pressure
evolution from the lichenized life style is not and acidity, and poor nutrient availability and
well supported. This could also be reflected by to survive high heat in a desiccated stage.
the differential preferences of lichenicolous Melanized fungi are also strikingly tolerant
fungi to the lichen symbionts. So far, lichen to ionizing radiation and have been found in
mycoparasitism has been shown microscopi- unusual habitats, such as Antarctic dry valleys
cally in the pleosporalean genus Pyrenidium (Onofri et al. 2007) or damaged nuclear reactors
(De los Rı́os and Grube 2000), but further and nuclear reactor cooling water (Zhdanova
study of more lichenicolous fungi in Dothi- et al. 2000), where they are able to survive con-
deales is required to clarify the preference for ditions analogous to outer space. This capacity
either of their host symbionts. Other species is a side effect of a perhaps widespread ancient
seem to show preference for the algal partner. adaptation to the rock habitat. It is therefore
Based on ascomatal characters, the lichenico- particularly interesting that RIF are found in
lous genus Zwackhiomyces is closely related to several unrelated lineages of Dothideomycetes
Anisomeridium (Monoblastiaceae, Dothideo- and could represent an ancestral trait; they also
mycetes) (M. Grube unpublished observation). form an early diverging group in the Arthonio-
Specific histochemical investigation for the mycetes (Ruibal et al. 2009). Recent analyses by
lichenicolous fungi revealed clear association Gueidan et al. (2011) support a much earlier
with the host’s unicellular coccal algae (Grube origin of the dothideomycetan rock-inhabiting
and Hafellner 1990). Several lichenicolous lifestyle than can be postulated in Chaetothyr-
Phoma species were described in the Phaeo- iales. The origin of RIF in Dothideomycetes is
sphaeriaceae (Lawrey et al. 2012). Like other estimated to date back to 309–420 million years
diverse lineages such as Lichenoconium (Law- ago in the late Devonian, but it is not possible to
rey et al. 2011), these species remain outside of fully rule out independent origins of RIF in
current familial and ordinal classification. It Arthoniomycetes and Dothideomycetes after a
should also be studied in this case whether the suggested diversification around 292–301 mil-
switch between lichenized and lichenicolous lion years ago in the Carboniferous.
life style coincides with a change of algal asso-
ciations. Several fungi grow as endophytes
inside lichens (endolichenic fungi) that include
an important subset of fungi in Dothideomy- IV. Modern Classification and
cetes (Arnold et al. 2009; Hofstetter et al. 2007; Phylogeny
U’Ren et al. 2010). Similarly, endophytic fungi
inside macroalgae include several Dothideomy- The earliest phylogenetic analysis performed
cetes (Zuccaro et al. 2008). These may very on a group or subgroup of dothideomyceta
was done on Arthoniales (Tehler 1990). The assemblage (Berbee 1996; Berbee and Taylor
author used 92 morphological, biological, and 1992; Spatafora et al. 1995) or monophyletic
other nonmolecular characters to run a cladis- (LoBuglio et al. 1996; Reynolds 1998; Winka
tic analysis on 20 members of Arthoniales to et al. 1998) but often with poor statistical sup-
determine relatedness within this group. A port. Although the orders Pleosporales and
more expansive analysis was run a year later Dothideales were almost always well resolved,
(Reynolds 1991) on a morphological data their shared monophyly was very dependent on
matrix representing the major ascostromatic the method of study and taxon sampling. All
and fissitunicate families and orders using 30 these studies generally supported the conclu-
characters. This included lichenized families sion that loculoascomyetes do not form a
such as Opegraphaceae and Trypetheliaceae. monophyletic group, and the subsequent use
An important conclusion was that loculoasco- of additional phylogenetic markers affirmed
mycetes are not monophyletic, but the author the preceding small subunit phylogenies,
also suggested that the use of molecular data which tentatively supported a monophyletic
would be crucial in order to elucidate poorly Dothideomycetes. The second largest subunit
resolved nodes on the tree. This set the stage of the RNA polymerase gene (RPB2) showed
forthe first phylogenetic studies using DNA strong bootstrap representation for a mono-
sequence comparisons. phyletic Dothideomycetes (Liu et al. 1999),
with similar results using the largest subunit
A phylogenetic analysis of dothideomyceta is shown in of the RNA polymerase gene (RPB1) (Lumbsch
Fig. 6.2. A maximum-likelihood RAxML run was per- et al. 2000). A more complete set of samples
formed on 400 taxa (using Pyrenula aspistea and Pyr- found support for a single ancestor to loculoas-
enula pseudobufonia as outgroups). Sequences were
taken from GenBank relying extensively on data pro-
comycetes in a phylogeny of RPB2 sequences
duced in Schoch et al. (2009a) and Ertz and Tehler (Liu and Hall 2004), but this was contrasted by
(2011). Alignments from five loci (fragments from a multigene study that included RPB2 (Lutzoni
large and small nuclear rDNA, mitochondrial small et al. 2004). The lack of reliable morphological
subunit rDNA, transcription elongation factor, and characters to indicate ancestral relatedness is
the second largest subunit of the RNA polymerase I
gene) were individually obtained with SATe (Liu et al.
challenging in dothideomyceta (Lumbsch and
2009). This was concatenated and analyzed with introns Huhndorf 2007). The first indications that mor-
and ambiguous regions excluded, resulting in 6,299 phological characters are informative above the
characters of which 56 % were missing. Phylogenetic level of family were provided by Berbee (1996),
analysis was done using the “bootstopping” criterion in who proposed that the presence of pseudopar-
RAxML (Stamatakis et al. 2008) under the CIPRES v. 3.1
Web portal (Miller et al. 2010). The analysis was per-
aphyses was a good monophyletic character to
formed applying unique model parameters for each delimit pleosporaceous taxa, and this was gen-
gene and codon, and the data set was divided into erally well supported in subsequent phyloge-
nine partitions, as previously described in Schoch nies (Liew et al. 2000; Silva-Hanlin and Hanlin
et al. (2009a). A general time-reversible model approxi- 1999) despite the fact that Leptosphaerulina
mation (GTRCAT) was applied, and the resulting tree
was compressed to indicate major lineages in MEGA
(lacking pseudoparaphyses) was shown to be
(Tamura et al. 2011). The alignment (and complete in the Pleosporales clade. The presence or
phylogeny) is deposited in TreeBASE (11726, www.tree- absence of pseudoparaphyses correlated with
base.org). a molecular derived phylogeny, which was sub-
sequently confirmed by additional authors
(Lumbsch and Lindemuth 2001) and resulted
A. Dothideomycetes in the designations of two subclasses, Pleospor-
omycetidae and Dothideomycetidae (Schoch
Phylogenetic analyses of single-gene data sets et al. 2006). A large number of new and existing
of the nuclear small ribosomal subunit never orders containing single families with relatively
resulted in unambiguous support for Dothideo- low coverage in taxon sampling and phyloge-
mycetes as a monophyletic entity with phylo- netic markers have been proposed elsewhere
genies indicating the class as a paraphyletic for Dothideomycetes. We refrain from utilizing
these names here until more analysis is possible ales, as are Hysteriaceae, Venturiaceae, and
(Hyde et al. 2013) Tubeufiaceae (Kruys et al. 2006; Schoch et al.
2006, 2009a; Zhang et al. 2009).
1. Pleosporomycetidae Familial designations in Pleosporales tradi-
tionally relied on distinctions of the ascomata,
The subclass Pleosporomycetidae was des- size, and shape variations in the ostiole as well
cribed (Schoch et al. 2006) based on phyloge- as the presence of periphyses and other cells in
netic comparisons of DNA and the presence the centrum. The lifestyles of different species
of a centrum with pseudoparaphyses. This were also considered informative. However, as
description included only the order Pleospor- noted earlier, DNA-based phylogenies exposed
ales and initially a single species outside of this several familial concepts that require readjust-
order, Lophium mytilinum. Since then, it has ment because several of these characters are
been expanded to include additional orders— shown to be highly convergent (Schoch et al.
Hysteriales, Mytilinidiales, and Jahnulales 2006). Taxon sampling of DNA data in the
(Schoch et al. 2009a). However, the placement order has been greatly expanded in the last
of Jahnulales in Pleosporomycetidae remains few years, and a more complete phylogeny
tenuous because DNA-based phylogenetic sup- could soon delineate 19 families with the tenta-
port for this relationship is still ambiguous tive inclusion of an additional 5 families (Zhang
(Fig. 6.2). et al. 2009). Currently, 28 families are recog-
Pleosporales. This is the largest order in nized in the order, with several of these taxon
Dothideomycetes, consisting of 332 genera concepts still not well supported in multigene
and more than 4,700 species (Kirk et al. 2008). phylogenies (Zhang et al. 2012). The most obvi-
The order was recently monographed by Zhang ous discrepancy is the treatment of Pleomassar-
et al. (2012), and readers are referred to that iaceae, which is accepted in Lumbsch and
work for a more detailed discussion of the Huhndorf (2010) but synonymized by Zhang
morphology and biology of the order. Ecologi- et al. (2009). It is indicated as being separate
cally, members subsist as epiphytes, endo- in Fig. 6.2 and now includes a number of
phytes, or parasites of plants. Several species recently described anamorph species with stel-
are saprophytes on dead plant material or late conidia in Prosthemium (Tanaka et al.
dung, and hyperparasites can occur on fungi 2010).
or insects in addition to a smaller number of In addition, several family-level branches in
lichenized species in Arthopyreniaceae. The the Pleosporales phylogeny remain unnamed
concept of Pleosporales has changed since it and await further sampling. One example is
was first proposed by Luttrell (1955) and sub- the unique branch supporting the anamorphic
sequently emended and validated by Luttrell rubber pathogen Corynespora cassiicola. This
and Barr (Barr 1987). The advent of molecular species may have associations with the teleo-
data has shown that a classification based on morph Corynasca, implying that the family
the distinction of pseudoparaphyses morphol- name Corynascaceae could be applied if that
ogy does not agree with DNA-based phyloge- is the case (Sivanesan 1996). Another well-
nies. Therefore, Melanommatales as described supported but separate lineage contains the
by Barr by relying on the shapes of pseudopar- marine mangrove-associated species Julella
aphyses (Barr 1990) are now considered synon- avicenniae (Schoch et al. 2009a). The genus
ymous with Pleosporales, while the type- Julella has since been shown to be polyphyletic,
bearing family Melanommataceae is currently with Julella fallaciosa showing a close relation-
accepted in a more restricted sense as part of ship to species in Trypetheliales (Nelsen et al.
Pleosporales (Zhang et al. 2009). In addition, a 2011). Julella avicenniae has since been recom-
number of families included in Barr’s 1987 bined as Halojulella avicenniae and the family
description have proven to be only distantly Halojulellaceae proposed (Hyde et al. 2013).
related to this order. Most notably, Botryo- Despite the high level of uncertainty
sphaeriaceae is now placed outside of Pleospor- remaining in family-level classification, recent
molecular data from multiple genes have families in Pleosporales, more narrow concepts
steadily improved the phylogenetic context. are now applied to these two families
Thus, the suborder Pleosporineae as circum- (Mugambi and Huhndorf 2009a ; Zhang et al.
scribed by Barr (1979) was emended to repre- 2011).
sent a well-resolved phylogenetic node showing Outside of Massarineae, the partially liche-
common ancestry for five families (Zhang et al. nized Arthopyrenia species are grouped with
2009). This represents a majority of important Roussoella and Roussoellopsis, species isolated
Dothideomycetes plant pathogenic families and from bamboo (Tanaka et al. 2009). These
includes the type-bearing family, Pleosporaceae groups have clear morphological differences,
(Kodsueb et al. 2006a). This family represents so this relationship is surprising, prompting
the most intensively studied clade of Dothideo- the need for more in-depth sampling and anal-
mycetes with genome data available for several ysis. The variety of fungi isolated from bamboo
species, including Cochliobolus (with Bipolaris is further seen in a new family, Tetraplosphaer-
and Curvularia anamorphs), Pyrenophora iceae, described for species predominantly
(with Drechslera anamorphs), and several spe- isolated from this host (Tanaka et al. 2009).
cies in the anamorph genus Alternaria (Ohm However, some studies could not corroborate
et al. 2012), some tied to Lewia teleomorphs. unambiguous support for this taxon (e.g.,
Other families in the suborder are Cucurbitar- Schoch et al. 2009a; Zhang et al. 2011).
iaceae, Leptosphaeriaceae, and Phaeosphaeria- Additional pleosporalean families recently
ceae, which contain multiple plant pathogens in described, including Aigialaceae and Lindgo-
Leptosphaeria and Phaeosphaeria associated mycetaceae, contain majorities of marine and
with Phoma and Septoria anamorphs (Aves- freshwater species, respectively (Shearer et al.
kamp et al. 2010; de Gruyter et al. 2009, 2012; 2009; Suetrong et al. 2009).
Zhang et al. 2009). Notably, the monophyly of The remaining ecological niche in Pleos-
Leptosphaeriaceae is only poorly to moderately porales comprises species growing in animal
supported in most recent molecular studies dung. This is the characteristic ecology in two
(e.g., de Gruyter et al. 2012; Zhang et al. 2009) families where coprophilic species are inter-
(Fig. 6.2). A fifth family, Didymellaceae spersed with saprobic lineages. In Pleosporales,
described with pseudoparaphyses deliquescing these species are found in Sporormiaceae and
at maturity, contains the majority of Phoma Delitschiaceae (Kruys and Wedin 2009; Kruys
anamorphs, although this large ubiquitous et al. 2006), and although the relation is only a
genus is interspersed throughout the suborder distant one, the related ascospores of both
(de Gruyter et al. 2009, 2012). Finally, a dark- Sporormiaceae and Delitschiaceae have germ
spored Botryosphaeria-like species, Dothidot- slits. In the first large-scale phylogenetic studies
thia, was recently removed from Botryosphaer- of Dothideomycetes, Delitschiaceae appeared
iales based on multigene phylogenies and as the earliest diverging lineage in Pleosporales
placed in a separate family, Dothidotthiaceae, (Liew et al. 2000; Kruys et al. 2006; Schoch et al.
within this suborder (Phillips et al. 2008). 2006). An isolate of Zopfia rhizophila has also
Outside of Pleosporineae, the recently been shown to be sister to this clade as repre-
emended suborder Massarineae (Barr 1979; sentative of the large and poorly defined family
Zhang et al. 2012) contains families Montagnu- Zopfiaceae, often isolated from soil and asso-
laceae, Massarinaceae, Lentitheciaceae, Moro- ciated with roots (Kruys et al. 2006). However,
sphaeriaceae, and Trematosphaeriaceae. These a more recent paper placed a well-sampled
families contain a majority of saprobic species, clade of Massariaceae as the earliest diverging
and some may occur in aquatic environments. clade in Pleosporales (Voglmayr and Jaklitsch
The type-bearing family, Massarinaceae, was 2011). Members of this family were shown to
traditionally treated as closely related to be weakly parasitic or hemibiotrophic and
Lophiostomataceae (Barr 1987). Phylogenetic were found to have high host specificity. As is
analysis shows that Lophiostomataceae is the case for several families across Pleospor-
more distantly related, and like most other ales (and Dothideomycetes), each of the
morphological characteristics used to define ceae can be accepted, and a new order, My-
Massariaceae also occurs in other, unrelated tilinidiales, was proposed for species in
groups. Therefore, characterization required Mytilinidiaceae with thin-walled mussel-
the combination of these characters with a shaped ascomata. A species, Rhytidhysteron
strong reliance on DNA sequence comparisons. rufulum, included in an unrelated family, Patel-
Hysteriales and Mytlinidiales. The hyster- lariaceae, was placed in Hysteriales, and many
iaceous fungi are characterized by apothecioid other genera were shown to be polyphyletic, for
ascomata that consist of persistent, carbona- example, Hysterium, Hysterographium, and
ceous structures that dehisce by a longitudinal Gloniopsis. In addition, some taxa previously
cleft that allows for the exposure of the hyme- included in Mytlinidiaceae based on these
nium and can be closed in response to changes morphologies may also reside in Hysteriales,
in humidity. This particular ascomatal struc- for example, Ostreichnon. Indicating additional
ture, with its boat-shaped outline, has been poorly sampled diversity, a number of species
referred to as a hysterothecium and has been could not be accommodated in the newly
described in several taxa now known to belong defined orders. Farlowiella and Glonium could
to Lecanoromycetes, Arthoniomycetes, and be placed as Pleosporomycetidae incertae sedis
Dothideomycetes. The convergence of this (Boehm et al. 2009b), but members of the Patel-
morphology is illustrated in a photo plate lariaceae (Patellariales) did not group with any
that shows ascomata from various unrelated defined subclass or order (Schoch et al. 2009a).
lineages in dothideomyceta (Fig. 6.3). Initially, An additional lineage of hysteriaceous species
taxa producing these structures were thought to previously described in Glonium was also
reflect a single genealogical group and classified placed in Pleosporales under the genus Ante-
under the family Hysteriaceae, later raised to aglonium (Mugambi and Huhndorf 2009b).
the order Hysteriales. Thus the original Members of this genus are now placed in the
description of Hysteriales by Lindau (1897) new family Anteagloniaceae (Hyde et al. 2013).
included four families of which only one has In addition, Cenococcum geophilum, one of the
bitunicate asci. Zogg (1962) monographed the most commonly isolated ectomycorrhizal fungi
remaining bitunicate species in the order and on a global scale, has a close relationship to
classified them in two families, Hysteriaceae Glonium species in the Gloniaceae (Schoch
and Mytilinidiaceae (as Lophiaceae). Because et al. 2009a; Spatafora et al. 2012). In conclu-
of similarities in the centrum, some authors sion, the latest phylogenetic results indicate at
have also placed Hysteriaceae under Pleospor- least two lineages corresponding to the orders
ales (Barr 1987) and Mytilinidiaceae in Mela- Hysteriales and Mytilinidiales, with the Hyster-
nommatales (Barr 1987, 1990), while others do iales as a sibling clade of Pleosporales and both
not accept a second family (Luttrell 1973). sharing a common ancestry with Mytilinidiales
The role that DNA-based phylogenies can (Schoch et al. 2009a) (Figs. 6.2 and 6.3).
play in revealing the unreliability of morpho- Jahnulales. Based on moderate phyloge-
logical characters is displayed well in the hys- netic statistical support and the presence of
teriaceous fungi. Earlier phylogenetic studies pseudoparaphyses, Jahnulales was tentatively
included a few of these taxa (Liew et al. 2000; added to subclass Pleosporomycetidae (Schoch
Schoch et al. 2006), but only recently could et al. 2009a), but more resolved phylogenies are
large-scale comparisons be made (Boehm required to verify this. Although the genus Jah-
et al. 2009a, b; Mugambi and Huhndorf nula was validated in 1936 (Kirschstein 1936), it
2009b). The shared conclusion of these recent was only by 1999 that additional species in the
papers was that a high degree of convergence genus were described (Hyde and Wong 1999).
occurs in virtually all important morphological In 2001, Aliquandostipite khaoyaiensis was
characters and that morphologically defined described with extraordinarily broad hyphae,
groups hide an unexpected genetic diversity. stalked ascomata, and the presence of pseudo-
The results were that only a narrow concept of paraphyses in its centrum, suggesting a link to
Hysteriales including the majority of Hysteria- Pleosporales. However, a DNA-sequence-based
phylogeny found no clear association with this being oriented toward the ostiolar region (Reynolds
order, and a new family was described, Ali- 1998). This convergent character is also shared with
unrelated loculoascomyetes in Chaetothyriomycetidae
quandostipitaceae. Subsequently, a new order (Eurotiomycetes).
was described, including Aliquandostipite, Jah-
nula, and Patescospora (Pang et al. 2002). The
latter genus was later synonymized with Ali- The diverse and expanded Capnodiales
quandostipite, and the ordinal description now includes the epiphytic sooty mold family
emended to include hyphae wider than 10 mm Capnodiaceae, rock-inhabiting and lichenized
and more variable ascospore morphology species, as well as foliar epiphytes and species
(Campbell et al. 2007). This small order now associated with human hair in the genus Pie-
contains more than 40 species that are almost draia. In Fig. 6.2, the earliest diverging lineage
always found in freshwater habitats. The spe- in Capnodiales represents a lichenized genus,
cies exhibit a range of variation, with ascos- Racodium (Muggia et al. 2008). Another liche-
pores filled with lipid guttules, equipped with nized species, Cystocoleus ebeneus, is repre-
a variety of gelatinous appendages and sheaths, sented as being unrelated to any of the known
and broad vegetative hyphae (10–40 mm) that families and in Schoch et al. (2009a) formed a
attach the fungi to submerged wood. In addi- single clade (clade C) together with a number of
tion to four possible lineages in Jahnulales, a rock-inhabiting species and other extremo-
mangrove-associated species, Manglicola gua- philes. Whether this represents a novel family
temalensis, which is able to grow in marine will require further analysis, but it appears
environments, was added and a new family, likely. RIF occur throughout the order Capno-
Manglicolaceae, described (Suetrong et al. diales and are diverse in both classes. Piedraia
2010, 2011). hortae, a specialized parasite of human hair in
the tropics, was shown by Schoch (2006) to
belong to Capnodiales. Piedraiaceae appeared
2. Dothideomycetidae to cluster within Teratosphaeriaceae (Schoch
et al. 2009a), but in some cases only with poor
Dothideomycetidae was emended in Schoch support (Crous et al. 2009a), and should likely
et al. (2006) from an earlier designation that still be regarded as Capnodiales incertae sedis.
used the subclass extension as applied to the Several additional lineages appear unresolved
whole Dothideomycetes (Kirk et al. 2001). This in Fig. 6.2.
means that Dothideomycetidae sensu Schoch Davidiellaceae was introduced for the
et al. (2006) is confined to the generally apar- genus Davidiella with Cladosporium ana-
aphysate orders Dothideales, Myriangiales, and morphs (Braun et al. 2003; Schoch et al. 2006).
Capnodiales. Cladosporium is one of the most commonly
Capnodiales. Capnodiales represents the isolated hyphomycete anamorphs in environ-
second largest order of diverse species in mental samples and currently contains only
Dothideomycetes after Pleosporales. The cur- approximately 100 species, many without any
rent concept of the order was expanded from sexual state (Seifert et al. 2011). It is one of the
Luttrell’s original, based on a multigene phy- most commonly isolated fungal forms from
logeny with the presence of ostiolar periphyses environmental samples and contains numerous
proposed as possible synapomorphy (Schoch species that are endophytic, fungicolous, and
et al. 2006). Like Dothideales, this order lacks pathogenic on humans and plants. Based on
pseudoparaphyses but does contain several molecular phylogenies, several important spe-
species with periphysoids and periphyses. cies, such as the tomato pathogen Cladospor-
ium fulvum (now Passalora fulva), were
The terms periphyses and ostiolar periphysoids are removed from this genus only in the past
sometimes applied interchangeably to the same cells
in species descriptions in Capnodiales. One reason for decade and are now classified in different
this is that periphysoids can be reoriented during ascus families (Thomma et al. 2005). The older
discharge from hanging downward in the ascoma to family name Cladosporiaceae was recently
reintroduced in place of Davidiellaceae (Hyde 2012). They are found in the same niches as the
et al. 2013). Dothideomycetes sooty mold lineages and
The original concept of the Capnodiales their main ecological distinctions from Cap-
was proposed based on the sooty molds, diverse nodiaceae are a lack of insect associations.
fungi that occur together in a common sooty Morphologically, these two families can be dif-
mass. It is now clear that this ecological guild ferentiated by the tendency for Chaetothyria-
consists of convergent but unrelated species. ceae to produce ascostromata containing
The order was proposed to consist of three multiple locules, as opposed to single locules
families, Antennulariaceae, Capnodiaceae, and in the ascomata of Capnodiaceae (Chomnunti
Coccodiniaceae (Woronichin 1925). The major- et al. 2011, 2012).
ity of sooty mold species with DNA sequence The majority of plant pathogenic species
data currently comprise the single family in Capnodiales reside in Mycosphaerellaceae
Capnodiaceae, while a number of additional (Arzanlou et al. 2008), with a smaller number
families previously named in this ecological in the more ecologically diverse Teratosphaer-
guild await sequence sampling. Members of iaceae (Crous et al. 2009b). DNA sequence com-
Coccodiniaceae were recently shown to belong parisons have revealed that several generic taxa
to Chaetothyriales, Eurotiomycetes (Crous et al. in both families are polyphyletic. For example,
2009a). This in contrast with an older study that the large genus Mycosphaerella is proposed to
placed a species in a clade now defined as consist of several distantly related species, and
Teratosphaeriaceae (Winka et al. 1998). Species some lineages have been proposed to be
in Capnodiaceae tend to occur on the leaf renamed based on anamorphs, for example,
surfaces of live plants where they absorb nutri- Ramularia, Zymoseptoria, and Pseudocercos-
ents from honeydew produced by insects and pora (Crous et al. 2009b; Quaedvlieg et al.
other nutrients in the phyllosphere. The group 2011). On a family level, the stability of both
is highly pleomorphic with several anamorphs Mycosphaerellaceae and Teratosphaeriaceae
with characteristically darkly pigmented also varies in different phylogenetic analyses
hyphae usually found on leaf surfaces (Hughes with nonoverlapping sample sets. This is evi-
1976). Anamorphic states tend to be mainly dent in Fig. 6.2, where Teratosphaeriaceae can
pycnidial with small hyaline conidia (Hughes be found with 98 % frequency in bootstrap
1976). Most sooty molds do not grow in axenic resampling but Mycosphaerellaceae are only
culture, and only a small number of them have found in 49 %. The opposite trend was true in
yielded DNA sequence data to date. An initial a previous study that sampled taxa broadly in
phylogeny based on 18S rDNA sequences indi- Capnodiales but only used nuclear ribosomal
cated that those species in Capnodiaceae form sequences (Crous et al. 2009a). Using a com-
well-defined groups with affinities to the other bined set of ribosomal and protein coding
aparaphysate orders (Reynolds 1998), and a sequences, Schoch et al. (2009) also indicated
monophyletic Capnodiaceae was recovered in that families containing the most economically
more recent analyses (Chomnunti et al. 2011; important plant pathogens (Mycosphaerella-
Crous et al. 2009a). The family also includes a ceae, Schizothyriaceae) may share common
lineage collected from ant nests. These unde- ancestry. This phylogeny suggested that Capno-
scribed species reinforce the nest walls and are diales is analogous to Pleosporales in the sense
nourished by the ants with honeydew, indicat- that most plant pathogenic lineages are found
ing an expansion of possible niches for these in derived nodes with saprobes and RIF dis-
fungi (Schlick-Steiner et al. 2008). Another fam- persed in early diverging lineages.
ily containing sooty molds, Chaetothyriaceae, Several other diverse lineages await compre-
was initially described as possibly related to hensive sampling. The RIF (discussed earlier)
Capnodiales by Woronichin (1925). Members contain numerous underdescribed lineages in
of this family are now classified in Chaetothyr- Capnodiales (Ruibal et al. 2009). Recent studies
iales with the help of some recently generated on the sooty blotch and flyspeck (SBFS) com-
DNA sequence comparisons (Chomnunti et al. plex that causes blemishes on apple and pear
fruit revealed a comparable level of poorly 3. Incertae Sedis Lineages

described complexity (Batzer et al. 2005, 2008;
Yang et al. 2010). The rediscovery and analysis A number of monotypic orders remain un-
of a previously described species, Torula comp- placed in any subclass. These include Acrosper-
niacensis (now Baudoinia compniacensis) (Scott males, a proposed order based on morphology
et al. 2007), has also yielded unexpected addi- only (Minter et al. 2007) (Fig. 6.2), with only
tions to this group. This is another extremophile one species included in Fig. 6.2. Patellariales
member of Teratosphaeriaceae with a high tol- was proposed in a similar fashion (Hawksworth
erance of ethanol. It is often found growing on and Eriksson 1986) and was briefly discussed
surfaces near the vents of distilleries, where it here under the apothecioid orders Hysteriales
metabolizes ethanol vapors. and Mytilinidiales. The remaining incertae
Dothideales and Myriangiales. These two sedis lineages are covered subsequently in
orders are well resolved as sister taxa in most more detail.
modern phylogenetic analyses (Schoch et al. Botryosphaeriales. The single-family Botryo-
2006, 2009a). Myriangiales has spherical asci sphaeriaceae, now containing 2,000 species
in monascous locules in pseudoparenchyma names, was described in 1918 (Theissen and
and consists of two families. Myriangiaceae is Sydow 1918). The members of this group gener-
saprophytic, often in association with scale ally have pseudoparaphyses present, but they are
insects, while Elsinoaceae is biotrophic, with not persistent and mature specimens may appear
several species involved in plant disease such to be aparaphysate. Because of this and other
as Elsinoe fawcettii and Elsinoe australis, the intermediate characters, members were initially
causal agents of citrus scab (Chung 2011). placed by different authors in either Pleosporales
Dothideales contains two families, Dothiora- or Dothideales (Kirk et al. 2001; Luttrell 1955).
ceae and Dothideaceae, which remain poorly Despite the fact that a new order, Botryosphaer-
resolved in DNA-based phylogenies (e.g., iales, was proposed (Schoch et al. 2006), the latest
Schoch et al. 2009a). Dothideales also contains classwide DNA sequence comparisons can still
several so-called black yeast anamorphs (de not support a clear relationship with either Pleos-
Hoog and Hermanides-Nijhof 1977) that are poromycetidae or Dothideomycetidae (Schoch
only distantly related. One of the most common et al. 2009a). The taxonomy of species in this
morphological types in this group is the species group has been much changed by the use of
in the Aureobasidium pullulans complex. It is a DNA-based comparisons. This resulted in a
genetically diverse set of similar morphological much narrower definition of the type genus
species (Manitchotpisit et al. 2009) that con- Botryosphaeria. Several new genera have been
tains producers of pullulan, a promising and proposed, such as Spencermartinsia and Barriop-
commercially valuable polysaccharide cur- sis, while other groups continue to be referred to
rently used for the packaging of food and by their anamorph names, such as Dothiorella
drugs, among other applications (Singh et al. and Diplodia (Crous et al. 2006; Phillips et al.
2008). Aureobasidium pullulans is a cosmopol- 2008).
itan species found on leaves and various sur- Species in Botryosphaeriales are often
faces such as concrete, wood, painted walls, and isolated as endophytes and can become causal
indoor environments. It has also been isolated agents of disease when plant hosts become
from extreme environments, including hyper- stressed. This suggests that species in this
saline waters in salterns, as well as glacial and group could be important indicators of climate
subglacial ice (Zalar et al. 2008). A comprehen- change (Slippers and Wingfield 2007). Several
sive phylogenetic treatment of this complex anamorphs are prominent as disease agents in
remains to be completed, with several genome woody hosts. Anamorph features have tradi-
sequences in preparation (P. Zalar personal tionally been the most informative to tax-
communication). onomists. They can be coelomycetes or
hyphomycetes, and conidial pigmentation and that thyriothecioid species represent a polyphy-
development, septation, and morphology have letic convergent group, but with very few DNA
been used to define genera. Several cryptic sequences obtained from any species, much of
species continue to be detected relying on the phylogenetic affinities remain uncertain.
molecular methods (e.g., Inderbitzin et al. The small numbers of molecular characters
2010), and it is clear that, given the amount of obtained so far have shown that some members
undescribed species, an initial single family of the thyriothecioid Schizothyriaceae and
classification understates the genetic diversity. Micropeltidaceae, for example, Stomiopeltis
Several family-level lineages have recently been versicolor and Aulographina pinorum, should
described, including Guignardia and its Phyl- be classified in Capnodiales, likely close to
losticta anamorphs (Phyllostictaceae), species Mycosphaerellaceae (Yang et al. 2010)
in Saccharataceae, Melanopsaceae (not shown (Fig. 6.2). A single representative was analyzed
in Fig. 6.2), and Aplosporellaceae (Slippers for Microthyriaceae, an unverified isolate of
et al. 2013). Mycrothyrium microscopicum (Schoch et al.
Microthyriaceae, Asterinaceae, and other 2009a) (Fig 6.2). This was placed outside of
thyriothecioid fungi. This group of species pro- the main subclasses and sister to Venturiaceae
duces small, shield-shaped, flat structures that with moderate support. More recently, DNA
develop superficially (thyriothecia) on a broad sequence data were expanded, underlining the
range of substrates, including living and dead high divergence for thyriotheciate species with
plant surfaces and other fungi. The ascomata morphologies resembling Microthyriaceae (Wu
can be removed from the substrate surface et al. 2011). Another species in Stomiopeltis, S.
without any damage and are often visible as beltulae, was shown to be likely related to
black specks resembling insect exudates. Thus Mycrothyrium microscopicum, and species in
they are broadly and indistinctly referred to as Micropeltis, Muyocopron, Neomicrothyrium,
fly speck fungi (Hofmann and Piepenbring Paramicrothyrium, and Tothia were all shown
2006). Many thyriothecioid species that are epi- to be related to divergent lineages.
phytic and biotrophic can be found in the tro- The Asterinaceae was recently placed in
pics and subtropics. A number of related Capnodiales (Kirk et al. 2008) but according
species that occur as hyperparasites and sap- to recent results should also be placed in a
robes also tend to occur in more temperate position similar to Microthyriaceae (Hofmann
areas. Traditionally, thyriothecioid species are et al. 2010; Wu et al. 2011). Unlike Hofmann
separated into several families based on ecology et al. (2010), we do not find support for a sister
and variations in the ascomata, and the largest relationship of Asterinaceae with Venturiaceae,
of these are Asterinaceae and Microthyriaceae. although such support is found for Microthyr-
It is thought that Asterinaceae and Microthyr- iaceae (Fig. 6.2). In any event, character and
iaceae obtain nutrients by penetrating plants taxon sampling requires expansion, and it
with hyphopodia forming specialized struc- appears likely that Asterinales (Barr and Huhn-
tures. An important morphological feature is dorf 2001) could be reinstated in a similar fash-
the upper layer of cells shielding the thyriothe- ion as Microthyriales. Both these taxa contain
cium, called the scutellum. Traditional taxon- large numbers of species, mainly segregated on
omy treated the variable ways in which the their isolation from various plant hosts, and
scutellum opens to release the ascospore as an this will have to be tested in more comprehen-
important character for distinguishing genera sive DNA sequence comparisons (Hofmann
and families in these fungi (von Arx and Müller 2009). A number of remaining families of thyr-
1975). Asterinaceae opens with star-shaped or iothecioid taxa occurring on plant leaves still
irregular fissures while Mycrothyriaceae has a requires DNA sequence data: Brefeldiellaceae,
central pore. Kirk et al. (2008) lists Microthyr- Engerulaceae, Parmulariaceae, Polystomella-
iaceae with Micropeltidaceae and Leptopeltida- ceae, and Vizellaceae (Hofmann 2009).
ceae as members of the order Microthyriales Tubeufiaceae. This family currently con-
[first introduced by Arnaud (1918)]. It is clear tains approximately 32 genera and more than
200 species (Kirk et al. 2008) and was first outside of Venturiales (Kruys et al. 2006;
described by Barr (1979) for species within the Winton et al. 2007). Multigene phylogenies
Pleosporales that possess superficial, often pal- confirmed the placement of several other spe-
lid to bright, perithecioid ascomata, which may cies previously placed in Venturiaceae, for
darken at maturity. Species in the family can be example, Venturia, Protoventuria, Metacoleroa,
found in diverse ecological niches and range Apiosporina, and Dibotryon (Crous et al. 2007a,
from being parasites on scale insects and b; Winton et al. 2007; Zhang et al. 2011). Recent
other fungi to growing on rotting plant phylogenies have also shown that Tyrannosorus
material, often in freshwater environments. pinicola, not previously placed in Venturiaceae,
Members of Tubeufiaceae frequently have belongs in this family (Zhang et al. 2011). This
anamorphs that produce distinctive coiled heli- species produces ascomata with remarkably
cosporous conidia. These can be found in the long, sharp spines and ascospores with multiple
genera Helicoma, Helicomyces, and Helicospor- germ slits and was isolated from Pinus wood
ium (Tsui et al. 2006). Although members of the (Untereiner et al. 1995). Outside of the Ventur-
family have a pleosporalean centrum, initial iales clade a number of lineages grouped with
DNA-sequence-based phylogenies could not good statistical support in recent large-scale
place them with certainty within Pleosporales phylogenies, for example, the coprophilic
(Kodsueb et al. 2006b), and more comprehen- fungi in Phaeotrichaceae and a single isolate
sive phylogenies now clearly place them outside from Microthyriaceae (Schoch et al. 2009a).
of Pleosporomycetidae (Schoch et al. 2009a). Shared ancestry with members of Asterinaceae
They may constitute a new order, but several is implied by some studies (Hofmann 2009; Hof-
genera as currently circumscribed remain poly- mann et al. 2010) but not supported in Fig. 6.2 as
phyletic (Promputtha and Miller 2010; Sánchez well as some recent publications (Zhang et al.
et al. 2012; Tsui and Berbee 2006; Tsui et al. 2011). Still, it appears that a core set of lineages
2007), and more descriptive work will be surrounding Venturiales may represent an addi-
required. tional subphylum taxon besides Pleosporomyce-
Venturiales and related species. Tradition- tidae and Dothideomycetidae. More complete
ally, members of Venturiaceae were placed sequence sampling including protein coding
under Pleosporales due to the presence of a genes would be required before proposing this
hamathecium of pseudoparaphyses that often taxon with a reasonable amount of confidence.
deliquesces at maturity. The order Venturiales Trypetheliales, Strigulaceae, and Mono-
was introduced for the core genera in Ventur- blastiaceae. These three lichenized lineages
iaceae with the description of a new family, may represent separate lichenization events in
Sympoventuriaceae (Zhang et al. 2011) (not Dothideomycetes, although their phylogenetic
indicated in Fig. 6.2). Species in Venturiaceae placement remains unresolved (Nelsen et al.
produce ascospores from small ascomata, often 2011). The order Trypetheliales is a monotypic
with greenish or olivaceous ascospores but order containing approximately 200 species of
sometimes becoming dark brown (Barr 1987). tropical and subtropical crustose pyrenocar-
Anamorphs include species in Fusicladium, pous lichens. The first unambiguous support
Spilocaea, and Stigmina. The type species of for a classification within Dothideomycetes
the family, Venturia inaequalis, is the causal was provided by Del Prado et al. (2006) after
agent of apple scab and therefore well studied initial phylogenetic results by Lutzoni et al.
as a plant pathogen (Bowen et al. 2011). Sym- (2004). Subsequently the order was erected
poventuriaceae contains the genera Sympoven- using morphological criteria only (Aptroot
turia as well as the anamorph-only genera et al. 2008) and was then corroborated to be
Veronaeopsis and Fusicladium (Zhang et al. distinct from other lineages in Dothideomy-
2011). cetes using DNA sequence comparisons (Nelsen
The use of DNA-sequence-based phyloge- et al. 2009, 2011; Schoch et al. 2009) (Fig. 6.2).
netics has now shown that the core set of taxa Members of this order have variable perithecial
traditionally placed in Venturiaceae resides morphologies but share a hamathecium of
thin, anastomosing pseudoparaphyses em- taxon) dothideomyceta (introduced in Schoch

bedded in a stiff gelatinous matrix. Hyaline et al. 2009b) to indicate the common ancestry of
ascospores with diamond-shaped septa are dis- Arthoniomycetes and Dothideomycetes. Mem-
tinctive for these groups but are often reduced bers of Arthoniomycetes were treated as a
or absent in species with muriform or multi- monotypic lineage with the sole order Artho-
septate ascospores (Aptroot et al. 2008; Nelsen niales until Ertz et al. (2013) described the
et al. 2009). In addition, a number of lichenized order Lichenostigmatales as a sister group of
lineages remain outside of Trypetheliales and Arthoniales. Lichenostigmatales (not shown in
placed incertae sedis in Dothideomycetes. Fig. 6.2) contain the lichenicolous fungus Liche-
They are noted as long branches of uncertain nostigma maureri (with its Phaesporobolus ana-
affinity in recent phylogenies but in all likeli- morph) and RIF. Typically, the species in
hood represent clades separate from the non- Lichenostimatales grow by budding of cells
lichenized dothideomycete orders (Nelsen (either representing black yeasts, or species in
et al. 2009). Strigulaceae (120 species) and which conidiomata and ascomata are made of
Monoblastiaceae (130 species) are mainly agglomerated spherical cells). This is in sharp
tropical families with a similar ascus type and contrast to the hyphal growth found in
generally one- or three-septate ascospores Arthoniales. Ertz et al. (2013) also found that
(Aptroot et al. 2008; Lücking 2008). One of other species of Lichenostigma belong to
the most striking shared morphologies for Dothideomycetes and are there grouped with
either family is related to their asexual states Lichenothelia, a genus also containing species
(when present). Monoblastiaceae contain spe- representing so-called borderline lichens
cies with unique pycnidia, but conidia are (Muggia et al. 2013).
embedded in a gelatinous matrix, while Strigu- The classification within Arthoniomycetes
laceae has conidia with terminal gelatinous was for a long time based on few phenotypic
appendices (Nelsen et al. 2009). The most cardinal characters, such as thallus, ascomata,
recent phylogenetic analysis showed that non- and ascospore morphology. However, Ertz and
lichenized saprobic species, Heleiosa barbatula Tehler (2011) noticed that many phenotypic
and Funbolia dimorpha, may also be members characters in Arthoniomycetes were of limited
of Monoblastiaceae (Nelsen et al. 2011). use in classification. They interpreted this lim-
itation as a possible result of a long phyloge-
netic history of adaptation and a slow
B. Arthoniomycetes evolutionary rate (according to the loci so far
studied). The hypothesis of slow evolutionary
Earlier studies of ascomatal ontogenies sug- rates was based on the idea that the precursors
gested that Arthoniales was a distinct entity to Roccella were already widely distributed
not closely related to the Dothideales or Myr- before the split of the Atlantic Ocean in the
iangiales (Henssen and Thor 1994, 1998). A Mesozoic (Tehler et al. 2009). Some newly dis-
separate class, Arthoniomycetes, was thus covered relationships are indeed in contrast to
described to account for the distinctiveness of previous classification. Growth form was earlier
this group (Eriksson and Winka 1997). Mem- seen as a family-separating character, but now
bers of Arthoniomycetes were also included in crustose and fruticose members are found in
the first phylogenetic analysis integrating liche- the same genus. As in other large groups of
nized and nonlichenized fungi (Gargas et al. Fungi, the phenotypic flexibility has been
1995), and their monophyly was later con- severely underestimated, and the notion of
firmed (Lutzoni et al. 2001). The class is now slow evolutionary rate may be revised with the
generally accepted (Kirk et al. 2008; Lumbsch analysis of genomic data in the future.
and Huhndorf 2010), and recent phylogenetic Recent decades have witnessed a trend
studies solidified the recognition of sister rela- toward reducing the number of families in
tionship with Dothideomycetes. Nelsen et al. Arthoniomycetes, as it has been commonly
(2009) applied the informal name (rankless accepted that the classification concepts of the
so-called Zahlbruckner era were artificial [e.g., which only differ in spore septation, are strik-
Zahlbruckner (1907) accepted nine families]. ingly similar in all other respects (e.g., Coppins
Grube (1998) presented a review of the order, and James 1979; Grube and Giralt 1996). There-
which comprised only three families: Arthonia- fore, Grube (2007) retained several Arthothe-
ceae, Chrysotrichaceae, and Roccellaceae. This lium species in Arthonia. However, recent
classification used ascus and secondary com- results suggest that the phenotypically distinct
pound differences to keep the classification as genus Cryptothecia, usually kept in a separate
clear and simple as possible. Recent molecular family, Cryptotheciaceae, is nested in a group
evidence again promotes the expansion of fam- comprising Arthonia and Arthothelium. Cryp-
ily numbers. The monophyletic Roccellaceae, tothecia is characterized by poorly delimited
with more or less cylindrical asci as accepted ascomatal aggregates and dispersed, globose
by Grube (1998), is now split up into several asci. Recent data show that Cryptothecia is not
families (Ertz and Tehler 2011). Their phyloge- monophyletic either (Frisch et al. 2014). In
netic study supports Opegraphaceae and Roc- comparison with other families in Arthoniomy-
cellographaceae as families. They also propose cetes, sampling is still limited in the early
Lecanographaceae as an additional family can- diverging groups consisting of the unresolved
didate in Arthoniomycetes, although it is still genera Arthonia and Arthothelium, and major
poorly supported as a monophyletic group. The rearrangements are expected. Current data
concept of Roccellaceae has become narrow indicate two major clades in Arthoniaceae,
again, and phenotypic recognition of families one containing lichenized and lichenicolous
appears more complicated now (compare species with generally blackish, adnate, moder-
Table 1 of Ertz and Tehler 2011). ately to strongly convex ascomata, and another
Arthoniaceae. Arthoniaceae are character- clade, which is morphologically more diverse
ized by globose to clavate asci with strongly and includes the genera Cryptothecia, Stirtonia,
thickened apical and side walls. All species and Herpothallon (Frisch et al. 2014).
develop thalli with crustose shapes, unless The ascomatal structures of Arthoniaceae
they are growing in other lichens or mosses, display a high degree of phenotypic heteroge-
for example, Dawsophila. Clavate asci are neity, especially within the bulk genus Artho-
known also from Chrysotricacaceae (see nia. For example, some tropical Arthonia and
below), whereas asci in other families are Arthothelium species with red ascomatal pig-
more cylindrical with thinner side walls. A few ments and hydrophobic ascomata were placed
representatives with passive spore release have in the genus Coniarthonia (Grube 2001b). The
asci with thin to evanescent walls. The type new genus Crypthonia (Frisch and Thor 2010)
genus Arthonia of Arthoniales is the most spe- lacks such red pigments but also has hydropho-
ciose. Approximately 500 species have been bic fruit bodies and is related to Herpothallon
described, although the number may increase (Frisch et al. 2014).
with ongoing taxonomic revisions. It is charac- With a broader sampling, characters of
terized by crustose thalli bearing rounded, ascomatal construction, for example hyphal
maculiform, or lirellate ascomata with poorly textures and locule arrangement, may appear
developed exciples and transversely septate as phenotypic markers of phylogenetic rela-
ascospores (e.g., Coppins and Aptroot 2009; tionships in Arthoniaceae. Arthoniaceae also
Grube 2007). The transversely septate ascos- includes Tylophoron, a peculiar genus with
pores of Arthonia species were used as a diag- mazaediate ascomata and passive spore dis-
nostic character to distinguish the genus persal (Nelsen et al. 2009). Recent data show
Arthothelium with muriform ascospores that the sterile, sporodochia-producing genus
(approximately 80 species). However, the valid- Blarneya is included in Tylophoron (Ertz et al.
ity of this concept has always been questioned. 2011). Additionally, a lichenicolous coelomy-
Apart from the septation of the ascospores, cete, which could be grown in pure culture,
other characters are shared among Arthonia was recently classified in Arthoniaceae. The
and Arthothelium species, and some species, new genus Briancoppinsia was introduced for
a species previously classified in Phoma (Die- family also includes the lichenicolous genera
derich et al. 2011), a coelomycete genus usually Plectocarpon and Phacographa (Frisch et al.
associated with members of Dothideomycetes. 2014).
Molecular data have also classified the hypho- Opegraphaceae. Recent phylogenetic stud-
mycete Reichlingia as a member of the Artho- ies suggest a substantial reclassification of Ope-
niaceae (Ertz and Tehler 2011). This species graphaceae. Opegrapha was the second largest
was earlier interpreted as a lichenicolous fun- genus in Arthoniales, and traditionally recog-
gus on an unidentified host but apparently nized by distinctly developed, often elongate
represents the interesting case of a lichenized and dark colored exciples (outer layers). In
hyphomycete related to Arthonia. their three-gene phylogeny study focusing on
Chrysotrichaceae. Previously, Chrysotri- Opegraphaceae, Ertz et al. (2009) found that
chaceae were principally distinguished from Opegrapha atra and O. calcarea are in fact
other families in Arthoniomycetes by bright closely related to the type species of Arthonia
yellow pigments, both in the thallus and in the and were thus transferred to this genus. This
ascomata (due to production of pulvinic acid placement is supported by similarities in asci,
derivates). Moreover, all species in Chrysotri- spores, and pigments. Other Opegrapha species
chaceae associate with coccal green algae and are now placed in Lecanographaceae (e.g., in
have crustose thalli. Therefore, it came as a Zwackhia and Alyxoria). Paralecanographa,
surprise when Nelsen et al. (2009) demon- Paraschismatomma, Paraingaderia, and Spar-
strated that Arthonia caesia with pruinose, but ria (for Arthonia endlicheri and Sclerophyton
not yellow, ascomata grouped together with cerebriforme) are new genera in this family.
Chrysothrix. However, this placement also Roccellographaceae. This small family,
agreed with the presence of coccal green photo- described by Ertz and Tehler (2011), com-
bionts. Despite the presence of coccal green prises—so far—crustose and subfruticose spe-
photobionts, the placement of Arthonia med- cies. It includes the new genera Dimidiographa
ialla in Chrysotricaceae is more surprising and Fulvophyton, in addition to the known
because this species has only dark, melaninlike genus Roccellographa (including Sclerophyton
pigments in the cell walls (Frisch et al. 2014). muriforme and Peterjamesia circumscripta).
Nevertheless, present data strongly support Roccellaceae. The concept of Roccellaceae
Chrysotrichaceae as a separate family and as a has significantly changed, and this family
sister group to all remaining Arthoniomycetes includes crustose and fruticose representatives
(Ertz and Tehler 2011; Nelsen et al. 2009). of quite different phenotypic appearance. The
Future work will show whether further species monophyletic grouping of Enterographa and
currently placed in Arthonia might actually Erythrodecton represents a basal lineage in the
represent lineages in or near Chrysotrichaceae. family according to Ertz and Tehler (2011).
This is at least the case with Arthonia leucopel- Enterographa is now accepted in a narrower
laea, a species with distinct ascomata, atypical sense than before (Ertz et al. 2009). Interest-
for Arthoniaceae (Frisch et al. 2014). ingly, Dendrographa, originally a fruticose
Lecanographaceae. Ertz and Tehler (2011) lichen, includes several newly transferred crus-
proposed Lecanographaceae as a family that is tose species of other genera (of Schismatomma
comprised of the genera Zwackhia and Alyxoria and Roccellina, as well as the sterile lichen
(with species previously assigned to Opegra- Lecanactis latebrarum) (Ertz & Tehler 2011).
pha) with the genus Lecanographa. Zwackhia The family and genus concept in Arthonio-
and Alyxoria have asci with slightly different mycetes has been changing significantly fol-
hemiamyloid (blue in alkaline Lugol’s solution) lowing the most recent phylogenetic studies,
internal staining, comprising the Vulgata and and accepted genera and families, which
Varia types, respectively (whereas the Grumu- turned out to be paraphyletic, are currently
losa type is found in Lecanographa). This being restructured. So far, the emerging
concept is hardly reflected by phenotypic car- represent swarms of multiple species the is
dinal characters. Moreover, the splitting of expected to be characterized by molecular data.
families raises the question of whether more
families should be accepted for basal clades
comprising phenotypically rather distinct
members of currently accepted families. Since V. Maintenance and Culture
a considerable number of phenotypically enig-
matic species have not yet been included in The majority of saprobic and plant-pathogenic
molecular phylogenies, further higher-level species in Dothideomycetes can be cultured on
classification should be undertaken with cau- synthetic media using techniques commonly
tion. Some of these species represent new used in other fungi (Jong and Birmingham
genera; for example, the monotypic genus 2001). Marine species, such as members of
Phoebus has opegraphoid ascomata and thalli Aigialaceae and Morosphaeriaceae, often
with an orange quinoid pigment (Harris and require the extra addition of seawater or aquar-
Ladd 2007). A serious drawback of the current ium salt and trace elements to grow optimally
concept is the poor representation of licheni- (Kohlmeyer and Kohlmeyer 1979). Other spe-
colous fungi so far, although some of them are cies, growing in extreme environments, such as
described as phenotypically distinct genera members of Teratosphaeriaceae, may require
(e.g., Phacothecium, Arthophacopsis, Paradox- additions to growth media, reflecting their
omyces, Perigrapha, Phacographa, Plectocar- growth habit. For example, primary isolations
pon). Recent analyses suggest that the lichen- of the Baudoinia compniacensis from habitats
parasitic lifestyle has emerged multiple times rich in alcohol vapors required the addition of
in Arthoniales and are so far found in four 5 ppm ethanol (Scott et al. 2007). However,
lineages within Arthoniaceae (Frisch et al. several large groups of species remain poorly
2014). represented in culture collections. One such
Two aspects of the evolutionary biology of group of species, commonly referred to as
Arthoniomycetes should finally be mentioned. sooty molds, only rarely grows in axenic cul-
The first concerns the species-pair concept, i.e., ture. As mentioned earlier, a large number of
two closely related species with vegetative and these species appear to reside in Capnodiaceae.
sexual dispersal strategies. This concept was Other diverse groups with poor representation
originally derived from observations on the in culture collections and DNA databases
geographic distribution of closely related (foli- include the biotrophic Asterinaceae, Micro-
ose) species in Lecanoromycetes, where the thyriaceae, and Parmulariaceae (Hofmann
primary sexual species have usually restricted 2009; Wu et al. 2011).
ranges and the vegetative secondary species is Lichenized species of Dothideomycetes and
widespread (Poelt 1970). Such a concept has Arthoniomycetes are also poorly represented in
not been confirmed in Arthoniomycetes (Teh- public culture collections, partly due to the
ler et al. 2009). The dispersal mode is modu- problems in initiating growth. Generally, axenic
lated by other processes but not sufficient to cultures of lichenized fungi are generated either
describe species. On the other hand, rapid radi- from ejected (asco)spores or from tiny frag-
ation has been suggested for the Roccella gala- ments of surface-sterilized thalli (after homo-
pagoensis group and a high fraction of endemic genizing the thalli or by microdissection from
taxa in Roccellaceae (Tehler et al. 2009). Geo- thalli), both of which are maintained on stan-
graphically limited ranges of species are also dard media. Ertz et al. (2009) made use of
found in Arthonia (Ertz et al. 2010; Grube and multispore cultures of Arthoniales to avoid
Lendemer 2009; Grube et al. 2004). It remains to the fairly common contaminations by other
be tested whether widespread phenotypically fungi, which co-occur with the species with
recognized species in Arthoniales (and particu- inconspicuous, bark-inhabiting thalli. Growth
larly polymorphic Arthonia species) actually of lichenized fungi can be improved when
their natural ecology is mimicked, for example, Eocene (55–35 MYA) (Mindell et al. 2007). Sev-
by supplying polyols to media. Some useful eral sooty mold species with likely members in
hints on culturing lichen fungi are found in Capnodiales have also been described from
Stocker-Wörgötter and Hager (2008) and refer- amber older than 100 million years ago
ences therein. (Schmidt et al. 2014). Calibrated molecular
Often the lack of robust cultures translates clock analyses point to a much older origin
into poor representation of reliable sequences for the shared ancestor of Dothideomycetes
in public DNA databases. However, techniques and Arthoniomycetes. Although fossil calibra-
to isolate DNA from herbarium and fresh speci- tion points remain scarce and the interpreta-
mens are steadily improving. Success from old, tion of their placement can be contentious, a
but well-preserved, specimens are encouraging, recent paper hypothesized a late Devonian ori-
and examples include sequences obtained from gin (Gueidan et al. 2011). Much remains uncer-
a more than 35-year-old Didymella species tain about the ancestral ecology that gave rise to
(Zuccaro et al. 2008) and a Septoria specimen these two classes, but RIF exhibit extremely
of more than a century old (Quaedvlieg et al. high diversity in dothideomyceta, and, being
2011). Nevertheless, the need for having highly tolerant to stress, they may represent
well-represented DNA databases tied to well- an ancient pool of diversity giving rise to vari-
vouchered specimens remains compelling able ecologies in extant lineages. This will have
(Pleijel et al. 2008). Several recent efforts to to be tested further with densely sampled phy-
extend this include epitypification (Hyde and logenies and well-documented biological data.
Zhang 2008) and DNA barcoding (Begerow Another important question to ponder will be
et al. 2010; Schoch et al. 2012). how evolution shapes morphological conver-
gence and plasticity. As mentioned earlier in
this review, several morphologies are shared
with members of Eurotiomycetes, and it
VI. Conclusions remains challenging to place some species in
either class using only morphology (Rossman
Dothideomyceta represents a well-resolved et al. 2010).
node on the fungal tree of life, still with some Finally, it is hoped that massive environ-
level of uncertainty as to which class is its mental sampling, barcoding, and comparative
nearest neighbor (Schoch et al. 2009b). Mem- genomics will rapidly fill in several gaps in our
bers of this group, consisting of sister classes knowledge of these fungi. Recent comparative
Dothideomycetes and Arthoniomycetes, share genomics studies are already uncovering a
distinctive morphological characters regard- potentially novel system of chromosomal regu-
ing their asci and ascomata but clear differ- lation unique to filamentous ascomycetes
ences in their ascoma, development, and major (Pezizomycotina). This is especially striking in
ecological roles. The classification for these Dothideomycetes. Mesosynteny (designated to
organisms remains in a transitory state, distinguish it from micro- and macrosynteny)
where several concepts proposed by earlier tax- indicates a pattern where genes are conserved
onomists relying on morphological, biochemi- within homologous chromosomes, but with
cal, and developmental comparisons are still randomized orders and orientations (Hane
being tested and adapted to correlate with phy- et al. 2011; Ohm et al. 2012). Additionally, a
logenetic hypotheses determined by compari- recent 18-genome comparison of Dothideomy-
sons of molecular characters. cetes found differences in gene numbers of
As the phylogenetic resolution improves, Pleosporales and Capnodiales, despite a con-
researchers can also begin to investigate the stant set of core genes (Ohm et al. 2012). Several
evolutionary history of this group. Dothideo- of these genes involved in pathogenesis are
myceta have been recognized infrequently in positioned close to areas enriched in transpo-
the fossil record, and one of the oldest known sable elements (Ohm et al. 2012). Other poten-
fossils likely belongs to Pleosporales from the tial evolutionary mechanisms to be investigated
with expanded genome data include the hori- Barr ME (1990) Melanommatales (Loculoascomycetes).
zontal transfer of genetic material (Richards North American Flora Series II 13:1–129
Barr ME, Huhndorf SM (2001) Loculoascomycetes. In:
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It should be obvious that continued study The Mycota, vol VII, Part A: systematics and evo-
in dothideomyceta, with its huge genetic and lution. Springer, Berlin, pp 161–177
ecological diversity, has tremendous potential. Batzer JC, Gleason ML, Harrington TC, Tiffany LH
A focus on the addition of data from under- (2005) Expansion of the sooty blotch and flyspeck
complex on apples based on analysis of ribosomal
studied saprobic and lichenized species will DNA gene sequences and morphology. Mycologia
enrich comparative studies and continue to 97:1268–1286
inform our concepts of fungal ecology, evolu- Batzer JC, Arias MM, Harrington TC, Gleason ML,
tion, and biology. Groenewald JZ, Crous PW (2008) Four species of
Zygophiala (Schizothyriaceae, Capnodiales) are
Acknowledgements We appreciate the photos provided associated with the sooty blotch and flyspeck com-
by Sabine Huhndorf (Field Museum) and Walter Ober- plex on apple. Mycologia 100:246–258
mayer (Karl-Franzens-University). We thank the Cen- Bellemère A (1994) Asci and ascospores in ascomycete
traalbureau voor Schimmelcultures (CBS) and Studies systematics. In: Hawksworth DL (ed) Ascomycete
in Mycology for allowing us to use illustrations from systematics: Problems and Perspectives in the
the 1975 publication by J. von Arx and E. Müller. nineties. Plenum, New York, NY, pp 111–126
Permission to use an adaptation from an illustration Begerow D, Nilsson H, Unterseher M, Maier W (2010)
presented in the Ph.D. thesis of Tina Hoffman (Johann Current state and perspectives of fungal DNA bar-
Wolfgang Goethe-University) is also appreciated. CLS coding and rapid identification procedures. Appl
acknowledges the Intramural Research Program of the Microbiol Biotechnol 87:99–108
National Institutes of Health, National Library of Med- Berbee ML (1996) Loculoascomycete origins and evo-
icine. We would like to thank Göran Thor (Swedish lution of filamentous ascomycete morphology
University of Agricultural Sciences) and Barbara Rob- based on 18S rRNA gene sequence data. Mol Biol
bertse (NCBI) for critical readings of the manuscript. Evol 13:462–470
Berbee ML, Taylor JW (1992) Two Ascomycete classes
based on fruiting-body characters and ribosomal
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Nomenclature and Documentation
7 The Shifting Sands of Fungal Naming Under the ICN and the One
Name Era for Fungi
ANDREW M. MINNIS1
CONTENTS IV. Conspectus and the Future of

Fungal Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
II. International Code of Nomenclature for Algae,
Fungi, and Plants (ICN). . . . . . . . . . . . . . . . . . . . . 180
A. Transition to One Name Per Fungus . . . . 182
1. History and Enactment of I. Introduction
Rule Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2. How One Name Per Fungus Will Happen
Under the New Rules . . . . . . . . . . . . . . . . . . 184 The accumulated knowledge about fungi and
B. Other Major and Minor Changes funguslike organisms is linked to names, and
in the ICN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 communication of this knowledge relies upon
1. Effective Publication via Electronic
Publication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 the use of these names. Naming of these organ-
2. Latin or English for Valid Publication of isms can be thought of simply as involving two
Names of New Taxa. . . . . . . . . . . . . . . . . . . . 190 principal components, taxonomy and nomen-
3. Registration of Fungal Names . . . . . . . . . 190 clature. Taxonomy is how a fungal entity is
4. Minor Changes . . . . . . . . . . . . . . . . . . . . . . . . . 191 defined and grouped with other fungal entities
C. Practical Notes on Using the ICN. . . . . . . . 193
1. How Do I Describe New Taxa at the Rank into a system of classification. Although there is
of Species? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 no universally agreed upon right or wrong way
2. How Do I Describe New Taxa at the Rank to do this, mycologists today generally base
of Genus? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 taxonomic concepts and classifications of
3. What Is an Ex-type?. . . . . . . . . . . . . . . . . . . . 195 fungi on evolutionary relationships inferred
4. How and When Do I Designate a Lectotype
for a Species? . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 from phylogenetic analyses. Nomenclature
5. How and When Do I Designate a Neotype includes the rules for formally establishing a
for a Species? . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 name, determining which is correct, and esta-
6. How and When Do I Designate an Epitype blishing a standard to determine the appli-
for a Species? . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 cation of a name, i.e., the type. The formal
7. How Do I Validly Publish New
Combinations? . . . . . . . . . . . . . . . . . . . . . . . . . 197 rules of nomenclature that govern the naming
8. How Do I Validly Publish a Replacement of fungi and funguslike organisms, excluding
Name Also Known as a Nomen Microsporidia, are found in the current version
Novum?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 of the International Code of Nomenclature for
9. How Do I Correctly Give Author Citations algae, fungi and plants, or ICN (Melbourne
for Taxa?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
III. Potential Changes to Nomenclatural Code) (McNeill et al. 2012). New taxonomic
Rules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 concepts often lead to name changes, and
newly recognized taxa are added as mycologists
document the enormous quantity of fungi and
funguslike organisms, estimated to include as
1
USDA-US Forest Service, Center for Forest Mycology
many as 5.1 million fungal species (Blackwell
Research, One Gifford Pinchot Dr., Madison, WI 53726, USA; 2011). Name changes also result from the appli-
e-mail: minnisncf@gmail.com cation of and changes to the rules of the ICN.

180 A.M. Minnis
Though the name of a fungus might change, the plants (McNeill et al. 2012). Modifications of
fungus itself remains the same. Thus, tracking and additions to the rules are considered, and
the different names used for a specific fungus new Codes are adopted via a democratic pro-
allows all of the accumulated knowledge about cess every 6 years at an International Botanical
it to be pooled, and in some cases misidentifi- Congress (IBC), the next of which will occur in
cations and changing concepts can be evaluated 2017 in Shenzhen, China. The Code may be
and information that is no longer clearly appli- thought of as a sort of gentlemen’s agreement
cable to a specific fungus can be identified and since it has no legal status; hence, it depends on
reconsidered. The value of fungal naming and users’ willingness to voluntarily follow its rules,
following the ICN thus lies in having one cor- which has largely been done over the years. The
rect name for each fungus and funguslike Code itself is divided into a number of parts,
organism that can be used for precise commu- and the organization and numbering of these in
nication and an associated standard for deter- the current ICN has changed substantially from
mining what organism that name represents. earlier Codes. Its language is complicated, and
In the first edition of The Mycota, Hawks- the finer details of the rules take a while to learn
worth (2001) provided a scholarly and detailed and appreciate; a single read is insufficient for
summary of fungal naming that provided guid- most mycologists to gain anything close to a
ance for using the version of the International basic degree of mastery. Typically, nomen-
Code of Botanical Nomenclature (Tokyo Code) clatural experts must read and reread and re-
(Greuter et al. 1994) that was in effect at that interpret the Code depending on what
time and integrated information about poten- nomenclatural procedures, some little used,
tial proposals for changes to fungal naming, and areas of interest have emerged during a
some of which were never adopted, e.g., the taxonomic project. Unfortunately, many
BioCode. Subsequent to the completion of the mycologists do not have time to learn the
original text of Hawksworth’s (2001) publica- Code because the rigors of keeping up with
tion, three different Codes have been used for other scientific matters in a quickly changing
fungal naming, and major changes to the pro- world take up a lot of time. It is hoped that the
cedures for naming fungi have been adopted, present chapter will serve mycologists as an
especially changes found in the current ICN elementary guide to the basic essentials of
(McNeill et al. 2012). Rather than restate the nomenclature.
historical aspects and basics of fungal naming Following a preface summarizing the
summarized by Hawksworth (2001), this chapter changes found in the current Code, a key
will provide an update and supplement to the to the renumbering of parts, and a list of
previous version. Much has been written on the important dates in the Code, the ICN begins
topic of fungal naming in recent years, and all of with a preamble that defines its purpose and
this will not be covered in detail here. The pres- the organisms it covers and summarizes its
ent chapter will focus on the transition to a one- parts, among other items. Next comes the
name-per-fungus system of classification, other first of three divisions of the ICN, Principles.
major changes to how fungi are named found in This single page contains the six major prin-
the ICN, and how to perform frequently used ciples that form the basis of nomenclature, not
nomenclatural procedures correctly. Some the least of which are determining the appli-
attention will also be given to a number of cur- cation of names by nomenclatural types,
rent proposals and ideas on changes to fungal priority of publication, and that each taxon
naming that may be adopted in the future. may only bear one correct name, with a few
exceptions.
The second division, Rules and Recommen-
dations, contains nine chapters, some of which
II. International Code of Nomenclature have additional sections. These chapters con-
for Algae, Fungi, and Plants (ICN) tain rules, which are set out in articles and
sometimes clarified with notes, and recommen-
The ICN is a set of rules for naming the organ- dations, and examples are added occasionally
isms traditionally treated as algae, fungi, or for illustrative purposes. Rather than detail
The Shifting Sands of Fungal Naming Under the ICN and the One Name Era for Fungi 181
all of these, a brief summary of important named fungus, for example, I have “Amanita
concepts and parts that should be useful for hibbettii Vilgalys 2011,” but I call it “Amanita
most mycologists follows. Chapter I covers justoi Minnis 2014.” The terms legitimate and
taxa and their ranks. The principal ranks illegitimate are also often misused by authors
include kingdom, phylum, class, order, family, despite being clearly defined in the various
genus, and species, which we have all learned Codes.
and loved. A bevy of secondary ranks are also Typification is one of the most important
presented for those inclined to produce compli- principles of the Code because a type is a stan-
cated classifications. None of these ranks is a dard used to determine the application of a
level, so studies that use the term “level,” such name, i.e., answer the question: what is this
as species level or level at other ranks, reflect fungus? An analogous standard is the Interna-
the authors’ nomenclatural ignorance. Such tional Prototype Kilogram (IPK), which is used
usage is best avoided by employing the term to ascertain the base unit of mass if and when
rank rather than level because there is no such questions about it arise (BIPM 2006). Needless
thing as the latter (at least under the Code). to say, the IPK is rarely handled and protected
Chapter II covers status, typification, and from damage for good reason. A nomenclatural
priority of names. Effective publication (getting type functions similarly for the name of a fun-
the word out) is defined as per the articles of gus. As a single standard, a type may not
Chapter IV, and this simply means that the encompass all of the variation of a biological
name of a taxon has been presented to the entity, and it may not be the most representa-
world by appropriate means, for example, in a tive element. A type is typically a specimen, but
printed book or journal that is widely illustrations may serve as types in some cases.
distributed rather than on a note left in a single There are a large number of different kinds of
university herbarium specimen. Valid publica- types and terminology, and some of them are
tion, which produces a valid name, means that reviewed later in this chapter. The name of a
the articles of Chapter V, hybrids ignored genus is typified by the type of name of a spe-
herein, were followed during the naming pro- cies, and this may be indicated by citation of the
cess. To be a name, a name must be valid, and species name. The name of a family is typified
valid simply means meeting the basic require- by the same type as the generic name on which
ments to exist or have status under the Code it is based, and this may be indicated by citing
and be recognized by the scientific community. the name of the genus. Above the rank of fam-
An invalid name does not have status (exist) ily, typification does not apply unless the name
under the Code and should not be recognized is based on a generic name, in which case the
by the scientific community because it does not type is automatically the same as that of the
meet the basic requirements of the ICN. The genus. Thus, the Agaricomycetes has the same
terms valid and invalid are misused by a num- type as that of the genus Agaricus, the type of
ber of authors, often as a way of indicating Agaricus campestris L.
disagreement with a particular taxonomic con- Priority applies to names at the rank of
cept of someone else. A legitimate name is a family and below where each name may bear
valid name that is in accordance with the rules, only one correct name, with a few exceptions.
while an illegitimate name is a valid one that is Names do not have priority outside of the rank
defined as such by the articles listed in ICN Art. at which they were published, and the correct
6.4. There are two common ways in which a name is the legitimate name that was effectively
name may be illegitimate. The first is by being a published first. Priority does not operate above
later homonym, or the same name validly pub- the rank of family. Generally speaking, there are
lished at a later date, like the imaginary cases of three major ways that the principle of priority
“Amanita hibbettii Vilgalys 2011” and the ille- may be limited for fungal names. For fungi, the
gitimate “Amanita hibbettii J.W. Taylor 2013.” valid publication of names is treated as begin-
The second is by being superfluous, which can ning on 1 May 1753 with Species Plantarum, a
be thought of as simply renaming an already work of Linnaeus, and fungal names that may
182 A.M. Minnis
have existed before this date have no status tional information on the ICN and matters not
under the Code. However, this has not always summarized herein, refer to the complete ICN
been the case, and the starting point for rusts, (McNeill et al. 2012) and the earlier chapter by
smuts, and gasteromycetes used to be Persoon’s Hawksworth (2001).
Synopsis Methodica Fungorum, while that of
other fungi (excluding slime molds) was Fries’
Systema Mycologicum. Because of the change in A. Transition to One Name Per Fungus
starting point for these groups and the resulting
and unfortunate need to change many names, Perhaps the most significant change to the
sanctioning was adopted to give priority naming of fungi found in the current ICN
against earlier names to names adopted in the (McNeill et al. 2012) is the elimination of provi-
works that used to be the former starting point sions that allowed multiple names for the same
for fungi; additional information relating to taxa of non-lichen-forming ascomycetous and
sanctioning and its history was summarized basidiomycetous fungi, sometimes referred to
by Hawksworth (2001). Conservation via ICN as dual nomenclature. The following sections
Article 14 is another procedure that limits pri- consider historical aspects and how the transi-
ority and is employed when strict application of tion to a one-name-per-fungus system of clas-
the rules results in disadvantageous changes. sification was implemented as well as the
Names may be conserved to have priority over specific rules of the ICN and how they will be
competing names or to have a different type used to make the transition happen. Terms
that preserves usage in order to serve stability. such as anamorph, teleomorph, and holo-
Rejection via ICN Article 56 is another proce- morph, though still present in the ICN, are
dure that limits priority. It is similar to conser- abandoned as much as possible in accordance
vation in some ways, but a rejected name may with the preferences of mycologists including
not be used and can be thought of as being Hawksworth (2013) and Amy Y. Rossman, my
taken out of play permanently, unless later con- recent mentor. Seifert (2014), however, has
served. Additional limitations to priority in- made a good case for the continued use of
volve the transition to a one-name-per-fungus these terms. It would be hard for new students
system of classification, which will be covered of mycology to understand the historical myco-
in more detail subsequently in this chapter. logical literature without these terms and
Chapters of Division II not previously men- knowledge of earlier Codes of nomenclature.
tioned in detail include Chapter III, which
shows how to construct names at the various 1. History and Enactment of
ranks, Chapter VI, which shows how to give Rule Changes
author citations that credit the appropriate
authors of names, Chapter VII, which covers Many fungi possess complicated life cycles
all forms of name rejection, Chapter VIII, that include a number of different states, both
which deals with the names of anamorphic asexual and sexual. These states may look
fungi or those with a pleomorphic life cycle completely different, some may be found rarely
(discussed under the transition to one name or seemingly not at all, and sometimes they
per fungus below), and Chapter IX, on the occur at different times and/or places. Thus,
orthography and gender of names, which sim- mycologists have not always known whether
ply means spelling and grammar issues. Divi- or not the variously observed states of fungi
sion III, the last one of the Code, includes represent the same organism, and multiple
matters relating to governance of the Code. names for the same fungus inevitably accumu-
The last items of the Code appear as Appendix lated in the literature. During the formalization
I on the names of hybrids, a glossary, and a of the earliest Codes of nomenclature, mycolo-
couple of indexes. Seven additional appendices gists were already linking the different states of
are to be published separately and will be a number of fungi. Nevertheless, and with less
available in print and electronically. For addi- than universal agreement, multiple names were
allowed for certain ascomycetous and basidio- as evidenced by the results of the nomenclature
mycetous fungi with so-called pleomorphic life questionnaire from the International Myco-
cycles in early Codes for the sake of conve- logical Congress (IMC 9) in Edinburgh (Norvell
nience of communication. Weresub and Piro- et al. 2010). In 2010, as the next IBC in Mel-
zynski (1979) presented a historical account of bourne was approaching, Redhead (2010b)
pleomorphic fungi and fungal naming. Over the reported on the efforts of the latest group to
years, a number of modifications and clarifica- formally consider the matter in a representative
tions to the rules of naming pleomorphic fungi way, the Special Committee on Nomenclature
and fungi known only from asexual states were of Fungi with a Pleomorphic Life Cycle. Red-
adopted. These changes included rules regard- head (2010b) summarized an ideological
ing, for example, the invalidation of species impasse in which no agreement could be
names if the state of a genus differed from the reached on a course of action and noted the
state of a newly described species that was anarchy that was occurring in mycological pub-
included in that genus, which changed to the lications regarding the naming of pleomorphic
mere illegitimacy of species names under the fungi. Following the report by Redhead
same circumstances, and went to legitimacy of (2010b), proponents of a one-name-per-fungus
species names but incorrect classification in the system of classification felt an immediacy to act
wrong genus. Terms like imperfect and perfect on nomenclatural reform. This act came in the
that describe asexual and sexual states, respec- form of the “Amsterdam Declaration on Fungal
tively, were replaced by terms like anamorph Nomenclature” (Hawksworth et al. 2011). Early
(asexual state), teleomorph (sexual state), and versions of the declaration were circulated to
holomorph (fungus in all of its morphs) in mycologists to garner their support, and
formalized Codes. Because of the frequency of despite significant support, a number of propo-
rule changes and their increasing complexity, nents for one name per fungus chose not to
many problems involving the names of fungi support the Amsterdam Declaration due to its
with pleomorphic life cycles were associated general lack of details and the inclusion of
with the rules that had been in place when a unrelated issues, such as governance of the
significant amount of work was being per- Code and the naming of environmental
formed on certain groups of fungi and how sequences. The published version of the
rules changes affected this work. Oftentimes, Amsterdam Declaration (Hawksworth et al.
rules changed again before mycologists were 2011) made a strong statement on the principle
able to address issues associated with the of moving to a one-name-per-fungus system of
names of groups of fungi that were not receiv- classification, but the rule changes by which
ing substantial attention. A study of rust this was to occur were not detailed, and the
nomenclature is particularly enlightening aforementioned unrelated rider issues were
when it comes to understanding the impacts excluded from the formal declaration but put
of variously changing Codes, for example, up for future consideration. With knowledge
Judith and Rossman (2014). that this issue was to be brought up on the
As DNA sequence data became more read- floor of the IBC in Melbourne, “A Critical
ily obtainable and used in fungal taxonomy, a Response to the Amsterdam Declaration” was
small but vocal group of mycologists came to circulated to gain support by advocates of a
the conclusion that multiple names for the continuation of multiple names per fungus
same fungus were no longer convenient who strongly opposed a transition to a one-
(Hawksworth 2011; Hawksworth et al. 2011; name-per-fungus system of classification, but
Taylor 2011). The debate about whether or not because of a lack of time before the IBC, it was
to transition to a one-name-per-fungus system not published formally by Gams and Jaklitsch
of classification and how to do it went on for a (2011) until after the IBC, where the response
number of years with the small but vocal group was presented as a handout. The IBC itself was
slowly increasing in numbers but never quite attended by a small number (13) of representa-
reaching a substantial majority of mycologists, tive mycologists and a large number of botanists
184 A.M. Minnis
(nearly all of the remaining 201 attendees). forming ascomycetes and basidiomycetes are
Following an inconclusive discussion and no longer allowed. Understanding how the
presentation of the competing principles on transition to a one-name-per-fungus system of
naming fungi with a pleomorphic life cycle, classification will occur, perhaps best thought
Redhead prepared to circulate the first of his of as the continuing taxonomic process,
three proposals that were hastily developed requires examination of basic nomenclatural
and not seen or considered by more than a mechanisms, special provisions for stability,
handful of mycologists, some of whom were and considerations on how to decide which
consulted in making them (Milius 2014; Norvell names will be correct. Links are provided to
2011b). The three Redhead proposals were articles of the Code for the sake of convenience,
arranged from the most extreme to a moderate and the associated explanatory text is best con-
proposal that was in line with the majority of sidered concurrently with the text of the Code.
mycologists on the Special Committee on Hawksworth (2012) and Rossman (2014) offer
Nomenclature of Fungi with a Pleomorphic additional guidance on dealing with and under-
Life Cycle (Redhead 2010b). The mostly bota- standing the rule changes. Braun (2012) does so
nists and other attendees at the IBC passed the as well using powdery mildews as an example.
first and most extreme proposal without even The previous Art. 59 has been replaced in
considering the latter two (Milius 2014; Norvell the ICN by the new Art. 59 (McNeill et al. 2012)
2011b). A number of additional proposals for in Chapter VIII, “Names of anamorphic
and against dual nomenclature with varying fungi or fungi with a pleomorphic life cycle”:
degrees and often involving a term called tele- http://www.iapt-taxon.org/nomen/main.php?
otypification had been made and considered page¼art59.
prior to the IBC in Melbourne (McNeill and The first thing to note about the new Art. 59
Turland 2011a; McNeill et al. 2011; Norvell is that the changes are retroactive via ICN Prin-
2011a), but these were not seriously considered ciple VI. This means that all previous myco-
at the IBC because most were withdrawn logical literature must be interpreted as if these
(McNeill et al. 2011). In this way, well over 100 rules had been in place at the time they were
years of fungal naming involving multiple names published. Of course, they had not been, and
for fungi with pleomorphic life cycles passed the mycologists of other times were doing their
into history, and the transition was made to a best to follow whichever rules were in place at
one-name-per-fungus system of classification. their respective times. This also means that all
previous literature must be reconsidered to see
how this will affect the fungal names. Indexes of
2. How One Name Per Fungus Will Happen names and other databases will take some time
Under the New Rules to update because the task is enormous, so
scientists will have to check names on which
In the International Code of Botanical Nomen- they would like to publish themselves, and little
clature (Vienna Code), or ICBN (McNeill et al. existing literature involving pleomorphic fungi
2006), immediately preceding the ICN (Mel- can be taken for granted as being nomen-
bourne Code) (McNeill et al. 2012), separate claturally accurate.
names for asexual and sexual states of non- For names of newly described taxa, authors
lichen-forming ascomycetes and basidio- should not publish more than one name for a
mycetes were allowed by a number of rules fungus regardless of the number of states in its
that governed the naming of fungi, and Art. 59 life cycle. This is because simultaneously pub-
of that ICBN included most of these rules. Red- lished names that are alternative names for
head’s floor proposal at the IBC in Melbourne asexual and sexual states are invalid if published
and subsequent editorial modifications have on or after 1 January 2013. Additionally, multi-
more or less replaced Art. 59 and related provi- ple state names published for named species,
sions in their entirety, and separate names for regardless of whether the named taxon at the
the asexual and sexual states of non-lichen- same rank was typified by a sexual state or
asexual state, will be illegitimate as superfluous In recent history, most authors gave priority to
in most cases if published on or after 1 January those typified by sexual states, and they were
2013. Such a fungal name would be superfluous used widely in accordance with previous Codes.
as defined by the ICN since an already named If a taxonomic study concludes that the follow-
fungus would be renamed. ing families represent the same family, and
For names published prior to 1 January even if these were used for separate asexual
2013 when multiple names were allowed for and sexual states historically, priority deter-
certain fungi and there was an intent or implied mines the correct familial name, except when
intent of their applying to or being typified by a this principle is limited:
particular morph or state, these may be valid
and legitimate, and if so, they will compete for Competing names Correct name
priority. If proposed at the same time, the Chloridiaceae Nann. Chloridiaceae
names for the different states are not alternative 1932 (asexual) Nann. 1932
names and are heterotypic. Priority (typically Chaetosphaeriaceae
being validly published first), except when this Réblová, M.E. Barr &
is limited, will determine the correct name of Samuels 1999
(sexual)
taxa at the ranks of family and below, and this
can be thought of as the basic default mecha-
nism that requires no action. Regardless of the
type of the state of the fungus, all names are
The situation at the rank of species is per-
treated equally, and this is a departure from
haps the most complicated because a large
earlier Codes that gave precedence to names
number of possibilities might occur, especially
associated with the sexual state when the
owing to the existence or potential creation of
whole fungus in all of its forms was considered.
homonyms. In general, a taxonomic study
The following examples will illustrate how this
looking at a fungus with existing names for
works.
multiple states should look to determine the
The Amsterdam Declaration (Hawksworth
correct name for each state and then use this
et al. 2011) and most mycologists currently
to determine the correct name for the species. If
working on the transition to one name per
a taxonomic study concludes that the following
fungus primarily focus on the rank of genus.
species represent the same species, and even if
If a taxonomic study concludes that the follow-
these were used for separate asexual and sexual
ing genera represent the same genus, and even
states historically, priority determines the cor-
if these were used for separate asexual and
rect species name, except when this principle is
sexual states historically, priority determines
limited:
the correct generic name, except when this
principle is limited:
Competing names Correct name?
Competing names Correct name Chloridium virescens
Chloridium Chloridium (Pers.)
Link 1809 Link 1809 W. Gams &
(asexual) Hol.-Jech. 1976
Melanopsamella (asexual)
Höhn. 1929 Melanopsamella
(sexual) vermicularioides
(Sacc. & Roum.)
Réblová, M.E. Barr &
Samuels 1999
Though little considered prior to enactment (sexual)
Four possibilities:
of the new rules, a number of familial names Chloridium virescens, based on the basionym,
apply separately to asexual and sexual states. Dematium virescens Pers. 1797
(continued)
186 A.M. Minnis
would apply to the sexual state and have prece-

Competing names Correct name? dence for the name of the whole fungus in all of
Chloridium vermicularioides its states. Under Art. 59 of the ICN (McNeill
Melanopsamella virescens et al. 2012), Example 2 shows how in this situa-
Melanopsamella vermicularioides, based on the
basionym, Eriosphaeria vermicularioides Sacc. & tion the new species name reverts to a new
Roum. 1883 combination, Mycosphaerella aleuritidis
In this case, we know that Chloridium has priority at (Miyake) S.H. Ou, and the type changes from
the rank of genus, and the species epithet comes a specimen with asci and ascospores to the
from Dematium virescens, which has priority at the older specimen with conidia that typifies the
rank of species:
Correct name basionym, a process I refer to as detypification
Chloridium and retypification. The specimen with asci and
virescens ascospores is no longer a type. It has been
(Pers.) suggested (Hawksworth 2012) that this situa-
W. Gams & tion has not occurred very often. In my work
Hol.-Jech.
1976 updating the fungal databases at the U.S.
National Fungus Collections (Farr and Ross-
man 2014, and ongoing), however, I found
this situation to be more common than easily
In this simple case, no new combination overlooked because of its rarity, especially for
was required. But this may not always be the rust fungi. Authors addressing taxonomy and
case. nomenclature should be aware of this phenom-
The principle of priority does not operate enon because it would be especially significant
above the rank of family. Suppose only the ordi- if the different types did not represent the same
nal name Chaetoshaeriales Huhndorf, A.N. Mill. fungus, which seems to happen from time to
& F.A. Fernández 2004 is available. An author time in the era of molecular splitting.
could publish an additional name, for example, Universal recognition of one aspect of tran-
Chloridiales Minnis 2014, and use this as the sitioning to a one-name-per-fungus system of
correct name at that rank if it was desirable. classification was given to the need to provide
Author citations and types of names asso- some means for having stability of fungal
ciated with pleomorphic fungi may be changed names when the basic nomenclatural mechan-
as a result of the deletion of various provisions isms did not allow for a smooth transition to a
of Art. 59.6 found in the previous ICBN one-name-per-fungus system of classification.
(McNeill et al. 2006) found here: http://www. Along with typical conservation against com-
iapt-taxon.org/icbn/main.htm. peting synonyms via ICN Article 14.1 and rejec-
In some instances, like Example 6 of the tion of names via ICN Article 56.1, additional
previous Art. 59.6 (McNeill et al. 2006), the provisions for the stability of fungal names
Code would automatically create a new name were included in the ICN. The three major
based on a type that matches the appropriate provisions follow.
morph or state. This would effectively change The first of these is ICN Art. 14.13 (McNeill
the supposed new combination Mycosphaerella et al. 2012): http://www.iapt-taxon.org/nomen/
aleuritidis (Miyake) S.H. Ou based on Cerco- main.php?page¼art14.
spora aleuritidis Miyake into a new species Under this rule, fungal names, excluding
name, for example, Mycosphaerella aleuritidis lichen-forming fungi, may be added to lists of
S.H. Ou, with a different author citation, and accepted names and treated as if conserved
the type would change from an old specimen following submission of these lists to the Gen-
with conidia to a newly created type that is a eral Committee (GC) of the International Asso-
different specimen with asci and ascospores. ciation of Plant Taxonomists (IAPT) and
Cercospora aleuritidis would remain typified subsequent review and approval by both the
by the specimen bearing conidia and only Nomenclature Committee for Fungi (NCF)
apply to the asexual state, and M. aleuritidis and the GC. Accepted names on approved lists
are then added to Appendices of the Code along ual state names, will have to proceed by a more
with competing names against which they are democratic process that involves the mycologi-
conserved. Subcommittees and international cal community. Historical precedence of teleo-
groups established by and in support of the morph-typified names is also continued to
NCF are to aid in the assembly of lists some degree by default. It is worthwhile noting
and make recommendations regarding their that the existing Example 3 involves two genera,
approval. Fortunately, proposals by Redhead Magnaporthe and Pyricularia, that are no lon-
(2010a) on having an option to publish some ger considered to be congeneric (Luo and
of the Appendices of the ICN separately and/or Zhang 2013), and ICN Art. 57.2 does not
in electronic format only were approved at the apply. The downside is that a liberal interpreta-
IBC in Melbourne (McNeill and Turland 2011b; tion of the phrase “widely used” will create a lot
McNeill et al. 2011) since the Appendices are of work for mycologists and the appropriate
likely destined to become rather large during committees.
the transition to one name per fungus! Though several options are available to
The second of the provisions is ICN Art. protect against undesirable changes, ICN Arts.
56.3 (McNeill et al. 2012): http://www.iapt- 56.1 and 56.3 should be employed judiciously
taxon.org/nomen/main.php?page¼art56. and only after careful consideration since
Under this rule, fungal names, excluding rejected names may not be used later if taxo-
those of lichen-forming fungi, may be added nomic revisions create a reason to do so with-
to lists and treated as if rejected under ICN out the names first being conserved under ICN
Art. 56.1. The approval process is the same as Art. 14.
the one that applies to Art. 14.13. Names on The NCF of the International Association
approved lists are also added to the Appendices for Plant Taxonomy (IAPT) plays a major role
of the Code. Names to be treated as rejected in reviewing and approving lists of accepted
may become eligible for use only by subsequent and rejected names, and the International Com-
conservation via ICN Art. 14. mission on the Taxonomy of Fungi (ICTF;
The third provision is ICN Art. 57.2 http://www.fungaltaxonomy.org/) is assisting
(McNeill et al. 2012): http://www.iapt-taxon. with the coordination of working groups on
org/nomen/main.php?page¼art57. specific fungal groups that are charged with
This rule addresses pleomorphic fungi, making these lists. Since it is desirable to
excluding lichen-forming fungi, that have both make the transition to one name per fungus a
widely used anamorph-typified and widely used community-wide and inclusive effort, authors
teleomorph-typified names prior to 1 January are encouraged to contact the NCF and/or the
2013. In such cases, anamorph-typified names ICTF before publishing major revisions of
with priority are not to take precedence over important taxonomic groups of pleomorphic
teleomorph-typified names unless and until fungi affected by the changes to a one-name-
either a proposal to reject the anamorph- per-fungus system of classification.
typified names via Art. 56.1 or to put them on In determining whether to accept the basic
a list to be treated as rejected via Art. 56.3 or a nomenclatural mechanisms or employ one of
proposal to conserve the teleomorph-typified the tools allowing for the stability of fungal
names via Art. 14.1 or to put them on a list to names, it is difficult but necessary to remain
be treated as conserved via Art. 14.13 has been without prejudice because it is often easy to
submitted and rejected. This rule is perhaps prefer certain names for arbitrary or personal
one of the most difficult to interpret because reasons. A number of considerations on name
no guidance is given as to what it means to be choice are listed in what follows.
widely used. In any case, the benefit of this is Should genus choice be correlated with
that any major revision of groups, where either higher ranks?
both asexual and sexual state names were fre-
Trichoderma Pers. 1794 versus Hypocrea Fr.
quent and where sexual state names were given
1825
priority because of their state over older asex-
188 A.M. Minnis
Trichodermataceae Fr. 1825 versus Hypocrea- man et al. (2013b), is one in which going with
ceae De Not. 1844 the later name based on an asexual state results
Hypocreales, Hypocreomycetidae in the fewest changes, Bipolaris has more
names, and the asexual state is found more
In this example, Trichoderma and Tricho- commonly in nature. Significant literature
dermataceae have priority at their respective regarding genetics and genomics, unfortu-
ranks, but higher rank classification is based nately, uses the name Cochliobolus (Rossman
on the name Hypocrea. Rossman et al. (2013a) et al. 2013b).
recently proposed the use of Trichoderma, an Should we prioritize priority?
asexual genus with priority, instead of Hypo- Scientific discovery is typically credited to
crea, a sexual genus used as the correct name the first person to find something new and
for the whole fungus under the previous Code, publish on it. One of the six major principles
when both were widely used. Rossman et al. of the ICN is the principle of priority, which
(2013a) also favored the name Hypocreaceae, means that names that were validly published
though it lacks priority. In this case, the first shall be correct in most instances and the
accepted name of the genus does not correlate author citations of these names are those of the
with higher ranks, and Hypocrea will not be authors that should receive credit. One of the
used at the rank of genus or below. issues with lists of accepted and rejected names
What if I don’t like the familial name for of fungi is that such lists totally disregard the
these fungi? principle of priority and the authors who origi-
Planistromellaceae M.E. Barr 1996 nally discovered and described fungi are not
Kellermania Ellis & Everh. 1885 credited for this work. For this reason, some
¼ Piptarthron Mont. ex Höhn. 1918 might not prefer to liberally or arbitrarily use
¼ Alpakesa Subram. & K. Ramakr. 1954 the lists of accepted and rejected names at all or
¼ Planistroma A.W. Ramaley 1991 for a large number of cases. Others may feel it is
¼ Planistromella A.W. Ramaley 1993 not necessary to give credit where credit is due
since other factors are believed to be more
In this example, if all of these genera are important.
treated as synonyms, as was done by Minnis What do users of fungal names such as
et al. (2012), the only available familial name is plant pathologists and medical mycologists
Planistromellaceae, which is not based on an prefer and how do we get the word out?
accepted generic name. Alternatives include Users of fungal names in more applied
living with this or describing the new family fields are often frustrated by frequent name
Kellermaniaceae Minnis 2014 and using one of changes associated with scientific progress.
the tools for fungal name stability such as ICN Perhaps it would be wise to consider how to
Arts. 14.1, 14.13, 56.1, and 56.3 to make the later make the transition to one name per fungus
name correct. that resulted from philosophical differences in
What results in the fewest changes or max- naming to be as painless as possible for users in
imum stability? these fields. Wingfield et al. (2012) and Zhang
Which genus has more species names in it? et al. (2013) have provided valuable expla-
Which genus appears most in the litera- nations of the transition to one name per fun-
ture and in the most significant literature? gus and the benefits of doing this to the
What morph or state is found most com- community of plant pathologists. Hoog et al.
monly in nature? (2014) have provided a similar resource for
All four of these are worthy considerations. the medical community.
The case of Cochliobolus Drechsler 1934 versus Some general observations and thoughts
Bipolaris Shoemaker 1959, as noted by Ross- on the transition to one name per fungus:
l Taxonomic revisions and new species and Turland (2011b), and Norvell (2011b). The
descriptions are hardly justifiable without following sections consider the most significant
looking at the whole fungus in all of its changes.
morphs or states, for example, all of those
existing names, and taxonomic integration
1. Effective Publication via Electronic
is essential.
Publication
l Mycologists must value the discovery and
description of the other morphs or states for Earlier versions of the Code did not allow for
named species as key taxonomic and myco- effective publication via electronic formats
logical contributions because the recognition since only printed matter was available for
for doing so may not be the same as in the most of the time that a formal Code has been
past. in existence. An urgent need to update the Code
l We will have what I refer to as “The rise of the resulted from high costs associated with pro-
anamorphs” since, in general, asexual states ducing printed formats, the proliferation of
for many groups are more common and con- electronic-only publications, and the issuance
spicuous than sexual states and generic con- of material in both printed and electronic mat-
cepts for them were elaborated more quickly ter, not always at the same time, for the same
[except when later discovered to be polyphy- publication. The Special Committee on Elec-
letic hodgepodges, e.g., Acremonium (Sum- tronic Publication carefully considered the
merbell et al. 2011)] and asexual state names issue and presented a series of proposals to
will tend to be favored by the basic nomen- address effective publication via electronic
clatural mechanisms. publication (Chapman et al. 2010), and these
l Mycologists need to work together to make were accepted at the IBC in Melbourne (McNeill
the transition to one name per fungus. Indi- et al. 2011) and implemented in the ICN. The
viduals should be discouraged from taking relevant changes regarding effective publica-
matters into their own hands and enforcing tion by electronic means are found in
their preferences on others from positions of Chapter IV of the ICN and are summarized in
power, for example, a journal editor. what follows. Knapp et al. (2011) provide addi-
l Some groups will require significant study tional guidance.
before a wise choice can be made. In rusts, Electronic material distributed on or after 1
for example, we have yet to phylogenetically January 2012 in Portable Document Format
place the type of the genus Uredo, Uredo (PDF) in an online publication with an Interna-
betae. Crous et al. (2014) have provided sug- tional Serial Number (ISSN) or an International
gestions for addressing types of genera and a Standard Book Number (ISBN) is effectively
call to address them. published, a key component of valid publica-
l Obscure groups may not be dealt with as tion. Electronic material distributed before this
quickly. date is not effectively published. Online is
l Groups such as the NCF and the International defined as being accessible via the World
Commission on the Taxonomy of Fungi will Wide Web. Only the final version of an elec-
make the process more democratic. tronic publication is effectively published. In
press or preliminary versions that may be sub-
sequently altered and are not considered as
B. Other Major and Minor Changes being the final versions by the publisher are
in the ICN not yet effectively published. The content of
electronic publications must not be altered
A number of other significant changes to the after effective publication, and any such
naming of fungi are found in the current ICN changes are not effectively published. Content
(McNeill et al. 2012), and these have been in external sources, such as something accessed
reported by McNeill et al. (2011) and summar- via a hyperlink, is not part of the publication.
ized variously by Hawksworth (2011), McNeill Content is that which stands alone as what the
190 A.M. Minnis
publisher considers to be final, and preliminary there was substantial support for modifying
page numbers or a lack of them does not pre- this requirement at the International Mycologi-
vent a publication from being effectively pub- cal Congress (IMC 9) in Edinburgh (Norvell
lished, even if page numbers are added or et al. 2010). Demoulin (2010) subsequently
altered later. This can be confusing in some acted on the support at the IMC 9 and proposed
cases because it must be determined which that Latin or English be acceptable for naming
version is considered final by a publisher and of new fungal taxa. A number of additional and
what must be cited as the correct page numbers related proposals were made, including that
and date of effective publication. The date of any language be allowed (McNeill and Turland
effective publication is when the printed matter 2011a; Smith et al. 2011), which was not sup-
or electronic matter became available, and this ported by the NCF (Norvell 2011a) and was
dictates the date used to determine priority rejected in the preliminary mail vote for the
of names. When both electronic and printed IBC in Melbourne (McNeill et al. 2011; Smith
materials are issued for the same publication, et al. 2011). At the IBC in Melbourne, the
the date of effective publication is treated as Demoulin (2010) proposal was adopted and
being the same and is that of whichever form modified so that the Latin or English require-
comes first. ment was broadened to include all organisms
covered by the Code and set to take effect on 1
January 2012 (Hawksworth 2011; McNeill et al.
2. Latin or English for Valid Publication of 2011; Norvell 2011b; Smith et al. 2011). New
Names of New Taxa names described from 1935 up until this date
in 2012 must still have descriptions and/or
ICN Art. 38.1 states, with a few exceptions and diagnoses in Latin in order to be valid.
among other things, that valid publication of
the name of a new taxon requires a description
and/or diagnosis of the taxon or a reference 3. Registration of Fungal Names
to an effectively published description and/or
diagnosis. In earlier versions of the Code, one The mycological literature has always been
of the other requirements was that the descrip- scattered, and taxonomists must be collectors
tion or diagnosis of new fungal taxa must be in if they are to be scholars. In the earliest days,
Latin, and the Preface of the ICN provides a relatively few copies of mycological publi-
brief history of this requirement and how its cations were made, and their distribution relied
effective date was set to 1 January 1935. This upon relatively primitive means of transport-
requirement has not always been unanimously ation. Due to the slowness in getting the word
supported, and several attempts to modify or out, species were often described multiple
remove this requirement, some with success, times. Early indexes of fungal names like Sac-
were made in subsequent years (Hawksworth cardo’s Sylloge Fungorum gathered lists of
2011; Smith et al. 2011). Like others who flouted described names, their place of publication,
the rules of the Code they did not like, Dearness and relevant information about the fungi in
(1941) refused to describe new species of fungi one place. Hawksworth (2001) provided a list
in Latin because it was a dead language and of many of the historical fungal indexes and
would take up too much valuable space in noted in particular the value of the Index of
Mycologia. This particular instance of open Fungi. Index Fungorum (http://www.indexfun-
disregard for the rules was noted during rou- gorum.org/), closely allied with the Index of
tine updating of the SMML Fungal Databases Fungi, has and continues to be a valuable
(cited currently as Farr and Rossman 2014), source as an online global fungal nomenclator.
and Braun et al. (2009) validly published the As with the issue of effective publication via
previously invalid species of interest to them in electronic publication, the proliferation of
accordance with the rules. Many mycologists large numbers of journals and in numerous
continued to detest the Latin requirement, and formats provided challenges to mycologists, in
this case in the assembly and awareness of fungal name, are given to repositories by the
published fungal names. I remember having authors. The NCF officially approved three
trouble with a conservation proposal due to repositories, Fungal Names, Index Fungorum,
not knowing about a recent article in a regional and MycoBank, that had agreed to coordinate
faculty journal, the Revista Facultad Nacional their efforts, and the NCF noted that a number
de Agronomı́a Medellı́n. As related challenges of details would need further consideration
grew, financial support of existing indexes (Redhead and Norvell 2013).
began to wane, and the necessary personnel
for upkeep became increasingly challenged by
the volume of work. The idea of a formal regis- 4. Minor Changes
tration of names as a component of valid pub-
lication has been around for a while, but many The Code gets a name change. Botany has his-
attempts to implement it were not successful torically included the study of more than just
(Hawksworth et al. 2010; Hawksworth 2011). In plants and was inclusive of, for example, algae
recognizing the problem, the large number of and fungi. As the phylogenetic differences
undescribed fungi, and the need to have help in between these groups became more and more
assembling the kind of index that mycologists emphasized and understood, botany tended to
need, MycoBank was launched as an experi- become more restricted to just plants, and some
mental and voluntary repository for newly pub- major departments even went so far as to
lished fungal names and associated data on change their name from Department of Botany
these fungi (Crous et al. 2004). A key compo- to Department of Plant Biology. Mycologists,
nent for success was requiring that the authors wondering where they might be included and
themselves be responsible for entering names feeling slighted, began to think that the name
and data. Use of MycoBank increased rapidly, International Code of Botanical Nomenclature
and several mycological journals required that might be misleading. Rambold et al. (2013)
authors of fungal names employ it (Hawks- have also discussed the importance of fungi
worth et al. 2010; Hawksworth 2011). Signifi- and the need for more recognition of the field
cant support for formally requiring the of mycology. To address this matter, Hawks-
registration of fungal names received support worth et al. (2009) made a series of proposals to
at IMC 9 in Edinburgh (Norvell et al. 2010). As a amend the ICBN that would include mycology
result, Hawksworth et al. (2010) formally pro- in the title, add fungus-related terms through-
posed changing the Code so that key informa- out, and transfer some aspects of governance of
tion would be entered into an approved fungal naming from the International Botanical
repository and that the identifier provided dur- Congress to the International Mycological Con-
ing this process must be included in the publi- gress. Hawksworth et al. (2009) also noted that
cation of fungal names as a requirement for some mycologists preferred to break off and
valid publication. The NCF recommended establish an independent code for mycology.
approval (Norvell 2011a), and registration of At the nomenclature session of IMC 9 in Edin-
fungal names was formally approved at the burgh, mycologists generally did not support
IBC in Melbourne (Hawksworth 2011; McNeill an independent code, provided the name of
and Turland 2011b; McNeill et al. 2011; Norvell the current ICBN was changed, but did support
2011b). Article 42 of the ICN (McNeill et al. transfer of governance to an IMC (Norvell et al.
2012) is the relevant article on the registration 2010). The NCF recommended approval of a
of fungal names (http://www.iapt-taxon.org/ name change, the addition of fungal termino-
nomen/main.php?page¼art42). Thus, all fungal logy, and the election of members of the NCF at
names published on or after 1 January 2013 an IMC, but the NCF did not support transfer of
must cite an identifier issued by a recognized governance (Norvell 2011a). Proposals on a
repository; otherwise, it is not valid. Identifiers name change and adding fungal terminology
are issued when minimum elements, for exam- were so well supported at the IBC in Melbourne
ple, those necessary for valid publication of a that algae were also included in the title, and the
192 A.M. Minnis
Code was renamed the International Code of (2009) discussed this and made formal propo-
Nomenclature for algae, fungi, and plants via a sals to modify the ICBN to do just that, and
floor proposal (Hawksworth 2011; McNeill and Demoulin (2010) provided another proposal to
Turland 2011b; McNeill et al. 2011; Norvell extend this idea to other organisms historically
2011b). Proposals on governance for fungal treated under other codes. These proposals
names were withdrawn on the understanding passed at the IBC in Melbourne (Hawksworth
that a subcommittee would examine the issue at 2011; McNeill and Turland 2011b; Norvell
a future date (Hawksworth 2011; McNeill et al. 2011b). Practically speaking for mycologists, if
2011; Norvell 2011b). Mycologists did seem to you want to examine nomenclatural issues of
get everything they really wanted or needed at Microsporidia and/or describe new taxa, learn
the IBC and did benefit from having assistance to use the ICZN (International Commission on
from numerous experts in nomenclature who Zoological Nomenclature 1999).
happen to study plants. As for the new title of Typification of sanctioned names. Under
the Code, apologies are extended to those who the ICBN (Vienna Code) (McNeill et al. 2006),
study slime molds and other funguslike organ- typification of sanctioned names was confus-
isms; lowercase fungi is the best that could be ing, and sanctioned names could be typified
done to cover these organisms (Hawksworth “in the light of anything associated with the
2011)! name in that work,” meaning the sanctioning
Microsporidia excluded from ICN. Micro- work. The protologue or original description of
sporidia are intracellular parasites that invade a sanctioned name often predated the sanction-
host cells via injection by polar filaments (see ing work, and other rules of the Code stated
Didier et al., Chap. 5, Vol. VII, Part A). These that a lectotype is to be chosen and designated
organisms are medically important and have from the original material (material associated
been studied historically by zoologists. It turns with the protologue) used originally to describe
out that they are fungi, and Redhead et al. a taxon if no holotype exists. However, material
(2009) discussed the nomenclatural issues associated with the sanctioning work may not
resulting from this realization. Basically, Micro- have been part of the original material, and it
sporidia had been described under the rules of was unclear as to what such a type, if designated
the International Code of Zoological Nomen- with later material, should be called and which
clature (ICZN). This was problematic because of the potentially conflicting rules should be
Latin diagnoses were not provided, as was followed when typifying a sanctioned name.
required for fungal descriptions, which meant Other complications related to this matter are
that names of numerous Microsporidia would not discussed here, but Redhead et al. (2010)
be invalid. Under the ICBN (Vienna Code) proposed a set of rule changes to address this
(McNeill et al. 2006), rules were put into place matter that would either delete the rule about
to address this situation, and these stated that if typifying in light of anything associated with
an author of a fungal name thought the organ- the name in that sanctioning work (ICBN Art.
ism was something covered by the rules of 7.8) (McNeill et al. 2006) or modify a number of
another code, then the name was validly pub- rules to create a new kind of type called the
lished as long as that other code was followed, sanctiotype for sanctioned names. Perry
even if the organism was a fungus. This seemed (2011) provided competing proposals that
to solve the problem, and a large number of would not employ the term sanctiotype. The
names of Microsporidia were saved from NCF recommended Redhead’s proposals relat-
being declared invalid, except that those working to the introduction of the sanctiotype (Nor-
ing with Microsporidia began to say that they vell 2011a). At the IBC in Melbourne, the
were fungi and still kept using the ICZN. Those proposers of these competing options and a
researching Microsporidia subsequently asked few others met to resolve differences, and
for them to be excluded from the ICBN and when it became apparent that the term sanctio-
covered by the ICZN, which could already do type was hated enough by most at the IBC to be
so under one of its articles. Redhead et al. rejected, a compromise was reached (Norvell
2011b). Under the ICN, a sanctioned name may nomenclatural procedures. For questions not
be typified by an element associated with the covered here, please consult an expert and/or
protologue and/or the sanctioning work, and the current version of the International Code of
this is a lectotype. Several works (Hawksworth Nomenclature for algae, fungi, and plants (ICN)
2011; McNeill et al. 2011; Norvell 2011b) pro- at http://www.iapt-taxon.org/nomen/main.
vide more details on the compromise and the php?page¼title. Following several changes that
changes to the typification of sanctioned were adopted at the IBC in Melbourne in July
names. 2011, this FAQ has been modified from earlier
Problems with fungal cultures as types. versions.
Living fungal cultures may not serve as types Note: In several examples that follow, the
of fungal names, and if they are proposed as the symbol is used to indicate a homotypic
type of a new fungal name, that name is invalid. (sometimes called a nomenclatural or obligate)
Cultures of fungi permanently preserved in a synonym, which is a synonym based on the
metabolically inactive state, however, are same type. The symbol ¼ should be used to
acceptable as types. Having read a number of indicate a heterotypic (sometimes called a taxo-
publications, I have found it depressing to note nomic or facultative) synonym, which is a syno-
how many fungal names are invalid because nym based on a different type.
authors overlooked this requirement of valid
publication. Additionally, many authors fail to
note the preservation status of cultures, which 1. How Do I Describe New Taxa at the Rank of
requires subsequent investigation. Nakada Species?
(2010) noted this problem and several other
issues such as culture collections periodically a) Validation
reviving cultures and cultures not being depos- Ensure that the new name is in Latin (or is
ited in an inactive state. To help clarify matters acceptably Latinized). Construct the name
related to valid publication, Nakada (2010) pro- according to ICN Arts. 23 and 60. Make sure
posed that a recommendation be added to the that the epithet conforms to recommendations
Code that the phrase “permanently preserved and conventions if dedicating the name to a
in a metabolically inactive state” be given when person or place or referring to growth on a
a culture is designated as a type. This proposal substrate or host. Pay special attention to the
was adopted at the IBC in Melbourne (Hawks- gender of generic names and corresponding
worth 2011; McNeill et al. 2011; Norvell 2011b). adjectival species epithets. (However, this rule
It is often easiest to dry a culture and deposit it does not apply to epithets that are nouns; they
in a herbarium as the type of a taxon and retain their own gender and never change their
submit a subculture obtained prior to drying endings.)
to culture collections as the ex-type. Provide an English or Latin description or
diagnosis for your taxon (or provide a full and
direct reference to a previously published
English or Latin description or diagnosis
C. Practical Notes on Using the ICN uniquely applicable to your fungus). Have the
The following notes on how to perform fre- diagnosis/description checked by an expert in
quently used nomenclatural procedures are the language that is used.
taken and modified from a document prepared l You may use a single English or Latin
by A.M. Minnis, K.A. Seifert, S.A. Redhead, description or diagnosis (i.e., a descriptio
and R.E. Halling for Mycologia (http://www. generico-specifica) for both a new genus and
mycologia.org/site/misc/FAQvers2.xhtml). a new species if there is a single species in the
Nomenclatural Procedures FAQ (ICN/Mel- new genus and both are new.
bourne Code): Help and Checklist for Authors.
This document is designed to help authors Designate a holotype (authors must use the
avoid common mistakes in frequently used word “holotypus” or “holotype,” alternatively
194 A.M. Minnis
“typus” or “type”) and cite the single herbar- with another of two repositories, Fungal Names
ium or place the specimen is housed. See Index or Index Fungorum, that have been officially
Herbariorum or other biorepository for proper approved for this purpose (Redhead and Nor-
institutional codes. The phrase “hic designatus” vell 2013) and the listing of the identifier
(designated here) is not required. provided by that repository in the protologue
is also acceptable for valid publication.
l Designate a single collection made at one
place and time represented as a single speci-
men in a single institute (list the collector,
date, and collection number); b) Legitimization
l or a permanently metabolically inactive cul- Ensure that you do not publish a later hom-
ture or tissue (e.g., frozen, dried, or pickled) onym, a name spelled exactly like an earlier
designated by a unique reference and in a valid name (regardless of whether this is legiti-
single institute (write “permanently pre- mate or illegitimate), or one confusingly closely
served in a metabolically inactive state” and spelled. Later homonyms are illegitimate.
indicate the method of preservation to ensure You can and should use MycoBank and
that readers know the type is not a living Index of Fungi or Index Fungorum to check
culture or one that is in a temporarily inactive for earlier potential fungal homonyms and
state); Index Kewensis and other sources via the Inter-
l or an effectively published illustration (con- national Plant Names Index or Tropicos or
currently or previously published) if and only AlgaeBase for many “botanical” (covered by
if there are technical difficulties preserving a the ICN) names. Check Index Nominum Gen-
collection of a microfungus. If previously ericorum to ensure that the generic name in
published, a full and direct reference to the which you are publishing your species is
place of prior publication is required. uniquely fungal. If there are other valid “botan-
ical” homonyms at the generic rank, consider
Do not indicate that the “holotype” is in that there is the potential for you to create a
several institutes. Duplicates of the holotype later homonym at the species rank to a species
collection are isotypes when deposited else- in that other genus. You should check GenBank
where or are otherwise separate from the holo- and the World Wide Web for any such uses
type. Cultures derived from the holotype, or regardless of whether they are valid or legiti-
used to generate the holotype, are themselves mate.
not types. Because preserved cultures can serve Homonyms for Bacteria and Archaea and
as “type,” do not indiscriminately cite both a for animals (including protozoa) are covered
specimen and a culture as type. Ensure one is by other codes. You may create homonyms,
specifically designated as holotype and specifi- but this should be avoided.
cally state where that single type is located.
(Otherwise the name will be invalid.) Example (fictional; not intended for valid publication):
Do not provide alternative Latin names for Lodgea pini E.E. Sm., sp. nov.
the same taxon. (Otherwise all will be invalid MycoBank MB9876543
unless allowed by ICN Art. 59 for pleomorphic English diagnosis: this species is distinguished
from others in the genus by its brown pileus and yellow
fungi until the end of 2012.) pore surface.
Do not suggest that your new scientific Typus: USA: Idaho, Valley Co., near McCall, on soil
name is tentative, provisional, a temporary fix associated with stands of Pinus sp., 07/17/1910, coll. E.
or express any other doubt about accepting a E. Smith, E.E. Smith 22 (F).
name for a new taxon. (Otherwise it is invalid.)
You must register your name with Myco- Seifert and Rossman (2010) have provided
Bank, obtain a MycoBank registration number, some additional guidance on describing new
and present it in the protologue. Registration fungal species.
2. How Do I Describe New Taxa at the Rank of 4. How and When Do I Designate a Lectotype
Genus? for a Species?
Construct the name according to ICN Arts. 20 A lectotype is designated when there was no
and 60. Follow recommendations 20A.1(h) and holotype in the original description or if it has
60B when dedicating the name of the genus to a been lost or destroyed. Rarely, a lectotype may
person. Check databases (see earlier discus- be designated when the holotype belongs to
sion) to make sure the generic name has not more than one taxon (see ICN Art. 9 for more
been used already. A MycoBank, Fungal Names, information).
or Index Fungorum registration number must A lectotype is a designated specimen or
be obtained and listed for all new names. illustration that is part of the original material.
An English or Latin diagnosis or descrip- Simply speaking, original material consists of
tion must be supplied when describing a new specimens and published or unpublished illus-
genus or any other taxon. Have the diagnosis/ trations that were definitely used in the original
description checked by an expert in the lan- description of a name. For sanctioned fungal
guage that is used. names, the former material and/or any element
Designate the type of the genus by citing the associated with the name in the sanctioning
name of one previously or concurrently validly treatment (equivalent to original material)
published species. Use the word “typus” or may be used for lectotypification (see ICN Arti-
“type.” cles 9.2, 9.3, and 9.10).
When designating a lectotype for a name
Example (fictional; not intended for valid publication): that is not sanctioned, priority must be given to
Lodgea E.E. Sm., gen. nov. the following types of materials in the order
MycoBank MB457896 given:
Latin diagnosis: Similis Hygrocybe sed hymenio
poroso differt. 1. Isotype (see ICN Art. 9.4);
Typus: Lodgea pini E.E. Sm. 2. Syntype (also possibly an isosyntype) (see
ICN Art. 9.5);
3. What Is an Ex-type? 3. Paratype (see ICN Art. 9.6);
4. Uncited specimen, uncited illustration,
A living culture obtained from a type may be
cited illustration.
referred to as an ex-type (see ICN Article 8 for
more information). It is linked to the type, but On or after 1 January 1990, the herbarium
it is not the same as the type. Depending on housing the specimen or unpublished illustra-
the nature of the type, it may be called, for tion must be cited, and on or after 1 January
example, an ex-holotype, an ex-neotype, or an 2001, the term “lectotypus” or “lectotype” must
ex-epitype. Such cultures, as well as the place be given along with the phrase “hic designatus”
where the living culture is preserved, should be or “designated here.” A full and direct reference
indicated in publications, especially for new to the place of publication of previously pub-
taxa. This information is often listed next to lished illustrations should be given, and it is
the type designation. ideal if the illustration can be reproduced in
the current work. Lectotypification is only
Example (fictional; not intended for valid publication): achieved through effective publication. In the
Lodgea pini E.E. Sm., sp. nov. case of accepted names based on a basionym
MycoBank MB457896 (legitimate, previously published name on
Latin diagnosis: Pileo brunneo. Poris luteis.
Typus: USA: Idaho, Valley Co., near McCall, on soil
which a new combination or name at a new
associated with stands of Pinus sp., 07/17/1910, coll. E. rank is based and that provides the final epithet
E. Smith, E.E. Smith 22 (F); ex-type CBS 4567493. or stem of such a name) or replaced synonym
196 A.M. Minnis
(valid name on which a replacement name is replaced synonym should be the name that is
based and that does not provide the final neotypified.
epithet, etc.), the basionym or replaced syno-
nym should be the name that is lectotypified. Example (fictional):
Amanita nyssae (Peck) Peck, Mycologia 5: 9. 1913.
Example (fictional): Agaricus nyssae Peck, Mycologia 2: 39. 1910.
Pseudocercospora nyssicola (Peck) Peck, Mycolo- Neotypus of Agaricus nyssae (hic designatus):
gia 3: 377. 1911. USA: Louisiana, near Baton Rouge, scattered, asso-
Cercospora nyssicola Peck, Mycologia 1: 100. ciated with Nyssa sylvatica, 10/25/2001, coll. Tulloss,
1909. Tulloss 2211 (NYS).
Lectotypus of Cercospora nyssicola (hic designa-
tus): USA: Louisiana, near LSU, on leaves of Nyssa, 6. How and When Do I Designate an Epitype for
07/12/1907, coll. Peck, Peck 1239 (BPI). a Species?
5. How and When Do I Designate a Neotype for An epitype is designated when the existing
a Species? nomenclatural type (holotype, lectotype, or neo-
type) or all the original material is not sufficient
A neotype is designated when no original mate- to allow for precise application of a name. An
rial (specimens and published or unpublished example of this would be an agaric species
illustrations that were definitely used in the where the stipe of the holotype is missing but
original description of a name) exists. See earlier the stipe is critical for species recognition. In
notes on sanctioned fungal names. With rare this case, an epitype with a stipe displaying the
exception, a lectotype designated from original critical features may be designated to support
material supersedes a neotype. Thus, it is impor- the existing holotype. Many mycologists work-
tant not to overlook any original material when ing with culturable fungi designate epitype spe-
considering a neotype designation. cimens associated with a separate living culture
A neotype is a specimen or illustration, so that DNA data and cultural characters needed
preferably the former. Special consideration to recognize a species are associated with the
should be given so that the designated neotype type of a name. Others use the epitype to link
matches the material described in the protolo- asexual and sexual states of the same fungus.
gue in nearly every regard. For example, a Puc- An epitype is a specimen or illustration, but
cinia on Rosa from China should not be chosen a specimen should nearly always be employed.
as a neotype specimen for a Puccinia species Only one epitype is allowed per name. Thus, it
described on Potentilla from Ireland since there must be carefully chosen, and authors should
is a significant risk that they may not represent ensure that the epitype represents the same
the same taxon. taxon as the type it supports.
On or after 1 January 1990, the herbarium For an epitypification to be effected, the her-
housing the specimen or unpublished illustra- barium housing the specimen or unpublished
tion must be cited, and on or after 1 January illustration must be cited or, in the case of a
2001, the term “neotypus” or “neotype” must published illustration, a full and direct biblio-
be given along with the phrase “hic designa- graphic reference must be given, and on or after
tus” or “designated here.” A full and direct 1 January 2001, the term “epitypus” or “epitype”
reference to the place of publication of previ- must be given along with the phrase “hic desig-
ously published illustrations should be given, natus” or “designated here.” Additionally, the
and it is ideal if the illustration can be repro- nomenclatural type (holotype, lectotype, or neo-
duced in the current work. Neotypification is type) that the epitype supports must be explicitly
only achieved through effective publication. In cited. Epitypification is only achieved through
the case of accepted names based on a basio- effective publication. In the case of accepted
nym or replaced synonym, the basionym or names based on a basionym or replaced synonym,
the basionym or replaced synonym should be the In the second example, the new combination
name that is epitypified. also changes the rank from variety to species.
Example (fictional):
Amanita nyssae (Peck) Peck, Mycologia 5: 9. 1913. 8. How Do I Validly Publish a Replacement
Agaricus nyssae Peck, Mycologia 2: 39. 1910.
Neotypus of Agaricus nyssae (designated by R.E.
Name Also Known as a Nomen Novum?
Tulloss, Mycotaxon 82: 54. 2002): USA: Louisiana, near Replacement names are similar to new combi-
Baton Rouge, scattered, associated with Nyssa sylvatica,
10/25/2001, coll. Tulloss, Tulloss 2211 (NYS). nations, but they are made in cases where
Epitypus of Agaricus nyssae (hic designatus): USA: there is an illegitimate later homonym or
Louisiana, near Baton Rouge, solitary, associated with when the epithet of the basionym is already
Nyssa sylvatica, 10/31/2007, coll. Methven, ASM 55891 occupied in the genus where a new combina-
(EIU). tion is required. The replaced synonym (not a
Notes: The stipe of the neotype is missing and its
preservation in chemicals prevents PCR amplification. basionym since the epithet is not being used
Here, we designate a supporting epitype with stipe that in the new name) must be cited with a clear
is associated with DNA sequence data. and direct reference to its place of valid pub-
lication. For this, authors making replacement
7. How Do I Validly Publish New names must include the journal and volume
Combinations? or book title, the page where the protologue
begins (be sure not to cite the entire pagina-
The rules for publishing new combinations are tion of the whole publication that includes the
covered in large part and in more detail in ICN protologue), and the date. Authors should
Arts. 35, 37, and, especially, 41. The basionym make sure that species epithets agree gram-
must be cited with a clear and direct reference to matically with the genus of their new name. It
its place of valid publication. For this, authors is also suggested that authors include a cita-
making new combinations must include the jour- tion including a full and direct reference
nal and volume or book title, the page where the for the earlier homonym or species name
protologue begins (be sure not to cite the entire already occupying a genus that necessitates
pagination of the whole publication that includes the replacement name. A MycoBank, Fungal
the protologue), and the date. Authors should Names, or Index Fungorum registration num-
make sure that adjectival species epithets agree ber must be obtained and listed for replace-
grammatically with the genus in making new ment names.
combinations (e.g., Agaricus americanus be-
comes Lepiota americana instead of Lepiota Examples (both fictional):
americanus). A MycoBank, Fungal Names, or Nectria peckii Rossman, nom. nov.
Index Fungorum registration number must be MycoBank MB124669
obtained and listed for new combinations. Nectria cinnabarina Peck, Mycologia 3: 377.
1911 (replaced synonym), non Nectria cinnabarina
(Tode : Fr.) Fr., Summa vegetabilium Scandinaviae 2:
Examples (both fictional): 388. 1849.
Alternaria nyssicola (Peck) E.G. Simmons, comb.
nov.
MycoBank MB124578 In the preceding example, there is an ille-
Stemphylium nyssicola Peck, Mycologia 3: 375. gitimate later homonym (Peck’s “cinnabar-
1911 (basionym). ina”) and a cited earlier legitimate homonym
Ulocladium nyssicola (Peck) Minnis, Mycologia
100: 22. 2008.
(Fries’ “cinnabarina”).
Pseudocercospora nyssicola (Peck) A.H. Sm., comb. & Phoma braunii Rossman, nom. nov.
stat. nov. MycoBank MB222223
MycoBank MB654826 Phyllosticta cinnabarina Peck, Mycologia 5: 123.
Cercospora apii var. nyssicola Peck, Mycologia 3: 1913 (replaced synonym), non Phoma cinnabarina Fr.,
376. 1911 (basionym). Summa vegetabilium Scandinaviae 2: 390. 1849.
198 A.M. Minnis
In the preceding example, the epithet “cin- debate about whether or not to transition to a
nabarina” is already occupied in Phoma. The one-name-per-fungus system of classification,
new combination “Phoma cinnabarina” based strong feelings about philosophical principles
on Peck’s species would create a later hom- of maintaining dual nomenclature or abandon-
onym. ing it were voiced. Unfortunately and in gen-
eral, relatively little attention was given to the
actual details of potential rules to make a tran-
9. How Do I Correctly Give Author Citations for
sition to one name per fungus happen, and
Taxa?
numerous experts of nomenclature that really
Complete details about author citations for taxa know the Code well, but opposed the transition,
are found in ICN Arts. 46–50. For existing fun- did not assist the voices calling loudly for
gal names, correct author citations may be change. Gams et al. (2012a, b) brought up a
found (usually) in Index Fungorum and Myco- number of considerations, challenges, and
Bank. In detailed taxonomic studies, authors needed clarifications about the adopted rules
should attempt to carefully verify that these for the transition shortly after it was passed,
databases are correct since they are not perfect. and many of the finer details may have to be
For new names, including new combinations, addressed through additional modification of
authors should include author citations for the ICN (Hawksworth 2014).
such taxa. These author citations are not neces- A number of possible proposals that would
sarily the same as the authorship for the whole tinker with the current transition to a one-
publication. Abbreviations for authors of fungi name-per-fungus system of classification have
and plants should follow the standards estab- already been presented. Hawksworth et al.
lished by the International Plant Names Index (2013) have suggested that if a new species
(IPNI), and in cases where a standardized name were erected for a morph or state of an
abbreviation does not yet exist, authors should already existing species name having the
still attempt to conform to IPNI practices. corresponding morph or state under dual
Authors should be linked by the use of an “&” nomenclature and the two species names
and the serial comma is not employed. shared the same epithet, the later name should
be treated as a new combination rather than a
Example (fictional): new species name and share the same type as
Chaetomium oregonense T.C. Harr., H.Y. Su & the earlier species name. The later type would
Spatafora, sp. nov. then have no standing under the ICN. This
approach may be overly complicated since it
requires an understanding of old rules that
III. Potential Changes to Nomenclatural have been deleted from the Code. There is a
Rules danger that the two species names do not rep-
resent the same fungus, and the application of
A number of possible modifications to the ICN the name based on the later type might be more
are currently being considered, and some of prevalent in the literature. In noting existing
these may be implemented in the future. In confusion about terminology associated with
the following text, some of these ideas and lists of fungal names to be treated as conserved
background information are given, but in gen- via ICN Art. 14.13 or treated as rejected via ICN
eral, details from cited works are not because Art. 56.3, Hawksworth (2014) has proposed that
the proposals are preliminary and have not yet the term “protected” be introduced for the for-
been adopted in the Code. mer situation and “suppressed” for the latter.
More changes relating to Art. 59 and the Hawksworth (2014) has also proposed that fun-
transition to one name per fungus: During the gal names listed via ICN Art. 14.13 be protected
against all unlisted names. Informal discussion gram of soil or piece of cow manure filled with
of the NCF also suggests that the term “widely large numbers of organisms, some as a spore or
used” and Art. 57.2 in general are likely to be two, can serve as a suitable standard for the
modified in the next Code. application of a name or names. Nevertheless,
Registration of typifications of existing some authors have begun to explore the follow-
names: The same problems of scattered myco- ing questions: What is the absolute minimum
logical literature and the proliferation of that is required to publish a new fungal name?
numerous journals that make it valuable to And can this be done with only DNA sequence
construct a fungal index through required data? The NCF is currently debating many com-
registrations of fungal names also plague later plicated aspects brought up by these questions
typifications, such as lectotypification, neotypi- in deliberations on whether or not some
fication, and epitypification, of existing names. recently and minimally described fungi are
Shortly after Hawksworth et al. (2010) pro- valid names (Tripp and Lendemer 2012). The
posed registration of fungal names as a require- online serial publication provided by Index
ment for valid publication, Gams (2010) Fungorum (http://www.indexfungorum.org/
proposed that a similar type of registration be Names/IndexFungorumRegister.htm) has
required for typification of existing names. The other similar examples. Large-scale naming of
NCF recommended that this proposal be fungi based on environmental sequences will
adopted (Norvell 2011a), but it was rejected at likely require modification of the ICN. Given
the IBC in Melbourne (McNeill et al. 2011). that next-generation sequencing technology is
Hawksworth (2014) has since then suggested advancing rapidly, the field of molecular eco-
draft proposals for registration of typifications logy is still in its relative infancy, and many
of existing names, and MycoBank now has a taxonomists feel that ITS data alone are entirely
utility for just such an action. inadequate, it may be hasty to rush into the
Naming of environmental sequences: With formal description and naming of a large num-
the emergence of new DNA sequencing tech- ber of environmental sequences based on one
nologies, the accumulation of large amounts of marker at this time.
DNA sequence data from environmental sam- Other changes being considered: Hawks-
ples in a short time has outpaced the speed at worth (2014) has offered a grab bag of possible
which taxonomists address the multitude of proposals for consideration that include issues
undescribed fungi, some of which are seem- such as extension of sanctioning to additional
ingly known only from environmental data. As works, extending conservation to additional
a result, a few mycologists have proposed that ranks, and more. These will not be considered
these sequences be formally named and have in detail herein. Kirk et al. (2013) have shown
proposed some possible mechanisms by which an interest in expanding the coverage of the
this might happen (Hawksworth et al. 2011; lists for dealing with the transition to a one-
Hibbett and Taylor 2013; Hibbett et al. 2011; name-per-fungus system of classification to a
Taylor 2011). Challenges include sequencing protected list for all fungal genera via existing
errors, incomplete sampling of named taxa, rules of the ICN, even though this was not the
and intragenomic variation of markers like the intent of Redhead’s proposal.
ITS, among others (Hibbett and Taylor 2013;
Hibbett et al. 2011; Lindner and Banik 2011;
Lindner et al. 2013; Taylor 2011). It has also
been proposed that fungal naming be
IV. Conspectus and the Future of
completely automated by computer programs Fungal Naming
(Hibbett and Taylor 2013; Taylor 2011). Typifi-
cation of taxa known only from environmental Precise communication about fungi and fun-
samples remains a problem because it is not guslike organisms relies upon the use of
clear whether such material or data obtained names, and the application of a name is deter-
from it would qualify as a type under the ICN, mined by means of a nomenclatural type, i.e., a
and many mycologists object to the idea that a standard. This chapter provides a basic treat-
200 A.M. Minnis
ment of changes to fungal nomenclature as ble transition to a one-name-per-fungus system

implemented by the current International of classification, and perhaps his most valuable
Code of Nomenclature for algae, fungi, and lesson for fungal systematists is to make sure
plants (Melbourne Code), or ICN. The transi- that the most productive times in a career are
tion to a one-name-per-fungus system of clas- spent doing exciting and worthwhile mycology.
sification has begun. Several working groups or In the words quoted by Walter J. Sundberg, my
subcommissions have already organized in sorely missed doctoral advisor, are we having
loose association with the International Com- fun yet? As for the future of fungal naming, I
mission on the Taxonomy of Fungi, and a num- guess we will find out what we make of it.
ber of them have made progress on lists of
names, such as that offered by the Leotiomy-
cetes Working Group (Johnston et al. 2014). References
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mendation. Taxon 59:1610–1611
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8 The Role of Herbaria and Culture Collections
GERARD J.M. VERKLEY1, AMY ROSSMAN2, JO ANNE CROUCH2
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
II. Best Practices for Fungal Herbaria. . . . . 207 Living cultures and dried reference specimens
A. Preparing Fungal Reference
Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 of fungi preserved in herbaria document the
B. Preparing Dried Culture Specimens . 209 occurrence of fungi over time and space; we
C. Numbering and Labeling Fungal will never be able to return to the past to deter-
Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 mine exactly which fungi were present (Ross-
D. Storage and Organization of Dried man 1996). Cultures and specimens from the
Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
E. Requesting, Annotating, and Returning past are the only true representation of what
Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 existed then and, thus, are very important. In
F. Use of Fungal Herbarium Specimens in addition to fungal cultures and specimens
Molecular Studies . . . . . . . . . . . . . . . . . . . . . 211 deposited in the past to reveal the historical
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 211 mycota, plant specimens, including those of
2. Contamination of Herbarium DNA
Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 fossils preserved with their fungal associates,
3. Extraction of DNA from Herbarium have proven to be a useful source for discover-
Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 ing rarely collected fungi on living plants, such
4. DNA Quality from Herbarium as Uredophila (Rossman 1987), determining the
Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 worldwide spread of plant pathogens (Ristaino
III. Maintenance of Living Cultures . . . . . . . . 214
A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 214 2006; Crouch and Tomaso-Peterson 2012), or
B. Metabolically Active Preservation . . . 215 discovering the past fungal diversity associated
1. Storage on Agar with Periodic with plant fossils (Bidartondo et al. 2011). One
Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 practical reason to preserve fungal specimens
2. Storage on Agar Under Mineral Oil when describing a new taxon is to fulfill the
and Distilled Water . . . . . . . . . . . . . . . . 216
C. Metabolically Inactive Preservation . 217 requirement of the International Code for the
1. Cryopreservation (Cryogenic Nomenclature of algae, fungi, and plants. The
Storage). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 code requires the deposition of a holotype spec-
2. Freeze Drying . . . . . . . . . . . . . . . . . . . . . . 220 imen in a recognized herbarium (McNeill et al.
IV. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 2006; Seifert and Rossman 2010). This will
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
allow future scientists to know the precise char-
acteristics of a specific taxon; thus, these type
specimens serve as the “standard” for defining
a fungal species. Having them available for
1 study is essential for increasing scientific
CBS Biodiversity Center, Uppsalalaan 8, 3584 CT Utrecht, The
Netherlands; e-mail: g.verkleij@cbs.knaw.nl knowledge, especially concerning fungal sys-
2
Systematic Mycology & Microbiology Laboratory, USDA- tematics. What a joy to encounter a plentiful,
ARS, Rm. 246, B010A 10300 Baltimore Ave., Beltsville, MD well-preserved type specimen with appropriate
20705, USA; e-mail: amy.rossman@ars.usda.gov; joanne.crou- morphological features! While it may not
ch@ars.usda.gov

206 G.J.M. Verkley et al.
always be possible to deposit such a specimen, a eases caused by fungi are reported regularly,
well-preserved slide as a type specimen serves with the causal agent identified by a scientific
the same purpose. Although a living culture in a name. Over time, the concept of that species
metabolically inactive state is allowed to serve may change with increased knowledge. As an
as a holotype specimen, a dried culture speci- example, the concept of Armillaria mellea has
men is also useful because after a period of changed significantly over the past 50 years as
time cultures in some groups of fungi will no this broadly conceived species has been divided
longer produce their diagnostic reproductive into many more narrowly circumscribed taxa
structures. that have a more specific biology (Coetzee et al.
At present, it is generally agreed that only 2003). Often the species concept is initially
5–10 % of the fungi that exist have been revised based on molecular sequence data, but
described and that the total number of fungal upon reexamination of specimens morphologi-
species may range from 1.5 to 5 million species cal clues to these newly recognized species are
(Blackwell 2011; Hawksworth 2004). With the found. Once found, previously deposited speci-
growing interest in biological diversity, many mens can be reidentified using the accurate
new species and habitats for fungi are being species concepts and the biology of these
discovered that should be documented with more narrowly defined species (Rossman and
herbarium specimens and living cultures. For Palm-Hernandez 2008).
fungi as for flowering plants, herbaria them- Most sequence data available for fungi
selves are a source of new knowledge of diver- today are generated from DNA extracts from
sity (Bebber et al. 2010; Brock et al. 2009), but fresh fruiting bodies of fungi collected in the
the data need to be digitized and disclosed for field or from isolates preserved in culture collec-
usage. Recent support from funding bodies, tions. Many different extraction protocols have
such as the National Science Foundation to been developed for these materials, and the
digitize 35 fungal collections in the USA, under- reader may consult other publications for
pins the importance of such collections. In more details (Eberhardt 2012). With the ability
addition, environmental sampling by extract- to extract DNA, fungal herbarium specimens are
ing, cloning, and sequencing DNA suggests becoming increasingly useful in, for example,
that the biological diversity of fungi is even precisely identifying type specimens or deter-
greater than has been hypothesized. Even new mining the historical distribution of specific
phylogenetic lineages are being discovered populations of plant pathogens. Ristaino (1998,
using these techniques (Schadt et al. 2003; 2006) successfully tracked the movement of
Jones et al. 2011). Although documented with populations of Phytophthora infestans through-
sequences, the environmental samples them- out the 1800–1900s using historical specimens
selves from which the DNA has been extracted from various herbaria (May and Ristaino 2002,
may be deposited in herbaria, where they will 2004; Ristaino et al. 2001). Later in this chapter
be made available for future analyses using as is a section with suggested protocols for DNA
yet unknown methods for describing biological extraction from herbarium specimens along
diversity. Thus, as for collections of all organ- with the problems that may be encountered.
isms, the role of fungal collections may be even This should only be done with permission
more important than currently recognized in from the herbarium that issued the loan and in
ecological and environmental research of the cases where the specimen is abundant.
future (Pyke and Ehrlich 2010). The protocols and techniques presented
Beyond systematics and biological diver- subsequently represent an overview of these
sity, fungal cultures and specimens should be subjects. More detailed accounts of these sub-
deposited to document fungal research of all jects have been published for herbaria in gen-
kinds, including ecology, genetics, and plant eral (Forman and Bridson 1989; Savile 1962)
pathology. Reference or voucher specimens and for fungal herbaria specifically (SAFRINET
are essential for the repeatability or later verifi- 1999; Wu et al. 2004). A useful overview of
cation of that research. For example, new dis- practical aspects in dealing with fungi is
The Role of Herbaria and Culture Collections 207
provided by Mueller et al. (2004) in which mea- A. Preparing Fungal Reference Specimens
suring and monitoring the biological diversity
of all kinds of fungi are considered. Hawks- When collecting a fungal specimen to be
worth (1974) provides a general account of the deposited in an herbarium, as much material
systematics of fungi. as possible should be gathered so that multiple
specimens can be sent to various institutions
for establishing the fungus in pure culture and
for DNA extraction. Fungal specimens includ-
II. Best Practices for Fungal Herbaria ing lichens should be timely and thoroughly
dried to prevent mold and bacterial growth
Fungal herbaria have historically been included prior to beginning the preservation process.
in botanical institutions. Each herbarium has Characteristics such as color, shape, size, and
an official acronym: for example, K stands for surface texture of fleshy fungi that will disap-
Kew Botanical Gardens and BPI is the official pear upon drying should be recorded and
acronym for the U.S. National Fungus Collec- included with the specimen. It is often useful
tions based on its previous affiliation with the to photograph specimens before they are dried.
USDA Bureau of Plant Industry. Information If material is to be used later for DNA extrac-
about fungal herbaria can be found at the tion, a portion of the specimen should be
Index Herbariorum Web site, http://sciweb. placed in a cryovial with or without a buffer
nybg.org/science2/IndexHerbariorum.asp, in- solution.
cluding listings of directors and associated Air-drying is usually sufficient to ade-
staff, herbarium Web sites, lists of major scien- quately dry small fungal specimens, but large
tists’ specimens deposited at those institutions, fleshy or woody fungi may require the use of a
and rules governing the loan of specimens. dehydrator or drying cabinet to prevent the
These data are continuously updated. This specimen from becoming moldy. Large poly-
Web site should be consulted before requesting pores and mushrooms can be cut in slices;
specimens on loan. Spooner and Cannon (2010) mushroom caps and stipes may be separated
recently coined the term “fungarium” for col- to fit more easily into specimen packets or
lections of dried fungal specimens, underlining boxes and save storage space. Care should be
the fact that the fungi are now generally taken to preserve diagnostic characters such as
accepted as a kingdom in their own right and the reproductive structures. In the case of deli-
not to be seen as part of the kingdom of plants. cate specimens such as myxomycetes or hypho-
Increasingly the information associated mycetes, material should be glued into boxes in
with fungal herbarium specimens, especially the field to prevent crushing.
types, is available online (Farr and Farr 2004), Fungi on living or dead leaves or other thin
and the herbarium Web sites with these data substrates and folious lichens should be
should be consulted prior to requesting a spec- pressed in paper towels and newspaper in a
imen. In addition, the specimens themselves plant press or under a heavy weight, such as a
along with the data are increasingly being digi- pile of books, similar to the procedure used for
tized and made available on the Internet, as for pressing and drying plants. The material should
example in the USA (Baker 2011). This allows be spread flat in one layer. Once dried, material
users to select the specimen most useful to can be cut with scissors or folded into packet-
them; in some cases, just seeing the specimen sized specimens, always taking care to protect
may be all that is required to answer the user’s the diagnostic structures. After drying, speci-
question. In addition to preventing damage to mens are ideally stored in a labeled glassine
the invaluable specimen itself, this also saves envelope, especially if the specimen is fragile.
time and money for both the herbarium man- It is useful to include a pressed voucher speci-
agement and user. men of the substrate when collecting fungal
specimens in order to obtain an accurate host The following items may be added to specimen packets
identification; thus, a plant press is extremely if necessary:
useful in the field.
Fungi on woody substrates or twigs should l A thin piece of cardboard in the packet for added
rigidity; crustose lichens should be attached to a card
be trimmed to a size that will fit in a packet or to keep the substrate from disintegrating and prevent
box, then dried. Unnecessary substrate should the specimen from sliding around in the packet.
be removed, taking care to preserve the diag- l Cotton wadding to preserve delicate structures or
nostic fungal structures. A knife or good clip- prevent the specimen from sliding to the bottom of
pers are useful for this purpose. For large the packet.
l Acid-free glassine envelopes to contain dusty or frag-
woody specimens, a bandsaw can be used to ile specimens, such as thin, dried leaves, within the
trim material to a reasonable size. paper packets.
For fleshy fungi or specimens preserved for l Zip-top polyethylene bags to store specimens inside
exhibition, freeze drying or lyophilizing paper packets.
reduces shrinkage and color loss. However,
freeze-dried specimens readily absorb moisture Variously sized specimen packets are glued
from the environment and must be maintained to standard herbarium mounting sheets using
at a low relative humidity. They are also fragile archival-quality adhesive. Mounting sheets can
and vulnerable to breakage (Moore 1999). For be obtained from an herbarium supply store. In
this reason, this practice is not recommended gluing a packet to a sheet, a small drop of
as standard procedure for fungal herbarium adhesive should be applied to the center of the
specimens. back of the packet so that the side flaps can be
All newly accessioned specimens should be unfolded to open the packet. A label with the
placed in a 20 C freezer for 3–5 days to kill specimen data generated from a database
potential pests before adding them to the col- should be attached to the front of each packet.
lection. Polypores and mushrooms are particu- Multiple packets of the same fungal species on
larly susceptible to insect infestation. This is the same host may be attached to one mounting
also good practice for specimens coming to or sheet to conserve space. A sheet with multiple
leaving other herbaria and even specimens that packets is then labeled with the fungus and host
have been removed from the herbarium envi- names in the bottom right corner.
ronment for a period of time. Large or heavy specimens, such as agarics
Unlike plant specimens, fungi are not and polypores, and delicate specimens that
directly mounted on herbarium sheets. Paper need protection from crushing, such as puff-
packets can be used to contain small speci- balls, should be placed in appropriately sized
mens. Machine-cut or prefolded packets, some- cardboard boxes. Myxomycetes are sometimes
times called bryophyte packets or fragment glued to box interiors, especially shallow boxes
folders, can be purchased from an herbarium such as matchboxes or pill boxes; these are then
supply company, or packets can be folded by placed inside specimen boxes or packets. Fungi
hand from 100 % cotton, acid-free, A4 (21.0 growing on insect hosts may also need to be
29.7 cm) paper. Whatever folding method is stored in boxes. Specimens may be stored in
used, the packets should stay closed without zip-top polyethylene bags inside boxes.
the use of clips, to prevent the loss of small Permanent slide mounts or dried cultures
pieces of the specimen, and open flat. Paper can be stored in cardboard slide mailers within
may be made into a packet by folding about specimen packets or boxes. If a separate slide
one-third of the paper from the bottom and a collection is maintained, slides should be cross-
short-third from the top, then folding back referenced with their corresponding specimen
about 2.5 cm on both sides. packets. Photographs, illustrations, descrip-
tions, spore prints, dried cultures, and any serving as an ex-holotype, and deposited in a
associated material should also be stored with publicly available culture collection.
specimens or cross-referenced.
C. Numbering and Labeling Fungal

Specimens
B. Preparing Dried Culture Specimens
When starting a fungal herbarium, it is impor-
Dried specimens of living cultures can be tant to decide on a numbering system and stick
included in a fungal herbarium. One technique with that system. The best approach is to decide
for making dried cultures requires a cardboard on or obtain a unique acronym from Index
slide mailer, water-soluble glue such as Herbariorum and start with number 1, giving
Elmer’s, and silica gel in a tight-fitting box. each specimen a unique number. In addition,
This technique was described by Rossman and entering specimen data into a database will
Simmons (1999) and is described and illu- greatly facilitate making labels, handling speci-
strated at http://www.ars.usda.gov/Services/ mens, and keeping track of loaned material.
docs.htm?docid¼9405. Some programs exist that do this, such as
SPECIFY, which is available from the Univer-
The steps are as follows: sity of Kansas: http://specifysoftware.org/.
1. Label a cardboard slide mailer with the name and At a minimum, packets and boxes should
number of the fungus to be preserved as well as the
kind of agar on which it is growing. If the fungus is
be labeled with the scientific name of the fun-
on only one kind of agar, use a single slide mailer, gus and host, complete collection locality data,
but if it is on two kinds of agar, a double slide collector name, determiner name, and collec-
mailer can be used placing a strip of fungus from tion date. Labels should be printed on acid-free
each kind of agar into each place of a slide. paper and attached to a packet or box lid with
2. Squeeze a line of glue directly onto the slide holder
and smear it around. Cut a slice of agar with the
archival-quality adhesive.
diagnostic features of the fungus from a Petri plate
and place on the thin layer of glue in the slide
mailer. D. Storage and Organization of Dried
3. Then place the slide mailer, lid open, in a container Specimens
with dried (blue) silica gel about 1 cm deep. Place a
tight-fitting lid on the container. Like plant collections, fungus collections
4. The glue and agar will dry at about the same rate, should be protected from excessive heat, mois-
becoming hard within 2–3 days. The slide mailer
with the lid closed to protect delicate structures
ture, and sunlight. Cabinets should be well
may then be considered as a separate specimen or sealed and collection areas kept clean to help
placed in a packet with the specimen from which prevent insect infestations.
the culture was derived. Specimen packets attached to herbarium
Other techniques exist for making dried cultures sheets can be stored in standard metal herbar-
(Wu et al. 2004). A culture in a plastic Petri plate may ium cabinets. As mentioned earlier, specimens
simply be allowed to dry up if the lid is left unsealed. of the same genus or species or species on a
The resulting flat circle of media with the fungus can be single host should be grouped in labeled folders
removed from the Petri plate and placed in a glassine
envelope. However, such cultures may stick to the plate
depending on the number of specimens pres-
and be difficult to remove without breaking. In addi- ent. Alternatively, each species can be placed in
tion, fragile and delicate aerial structures may be a separate folder, but this requires more space
crushed in the packet. than grouping specimens of several species into
generic folders. Specimen boxes may be placed
Although a metabolically inactive culture in larger boxes or trays that fit on herbarium
may serve as a holotype specimen, usually a cabinet shelves, with the larger boxes labeled
dried reference specimen from a culture is with the range of specimens they contain.
designated as the holotype, epitype, or other Another approach to storing fungal speci-
kind of type specimen, with the living culture mens involves storing individual specimen
packets and boxes vertically in cabinets with E. Requesting, Annotating, and Returning
drawers, such as 10.215.2 cm card file Specimens
drawers. While this storage method is space
efficient and might provide easier access to Herbarium specimens may be requested on
specimens, it can cause damage to fragile speci- loan from herbaria throughout the world for
mens that fall to the bottom of the packet. In use in studies of fungi. Usually specimens are
addition, it does not allow for the placement of loaned from one herbarium to another with the
variously sized packets and boxes in the same request agreement communicated between the
place in the herbarium. directors of the respective institutions. When
No standard arrangement scheme exists requesting a specimen, the requestor should
for fungal herbaria. The best arrangement for include as much information as possible about
an herbarium depends on the size of the her- the specimen desired. If the specimen data are
barium and the needs and knowledge level of available on the Internet, specimens should be
the users. For example, alphabetical arrange- requested by number as well as fungal name. If
ment by genus and species might work well type specimens are being requested, the scien-
for a small collection or a collection used by tific name of the species, any synonyms, its
nonspecialists, but a large collection might place of publication, and the data associated
benefit from having the specimens placed in with the type specimen should be included in
taxonomic order so that users do not have to the request. This will assist herbarium person-
move to different parts of the herbarium to find nel in locating the specimen. The institutions
specimens in related groups. Reference tables where specimens of important mycologists are
and labels should be posted on cabinets to aid housed are listed in Kirk et al. (2008) under
in filing and finding specimens. “Author Names” using the abbreviations in
Taxonomic and alphabetical arrangements Index Herbariorum.
can be combined, as in the following examples: In requesting specimens for loan, a letter or
e-mail from the director of the requesting her-
l Arrange fungal genera in taxonomic order by barium should be addressed to the director of
major groups. the loaning herbarium, with a brief statement
l Arrange fungal species in alphabetical order about who will be using the specimens and for
within genera. what purpose. Each specimen requested should
l Arrange multiple specimens of a single fungal be listed with details so that the specimens can
species in alphabetical order by host genera be easily located. Most herbaria loan specimens
and species. for a period of 6 months to a year, although the
This system can be altered so that fungal time period for the loan of type specimens may
genera are sorted alphabetically under classes be shorter. It is critical that specimens be
or families, which are arranged taxonomically returned in a timely matter. Return of overdue
following a recent classification scheme such as loans has been the source of considerable con-
that of Lumbsch and Huhndorf (2010) for asco- sternation in the herbarium community. At one
mycetes. For groups of parasitic fungi with nar- point in time, personnel at the invaluable Sac-
row host ranges such as rusts, arranging cardo herbarium (PAD) in Padova, Italy,
specimens of a single species by host family is refused to loan specimens until the fungal com-
useful. Within genera or species, specimens munity as a whole returned their overdue speci-
might also be arranged geographically, as is mens. Often the only way to obtain overdue
commonly done for plant specimens. For loans is to decline requests for loans of speci-
example, separate sheets or folders can be mens to others at the same institution.
used for specimens from the main region of When examining specimens on loan from
interest, with regions less well represented other institutions, the regulations provided by
grouped together. that institution must be followed. Usually
these regulations require that specimens be the use of specimens for molecular analysis
stored in a safe place such as a metal herbarium need to be carefully considered to maintain a
cabinet. When examining the specimen, the balance between preserving valuable resources
smallest amount of material possible should and enabling robust scientific inquiry. A work-
be used in order to preserve the specimen for ing knowledge of the many factors that influ-
the future. This is especially true for type speci- ence the successful outcome of research using
mens that can be degraded to nothing after aDNA, as detailed in the following sections, will
being examined by a few mycologists. In the assist in the development of effective policies in
case of type specimens, most institutions this emerging area.
require that all portions of the specimen, Specific policies for destructive sampling
including the microslides, be returned with of fungal reference specimens for the purpose
the specimen. The problem of destroyed speci- of aDNA analysis vary depending on the indi-
mens has become so dire that some type speci- vidual herbaria. Most generally, when permit-
mens, such as those of Fries, are no longer ted, sampling for DNA extraction must be
available for examination. preapproved in writing by curatorial staff. The
Examined herbarium specimens should majority of herbaria have strict policies prohi-
always be annotated with a small slip of paper biting destructive sampling of type specimens
that includes an evaluation of its identity, as and important historical samples but are gen-
much as can be determined. Annotations erally more permissive with other samples.
should be typed or made in legible handwriting
on small pieces of paper that include the her- At the U.S. National Fungus Collections and other her-
barium and specimen number, an evaluation of baria, decisions regarding destructive sampling for
the identity of that specimen, and the exami- aDNA extractions are made on a case-by-case basis
and are influenced by many factors, including the
ner’s name and date. Annotations provide a quantity and quality of material available for analysis,
history for future users and may serve to pre- the availability of other suitable material, the experi-
vent further destruction of that specimen. ence of those requesting permission, and the likelihood
of success. Some institutions, such as the Wageningen
Herbarium (WAG), explicitly limit sampling to a maxi-
mum of 5 % of available tissue up to a maximum of
F. Use of Fungal Herbarium Specimens in 50 mg (Telle and Thines 2008). Others, such as the
Molecular Studies Harvard University Herbaria, require researchers to
submit a written protocol for proposed molecular anal-
1. Introduction ysis in advance of loan approval (Wood et al. 1999).
In recent years, molecular systematists have Frequently, material to be used for aDNA
been increasingly using DNA extracted from extractions will be excised by herbarium per-
fungal herbarium specimens as part of their sonnel before loans are provided to scientists.
research. The widespread use of so-called Alternatively, scientists may perform the exci-
ancient DNA (aDNA) for molecular studies sion of tissue on site at the herbarium under the
arose shortly after the development of the poly- supervision of curatorial staff. Digital imaging
merase chain reaction (PCR), which allowed of specimens prior to the loaning of material is
scientists to perform selective enrichment of a labor-intensive practice but may be reluc-
targeted DNA sequences using very small quan- tantly adopted by some institutions in an
tities of heterogeneous DNA (Saiki et al. 1985, attempt to safeguard collections from unautho-
1988), even from poorly preserved sources, rized sampling.
such as fossils, museum specimens, and archae- At the completion of the research, from 50
ological remains (Pääbo et al. 2004). The to 100 % of the extracted aDNA is typically
importance of DNA analysis in modern system- returned to the herbarium because the loaning
atics is placing additional demands on herbar- institution retains ownership of all material
ium resources and has introduced a new set of derived from the loaned specimen. The myco-
challenges for institutions. Decisions regarding logical specimen associated with the aDNA
extraction is annotated with details of the data cillium rather than the targeted specimen
generated from the research, including the suc- (Brock et al. 2009). At present, there is no way
cess or failure of DNA extraction attempts and to know whether this level of contamination is
GenBank accession numbers of sequence data. representative; comprehensive studies on this
Although uncommon, some institutions, such topic are needed. Given the capacity of fungal
as WAG, are beginning to provide access to spores and other structures to travel unseen
stores of aDNA extracted from their collections through air currents or to move as passengers
to the scientific community. on, for example, hands, clothing, or tweezers,
many fungal herbarium specimens may contain
some level of cross contamination from the
2. Contamination of Herbarium DNA Samples environment. The typical fungal herbarium
environment seems especially vulnerable to
The potential for contamination of aDNA from cross contamination since samples are rou-
external sources is a serious concern that must tinely stored in close proximity to one another
be taken into consideration before embarking and multiple specimens are often stored in
on any study that uses herbarium-derived adjacent packets on a single herbarium sheet.
DNA. Researchers should be prepared to incor- The occurrence of mites or insect pests, and
porate rigorous controls to minimize sources their movement from one specimen to the
of error derived from contaminant DNA next, may add to the problem of contaminant
(Cooper and Poinar 2000; Pääbo et al. 2004). transfer. For obscure specimens that have
Contamination of ancient samples with con- remained unopened since their initial collec-
temporary DNA as a complicating factor was tion, the potential for the introduction of inter-
notorious in early studies of macroorganisms mediate contaminants may be nominal. In
(e.g., Zischler et al. 1995) and remains pervasive contrast, specimens that have been subjected
in studies involving microbial pathogens (e.g., to repeated examination over the course of
Gilbert et al. 2004). There are several points at decades or centuries are more likely to be con-
which herbarium specimens targeted for taminated. Likewise, specimens on loan with
molecular analysis may become contaminated specialists may be exposed unintentionally to
by external sources: (1) during initial collec- any number of fungal contaminants from the
tion, (2) during storage in the herbarium, (3) environment and run the risk of exposure to
during examination while on loan, and (4) dur- similar species that might go undetected if
ing the DNA manipulation process. Regardless appropriate experimental control measures
of when the contaminant is introduced, the are not implemented.
high level of sensitivity of the PCR process
used in molecular research ensures that the
presence of even small amounts of foreign 3. Extraction of DNA from Herbarium
DNA can be detected, compromising research Specimens
conclusions. For example, some PCR assays are
sensitive enough to reproducibly detect the A strict set of best practice criteria for the gen-
presence of just two fungal spores, as demon- eration of authentic, uncontaminated aDNA
strated from studies of the rust fungus Phakop- data has been proposed and refined over the
sora pachyrhizi (Barnes et al. 2008). past three decades. Though it is impossible to
How pervasive is contamination of myco- eliminate past contaminants introduced during
logical herbarium specimens? A recent survey routine storage and handling, careful adher-
of specimens at the fungal herbarium at the ence to aDNA authentication criteria can help
Royal Botanic Gardens in Kew, UK, collected researchers identify the presence of contami-
between the 1950s and the present day showed nants and minimize the introduction of new
that contamination can be a relatively common contaminants during handling. A discussion
occurrence, with 11 % of 263 ITS sequences of the experimental controls required to pro-
derived from contaminant fungi such as Peni- duce robust, authenticated aDNA data is
beyond the scope of the present review, and tissue performed on a sterile surface, such as a
readers are referred to detailed summaries of disposable plastic Petri plate.
these approaches presented elsewhere (Cooper The quantity of tissue excised from the
and Poinar 2000; Pääbo et al. 2004). The follow- specimen for DNA extraction may be dictated
ing section is limited to the technical consid- by the herbarium but in all cases should cause
erations involved in extracting aDNA from as little destruction to the sample as possible.
mycological specimens. Tissue is always excised from the least obvious
New contaminants can take the form of area of the specimen and must never destroy
living environmental sources such as spores unique morphological structures or features.
or previously amplified PCR products that For plant-associated fungi preserved in a host
may be present in the lab. During the process plant matrix, avoid the “best” lesions, pustules,
of extracting DNA from herbarium specimens, or other structures that serve as visual exem-
it is essential that work be performed in a spe- plars. Instead, sample from less striking areas
cially prepared clean area using specially or from broken pieces of lesser value. For plant-
prepared tools. Ideally, this will mean working associated fungal specimens, both fungus and
in a laboratory space where the experimental host tissue are included in the extraction pro-
organism has never been present, distant from cess, with the plant tissue serving as a DNA
places where contemporary sources of the carrier, although inclusion of excess, uncolo-
organism might be encountered. Ventilation nized plant tissue should be avoided when
systems for heating and air conditioning may possible. For specimens of both fleshy and
provide unanticipated connections between plant-associated fungi, a 2–4 mm3 section may
seemingly isolated laboratories in a single be more than adequate for many specimens and
building or introduce contaminants from adja- will allow the extraction process to be per-
cent environments such as greenhouses and formed in a single microcentrifuge tube. If the
experimental plots. amount of tissue for a specimen is minimal, less
Throughout all experiments, work should tissue must be used, and never more than 5 %
be performed on a surface thoroughly cleaned of the total specimen must be removed. Indeed,
with oxidants (bleach solution, UV light) within it has recently been demonstrated that only
an environment such as a laminar flow hood or 2 mg of grass tissue infected with either Scler-
PCR hood where air flow is restricted and con- ospora graminicola or Albugo sp. was needed
tinuously filtered to eliminate spores, dust, and for successful aDNA extractions (Telle and
other contaminants. The work area should con- Thines 2008). Excised tissue is cut into small
tain only the tools needed to perform the sections, then placed in a sterile 1.5 mL micro-
extraction. Exterior surfaces of specimen pack- centrifuge tube until needed.
ets are wiped with a dry, lint-free towel to dis- As with any DNA extraction from fungi, the
lodge any spores or other contaminants before first step is cell lysis to release the DNA from
introducing the sample into the clean work the confines of the cell wall, a process that is
area. Only one sample is handled at a time accomplished through mechanical, chemical,
within the prepared work space using fresh, or enzymatic disruption of the cells. Countless
sterile tools, generally a pair of tweezers, and a different protocols are available, with the
sharp knife or scalpel with a fresh blade. Loose choice of methodology dictated by the organ-
or long-sleeved clothing that could harbor con- ism being studied and the DNA extraction pro-
taminants should not be worn, and hands and tocol used. Chemical or enzymatic lysis may be
lower arms are to be thoroughly washed with difficult to achieve for some fungi, although
soap and water before manipulating specimens. many successful examples are found in the lit-
Fresh disposable latex or nitrile laboratory erature (e.g., Fredericks et al. 2005; Ristaino
gloves should be worn and changed between et al. 2001). Some laboratories grind the
specimens. Specimens should only be manipu- prepared tissue into a fine powder inside the
lated using fresh, sterile tools and excision of microcentrifuge tube using liquid nitrogen and
a glass rod, although this can result in the loss extracted from fresh samples, herbarium DNA
of valuable tissue if the liquid nitrogen is is shorter in length due to postmortem strand
allowed to accidentally bubble out of the tube. breakage caused by enzymatic degradation,
Increasingly, bead beating using silica or iron single-stranded nicks caused by nonenzymatic
beads and specialized vortexing equipment are hydrolytic cleavage of phosphodiester bonds in
used for fungal cell lysis (e.g., Crouch and the phosphate–sugar backbone, and strand
Szabo 2011; Fredericks et al. 2005; Telle and breakage due to chemical changes at abasic
Thines 2008). Regardless of the method sites (Friedberg et al. 1995; Lindahl 1993). On
employed, clean work areas as described in average, the length of ancient DNA is between
the preceding paragraphs must be maintained 100 and 500 bp (Hofreiter et al. 2001). Some
throughout the entire extraction process. It is publications report fragments as long as 1 kb
advisable to dedicate pipetters and other equip- (e.g., Lambert et al. 2002; Telle and Thines
ment in the laboratory as DNA-free and utilize 2008); however, many researchers interpret
them only for transferring reagents. Barrier tips the presence of lengthy fragments as an indica-
should always be used for transfers of DNA to tor of contamination (Gilbert et al. 2004), and
minimize the risk of cross contamination. others provide evidence that long fragments
Researchers currently make use of several may be artifacts (Lister et al. 2008).
different approaches for extracting DNA from In addition to the universal postmortem
fungal herbarium specimens, including con- degradation processes experienced by all
ventional or optimized CTAB-based methods organisms, the quality of herbarium specimen
(Brock et al. 2009; Cubero et al. 1997; Ristaino DNA may be further degraded as a result of
et al. 2001; Telle and Thines 2008) or, increas- unique storage and maintenance practices.
ingly, solution- or column-based commercial Specimens that have been air dried, stored at
kits (Crouch and Szabo 2011; O’Gorman et al. temperatures below 42 C, and not exposed to
2008; Sokolski et al. 2004; Telle and Thines chemical treatment will yield higher-quality
2008). Unfortunately, systematic comparisons aDNA than tissues that have been treated oth-
of different methodologies for extracting DNA erwise (Taylor and Swann 1994). Tissues pre-
from fungal herbarium specimens are rare. In served in silica gel or anhydrous CaSO4 may
the single study where a range of common contain highly degraded aDNA, as will speci-
extraction methods were tested using samples mens exposed to high temperatures. Fumiga-
of the oomycetes Sclerospora graminicola and tion and other disinfecting treatments, such as
Albugo sp., it was shown that the extraction the application of mercuric chloride and micro-
method plays a significant role in the quantity waving, may also negatively impact aDNA qual-
and quality of yield and affects the success of ity (Hall 1981; Hill 1983; Metsger and Byers
downstream PCR amplification (Telle and 1999; Taylor and Swann 1994).
Thines 2008). Similar research for the true
fungi is needed before recommendations can
be made for the ideal approach to maximize
yield, minimize failures, and conserve valuable
III. Maintenance of Living Cultures
specimens.
A. Introduction
Fungi that can be successfully isolated in axenic
4. DNA Quality from Herbarium Specimens culture open a plethora of possibilities to study
The degradation of DNA that occurs through life cycles, metabolism, antimicrobial activity,
enzymatic and microbial processes after an and many other aspects of their biology, includ-
organism dies is well documented and presents ing taxonomy. When the original source speci-
problems for researchers working with herbar- men for such cultures is lodged in an
ium specimens. This topic is reviewed in detail herbarium, the culture is even more valuable
by Pääbo et al. (2004). Compared to DNA to taxonomic studies and can, for example, also
serve as material to be selected for diagnostic the country of origin of the material. The con-
tools such as DNA barcoding. sequences for culture collections of the recently
This section is primarily intended for scien- published Nagoya Protocol on Access to Genetic
tists who want to establish or improve their Resources and the Fair and Equitable Sharing of
own working collections of living strains and Benefits Arising from Their Utilization to the
focuses on preservation methods. Handling Convention on Biological Diversity are currently
live cultures of human and animal pathogens being discussed in the community of microbial
is subject to national legislation for biosafety, resource centers with the aim of proposing
and dispatch of these organisms is strictly workable solutions that will contribute to the
bound to transport regulations. Plant- conservation and sustainable use of microbial
pathogenic fungi can be subject to national diversity in the spirit of the CBD, without
phytosanitary regulations and international seriously hampering international access to cul-
transfers to quarantine regimes. Culture collec- tures for taxonomic and noncommercial
tions that offer or plan to establish public ser- research.
vices for distribution or accession of cultures Living fungi can be preserved as cultures
should consider applying for a membership in a in either a metabolically active or metaboli-
national or international organization for cul- cally inactive state, as described in what fol-
ture collections, such as the World Federation lows. Fungal cultures that are intended as the
of Culture Collections (WFCC, http://www. actual holotype specimens must be stored in a
wfcc.info/). The World Data Center for Micro- metabolically inactive state and indicated as
organisms (WDCM), maintained by WFCC, such when designated (McNeill et al. 2006).
contains information on 585 culture collections
established in 68 countries related to their orga-
nization, management, services, and scientific B. Metabolically Active Preservation
interests. Regional networks, such as the Asian Metabolically active preservation methods are
Biological Resources Center Network (http:// used widely to preserve fungi, and although
www.abrcn.net/) and the European Culture some strains may survive for decades, most
Collections Organisation (ECCO, http://www. fungi cannot be expected to be kept viable for
eccosite.org/) regularly organize scientific more than 5–10 years using these methods. For
meetings to exchange information and discuss some methods that use refrigeration (5 C),
common problems and strategies. Most net- metabolic activity is strongly reduced yet
works also provide practical training courses never completely arrested. A number of other
for collection staff on topics like collection methods suitable for particular fungal groups
management, quality standards, and preserva- have been described in detail by Jong and
tion. The OECD Best Practice Guidelines for Birmingham (2001) and Nakasone et al.
Biological Resource Centres (OECD 2007) pro- (2004), including a method for the production
vides a baseline for the operation of service and storage of sclerotia-forming fungi or other
collections and deals with important issues resting structures and methods based on the
such as quality control, data management, colonization of organic (wood chips) and inor-
traceability, biosafety, and biosecurity. ganic (sand, soil, silica gel) substrata, which can
Many service collections furnish their be refrigerated or frozen to 20 C. Below the
strains under a material transfer agreement most frequently used methods are briefly
(MTA), which clarifies the qualifications and described and discussed.
responsibilities of the recipient and settles with
the recipient the terms of use, matters of intel- 1. Storage on Agar with Periodic Transfer
lectual property, and warranties. Most MTAs
also cover the obligations of the Convention Pure cultures can be maintained actively grow-
on Biological Diversity (CBD, http://www.cbd. ing on agar slants by periodic subculturing.
int/convention/default.shtml/) on matters of No expensive equipment is required, and this
access and benefit sharing (ABS) in relation to practice may be convenient when inoculum is
used regularly. But there are disadvantages; the plugs and refrigerator walls, especially when
method is labor-intensive. Moreover, the risk of humidity is high.
contamination is comparatively high, and most Smith and Onions (1994) and Jong and
fungi that are kept actively growing will show Birmingham (2001) discussed the problems
signs of degeneration sooner or later (Jong and related to mite infestations and provided sug-
Birmingham 2001; Nakasone et al. 2004). gestions for control. Natural plant and soil sam-
Unwanted selection effects may occur after ples received in the laboratory are a potential
spontaneous mutations, and fertility and viru- source of mites and should be kept separate
lence are often lost after multiple transfers. from cultures at all times. Mites introduced
These negative effects can largely be avoided with incoming cultures are the most difficult
if metabolism is completely suppressed, as is to control because they are adapted to living
the case in long-term preservation by freezing on cultured fungi. If the unwanted intruders are
and drying (see below). Some fungi are recalci- not stopped immediately from spreading and
trant to these methods, and periodic transfer on reproducing, an entire collection can be
agar media is the only feasible way of keeping destroyed by contaminations within a surpris-
them. ingly short period of time. An effective way of
Cultures are best maintained on agar protecting actively growing cultures from
slants in glass bottles or tubes. The containers mites while allowing normal air exchange is
can be closed with cotton plugs or various types by the use of tubes that are closed with cotton
of commercial cellulose or plastic plugs and plugs soaked in a substance that is highly toxic
metal (screw) caps. When keeping an agar col- to mites (Crous et al. 2009; Smith and Onions
lection, ideally two lines per strain should be 1994), for example, 25 % benzyl benzoate with a
maintained and each line alternatingly trans- colored dye added for visibility.
ferred to two kinds of medium, for example,
one nutrient-rich and one nutrient-poor Caps designed to allow air exchange are inadequate,
medium, but both should support growth of and parafilm does not block the passage of mites. Plates
the species. For a short period after transfer to are also impossible to seal completely from mite infes-
tations. If fungi need to be grown on plates, these
fresh media, the fungus is allowed to grow at its cultures should always be stored away from the tubes
optimal temperature for growth, after which it (preferably on islands in water or oil baths that block
can be stored at a lower temperature to maxi- mite migration) and never kept longer than absolutely
mize the time between transfers by reducing necessary.
metabolism and the evaporation of water.
Most filamentous Ascomycota can be stored at 5 C in

the dark and only need to be transferred every 6–12 2. Storage on Agar Under Mineral Oil
months, but at room temperature usually no longer and Distilled Water
than 3 months, depending on the volume of agar in
the container. Other important groups of fungi that The workload of maintaining an agar collection
should be stored at 17 C can, as a rule of thumb, be can be considerably reduced by adding an over-
transferred as follows: Oomycota, including Pythium lay of high-quality mineral oil or liquid paraf-
and Phytophthora, every 9 months; Chytridiomycota,
every 2 months; saprotrophic Basidiomycota (e.g.,
fin (Paraffinum perliquidum, 60–80 mPa.s) to
Coprinus,Coprinopsis, Conocybe, Mycena, Mycenella), the agar tubes. By this method most filamen-
every 2 months; mycorrhizal Basidiomycota (Amanita, tous Ascomycota and Basidiomycota can be
Boletus, Cortinarius, Lactarius, etc.), every 6 weeks. kept viable for at least 10 years without having
to transfer to fresh media, and mite infestations
Compact, refrigerated storage of cotton- can be prevented. Results for preserving Oomy-
plugged containers should be carefully cota by the oil overlay technique are generally
inspected regularly because cultures may be poor. Metabolism is reduced considerably with
prone to contamination by the growth of cold- this method but not completely stopped, so
tolerant fungi on the outside of containers, in negative effects such as those described earlier
or unwanted selection for conditions imposed long-term preservation for living cultures of
on the fungus may still occur. fungi (Stalpers et al. 1987). It can be applied to
After transfer to fresh agar slants the cul- both sporulating and nonsporulating cultures.
ture should have a chance to develop sufficient A large proportion of fungal taxa obtained in
mycelium (1–2 weeks), after which all myce- pure culture today can survive optimized pro-
lium and agar should be completely submerged cedures for subsequent freezing and thawing
in autoclaved oil and the tube stored in an and will retain most of their properties if
upright position at room temperature (15– stored at temperatures below 139 C as
18 C). Especially in the first 1 or 2 weeks the provided by liquid nitrogen. Below this tem-
oil level may drop, and extra oil should be perature, water activity is zero, no ice-crystal
added if necessary. Reviving cultures requires dynamics occur, and all cell metabolism is
special attention. A first attempt is performed stopped. Assuming that all other potentially
by aseptically taking some mycelium from the harmful influences, such as radiation energy,
culture with a long sterile needle, allowing the can be ruled out completely, this would mean
oil to drain, and inoculating on a fresh slant of that these cultures could be stored indefinitely
the same agar medium. In cases where no (Jong 1989). For these reasons, storage in
growth occurs using this method, the old oil mechanical freezers at 80 or 20 C is less
tube is turned upside down and the oil allowed optimal. Some fungi are recalcitrant to cryo-
to drain for 24 h, after which a new transfer is preservation, and several Basidiomycota and
attempted in the same way. If the fungus still Ascomycota may lose some morphological
does not grow, the old agar in the original oil characteristics, for example, showing a waxy
tube can be overlaid with a thin, fresh layer of or reduced growth after preservation, or will
hand-warmed medium of the same type and no longer sporulate. Only relatively small num-
checked for growth after several days. bers of fungal taxa do not survive cryopreser-
Storage in distilled water has been applied vation at all. Oomycota or water molds, which
to many different fungi, including oomycetes, are not fungi but funguslike Straminipila, need
ascomycetes, and basidiomycetes, commonly to be treated with special care (see below).
with good recovery after 2 years (Johnson and The disadvantages of cryopreservation
Martin 1992; Smith and Onions 1994). An eco- include the high costs of storage equipment
nomical method often used is to store the fungi and liquid nitrogen for cooling. The availability
at 4 C in screw-cap cryovials (1.5–2.0 mL) con- of liquid nitrogen may not always be guaran-
taining a few small discs of freshly grown cul- teed under all circumstances. An additional
tures topped with sterile distilled water disadvantage for service collections is that cul-
(Nakasone et al. 2004). tures need to be reactivated and grown on agar
medium before they can be shipped, in contrast
to freeze-dried ampoules, which can be sent
C. Metabolically Inactive Preservation immediately. Although shipment of frozen cul-
tures is not impossible in most situations, it is
This section highlights the methods that are generally considered too expensive.
used widely for the preservation of fungi for Cryogenic storage can be in liquid nitrogen
periods longer than 5 years. Although prefera- at ca. 196 C or in the vapor phase generated
ble in regards to preserving fungi, these meth- by liquid nitrogen. The disadvantages of plac-
ods require more equipment and are more ing cryovials, particularly glass vials, in the
labor-intensive than those described earlier. liquid phase have been addressed by Stalpers
et al. (1987) and Jong and Birmingham (2001).
1. Cryopreservation (Cryogenic In storage tanks with a classic design (a certain
Storage) level of liquid nitrogen at the bottom over
which the samples are stored), a temperature
Cryogenic storage is the most universally appli- gradient may develop in the vapor phase, and if
cable and one of the most reliable methods of the storage tank is not opened for a long time,
vapor temperatures can even rise as high as mum period and temperature of handling prior
130 C just below the lid. Therefore, special to storage at ultralow temperatures (Tan 1997).
storage systems have been designed in which Vials used for cryopreserving fungi include
the vapor storage compartment is surrounded glass tubes, plastic screw-cap cryotubes (1–
by a mantle of liquid nitrogen at a high level. 2 mL), or straws. Vials should be stabile at
The liquid is contained between the inner wall storage temperatures and during freezing or
of the tank and outer wall of the storage com- warming and closed tightly to avoid any leakage
partment, and the coldest vapor is only allowed or contamination. Most labs use plastic cryo-
to drop over the top of this wall, creating a tubes or drinking straws, but these vials are
dynamic vapor phase by continuous mixture less suitable for storage in the liquid phase
of the cold and warmer vapor anywhere in the (Elliot 1976) because of the risk of leaking of
storage compartment. At CBS, fungal cultures liquid nitrogen and splitting of the straws. The
are stored in storage tanks (Taylor-Wharton, choice for a particular type of cryovial may
K-series) of such design, where all samples further depend on available storage space and
can be kept constantly below 175 C in the properties of the organisms. Several procedures
dynamic vapor phase. using polyvinylchloride or polypropylene
To survive cooling and freezing, the cells straws have been described (Elliot 1976; Elliott
must be protected from the formation of large and Challen 1979; Hoffmann 1991; Stalpers
intracellular ice crystals, which would cause et al. 1987). Straws may be completely sealed
lethal damage to the cell membranes and orga- and used as the sole container, or several straws
nelles (Calcott 1978; Heckly 1978). Most proce- may be partially or completely sealed and kept
dures developed for fungi make use of chemical in a sterile cryotube (Hoffmann 1991). At CBS,
cryoprotectants that partly penetrate the living fungal cultures are preserved mainly in poly-
cells where they prevent or reduce the growth propylene drinking straws. Because of their
of intracellular ice crystals. They can be added small size, eightfold storage per strain is rea-
to the medium used for growing the fungus or lized using much less space and consuming
added to the harvested mycelium shortly before much less liquid nitrogen than would be
freezing. Most widely used cryoprotectants are required for storing the same amount of sam-
glycerol and dimethyl sulfoxide (DMSO) ples in glass vials.
(Hoffmann 1991; Jong 1989; Smith 1983), usu- Cultures are inoculated on solid agar media
ally at final concentrations of 10–20 % (w/v) in on plates or in liquid cultivation medium in
the case of glycerol and 10–12.5 % for DMSO. tubes (yeasts). After incubation under optimal
conditions for growth, the cultures are checked
Mixtures of 10 % DMSO and 8 % glucose have proved for purity and identity. The fungal material is
more effective in protecting fungi than 10 % glycerol,
including some fungi that seem to be more sensitive to
preserved in commercial drinking straws 4 mm
damage during cooling than most other fungi (Smith in diameter (cut to pieces approximately 45 mm
1983), but since DMSO is toxic to humans, glycerol is long with a standard office paper cutter) that
usually preferred for standard cryopreservation proce- are closed at one end using an industrial heat
dures, and most fungi are not less well preserved with sealer (Audion Electro, the Netherlands) with
glycerol than with DMSO. For some groups of fungi
these cryoprotectants still give insufficient survival, and
adjustable sealing temperature and pressure
other chemicals can be used, for example, ethylene (Stalpers et al. 1987). The straws are then put
glycol for the preservation of zoospores of rumen chy- into a bacteriological screw-cap bottle and
tridiomycetes (Sakurada et al. 1995). autoclaved at 121 C for 20 min. Under aseptic
conditions in a safety cabinet, straws are half
Chemicals that are more effective in enter- filled with sterilized 10 % aqueous glycerol as
ing living fungal cells generally also show an the standard, but for some fungi 10 % aqueous
increased toxicity, and exposure time to the DMSO is used as the cryoprotectant. Pieces of
cryoprotectant can affect postpreservation sur- agar with mycelium are punched out of the
vival (Souzu 1987). When toxic cryoprotectants culture plate with a metal cork borer with a
are used, it is advisable to determine the maxi- pin and transferred to the straws. For each
strain 10–12 straws are thus filled, after which growth. After incubation under the right con-
they are labeled and sealed completely. Sealing ditions, the strain is checked for viability, iden-
quality is crucial and should always be checked tity, and purity. Many strains start growing
by squeezing the straws firmly with a pair of within 48 h after opening the straw, but some
flat-beaked forceps. For yeast, straws are filled only do so after a lag of up to 7 days.
with 60 % glycerol, and using a pipette the The straw technique is less suitable for
volume is supplemented with ca. 1.5 mL of the some fungi, particularly ectomycorrhizal basi-
gently mixed yeast culture. diomycetes, which may be explained by the fact
In a programmed freezer (e.g., ice cube), that these fungi are more sensitive to the physi-
the filled straws are frozen from room temper- cal damage impacting on the hyphae during
ature to 40 C at a rate of approximately preparation. Stielow et al. (2012) describe a
1 C/min, then more rapidly to 80 C. Alter- new technique in which mycelia are allowed to
natively, the samples can be frozen in a plasma grow over charcoal filter paper strips (CFSs).
freezer to 40 C and then transferred to Inoculum can be placed centrally on the CFSs,
80 C in a mechanical freezer or placed in and in the case of extremely slow growing fungi
(the vapor of) liquid nitrogen. The plasma the entire colonies will be used. After the fungus
freezer should be checked regularly for cool- has sufficiently grown, the strips are carefully
ing rates using an external calibrated ther- removed with forceps and placed in a sterile
mometer. When the frozen material is placed Petri dish and flooded with 10 % (v/v) glycerol
in storage or taken out for use later, it should solution and incubated for 1–2 min. Then the
always be adequately protected from excessive strips are taken out, with the excess liquid being
warming. allowed to drip off, and transferred to cryovials
and directly placed in the vapor phase of liquid
Sometimes precooled blocks are used to handle vials, nitrogen for freezing. For reactivation, a strip
but at CBS racks and drawers are taken from the cryo- can be taken out aseptically and placed on a
genic system and transferred to a mechanical 80 C
freezer with a modified interior, where the material fresh Petri dish with suitable medium for
remains at temperatures below 65 C while vials are growth. If the remaining straws in the cryovial
collected or stored. are kept frozen during this procedure, they can
be used later.
To check the viability, identity, and purity If a programmed freezer is not an option,
of the cryopreserved strains, one straw is another convenient technique for cryopreser-
opened a minimum of 7 days after preservation, ving fungi that sporulate readily is freezing in a
and a second check after 3–5 years is advisable. 80 C freezer using Nalgene Cryo 1 C freez-
Frozen straws need to be thawed suffi- ing containers (Nalgene, Rochester, NY). Agar
ciently rapidly to avoid cell damage by recrys- slices cut from actively growing colonies are
talization of water. This can be done by placing placed in sterile plastic cryogenic vials in 20 %
them in warm water at around 35 C for glycerol. After 1 or 2 days at room temperature,
approximately 5 min until all contained mate- which allows the glycerol to penetrate the fun-
rial has thawed. Caution should be taken not to gus, the cryovials in cryogenic containers are
overheat the material. Oomycota are thawed in placed in a 80 C freezer. To have some con-
water at 20 C because they are very sensitive to trol over the freezing process in cryovials when
overheating and normally do not survive tem- placed in the 80 C freezer, these vials are
peratures above 25 C. Before opening, the sur- placed in Nalgene Cryo 1 C freezing containers
face of the straws is sterilized in 70 % ethanol. that contain a reservoir for isopropyl alcohol
Then the straws are cut open with sterile scis- providing the controlled cooling. Once frozen,
sors aseptically in a safety cabinet, and the the cryovials can then be transferred to trays for
content is taken out with a sterile needle and storage in liquid nitrogen storage facilities
placed on fresh agar medium suitable for where available.
2. Freeze Drying penter and Crowe 1988; Carpenter et al. 1991).

Lactose and other disaccharides have been
Freeze drying, or lyophilization, is a widely found to be very good protectants. Trehalose,
used method for long-term preservation of which is known to be produced naturally by
fungi that has several advantages over cryo- some drought-tolerant filamentous fungi and
preservation. Adequately freeze-dried cultures yeasts (Thevelein 1984; Wiemken 1990), has
generally maintain their morphological, physi- been found to be optimal, and this disaccharide
ological, and metabolic properties and can can therefore be added to the suspension of
remain viable for more than 40 years (Hessel- skim milk (Berny and Hennebert 1991; Tan
tine et al. 1960). Storage of cultures can be et al. 1995). At CBS, trehalose is a standard
economical with regard to space and equip- additive in skim milk suspensions used in
ment, and the ampoules containing the freeze- freeze drying large or multicellular spores.
dried product can be quickly and more easily An optimal freeze-drying process involves
dispatched than cultures that need to be reacti- (1) dehydration of the propagules in the cool-
vated before shipment or kept at low tempera- ing phase prior to desiccation, (2) sublimation
tures during transport, and this is an obvious of ice from the frozen suspension during a
advantage for service collection. However, not phase of primary drying, followed by (3)
all fungi can be freeze dried successfully. removal of chemically bound water from the
Although nonsporulating mycelia can be freeze cells as well as from the protectant in a second-
dried with some success (Pertot et al. 1977; Tan ary drying phase (Tan 1997; Tan et al. 1994).
et al. 1991), the method is predominantly The freeze-drying process can only be con-
applied to yeast cells and propagules of fila- trolled sufficiently in a commercial freeze
mentous fungi and bacteria (Tan 1997, 2011). dryer. The optimal cooling rate for phase 1
Critical parameters for the survival of lyo- depends on the properties of the spores (see
philized fungal propagules are the composition below). As the extracellular water is frozen
of the lyoprotectant (Berny and Hennebert into ice crystals, the remaining lyoprotectant
1991; Tan et al. 1995), the cooling rate during becomes increasingly viscose, leading to the
the cooling phase (Smith et al. 1986), the size of removal of unbound water from the cells. In
the propagules, and the thickness of the cell the primary drying phase that follows, the vis-
wall (Tan et al. 1994). Residual moisture and cosity of the lyoprotectant solution is so high
storage temperature also influence the shelf life that it is a glass, viz., a liquid in which the
of the dry product. molecules are immobilized (Franks 1990; Tan
The use of lyoprotectants protects the liv- 1997). To keep a formulation stable, it should
ing cells from possible damage caused by ice- be maintained at a temperature below the glass
crystal formation during cooling and destabili- transition temperature (Tg). The Tg of a for-
zation of membranes and cellular proteins dur- mulation depends on the composition of the
ing subsequent stages of the freeze-drying protectant and the residual moisture content
process. Various media have been tested for (rmc) of the glass (Hatley 1990); the drier the
their suitability as lyoprotectants in fungal glass, the higher the temperatures at which it
spore suspensions, including sodium gluta- can be stable. The Tg of a formulation can be
mate, peptone, various mixtures of saccharides, determined by differential scanning calorime-
and skim milk (Berny and Hennebert 1991; Tan try (DSC) (Hatley 1990). A glass will melt at the
et al. 1995). Tg, and the product will rapidly deteriorate. In
An aqueous solution of skim milk (10 or the primary drying phase, usually ca. 20 C
20 %) has proved very suitable and has been for skim milk, the ice crystals are evaporated
used widely to prepare spore suspensions for under a vacuum of ca. 0.30 mbar, leaving a glass
freeze drying. Saccharides in the milk protect with numerous channels. In the secondary dry-
cellular membranes and membranes of orga- ing phase, the temperature can be slowly
nelles during cooling and drying and play a increased to +25 C, depending on the lyopro-
role in stabilizing dried cellular proteins (Car- tectant, and the vacuum brought to 0.03 mbar
so that bound water can be removed more To start the freeze-drying process, frozen
quickly from the cells and the surrounding ampoules are placed in a freeze dryer on a shelf
cake in order to obtain a sufficiently stable precooled to 40 C, or they can be stored in a
product at the desired storage temperature. mechanical freezer (80 C) for some time if
Cultures for freeze drying are inoculated on freeze drying cannot be started immediately.
fresh media that favor good sporulation and Shelf (sample) temperature and pressure can
incubated under optimal conditions. The CBS be optimized for the primary and secondary
homepage and strain catalog provide informa- drying phase. For example, in the primary
tion on media and conditions for sporulation drying phase, the frozen water can be subli-
for many fungal species (http://www.cbs.knaw. mated at ca. 20 C under high vacuum
nl). For yeast and most filamentous fungi, agar (0.30–0.40 mbar), while in the secondary
slants or plates are suitable. Spores should be phase the samples can be warmed to ca. 25 C
harvested at the right moment and be suffi- under an ultrahigh vacuum (0.10–0.02 mbar) to
ciently mature but not too old. After the culture remove the bound water from the cells and the
has been checked for sporulation, purity, and surrounding protectant (ice condenser temper-
identity, spores can be harvested by aseptically ature 60 to 80 C). It is important to docu-
and slowly pouring 5 mL of tyndalized skim ment the main process parameters during
milk into the culture tube or plate and gently drying (shelf and condenser temperatures,
scraping the colony surface with a pipette or pressure), which should be fully automated in
needle. With the pipette the suspension can be modern commercial freeze dryers.
transferred back to a tube with skim milk and
mixed carefully. Aliquots (0.05–0.2 mL) are The best types of vials are those that can be stoppered
then transferred to glass ampoules for freeze with cotton plugs (with or without silicon rubber stop-
drying. A concentration of ca. 106 spores/mL is pers), minimizing risk of contamination (of the vial
content and the equipment used!). The size and shape
generally required. Filled ampoules must be of the vials matter; in particular, the surface of the pellet
refrigerated (5–7 C) and processed within 1– in relation to its volume influences the efficacy of the
2 h to avoid germination. CBS uses two types of drying process and, ultimately, the viability of the end
ampoules and two basic methods of cooling, product.
depending on the properties of the fungal
spores. Thin-walled, one-celled spores dehy- Trace amounts of oxygen or water intro-
drate quickly and can therefore be instantly duced when sealing the ampoules after drying
cooled by plunging the ampoules (simple cylin- can cause the deterioration of samples during
drical ampoules 1106 mm in diameter, non- storage. The ampoules therefore need to be
prescored) into a bath of acetone cooled to sealed under high vacuum or filled with a dry
80 C with a cooling device (e.g., Cryocool, inert gas (nitrogen) before closure. Vials made
Neslab). Multicellular or thick-walled spores of borosilicate glass are easiest to heat-seal
dehydrate less quickly and should be frozen at securely. Freeze-dried samples survive storage
a lower rate, optimally around 1 C/min, in in the dark at room temperature (20 C) for
order to avoid intracellular ice-crystal forma- periods of up to 25 years when dried to a rmc
tion, which is lethal. For cooling of these spores below 5 % (Tan et al. 1991). Shelf life is
a plasma freezer or programmed freezer (e.g., extended considerably if the samples are drier
ice-cube) can be used. (2–3 % rmc) and can be stored under controlled
temperature, preferably 4–6 C. One ampoule
For these more sensitive spores CBS uses prescored of each series of dried spores should be opened
flat-bottom glass “vampoules” (Wheaton, 86 immediately after freeze drying to check viabil-
12 mm), which can be half closed with rubber stoppers ity, purity, and identity.
during the drying phases and shut airtight before tak-
ing them out of the freeze dryer at the end of the
Reactivation of freeze-dried material is
secondary drying phase. simple, but the procedures recommended by
the supplier of the material should always be culture collections not only provide the neces-
carefully followed, particularly when rehydrat- sary basis for fungal systematics and biodiver-
ing more sensitive fungal spores. Freeze-dried sity research but are equally important for
cells should be given sufficient time to rehy- documenting research in ecology, genetics,
drate before being transferred to a solid and plant pathology. For example, phytosani-
medium. After disinfecting the outer surface tary regulations for the protection of food crops
of the ampoule with 70 % ethanol, it should be against fungal pathogens depend on properly
opened aseptically (preferably in a safety cabi- documented reference material in order to be
net), suspending the freeze-dried material by effective.
pouring the full content into a tube containing The value of herbarium specimens and cul-
1–2 mL of sterile water or sterile malt-peptone tures as reference material depends strongly on
solution. The tube is then shaken gently and left the quality of the preservation methods and
at room temperature for 4–12 h before the sus- management of the associated data. Mycolo-
pension is poured onto a solid agar medium gists who have performed DNA sequencing on
suitable for (vegetative) growth. The remainder herbarium specimens and cultures from culture
of the suspension can be stored in a refrigerator collections have demonstrated repeatedly that
for up to 2 days for use in case the first attempt they provide a rich source of undescribed taxa.
fails. Optimal conditions for growth should be To fix the application of already published
applied, noting that not all media suitable for names in the future, living cultures and vou-
sporulation are equally fit for reactivation of chered herbarium material have been selected
freeze-dried material. Most fungi will start by taxonomists as epitypes, demonstrating
growing within 1–2 days, but some, particularly again the importance of these resources.
certain slow-growing fungi, need more time to Early fungal genome sequencing projects
grow. Yeasts cells that are suspended in water have relied sometimes completely on isolates
or malt peptone can normally be poured imme- kept in laboratories lacking adequate proce-
diately onto solid agar medium, and suspen- dures for quality control and preservation.
sions in malt peptone can be stored in a Fungal cultures accessioned in public culture
refrigerator for up to 14 days. If survival is collections that have methods in place for
expected to be low, it is advisable to revive long-term preservation, state-of-the-art
cells in 1.2 M sucrose to dilute the protectants authentication, and dispatch will form an indis-
and stimulate the efflux of the protectants from pensable resource of material for global initia-
the cell while minimizing osmotic expansion tives, such as DNA barcoding and genome
(Tan 1997; Tan and Stalpers 1996). sequencing projects. Herbarium specimens
Even if the crucial parameters for freeze associated with these cultures will be of equal
drying are carefully optimized and controlled, importance for future reference.
some fungi will still show loss of viability
immediately after drying or considerable Acknowledgements Collections Manager Shannon
Dominick is thanked for her useful comments on best
decline during later storage. This may be herbarium practices as well as her conscientious man-
caused by the lesser condition of the strain, agement of the U.S. National Fungus Collections (BPI).
which may have deteriorated from having Pedro W. Crous is kindly acknowledged for comment-
been kept in active growth for a longer time ing on the manuscript.
before being first freeze-dried or because of
unknown properties of the spores that render
them recalcitrant to freeze drying.
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Evolution
9 Subcellular Structure and Biochemical Characters in Fungal
Phylogeny
DAVID J. MCLAUGHLIN1, T.K. ARUN KUMAR2, MEREDITH BLACKWELL3,4, PETER M. LETCHER5,

ROBERT W. ROBERSON6
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
II. The Role of Subcellular Structure in Fungal Structural and biochemical characters have
Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
III. The Role of Biochemical Characters in played an important role in determining phylo-
Fungal Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . 233 genetic relationships among Fungi since the
IV. Character and Character State Definition beginning of their modern classification in the
and Refinement . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 mid-nineteenth century. It was only with the
V. Structural and Biochemical Database . . . . 238 introduction of ultrastructural methods and
VI. Structural and Biochemical Database in
Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . 239 improved biochemical analyses in the 1950s
VII. Development of the Fungal Subcellular that a clearer understanding of structural vari-
Ontology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 ation and its phylogenetic implications became
VIII. Specimen Preparation and Evaluation for possible (McLaughlin et al. 2009). Subcellular
Subcellular Structure. . . . . . . . . . . . . . . . . . . . . . 242 structural data complemented biochemical
A. Specimen Preparation and Evaluation . 242
1. Fixation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 studies on cell walls and enzymatic pathways
2. Standard Infiltration and that began to delimit a monophyletic kingdom
Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Fungi from organisms that are similar to Fungi
3. Microwave Fixation and Embedding . . 246 in form and ecology (Bartnicki-Garcia 1970,
4. Section Staining and Microscopy 1987). Early subcellular studies of Fungi
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
B. Methods for Selected Cellular Features. . 247 resulted in an extensive literature in the 1960s
1. Nuclear Division Studies . . . . . . . . . . . . 247 through the 1980s, but the number of studies
2. Apical Organization Studies . . . . . . . . . 248 declined with the introduction of more
3. Flagellated Fungi Studies . . . . . . . . . . . . 249 demanding cell preparation procedures devel-
IX. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 oped in the 1980s (Hoch 1986; Howard and
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
O’Donnell 1987). These new cryofixation meth-
ods also added confusion as to which methods
provided reliable data and discouraged
researchers who worked with taxa that were
1
Department of Plant Biology, University of Minnesota, St.
not suitable for preparation by cryofixation
Paul, MN 55108, USA; e-mail: davem@umn.edu methods. As a result, the number of fungal
2
Department of Botany, The Zamorin’s Guruvayurappan College, taxa studied ultrastructurally is still very lim-
Calicut, Kerala 673014, India; e-mail: tkakumar@gmail.com ited and does not provide a meaningful under-
3
Department of Biological Sciences, Louisiana State University, standing of subcellular structural variation in
Baton Rouge, LA 70803, USA; e-mail: mblackwell@lsu.edu
4 Fungi.
Department of Biological Sciences, University of South Car-
olina, Columbia, SC 29208, USA; e-mail: mblackwell@lsu.edu Molecular phylogenetics and phyloge-
5
Department of Biological Sciences, University of Alabama, nomic data are currently providing a better
Tuscaloosa, AL 35487, USA; e-mail: letch006@bama.ua.edu understanding of fungal relationships and
6
School of Life Sciences, Arizona State University, Tempe, AZ, revealing a large number of fungal clades for
85287, USA; e-mail: robby2@asu.edu

230 D.J. McLaughlin et al.
which little or no subcellular or biochemical Character states for structural characters in Fig. 9.1:
data exist (Hibbett et al. 2007; James et al. Motile Cell
1. Centriole absent, 0; present, 1.
2006a; McLaughlin et al. 2009). The AFTOL 2. Nonflagellated centriole absent, 0; present, 1.
Structural and Biochemical Database (SBD) is 3. Kinetosome absent, 0; short, <0.5 mm, 1; long,
intended to clarify the limits of our knowledge >0.5 mm, 2.
of subcellular characters and provide a ready 4. Organization of microbody lipid complex (MLC)
reference for comparison of structural data to absent, 0; MLC cisterna backs lipid, 1; MLC cisterna
backs microbody, 2.
reach a meaningful level of understanding of 5. Rhizoplast cap absent, 0; present, 1.
structural evolution in Fungi (Kumar et al. 6. Flagellar props absent, 0; present, 1.
2013). Molecular phylogenies have permitted Nuclear Division and Spindle Pole Body
a reevaluation of both biochemical and subcel- 7. Basic spindle pole body form centriole-associated
lular characters and provided a new under- extranuclear and intranuclear components with intact
nuclear envelope, 0; centriole-associated material with
standing of their evolution (McLaughlin et al. open nuclear envelope, 1; small amounts of extranu-
2009; Weete et al. 2010). This chapter aims clear and intranuclear material with intact nuclear
to give the reader guidance on how to acquire envelope, 2; notched ring with persistent half middle
and evaluate subcellular characters and on piece plus intranuclear component, 3; plaque or disc, 4;
the role of the SBD and Fungal Subcellular globular, 5; ring(s) containing microtubules but lacking
centriole nine-fold symmetry, 6; bar, 7.
Ontology (FSO) in fungal systematics and phy- 8. Metaphase nuclear envelope intact, 0; intact with
logeny. The structural data also have functional small polar fenestrae plugged by the spindle pole bod-
significance and provide guidance in cell and ies, 1; loose polar fenestrae, including extensions of
developmental studies on character diversity nuclear envelope into the cytoplasm at the spindle
and evolution within clades and across the pole, but mainly intact, 2; partially dispersed, 3; nearly
or entirely dispersed, 4; intact with polar fenestrae
Fungi. adjacent to centrosome containing paired centrioles, 5.
Septum
9. Uniperforate septum absent, walled-off pore, or
apparently walled-off pore, 0; a single central pore, 1.
II. The Role of Subcellular Structure 10. Multiperforate septum absent, 0; simple pores
in Fungal Phylogeny with uniform pores not solely adjacent to hyphal wall,
1; simple pores with variable-sized large pores adjacent
to hyphal wall, 2; plasmodesmata, 3; thickened septum
The relatively simple structure of Fungi masks with central pore closed by plasmodesmata, 4.
many instances of homoplasy (e.g., convergent 11. Uniperforate septal pore margin uniperforate
evolution). Light microscopy may suggest that septal pore absent, 0; unelaborated margin, 1; uniper-
two organisms are not closely related as, for forate septal pore absent but with some type of discon-
tinuity within septum, for example, disruption of
example, in the organization of zoospores, or central layer of cross wall, wall swelling, or deposits
specialized cell types, such as meiosporangia within cross wall, suggestive of a blocked or disrupted
and spores, but light microscopic images are pore, 2; septal pore swelling, 3; with lenticular cavity
best understood after the vastly improved reso- (bifurcate), 4.
lution of ultrastructural examination. Although 12. Membrane-bound bodies associated with septal
pore(s) absent, 0; Woronin bodies, 1; microbodies, 2.
Fungi contain a broad array of subcellular 13. Septal pore cap absent, 0; present, 1.
structures (Beckett et al. 1974; Bracker 1967; Apical Organization
McLaughlin et al. 2001), relatively few provide 14. Apical organization of hypha or germ tube
synapomorphies for kingdom Fungi. Excep- absent, 0; loose vesicle cluster, 1; apical crescent, 2;
tions include flattened mitochondrial cristae, Spitzenkörper, 3.
reduced Golgi bodies in most and in certain
forms, motile cells with a single posterior fla- Light microscopy initially revealed the diver-
gellum, and similar flagellar apparatus (Baldauf sity in zoospores of Fungi and fungus-like organ-
et al. 2004). Motile cells, septa, and cytological isms, and zoospores subsequently became the
features of dividing nuclei have provided clues focus of ultrastructural studies (Barr 1981; Fuller
to taxonomic relationships within the Fungi 1977). The structure of the transition zone of the
(Fig. 9.1). flagellar apparatus (Barr 1992) and of the flagellar
Subcellular Structure and Biochemical Characters in Fungal Phylogeny 231
Fig. 9.1 Distribution of 14 characters used in differen- Structural and Biochemical Database https://aftol.umn.
tiating subphyla and classes of Fungi. Taxa with motile edu/. Asterisk (*) indicates that the character state is
cells are indicated by flagellated cell diagrams, and based on information from a single taxon. See text for
zygosporic fungi by zygospore diagrams. Characters character state descriptions. Fungal phylogenetic tree
1–6 associated with flagellum apparatus of motile data are based on James et al. (2006a), White et al.
cells; characters 7 and 8 associated with the nucleus; (2006), and Hibbett et al. (2007). aUnpublished.
b
characters 9–13 associated with the septum; character Germ tubes only
14 associated with the hyphal apex. Data from AFTOL
roots within the zoospore (Barr 1981) separated basal Fungi, although relatively few taxa have
early diverging Fungi from other fungus-like been studied within each lineage (James et al.
groups with motile cells that are more closely 2006b).
related to stramenopiles or Rhizaria (Braselton The septal pore structure was first observed
2001; Cavalier-Smith 2001; Dick 2001; Fuller with light microscopy, but septum diversity was
2001) (see Beakes et al., Chap. 3, and Bulman not fully understood until septa were examined
and Braselton, Chap. 4, Vol. VII. Part A). The ultrastructurally (Bracker 1967). Hemispherical
systematic importance of zoospore structure pads at the septal pore were first reported by
has been supported by recent studies that light microscopy in Basidiomycota (Buller
revealed multiple monophyletic lineages within 1933). The pads, termed dolipores when
examined ultrastructurally (Moore and McAlear Specialized cell types, such as cystidia and para-
1962), helped to distinguish some Basidiomycota physes, are also potential sources of characters,
from the Ascomycota. It should be noted, how- but homoplasy may be a problem with interpre-
ever, that the flared dolipore septum may be an tation at certain taxonomic ranks (Bellemere
artifact consistently seen with chemical fixation 1994; Jenkinson et al. 2008; Padamsee et al.
and is not pronounced with cryofixation (Hoch 2008; Pfister and Kimbrough 2001) but are
and Howard 1981). The septal pore apparatus, incompletely surveyed. Host–parasite interac-
i.e., the septum and cytoplasmic organization tions show remarkable structural variations in
around the pore (Bracker and Butler 1963), some fungal groups and have been used for tax-
lacked dolipores in some Basidiomycota, and onomic purposes (Bauer et al. 2001; Bergerow
these simple septate taxa share plesiomorphic et al. 2006). Dictyosomes, consisting of stacked
septal characters with Ascomycota (Celio et al. cisternae, are restricted to a few basal fungi and
2006). Similarly, in some zygosporic fungi, light replaced by less morphologically complex Golgi
and the earliest electron microscopy revealed a equivalents, comprised of only a single cisterna,
distinctive pore with a lenticular cavity (Benjamin in other fungi including many flagellated taxa
1959; Benny 1972; Reichle and Lichtwardt 1972), (Roberson et al. 2010). Other subcellular features
which was subsequently shown to be synapo- may also be of phylogenetic interest at the ordi-
morphic for the Kickxellomycotina (Celio et al. nal or lower taxonomic ranks, such as hyphal
2006; Hibbett et al. 2007). branching involving the disruption of the outer
The fundamental nature of mitosis and wall (McLaughlin et al. 1995b), the microscala
meiosis to eukaryotic cells made the microtu- (McLaughlin 1990) or symplechosome (Bauer
bule organizing center at the spindle pole, i.e., and Oberwinkler 1991; Oberwinkler and Bauer
the centriole and its associated material or the 1989), which consists of endoplasmic reticulum
spindle pole body (SPB) in Fungi that lack or mitochondria cross-linked by regularly
motile cells, and changes in the nucleoplasm, spaced filaments, or unusual vesicles, such as
nucleolus, and nuclear envelope during divi- those with tubular invaginations (McLaughlin
sion, a focus of study for phylogenetic charac- et al. 2008). The continual addition of new cell
ters that were used to separate Fungi from structures, such as a new septal pore cap type
fungus-like organisms (Heath 1980, 1986). (Padamsee et al. 2012) and type of septal pore
Cryofixation resolved concerns about the valid- organization (Healy et al. 2013), suggests that the
ity of using membrane fragmentation during Fungi are still poorly understood ultrastructur-
nuclear division as a structural character. ally.
Septa, nuclear division, and SPB characters Many of the structural studies by system-
provide synapomorphies for some clades atic mycologists interpret cellular components
within Fungi (Celio et al. 2006). on a morphological basis only. Cell biological
Other subcellular features also may have analyses are needed to understand these cell
phylogenetic significance. The growing hyphal components. Differentiating Woronin bodies
apex forms a Spitzenkörper in many Fungi, but it in Ascomycota, a specialized form of peroxi-
appears to be absent among most zygomycete some in Pezizomycotina, from microbodies at
taxa (Roberson et al. 2010). The hyphal apical the septal pore in other Ascomycota and Basi-
organization is currently being examined in diomycota required cytochemical localization
unstudied major fungal clades (Bentivenga et al. for HEX-1 protein (Dhavale and Jedd 2007;
2013). Septa in ascogenous hyphae and the ascus Roberson et al. 2010). Similar studies are
have been the source of subcellular characters, as needed for other cell structures, such as the
has the ascus apex (Bellemere 1994; Cole 1979; atractosome, which exhibits a morphology
Kimbrough 1994). Structural variations in the that varies among species (Oberwinkler et al.
developmental stages of basidiospores are poten- 2006; Weiss et al. 2004). This microbody-like
tial sources of phylogenetic characters, but addi- structure is found around the septal pore
tional studies are needed to assess their in some Pucciniomycotina and contains
phylogenetic potential (McLaughlin et al. 1985; peripheral electron-opaque material or curved
Miller 1988; Yoon and McLaughlin 1986). cisternae.
fungi, biochemical and physiological characters

were used to distinguish among taxa.
Nineteenth-century mycologists used stains
and dyes that yielded color reactions that were
used to obtain information for lichens and
other ascomycetes (Nylander 1865; Rolland
1887). For lichens, simple spot tests based on
reactions of metabolites to KOH and Ca(ClO)2,
Fig. 9.2 Drawings comparing spindle pole body– at first used without knowledge of the basis for
nuclear envelope relationship at metaphase–anaphase the reaction, helped to differentiate the pro-
in smut fungi: (a) Ustilago maydis (Ustilaginomyco- ducts produced by specimens with similar
tina) at meiosis and (b) Microbotryum violaceum (Puc- appearance (Nylander 1867). In the early nine-
ciniomycotina) at mitosis. In U. maydis the SPB is
subglobular with a convex internal layer, while in M.
teenth century, however, chemists were inter-
violaceum it is subglobular with a flat internal layer and ested in determining the nature of the
resembles a modified disc. Scale bar¼0.2 mm. U. may- compounds. For example, Pereira (1854) dis-
dis after O’Donnell and McLaughlin (1984); M. viola- cussed using lichens such as species of Roccella
ceum after Poon and Day (1976) to obtain litmus. Fluorescence and thin-layer
chromatography were more sophisticated
There are limitations to the use of struc- means developed later to obtain similar infor-
tural characters for phylogenetic reconstruc- mation.
tion without guidance from an independent Color tests continue to be used in identifi-
and data set such as molecular sequences. cation keys for many fungi, including lichens,
While nuclear division and SPB and/or septal yeasts, other ascomycetes, and basidiomycetes
characters can be used independently to pro- (Ammirati et al. 1985). The well-known Mel-
duce phylogenetic trees (Lutzoni et al. 2004; zer’s reagent (Melzer 1924), a modification of
McLaughlin et al. 1995a), guidance from molec- earlier formulations of iodine, produces a blue
ular data is desirable to recognize structural staining (amyloid reaction due to fungal starch)
homoplasy and to determine character polarity, of certain basidiospores and ascus tips of apo-
i.e., its plesiomorphic or apomorphic status. thecial fungi, such as Peziza by Melzer’s iodine,
Many unexpected taxonomic relationships and the red staining (dextrinoid reaction due to
were revealed with molecular sequence data glycine betaine in some fungi) of hyphidia of
and led to a new understanding of structural Vararia and other members of the Lachnocla-
characters, for example, motile cells of Blasto- diaceae (Baral 1987; Dodd and McCraken 1972;
cladiomycota and Chytridiomycota (James Kuo 2006; McCracken and Dodd 1971). A more
et al. 2006b) (see Powell and Letcher, Chap. 6, recently developed spot test, Diazonium Blue B
and James et al., Chap. 7, Vol. VII, Part A), and (DBB), has been used to distinguish basidiomy-
SPB form among certain smut taxa, which are cete (red staining) from most ascomycete (yel-
now classified in different subphyla (Celio et al. lowish or nonstaining) yeasts (Kurtzman et al.
2006; McLaughlin et al. 1995b) (Fig. 9.2) (see 2011) but is of more limited use in distinguish-
Aime et al., Chap. 10, Vol. VII, Part A). Thus, ing filamentous fungi (Hutchison and Summer-
sequence data aid in polarizing often autapo- bell 1990). DBB apparently links to tyrosine and
morphic structural characters. histidine residues of wall proteins of basidio-
mycetes (Tevayanond 1981).
In addition to DBB, a wide array of bio-
chemical and physiological tests have tradition-
III. The Role of Biochemical Characters ally been used in yeast taxonomy. Yeasts have
in Fungal Phylogeny few morphological characters for distinguish-
ing taxa, so bacterial methods were adapted for
Before effective microscopic methods were distinguishing them. Approximately 80 tests for
available to search out distinctive features of determining the assimilation and fermentation
Table 9.1 Major chemical characters used in distinguishing Fungi from other major groups of organisms (Vogel
1960, 1965; Alexopoulos et al. 1996; Prillinger 2002; Torruella et al. 2009)
Storage
Group Lysine pathwaya compound Cell wall Sterol
Plants Diaminopimelic Starch Cellulose, hemicellulose, Stigmasterol, brassicasterol,
(DAP) (Vogel lignin others
1960, 1965)
Animals – Glycogen, lipids, – Cholesterol
trehalose
Fungi ∂-aminoadipate Glycogen, lipids, b-1,3 and 1,6 glucans+chitin Ergosterol, others including
(AAA) trehalose (b-1,4-N- cholesterol, 24-methylene
acetylglucosamine cholesterol, 24-ethyl
polymer), chitosan in cholesterol,
zygomycete fungi (b-1,4- brassicasterol
glucosamine polymer),
mannoprotein or
polysaccharide matrix
Oomycota DAP Mycolaminarins, b-glucan, hydroxy proline Fucosterol or acquired
soluble b-1,3- and cellulose
glucans
Myxomycetes DAP Glycogen b-galactose Campesterol [(24R)-24-
Methylcholestan-3b-ol]
Dictyostelids DAP Glycogen, Cellulose ∂-22-stigmasten-3b-ol
trehalose in
mature
spores?
a
See text for discussion of genes involved in lysine synthesis
potential of carbon and nitrogen sources, water As late as the mid-twentieth Century, G. W.
potential, and vitamin production are com- Martin, a prominent mycologist, asked, “Are
monly used. Ubiquinone (CoEnzyme Q) also fungi plants?” He thought that biologists
has been used in yeasts, as well as for some would answer with “an unqualified affirmative”
filamentous fungi, but is variable within many (Martin 1955). Alexopoulos (1952), however,
lower-rank taxa. Although such characters are had already accepted the “strong and growing
no longer used solely to identify yeasts, they are opinion among mycologists that the fungi may
used in conjunction with other characters and have originated from animal-like forms.” As
are important in determining the potential for more was learned about biochemistry, other
substrate utilization in the natural environmen- characters, such as the fungal storage product
tal (Kurtzman et al. 2011). glycogen, lysine synthesis pathway, and the
In addition to using biochemical attributes ergosterol membrane sterol (Weete et al.
to distinguish taxa, they also were used as some 2010), were used to distinguish fungi from
of the only characters that extended across taxa other walled organisms (Alexopoulos et al.
for use in hypothesizing higher relationships or 1996) (Table 9.1).
distinguishing Fungi from other major groups Walls of fungi in particular have been used
of organisms (Table 9.1). Linnaeus (1753) con- successfully in distinguishing Fungi from the
sidered fungi to be plants and placed them in other major filamentous organisms, for exam-
class Cryptogamia with ferns, bryophytes, and ple the Oomycota, and later were to become
algae based on the presence of a cell wall. Later important as a target for the action of antibio-
the chemical composition of the wall became a tics. The classic studies of Bartnicki-Garcia
main character to distinguish among the wide firmly established the differences between the
variety of unrelated organisms with cell walls. cell wall chemistry of oomycetes and Fungi,
including uniflagellated forms (e.g., Bartnicki- ergosterol in soils (Grandmougin-Ferjani et al.

Garcia 1970, 2006), and this work has been 1999). Exceptions to ergosterol membranes also
tested many times by DNA analyses to show exist within the Ascomycota and Basidiomy-
the distance between the two groups. Cell wall cota, although most phylum members have
chemistry also has been used to support dif- ergosterol membranes. For example, Puccinio-
ferences among fungal groups. Prillinger and mycotina have distinctive membranes of either
his colleagues (e.g., Prillinger et al. 1993, 2002) 24-ethyl-cholesta-7,24(28)-dienol (stigmasta-
used a gas chromatography method (Prillinger 7,24(28)-dienol) or 24-ethyl-cholest-7-enol
et al. 1990) to analyze the cell wall carbohydrate (stigmast-7-enol) intermediates in the 24-ethyl-
composition and compare 250 species of asco- cholesterol pathway. The synthesis of these
mycete and basidiomycete yeasts. The differ- sterols is separated by a single step in their
ences in neutral sugars of the groups indicated pathway (Weete et al. 2010). Among the asco-
that several patterns of wall carbohydrates were mycetes, the Erysiphales differ from other mem-
consistent with phylogenetic analyses based on bers of the Pezizomycotina by production of 24-
DNA as well as septal pore margin and septal methyl-cholesta-5,24(28)-dienol (24-methylene
pore cap characters. Ascomycetes with simple cholesterol) (Fig. 9.3).
pores and no associated membranes typically A major problem inherent in using the
have a predominance of glucose with some presence or absence of biochemical and physi-
mannose and galactose. Pucciniomycotina ological characters such as sterols is whether a
with simple septal pores and lacking a septal feature is absent or merely not expressed. Cer-
pore cap had cell walls that were predominantly tain examples need clarification to fully utilize
mannose but also with glucose, galactose, and the information from the expression of sterol
fructose. Ustilaginomycotina with septal pore membranes as a phylogenetic character. There
margin flared or not and no septal pore cap is some indication that certain parasitic fungi
had walls that were predominantly glucose but closely associated with plants and animals scav-
also often containing mannose and galactose enge membrane sterols from their hosts. Such
and lacking xylose. Members of Agaricomyco- information is vital beyond its use in evolution-
tina had cell walls predominantly of glucose ary studies because of the common treatment of
with mannose and xylose, and these findings many fungal infections using antifungal drugs
also corresponded with the presence of doli- that target sterol synthesis. Pneumocystis cari-
pore septa and a septal pore cap. nii has been reported to obtain membrane ster-
More recently, the predominant type of ols, i.e., cholesterol, from its host. Other reports
sterol present in fungal membranes has been indicate that P. carinii has genes for the synthe-
surveyed to compare sterol types and path- sis of several sterols based in part on genome
ways with recent phylogenetic information scans (Giner et al. 2002; Joffrion and Cushion
that was not available when much of the sterol 2010; Kaneshiro 2002). Is the major membrane
data were collected (Weete et al. 2010). Most of sterol, brassicasterol, in the membranes of
the fungi in the Ascomycota and Basidiomycota other symbiotic fungi including Tuber, Terfe-
have membranes that are distinctive from other zia, and the Taphrinales accumulated or is it
groups of organisms in that they contain ergos- synthesized?
terol, long considered to be the “fungal sterol.” Lysine synthesis has been used as a major
Among the early diverging fungal lineages, distinguishing feature between Fungi and all
however, there is variation in the membrane other major groups of organisms. The method
sterols (Fig. 9.3). Membranes of Glomeromy- of lysine synthesis has become murky because
cota tested contain 24-ethyl cholesterol, and it has recently been found that the ∂-amino-
this finding has implications for previously adipate reductase (AAR) gene, a core gene of
used research protocols based on mycorrhizal the AAA pathway, and lysA gene, a core gene of
biomass measurements by the presence of the DAP pathway, are both sometimes present
Fig. 9.3 Sterol distribution in Fungi. Taxa with motile methyl cholesterol and 24-methylene cholesterol; 14
cells are indicated by flagellated cell diagrams, and Basidiobolus, one species only; 15 Catenaria anguillu-
zygosporic fungi by zygospore diagrams. Asterisks lae; 16 Allomyces macrogynus and Blastocladiella emer-
indicate the predominant sterol present in a terminal sonii; 17 other chytrids; 18 only Monoblepharella sp.
taxon (usually a phylum). Distinctive sterols are pres- was sampled. Basal fungal groups and fungal sister taxa
ent in some clades within phyla: 1 Erysiphales; 2 Tuber are poorly sampled or completely unsampled. Fungal
and Terfezia; Tuber also contains mostly ergosterol; 3 phylogenetic tree based on James et al. (2006a), White
Pneumocystis; 4 Schizosaccharomyces; 5 Taphrina and et al. (2006), and Hibbett et al. (2007). Sterol data after
Protomyces; 6 several Polyporaceae; 7 Pucciniales; Weete et al. (2010). aPneumocystis spp. may scavenge
8 members of Mortierellales; 9 some Mucorales; 10 membrane cholesterol from hosts, although recent
Kickxellales; 11 Dimargaritales; 12 Smittium spp.; 13 studies indicate that it has genes to synthesize a variety
most members of Entomophthorales contain 24-methyl of D7 and D8 24-alkylsterols (Kaneshiro 2002; Giner
cholesterol, although several are known to have choles- et al. 2002; Joffrion and Cushion 2010)
terol or 24-methylene cholesterol or mixtures of 24-
in the same organisms, including all fungi that genes. Urbina and Blackwell (2012) compared
have completed or nearly completed genomes several species of yeast that assimilated D-
(Torruella et al. 2009). In another example, xylose and xylitol as sole carbon sources.
xylose fermentation is a rare ability of fewer Some of the yeasts fermented xylose, but others
than 20 of approximately 1,000 described did not. Fungi competent in xylose fermenta-
yeasts, determined by the presence of three tion had three intact genes [xylose reductase
(XYL1), xylitol dehydrogenase (XYL2), and ing homologies based on comparative methods
xylulose kinase (XYL3)] that functioned in and has been especially useful in reevaluating
xylose fermentation. In the yeasts that had the conclusions drawn from comparative methods.
ability to assimilate but not ferment xylose, For example, the determination, using molecu-
however, the xylose reductase gene (XYL1) lar methods, that smut fungi consisted of two
had a modified motif that may prevent the distantly related groups prompted a reevalua-
correct 3-D configuration and functioning of tion of SPB characters that revealed subtle
the gene. A new era in the interpretation of structural differences between SPBs of smuts
characters of organisms, comparative genomics in two subphyla of Basidiomycota (Fig. 9.2)
(Stajich et al. 2009), allows one to determine (McLaughlin et al. 1995b). Although structural
whether genes controlling certain factors are characters provide additional strong support
present or absent or nonfunctional and, per- for phylogenies, a stable phylogeny based on
haps, even why they are nonfunctional (see DNA sequences is required to determine rela-
Stajich, Chap. 11, this volume). tionships among taxa and to polarize the struc-
tural and biochemical characters.
A variety of structures are associated with
the septal pore, including variations in pore
IV. Character and Character State plugs and several types of vesicles. These char-
Definition and Refinement acters have presented problems in determining
homologies (Celio et al. 2006). Pore plugs may
A broad range of subcellular and biochemical vary with the developmental stage of the cell
characters are potential phylogenetic markers and with functional changes in vegetative and
within the Fungi at a variety of taxonomic reproductive hyphae. The characters associated
levels. The major focus for structural characters with pore plugs usually have homologies lim-
for the AFTOL Structural and Biochemical ited to specific taxonomic groups. When they
Database (SBD, see Sect. V) thus far has been were applied across phyla, as in the type of pore
those characters associated with nuclear divi- plug observed in some Pezizomycotina and
sion and the SPB, septa, and motile cell. Addi- Basidiomycota, new structural data revealed
tional characters currently being assessed for differences between the pore plugs that permit-
their phylogenetic utility include hyphal apical ted a redefinition of the character state
organization, and some meiosporangium and (Fig. 9.4) (Kumar et al. 2012). Similarly, it was
cystidial features, but other sources of potential unclear whether Woronin bodies (Ascomycota)
characters include other sterile cell types and occur within Pucciniomycotina where micro-
haustoria. Several promising biochemical char- bodies are associated with the septal pore; how-
acters, including membrane sterols and cell ever, the determination that the Woronin-
wall components, are being developed with body-specific protein HEX-1 is restricted to
the aid of genomics. Pezizomycotina clarified the character state
To determine whether structural characters definitions (Dhavale and Jedd 2007). The isola-
are evolutionarily significant and have phyloge- tion of specific proteins that localize at septal
netic application, the homology of characters pore caps and pore plugs (van Driel et al. 2008;
and their states must be assessed (Celio et al. van Peer et al. 2010) may provide a more pre-
2006). Comparative methods in which the same cise understanding of the septal pore apparatus
or similar character states occur in related and character evolution.
organisms at the same developmental stage Character homology requires continued reas-
have been the most direct way to support sessment as additional data become available to
hypotheses of homology. A variety of data accurately trace character evolution within and
sources, such as molecular phylogenies, new among fungal clades and unravel the structural
structural data, and cell biological data, can changes that accompany functional evolution.
aid in clarifying homologies. The advent of Cell biological approaches, involving protein iso-
molecular phylogenetic data has permitted test- lation and localization within cells, hold promise
Fig. 9.4 Comparison of septal pore plugs in (a) hypha between these subphyla. SPC septal pore cap, SS septal
of Exidia crenata (G. Thorn, pers. comm.) (Agaricomy- pore swelling, WB Woronin body. Scale bar¼0.2 mm.
cotina) and (b) excipulum of Orbilia sp. (Pezizomyco- (a) from Lü and McLaughlin (1991) published as Aur-
tina) reveals a thin plate in the former and a thickened icularia auricula-judae; (b) from Kumar et al. (2012).
plate in the latter (arrows). These pore plug differences Figures used with permission of Mycological Society of
are consistent and clarify the structural differences America
in gaining an understanding of character states states to guide users unfamiliar with subcellu-
and their relationships. lar data interpretation. The production of data
matrices in NEXUS format is a key feature for
analyses, including tree reconstructions and
ancestral state reconstructions, thereby
V. Structural and Biochemical enabling independent morphological or com-
Database bined morphological and molecular phyloge-
netic analyses.
The SBD, developed and maintained by the The SBD consists of a number of data tables
Assembling the Fungal Tree of Life (AFTOL) for characters and their states; ancillary infor-
consortium, is a searchable Internet resource mation including bibliographic information,
publicly available at https://aftol.umn.edu. The fixation methods, and data quality; and voucher
SBD compiles new and published subcellular information. The focus of the database is the
and biochemical data on Fungi, supplemented table linking species, cell type, and character
by images and literature links, to serve as a state. See Celio et al. (2006) for a diagram of
phyloinformatics tool (Kumar et al. 2013). the database design.
The development of a comprehensive During database development, the follow-
repository that provides subcellular and bio- ing data mining and entry steps were followed:
chemical data on Fungi was initiated during subcellular and biochemical data were collected
the first phase of the AFTOL project from published studies and new research and
(AFTOL1), and additions and improvements their quality and phylogenetic informativeness
to the database are being made during were assessed and recorded in Microsoft Excel
AFTOL2. The main goals were to bring together files (Microsoft Corp., Redmond, WA, USA)
data widely scattered in the literature, thereby prior to entry in the database. Simultaneously,
enhancing information retrieval, provide stan- characters and character states were defined,
dardized data interpretation and uniformity in modified as data were accumulated, and re-
terminology for detailed comparison, evaluate corded in accompanying Microsoft Word files.
image quality, identify phylogenetically infor- Quality published/unpublished images (with
mative characters and assign character state copyright permissions) supporting the charac-
coding, and provide illustrated character ter states were scanned at 1,200 dpi, saved in
JPEG/TIFF formats, and uploaded to the data- Many studies of character evolution have
base. Characters and character states illustrated employed PAUP* (Swofford 2002), MacClade
with photographs and interpretive line draw- (Maddison and Maddison 2000), and, more
ings for nuclear division and SPB, septum/pore recently, Mesquite (Maddison and Maddison
cap, motile cells, and sterols were assembled, 2007) and either a summary phylogenetic tree,
coded, and compiled as lists in downloadable combining the results of several analyses (Celio
PDF format. The line drawings are either from et al. 2006), or a single tree, for example, Kumar
published literature or generated from hand et al. (2012). The phylogenetic programs aid in
drawings using Adobe Photoshop CS3 and determining phylogenies or ancestral or derived
Adobe Illustrator CS2 programs (Adobe Sys- character states, but the limited structural and
tems, San Jose, CA). The nonproprietary Ruby biochemical data sets available for Fungi can
on Rails Web framework and the Ruby pro- present difficulties in drawing conclusions
gramming language were used to rebuild the (Celio et al. 2006; Kumar et al. 2012).
database application and Web site in AFTOL2. The phylogenetic analysis programs have
The data in the SBD are continuously different uses and limitations. PAUP* con-
checked and modified as required. New data structs phylogenetic trees using structural or
submissions pass through a review process biochemical characters alone or in combination
and are checked and approved by the database with molecular characters. It has a full range of
administrator before they appear online. parsimony options, such as irreversible or
Images deposited in the database eventually Dollo characters, that can be used to code char-
may be linked to and stored in a central reposi- acters for analyses. MacClade 4 and Mesquite
tory, such as Morphbank, from which they can 2 are not designed to infer phylogenetic trees;
be retrieved. Acquiring copyright permission instead, they use an imported tree or trees in
for reuse of images from journals with predom- studies of character evolution. MacClade is the
inantly commercial interests, however, remains easier of these programs to use, and it provides
a major impediment to providing access to some parsimony options not available in Mes-
images for the scientific community. This quite. Mesquite is being developed as a plat-
brings up an important issue that authors of form to allow many different types of analysis,
scientific works need to consider carefully. We including experimental ones. It is more com-
appeal to authors who wish their data to be plex to use but includes likelihood reconstruc-
made available to the scientific community to tions not available in MacClade.
choose journals with appropriate copyright Incorporation of structural characters into
policies on reuse of images or to retain copy- molecular phylogenetic studies is little used
right to their images. because it is not clear how to weight the struc-
tural characters for a combined analysis. If
structural characters represent multiple genes
under evolutionary constraint, they may pro-
VI. Structural and Biochemical vide a strong phylogenetic signal, which can be
Database in Phylogenetic Analysis swamped out when treated as the equivalent of
a single character in a molecular data set. Some
The SBD provides sets of character states in structural characters provide a very strong phy-
NEXUS files, which can be analyzed in various logenetic signal, for example, some meiospor-
ways to study character evolution or to perform angium types (ascus, basidium), or the septal
phylogenetic analyses. Structural characters, for pore apparatus, which act as stable markers at
example, spindle pole organization during phylum or lower taxonomic ranks. For charac-
nuclear division, presumably represent the ter evolution studies the best approach at pres-
expression of multiple genes under evolutionary ent appears to be to use well supported
constraint and thus may suggest alternate phylo- molecular phylogenies to trace structural and
genies to those derived from phylogenies based biochemical evolution while the database of
on a limited gene data set (Celio et al. 2007). these characters is gradually assembled, and to
be alert to clues provided by new data that may

challenge current phylogenetic interpretations.
VII. Development of the Fungal

Subcellular Ontology
The reconstructed SBD aims to provide a major

resource that manages morphological and bio-
chemical information on Fungi and serves as a
phyloinformatics tool. However, the extent to
which data deposited in the SBD are utilized by
other biological and genetic databases is cur-
rently limited because of the lack of defined
logical relationships between the characters
and terms and because of terminology pro-
blems that are associated with many fungal
subcellular terms (Kumar et al. 2011). Unifor-
mity in the use of terms, along with defining
them in ways that are meaningful and repre-
sented by class/subclass relations, thereby
revealing the hierarchical connections between
terms, is now increasingly used to help software Fig. 9.5 Graphical representation of part of fungal sub-
programmers and researchers in other special- cellular ontology, displaying subclass relationships
ties establish links between diverse databases. under higher-level class terms, biological process, and
cellular structure character
Such a “controlled vocabulary that describes
objects and the relations between them in a
formal way” (Hughes et al. 2008) is termed an not reveal the full range of diversity among the
ontology and is now being used by biologists Fungi, and many terms that are evolutionarily
who wish to make their data accessible to significant are absent. Hence, an ontology
researchers in other related disciplines. developed by fungal evolutionary biologists
The Fungal Subcellular Ontology (FSO) will be of use to fungal systematists and can
provides a controlled vocabulary describing be easily adapted by others, especially the geno-
subcellular structures unique to Fungi. The mics community. The FSO covers both model
FSO establishes a full complement of terms and nonmodel fungi and is freely downloadable
that provide an operating ontological frame- in OBO-Edit format at http://aftol.umn.edu/
work for the SBD. The FSO was developed ontology/fungal_subcellular.obo.
using a phylogenetic perspective for character Character data for (1) septum and septal
selection and definition and includes charac- pore organization, (2) nuclear division, (3)
ters from across kingdom Fungi. Characters SPB form and development, and (4) the motile
included in the FSO have inherent phylogenetic cell structure of Fungi have been included in the
signal and reveal the extensive character state SBD and form the primary source of terms for
diversity within the Fungi. In the existing, the FSO. The ontology terms were categorized
mostly gene-centric, public ontologies under two higher-level classes: biological pro-
registered with Open Biological and Biomedical cess character and cellular structural character
Ontology (OBO) Foundry, a consortium that (Figs. 9.5 and 9.6). The FSO covers characters
provides coordination among the ontologies from across eight phyla (Ascomycota, Basidio-
registered with it, the terms that describe the mycota, Blastocladiomycota, Chytridiomycota,
structural diversity found among Fungi have Entomophthoromycota, Glomeromycota,
definitions limited to model organisms and do Monoblepharidiomycota, Neocallimastigo-
Fig. 9.6 Ontology tree in OBO-Edit format showing part of fungal subcellular ontology structure with expanded
nodes showing is_a [I] and part_of (P) biological relationships
mycota) and four unplaced subphyla (Kickxel- attributes of plant, animal, and microbial genes
lomycotina, Mortierellomycotina, Mucoromy- and gene products (Barrell et al. 2009; Camon
cotina, Zoopagomycotina). The highly derived et al. 2004; Gene Ontology Consortium 2000,
Microsporidia have not been included. For 2004, 2006, 2008). The GO provides a compre-
details of the methodology of FSO development, hensive platform for cellular, molecular, and
see Kumar et al. (2011). biological process-related terms for model
The FSO complements existing biological organisms across the living world. The Fungal
ontologies, such as the Gene Ontology (GO), Anatomy Ontology (FAO; http://yeastgenome.
which is the most popular of the biological org/fungi/fungal_anatomy_ontology/) and
ontologies. GO is an organism-independent Ascomycete Phenotype Ontology (APO) are
ontology that was developed to describe the other publicly available fungal-oriented ontolo-
gies. The FAO describes the gross structural goals of the study (e.g., structural or cytochem-
anatomy of model fungal species. The APO is a ical) and type of sample one is studying. It
specialized ontology for the phenotypes of asco- should also be noted that using both chemical
mycetes. and cryogenic procedures in a study may be
beneficial because some components of the
VIII. Specimen Preparation and cell may be better preserved with one proce-
Evaluation for Subcellular dure than the other (Heath and Rethoret 1982;
Structure Mims et al. 1988). Here we limit our discussion
to those protocols that are designed to maxi-
mize the structural integrity and resolution of
Knowledge of fungal cell structure and how to fungal cells and tissues.
interpret it has declined with the emphasis on A critical step that is often overlooked is to
DNA analyses and the improvements in molec- maintain a specimen under optimal conditions
ular methodologies that have permitted the up to the point of fixation. If the tissue is har-
rapid expansion of sequence data. Therefore, to vested directly from the environment, it must
provide guidance on fungal cell studies, we probe held under appropriate physiological and
vide an overview of the methods and specialized environmental conditions during transporta-
techniques used to obtain ultrastructural data tion back to the laboratory for fixation or, in
for selected cell features or traits. some situations, specimens may be fixed on site
if chemical fixation protocols are used. If cells
or tissues are maintained in the laboratory, they
A. Specimen Preparation and Evaluation must be grown on an appropriate medium
1. Fixation under correct environmental conditions.
As a rule, before starting a new study one
To examine subcellular structures using trans- should consult references on related fungi to
mission electron microscopy (TEM), regardless find fixation protocols and procedural hints
of what organism is being studied, samples that may work for a species or cell type to be
must be fixed (to kill rapidly and preserve), studied. For example, if one is interested in the
embedded (to stabilize and support), thin- study of hyphal growth, fusion events, or myco-
sectioned (to provide a thin slice of specimen parasitism, and chemical fixation protocols are
allowing electrons to pass), and stained (to appropriate, cultures growing on agar can be
enhance cellular contrast). Two fixation quickly and easily fixed with little or no inter-
approaches commonly used in the preparation ruption events prior to fixation by simply pour-
of cells are the chemical and cryogenic (i.e., low ing the fixative directly onto cultures in Petri
temperature) preparations. Both protocols use dishes (Bracker and Grove 1971; Bracker et al.
multiple steps and chemicals that together work 1996; Grove and Bracker 1970; Hoch and Fuller
to achieve the common goal of microscopists 1977).
for sample preparation: the maintenance of cel-
lular structure in as close to the living state as a) Chemical Preparation
possible, i.e., normal in structure and free from Processing fungal tissues for ultrastructural
artifacts of preparation (see below). This goal studies using chemical fixation protocols has
is, strictly speaking, unattainable (Bracker followed similar procedural milestones, as
1967). Thus, it is the responsibility of micro- have plant and animal studies. Seminal articles
scopists to recognize preparation artifacts and that represent this development are Hawker
to minimize or eliminate them when present by (1965), Bracker (1967), Girbardt (1969), and
protocol modification or, if unavoidable, to Grove and Bracker (1970). Here we describe
interpret the data in light of them. It should briefly the typical stages in the process of chem-
be noted that there are many approaches to ical fixation.
preparing samples for electron microscopic Primary fixation: The standard procedures
investigation that differ depending on the for chemical fixation start with an initial (pri-
mary) fixation in glutaraldehyde that is buff- The secondary fixative is then washed thor-
ered appropriately in either phosphate or caco- oughly with distilled water, and the sample is
dylate buffers. Glutaraldehyde is an excellent processed further.
compound for primary fixation of cells and En bloc staining: The next step, en bloc
tissues because of its ability to cross link pro- staining, is often necessary to enhance general
teins, forming a stable polymeric complex. Pen- cytoplasmic staining (Bracker 1967). Here, the
etration rates of the fixative, however, can be tissue is exposed to 0.5–1 % uranyl acetate in
rather slow depending on the cell/tissue type, water for 1 or 2 h in the dark at room tempera-
resulting in moderate to severe artifacts in ture or overnight at 4 C. Because of the positive
some cases. Thus, to optimize penetration charge of the uranyl ions, they bind tightly to
rates (faster penetration results in better repre- negatively charged elements of membranes,
sentation of healthy living samples), tissues nucleic acids, and proteins to provide enhanced
must be cut into small pieces. Additionally, a contrast. Uranyl acetate is also thought to stabi-
mixture of aldehydes can be used such as a lize nucleic acids and phospholipids and is con-
combination of glutaraldehyde and formalde- sidered a specialized fixative for these structures.
hyde that can provide excellent results for Dehydration: After the specimen has been
dense, hard-to-penetrate samples. Fixation is chemically fixed and thoroughly washed, it
often carried out for 1–6 h at room temperature must be dehydrated using ethanol or acetone.
or, depending on the sample, at 4 C. For some Dehydration is required because the specimens
difficult-to-fix fungal material such as thick- must ultimately be embedded in a solid matrix
walled teliospores and haustoria in mature leaf so that they can be sectioned. The embedding
tissue, fixation times of 1–6 days have been components used for structural studies are
used with good results (e.g., Roberson and Lut- often epoxy-based resins that are not soluble
trell 1987; Roberson et al. 1990; Taylor and in water and will not polymerize, i.e., harden,
Mims 1990). Furthermore, it is at times advis- properly in the presence of any water.
able to add a small amount of detergent (0.1 %
Triton X-100) to the primary fixative when Dehydration of specimens is usually carried out slowly
working with samples that have a hydrophobic in graded stages to prevent the collapse or extraction of
exterior. This helps such tissues to sink beneath cell components. Even though the tissues are well fixed
at this point, they are still susceptible to these artifacts
the surface of the fixative and facilitates good during dehydration. Ethanol is the dehydration agent
fixation. After the primary fixation, the tissue is typically used. To allow a gentle removal of water, the
then washed thoroughly in buffer without fixa- specimen is moved through a graded series of aqueous-
tive before proceeding. Aldehydes do not con- based ethanol solutions such as 10, 30, 50, 70, and 90 %,
tain a heavy metal and, therefore, do not impart and finally through two changes of 100 % ethanol. The
tissues should remain in each solution for 10–15 min. It
amplitude contrast, or so-called electron den- should be noted that flexibility can be tolerated; for
sity, to the sample. example, some workers will hold tissues at 50 % ethanol
Secondary fixation: Commonly, after the overnight at 4 C or longer with few noticeable effects
primary fixation and washing, cells are treated on the structure. Dehydration is typically performed at
with osmium tetroxide (OsO4) as a secondary room temperature, though dehydrations at cooler tem-
peratures, such as 4 C, are thought to reduce artifacts.
fixative. Osmium tetroxide is a strong oxidizing To facilitate the dehydration process, the samples can
agent that reacts with and cross links proteins be placed on a rotating rack that continually mixes the
and lipids, especially targeting double bonds. solutions.
This characteristic makes the fixative important
for stabilizing unsaturated fatty acid tails of At the end of the dehydration steps, the
membranes. Not only does the fixative stabilize sample is ready to be transitioned into the
the tissue structure, but the reduced osmium stages for resin infiltration and embedment
resulting from fixation acts as a heavy metal (see below).
stain to provide electron density. Secondary
fixation is commonly carried out with 1 % b) Cryogenic Preparation
OsO4 in either buffer (typically) or distilled Cryogenic methods for preparing tissues for
water for 2 h at room temperature or 4 C. electron microscopy are a major alternative to
chemical preparation protocols. These methods layer or smaller. Exceptions to this size require-
can be more costly, result in lower throughput ment are rare, but they do occur. For example,
than chemical preparation, i.e., there can be in Auriscalpium vulgare (Celio et al. 2007) and
significant loss of ultrastructural information Ascodesmis nigricans (Mims et al. 1990) work-
due to freeze damage, and are not amenable to ers were able to plunge freeze complex tissues
field-collected samples that cannot be trans- with minimal to no detectible ice damage. Sec-
ported back to the lab. However, the benefits ond, samples must be rapidly plunged into a
can include much improved preservation and cold liquid (i.e., cryogen) that has the charac-
the elimination of artifacts associated with teristics of remaining liquid at very low tem-
chemical fixation (Gilkey and Staehelin 1986; peratures (185 to 190 C) and absorbing
Hoch 1986; Howard and O’Donnell 1987; heat rapidly without boiling. Hydrocarbons
Moor 1987). Cryogenic methods can be divided meet these requirements, and liquid propane
into two parts: cryofixation and freeze substitu- is the most commonly used cryogen because it
tion. is widely available and inexpensive. Liquid pro-
Cryofixation: The initial step of freezing pane or a combination of liquid propane and
cells and tissues is analogous to the primary ethane are also popular alternatives; however,
fixation step described earlier with respect to hydrocarbons are explosive.
chemical preparation methods. A significant
obstacle in cryofixation is avoiding the forma- As mentioned earlier, for plunge freezing the sample
tion of ice crystals during the freezing process. must be thin. Hyphae can often be grown over thin,
When freezing rates are too slow, ice crystals water-permeable membrane supports (e.g., dialysis
membrane, cellophane) covering the surface of a semi-
can grow to sizes large enough to disrupt tissue solid growth medium. Once growth is established, a
structure at the resolutions used for electron selected region of cells and supporting membrane are
microscopy. Three common methods to avoid cut to approximately 47 mm; cells are then allowed
ice crystal growth are to use ultrarapid freezing time to recover (typically 30–60 min) from the wound-
methods that are so fast that ice crystals do not ing before freezing. For more details see Hoch (1986),
Howard and O’Donnell (1987), and Chandler and
have time to grow, use high pressure to slow the Roberson (2009). An air-driven stirrer is positioned in
rate of ice crystal growth, and use cryoprotec- the condensed propane in order to prevent temperature
tants that nucleate small crystals and slow their gradients from forming and to aid in heat transfer. It is
growth. The first two methods are most desir- also advisable that a gentle stream of nitrogen gas be
able and will be discussed in what follows. directed across the propane surface to provide an insu-
lating barrier to oxygen. As with chemical preparation,
There are three ultrarapid freezing meth- the specimen to be frozen is kept in appropriate growth
ods: immersion (plunge and spray), propane conditions at all times just prior to the instant of freez-
jet, and metal mirror freezing (Chandler and ing to insure that the cells are as normal in function and
Roberson 2009). For the Fungi, plunge and, to structure as possible. Just prior to freezing, the speci-
a lesser degree, spray immersion freezing have men is grasped with forceps and, with a smooth quick
motion, plunged into the liquid propane. The frozen
had well-documented success (Hoch and specimen is then transferred to a temporary holding
Howard 1980; Hoch and Staples 1983; Howard basket, kept in the propane bath. Once 15–20 samples
1981; Howard and Aist 1979; Mims et al. 1988; have been frozen, the basket is quickly transferred to
Roberson and Fuller 1988; Roberson et al. 2011; the freeze-substitution solution.
Shields and Fuller 1996; Vargas et al. 1993).
Plunge freezing will be discussed here. Ultra- Freezing samples at high pressure is useful
rapid freezing rates are achieved when heat is because ice crystal growth is significantly
withdrawn from cells at rates of 5,000– reduced, which permits large tissues and other
10,000 C/s. At these speeds, cellular motion is difficult to freeze cells, for example, spores, to be
stopped within milliseconds, and little or no frozen successfully. The pressures required for
damaging ice crystals form. However, such this to occur are around 2,100 atm, which repre-
rates of heat loss may be obtained only when sents a technical challenge to achieve and
certain conditions are met. First, samples must requires the use of a hydraulic piston and
be 10–12 mm thick or less, meaning a single cell sophisticated delivery mechanism that function
within fractions of a second. Equipment such as These freeze-substitution protocols can be

the Baltec HPM 010 High-Pressure Freezer (Bal- carried out automatically by dedicated freeze-
tec Products, Middlebury, CT) or Leica EM HPM substitution units or by hand moving the sample
High-Pressure Freezer (LEICA Microsystems from one cold chamber to the next. The washed
Inc., Buffalo Grove, IL) is required for this specimen is then infiltrated and embedded in
mode of freezing. For this procedure, the speci- epoxy resin, sectioned, stained, and viewed.
men is typically positioned in a double planchet There are numerous ways in which the
or specimen platform that is 3 mm in diameter. freeze-substitution process can be modified to
The planchet and specimen are inserted and meet the needs of a study. The more common
locked into the ballistic chamber, which is rap- modifications center on changes in the chemis-
idly pressurized, followed milliseconds later by a try of the freeze-substitution solutions. For
burst of liquid nitrogen. At these pressures, liq- structural studies, for example, the use of tan-
uid nitrogen is a good cryogen (unlike at atmo- nic acid, glutaraldehyde, or potassium perman-
spheric pressure), and freezing is accomplished ganate have been reported to provide good
within 100 ms. High-pressure freezing has been results (Giddings 2003; Howard and Aist 1979;
used successfully in freezing fungal spores (Bon- McDaniel and Roberson 2000; Winey et al.
fante et al. 1994; Mims and Richardson 2005; 1995). When studies call for the localization of
Roberson 1993), host–pathogen associations proteins or carbohydrates using antibodies, lec-
(Celio et al. 2000; Harding et al. 1999; Mendgen tins, or other cytochemical approaches, addi-
et al. 1991; Mims et al. 2002, 2003), septal pore tional changes in protocol are typically
complexes (van Driel et al. 2009), and zoospor- required, both in the freeze-substitution
angia (Fisher et al. 2000). makeup and type of embedding resin used
Freeze substitution: After cryofixation, the (e.g., use of acrylic-based instead of epoxy-
samples can be stored in liquid nitrogen or based resin) in order to preserve the chemical
processed in one of a number of ways. Freeze nature of the cell (Bourett and Howard 1991,
substitution is the approach followed if the 1994; Bourett et al. 1993; Buser and McDonald
researcher wishes to examine the material in 2010; Roberson and Vargas 1994).
thin section using TEM. The objectives of freeze
substitution are to gently dehydrate the sample
while simultaneously exposing it to secondary 2. Standard Infiltration and
chemical fixation and en bloc staining at low Embedding
temperature prior to embedding in resin at
room temperature. The advantages of this strat- Prior to obtaining thin sections for TEM analy-
egy are that many of the artifacts caused by sis, the sample must first be thoroughly infil-
chemical fixation and dehydration at room trated with liquid components of an embedding
temperature, for example, shrinkage and mem- resin followed by the polymerization (harden-
brane wrinkling, are eliminated. What follows ing) of the resin (typically by heat), creating a
is a brief description of a freeze-substitution highly cross-linked matrix of which the sample
protocol used for persevering fungal structure. is a part. If these procedures are not performed
adequately, the sample may suffer from severe
distortion artifacts during sectioning or may be
The frozen specimen is first placed in the cold (85 to
90 C) substitution fluid, which is typically composed unsectionable.
of acetone, 1 % osmium tetroxide, and 0.05 % uranyl Resins that are used most commonly for
acetate. The specimen is held at these low temperatures structural studies in the Fungi are low-viscosity
for 24–48 h while the ice in the specimen is slowly and epoxy-based, for example Spurr’s, or epoxy/
gently replaced by substitution fluid and dehydration is ethylene glycol diglycidyl ether-based, for
complete. The specimen is then warmed to 20 C for
2 h, during which time the osmium tetroxide becomes example Quetol 651, resins. These low-viscosity
increasingly more chemically active. The specimen is resins polymerize into a hard matrix that is
then warmed to 4 C for 2 h, and then to room temper- very well suited for infiltrating and sectioning
ature for 1 h, after which it is washed in 100 % acetone. cells bound by walls. Nonetheless, extended
resin infiltration schedules are recommended cess that give reproducible results and modified
to ensure complete resin penetration into protocols, like vacuum microwave processing
most fungal cells and tissues (see below). Typi- (Giberson et al. 1997), have been reported to
cally, four liquid components are mixed save more time and improve overall quality
together in certain ratios to achieve the desired (Giberson and Demaree 1995; Giberson et al.
hardness. For most fungal samples hard resins 1997).
are favored because they provide the best char- Microwave fixation and embedding for
acteristics for sectioning through cell walls. fungi is relatively new. The Vacuum Microwave
Processing Protocol (Ted Pella, Inc., Redding,
Resin infiltration begins immediately after dehydration CA, USA) has been used to process samples in a
when the samples are transferred from absolute ethanol specially designed laboratory microwave with a
into 100 % acetone. Samples are exchanged two to three vacuum chamber and a temperature probe to
times and held in each change for no longer than
15 min at room temperature. For infiltration of resin,
monitor and control heat inside the embedding
the sample is incubated in resin:acetone mixtures at solution. Infiltration of samples (e.g., thin fruit
ratios of 1:3, 1:1, 3:1, and then 100 % resin. Incubation body pieces, mycelium, germinating spores on
times are typically 8–12 h for each step. It is advisable to agar) is quick and requires relatively small
place the specimen vials on a rotating rack for slow amounts of chemicals. It works fairly well with
continuous mixing throughout the infiltration steps.
Several changes of 100 % resin are desirable to ensure
fungal samples; however, reproducibility of
that no traces of acetone remain in the solution, which results is a concern with thick tissue samples
could prevent complete polymerization and make sec- and cells with very thick walls.
tioning difficult or impossible.
Embedment is the next step and involves 4. Section Staining and Microscopy Methods
placing the specimens in molds that will deter-
mine the shape and size of the final resin block. Because standard TEM requires sections rang-
The final selection of the type of mold (usually flat ing from 60 to 80 nm thick in order to allow
sided or cylindrical) is dependent on the speci- electrons to pass, they are referred to as thin or
men and its desired specimen orientation during ultrathin sections and are cut on an instrument
sectioning. The molds are placed into an oven at called an ultramicrotome.
the temperature and time specified for the resin. Epoxy embedded tissues (or blocks) are
hard and must be cut using a glass or diamond
knife. A diamond knife is preferred because of
3. Microwave Fixation and Embedding its constancy in cutting characteristics, ease of
Microwave-assisted processing of biological use, and high-quality cutting edge. Diamond
specimens for electron microscopy has recently knives are extremely hard and will remain
attracted attention as a tool to achieve rapid sharp through thousands of cuttings if they
specimen fixation and embedding. Microwave- receive proper care. These knives, however,
processing protocols have also been used for are expensive, and once the edge is damaged,
specimen staining for electron microscopy, it must be professionally sharpened. After the
immunolabeling, antigen retrieval, and in situ block has been trimmed, it is mounted into an
hybridization. Reduced processing time, ultramicrotome and the knife is secured in place.
improved preservation of morphology, and Cutting thin sections is a time-consuming and
the antigenicity of specimens subjected to tedious job, but given the proper training,
microwave processing have been highlighted patience, and good modern equipment, the pro-
as significant benefits over the conventional cess is relatively easy to carry out.
specimen-preparation methods (Webster
2007). Specially designed laboratory microwave After thin sections are cut, they are collected on grids.
Grids come in many patterns and materials, but all are
processors for handling hazardous materials 3 mm in diameter and stamped out of a thin metallic
are used to carry out the protocols. Better foil (copper, gold, nickel). The sections must now be
knowledge of the variables involved in the pro- stained (poststained) with heavy metals such as ura-
nium or lead. This is accomplished by floating the grid,

section side down, on a drop of 2 % uranyl acetate (wt/
vol) in water or ethanol (50 %) for approximately
10 min, followed by a thorough washing in water. This
is followed by staining on a drop of aqueous lead citrate
for 5 min and another washing. Depending on the resin
and tissue, other poststaining schedules may be needed.
Grids are then allowed to dry and are stored in a grid
box until imaged with TEM.
B. Methods for Selected Cellular Features

1. Nuclear Division Studies
Analysis of nuclear division and SPB cycles
requires capturing specific division stages to Fig. 9.7 (a) Light micrograph of dikaryotic nuclei at
fully understand the changes that occur during base of a branching hypha of Helicobasidium mompa
(Basidiomycota) at mitotic metaphase–anaphase
meiosis or mitosis, but finding dividing nuclei stained with a DNA-specific fluorochrome. (b) Trans-
can be challenging. The number of nuclear divi- mission electron micrograph of same cell selected with
sion studies is limited; the best-studied group is fluorescent microscopy during primary fixation and
the Basidiomycota, with the Ascomycota seri- serial sectioned after processing for TEM. Nuclei divide
ously understudied for a phylum that constitutes at the base of new branches. Note the wall break on
branching (short arrows), a characteristic of some Puc-
more than half of the known Fungi. The gaps in ciniomycotina clades. Nu nucleolus, S spindle, SPB
our knowledge make generalizations about spindle pole body. Scale bar¼2 mm. From Bourett and
character distribution and evolution specula- McLaughlin (1986), used with permission of Canadian
tive (Fig. 9.1). Some phyla/subphyla present Journal of Botany
great difficulties in handling cells for cytologi-
cal study, for example, Glomeromycota, or do challenges in nuclear division studies, although
not respond well to the usual methods for cell in yeasts and germinating spores, concentrat-
preparation and selection, for example, zygomy- ing large numbers of cells for sectioning helps
cete fungi, and remain unstudied or little stud- to overcome this impediment (e.g., Frieders
ied. Meiotic studies have advantages over and McLaughlin 1996; Heath and Rethoret
mitotic ones in that the SPB may be larger and 1982). The method developed by Taylor (1984)
more differentiated and goes through a series of and modified by Bourett and McLaughlin
changes involving SPB fusion and separation (1986) is applicable to cultured hyphae. It
that results in distinctive structural features of involves growing mycelium on specially
potential phylogenetic significance (McLaughlin prepared cover slips in a thin layer of agar,
et al. 1995b). carrying out an initial chemical fixation, stain-
Choosing places in the life cycle where ing with a DNA-specific fluorochrome, and
nuclear divisions are known to occur optimizes photographing and selecting nuclei by scribing
the study of nuclear division and SPB cycles. the cover slip with a diamond scribe. The cover
Analysis of fixed cells with a DNA-specific fluo- slip is then processed to complete the postfixa-
rochrome can aid in finding sites of dividing tion and flat embedding between layers of
nuclei and can be combined with cell selection, Teflon-coated Mylar (Kleven and McLaughlin
either during the initial fixation process 1989). Selected cells are recovered by relocating
(Fig. 9.7) or after embedding, to select specific the scribed cells and mounting them for sec-
cells for serial sectioning, thereby maximizing tioning. This method works well for Fungi that
data acquisition (Bourett and McLaughlin 1986; will grow within the agar layer and that form
McLaughlin et al. 1996; Swann et al. 1999). limited aerial hyphae.
Very small nuclei and those in which chroma- Methods involving dispersal of meiospor-
tin condensation is not easily observed present angia such that individual cells can be selected
at different stages of meiosis can also be very monolayers over cellulose membranes on the
effective, for example, germinating rust telios- surface of nutrient agar medium and are often
pores (O’Donnell and McLaughlin 1981), and thin enough (<10–12 mm) to allow the rapid
these methods have been applied to asci. Alter- freezing rates required to achieve high-quality
natively, intact hymenia can be used by cutting ultrastructural preservation (Hoch 1986;
thick sections and staining them with toluidine Howard and O’Donnell 1987). Other freezing
blue O to locate desired areas with the light methods are less suitable primarily because of
microscope for thin sectioning. Finding divid- the prefixation handling of the sample that is
ing nuclei is especially aided by morphological required. Over the past several decades, the use
markers, such as branching or budding, in cells of cryofixation protocols has become the
under study that allow prediction of where to method of choice for preserving the ultrastruc-
find dividing nuclei in fixed and flat-embedded ture of the hyphal tip, as well as other structures
cells (Fig. 9.7) (Bourett and McLaughlin 1986; when applicable. The reasons for this are clear
Swann et al. 1999). No single procedure can be when the results obtained with cryofixation/
applied to all organisms. Each organism or freeze substitution are compared with those
group needs preliminary study to ascertain the obtained with chemical fixation procedures
best way to proceed. (Hoch and Howard 1980, 1981; Howard and
Aist 1979; Mims et al. 1988). When using cryo-
preparation methods the cytoplasm is pre-
2. Apical Organization Studies served with increased clarity, cytoskeletal
elements are well maintained, and membrane-
It is desirable when possible to utilize both associated structures (e.g., vesicles, vacuoles,
light microscopy and TEM in studies of hyphal mitochondria, Golgi apparatus, endoplasmic
apical organization. The major advantage of reticulum) appear rounded or with smooth
using light microscopy is that hyphae can be profiles. In contrast, similar structures
examined in their living state, and thus the prepared using chemical fixations may appear
dynamics of growth can be coupled with struc- disorganized and distorted as a result of the
ture. However, this can also result in challenges effects of postfixation rearrangements, osmotic
for those fungi with hyphae that are susceptible incompatibilities, or harsh dehydration.
to the stress of being grown and examined for Because well-frozen cells are immobilized in
light microscopy studies. Furthermore, the milliseconds and dehydration occurs slowly at
small diameter of some hyphae makes light low temperatures, the cytoplasmic structure is
microscopy examination less valuable. Girbardt preserved at a high level of fidelity.
(1957), Grove and Bracker (1970), and López- Although there are significant advantages
Franco and Bracker (1996) have demonstrated to using cryomethods for studies of hyphal
the utility of phase-contrast light microscopy in apices, the issue of inconsistent freezing rates
studies of the hyphal apex. Studies using the resulting in artifacts, such as minor to severe
membrane-selective fluorescent vital dye FM4- ice-crystal damage and fracturing of cells, is
64 have also been shown to be useful in studies common to all methods of freezing. The best
of hyphal apices (Fischer-Parton et al. 2000; way to avoid sectioning poorly frozen hyphal-
Roberson et al. 2011). tip cells is to carefully screen and select cells
Many components of the apical cytoplasm before sectioning. For this, the hyphae must be
in actively growing hyphae (vesicle and cyto- flat embedded between a Teflon-coated micro-
skeletal organization and appearance) are quite scope slide and Mylar strip (Kleven and
delicate and susceptible to disruption from McLaughlin 1989). After polymerization of the
prefixation handling and fixation artifacts resin, the Mylar strip is removed and the cells
when chemical fixation protocols are used to now embedded in a thin layer of resin are
prepare samples for TEM. Fortunately, most imaged with phase contrast light microscopy
fungal hyphae are well suited for cryofixation using an oil-immersion 100 objective.
by plunging because they can be grown easily as Severely damaged cells are quite easy to detect
Fig. 9.8 Transmission electron micrographs of near mitochondrion (m). Scale bar¼0.3 mm; micrograph by
median sections through hyphae illustrating apical Matt Garret. (b) Coemansia reversa (Kickxellomyco-
organization. Hyphae fixed using plunge-freezing and tina) illustrating apical vesicles (arrows) arranged in
freeze-substitution methods. (a) Fusarium solani crescent pattern beneath apical plasma membrane.
(Ascomycota) with organized Spitzenkörper com- Such a vesicular arrangement at the hyphal apex repre-
posed, in part, of spherically arranged apical vesicles sents a significant difference from that observed in
(arrows) and cluster of ribosomes (asterisks). Central ascomycete and basidiomycete hyphae. Membrane net-
core region, typical of the ascomycete Spitzenkörper, is work of Golgi equivalent at arrowhead. Scale bar¼
not present in this section. Tangential section through 0.35 mm
and avoid, while mild freeze damage is more describing the fundamental organization of
difficult to distinguish at the light microscope the hyphal apex in septate and nonseptate
level (Hoch 1986). This screening method is fungi (Fig. 9.8) (Amicucci et al. 2010; Harris
critical not only for avoiding cells that contain et al. 2004; Hoch and Howard 1980; Hoch and
obvious freeze damage but also for selecting Staples 1983; Hohmann-Marriott et al. 2006;
cells that show no evidence of prefixation Howard 1981; Howard and Aist 1979; Köhli
growth stress due to handling of the samples et al. 2008; Newhouse et al. 1983; Roberson
just prior to cryofixation. Stress is most easily and Fuller 1988; Roberson et al. 2011;
identified as a swelling of the apical region or Srijayanthi et al. 1994; Vargas et al. 1993) and
lack of a well-organized vesicle cluster, i.e., the effects of growth inhibitors and mutations
Spitzenkörper, in basidiomycetes and ascomy- on the hyphal apex organization (Howard and
cetes. Well-frozen cells appear as they would if Aist 1980; McDaniel and Roberson 2000;
imaged in their living state, for example, well- Riquelme et al. 2002; Roberson and Fuller
defined organelles in a clear cytosolic matrix. 1990).
After hyphae are selected for sectioning, the
small region containing the cells and surround-
3. Flagellated Fungi Studies
ing resin is removed using a sharp razor blade
and glued to the top of a blank resin stub using The vast majority of taxa in Chytridiomycota
superglue (Howard and O’Donnell 1987). After are microscopic with relatively simple thallus
the block is trimmed, cells are ready for sec- morphology. Subcellular characters associated
tioning. with posteriorly uniflagellate zoospores are
The methods of cryofixation and freeze informative at higher taxonomic ranks.
substitution have been used successfully in Chytridiomycota phylogeny is currently based
Fig. 9.10 Daughter nuclei (N) following a mitotic divi-

sion in a sporangium of Angulomyces argentinensis
Fig. 9.9 Longitudinal section through mature sporan- (Chytridiomycota). Nuclei contain dispersed chroma-
gium of an unidentified member of Rhizophydiales tin (Cr); nucleus on right illustrates invagination of
(Chytridiomycota), illustrating cleaved zoospores (Z), nuclear envelope (E) with adjacent paired centrioles
sporangial wall (W), discharge pore (P), and endo- (Ce) and cytoplasmic microtubules (Mt). Scale bar¼
operculum (Op). Scale bar¼5 mm 0.5 mm
on molecular sequences, while support from TEM provides thallus ultrastructure infor-
common features of higher-rank taxonomy uti- mation related to wall construction and orna-
lizes zoospore ultrastructure. For example, mentation, discharge apparatus (Fig. 9.9),
until recently the order Chytridiales was the septal characteristics, mitosis (Fig. 9.10), zoo-
largest order in Chytridiomycetes, but it was spore development, and rhizoid structure. TEM
considered to be polyphyletic. Combined mole- is also the primary tool for revealing zoospore
cular and ultrastructural studies have resulted ultrastructural characters and character states.
in establishing the limits of a monophyletic An adequate quantity of thalli and zoospores
Chytridiales (Vélez et al. 2011) and in the cir- must be obtained for subcellular studies. A
cumscription of the orders Rhizophydiales number of Petri dishes with a suitable nutrient
(Letcher et al. 2006), Cladochytriales (Mozley- agar are inoculated with either chytrid thalli or
Standridge et al. 2009), and Lobulomycetales a zoospore suspension obtained from pure cul-
(Longcore and Simmons 2012; Simmons et al. ture and incubated at room temperature.
2009). Caution is recommended, however, Within 1–5 days, mature thalli that cover the
because relatively few species within each dishes may be harvested, or the dishes may be
higher taxon have been examined. Additional flooded with distilled water or dilute salts
taxa at the generic and species levels have also (Fuller and Jaworski 1987) to obtain zoospore
been delineated on the basis of combined release. Thalli or zoospores are concentrated in
molecular and ultrastructural analyses (Letcher an aqueous suspension and then preserved
et al. 2008; Picard et al. 2009; Powell et al. 2011). through chemical fixation.
Thus, one aspect of obtaining robust chytrid Because the procedure is relatively straight-
phylogenies is reliable preservation, fixation, forward and reliable, chemical fixation is the
and observation of biological specimens to preferred method for specimen preparation for
complement molecular data. both chytrid thalli and zoospores. Vehicles for
Because of the microscopic nature and size fixatives include phosphate, cacodylate, and
limits of most chytrids, obtaining information collidine buffers (Glauert 1991). Phosphate buf-
regarding aspects of thallus morphology and fers are nontoxic and most closely mimic extra-
zoospore ultrastructure is enhanced by elec- cellular fluids, although precipitates often occur
tron microscopic analyses. during fixation and preservation of microtu-
bules may be less than optimal. Cacodylate

buffers are easily prepared, although the buffers
contain arsenic, which may act as a fixative.
Sym-collidine buffers, although toxic, are easily
prepared, stable at room temperature, and exist
at pH 7.4 when neutralized with strong acid
(Glauert 1991).
Glutaraldehyde is the primary fixative of
choice, and specimen fixation and preparation
for TEM analysis follow procedures described
in Letcher and Powell (2005). Although ethanol
and acetone are widely used for dehydration
and embedding, for zoosporic fungi, acetone
is preferred. Dehydration and specimen
embedding follow procedures described in
Letcher and Powell (2005). After thorough dry-
ing, grid-mounted sections are stained to
obtain sufficient contrast (Lewis and Knight
1977).
If nickel rather than copper grids are used, sections are

initially treated by being floated on a drop of 1 %
periodic-acid solution for 5 min to demonstrate the
presence of glycogen and then washed twice with fil- Fig. 9.11 Longitudinal section through zoospore of an
tered deionized (FDI) water. A periodic-acid stain can- unidentified member of Rhizophydiales (Chytridiomy-
not be used with copper grids because the acid will etch cota) illustrating organization of ultrastructural com-
the copper. The periodic-acid oxidation is followed by ponents. F flagellum, K kinetosome, KAS kinetosome-
immersion of sections in saturated uranyl acetate in associated electron-dense structure, M mitochondrion,
70 % ethanol for 8–10 min to enhance contrast (Silva N nucleus, R aggregated ribosomes, Vac vacuole, Ves
et al. 1968), followed by a wash with 70 % ethanol to vesicles in peripheral cytoplasm. Scale bar¼1 mm
dissolve residual uranyl acetate crystals and a wash
with FDI water. Sections are then stained for 4–6 min
in lead citrate (Reynolds 1963) in the presence of
sodium hydroxide pellets and then washed once with tigomycota motile cells for electron microscopy is
0.1 M sodium hydroxide and once with FDI water. similar to that for other groups (e.g., Gold et al.
1988; Heath et al. 1983; Li and Heath 1991).
When thoroughly dry, sections may be Members of the genus Olpidium (O. brassicae
examined by TEM at 60–80 kV. Random sec- and O. bornovanus), although now indicated to
tions on mesh grids are most commonly be affiliated with the nonmotile zygomycete
observed for obtaining images of randomly ori- fungi, have a motile spore, and treatment of
ented features (Fig. 9.11). Serial sections those organisms for ultrastructural analysis is
mounted on slot grids are observed to obtain similar to treatment of other organisms with
progressive imaging through selected struc- motile spores (e.g., Barr and Hartmann 1977;
tures such as nuclei undergoing mitosis or the Lange and Olson 1978).
flagellar apparatus (Fig. 9.12).
Culturing methods and zoospore fixation for
electron microscopic analysis for Blastocladiomy- IX. Conclusions
cota are similar to those for Chytridiomycota
(e.g., Chong and Barr 1974; Dewel and Dewel Structural and biochemical characters have a
1990). Culturing methods for the anaerobic Neo- great deal to tell us about the evolution and
callimastigomycota have been summarized by diversification of the Fungi. Our appreciation
Rezaeian et al. (2004). Treatment of Neocallimas- for how these characters have evolved is chang-
Fig. 9.12 Transverse serial sections through kinetoso- gellated centriole connected by a fibrillar bridge (FB),
mal region of a zoospore of Terramyces sp. (Chytridio- and a kinetosome-associated electron-dense structure
mycota). (a) Posterior end of kinetosome (K) with (KAS) adjacent to the kinetosome. Scale bar in (a)
flagellar props (P). (b) Kinetosome and adjacent non- ¼0.25 mm
flagellated centriole (NfC). (c) Kinetosome and nonfla-
ing dramatically as molecular phylogenetic data (2010) Hyphal and cytoskeleton polarization in
provide a well-supported Fungal Tree of Life. Tuber melanosporum: a genomic and cellular anal-
ysis. Fungal Genet Biol 48:561–572
We are entering a new era of scientific under- Ammirati JF, Traquair JA, Horgen PA (1985) Poisonous
standing with the advent of phylogenomic ana- mushrooms of the northern United States and
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National Science Foundation Assembling the Fungal sporic fungi. Biosystems 14:359–370
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10 Fungal Diversity in the Fossil Record
THOMAS N. TAYLOR1,2, MICHAEL KRINGS1,2,3,4, EDITH L. TAYLOR1,2
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
II. History of Fossil Fungi. . . . . . . . . . . . . . . . . . . . 260 Fungi that are both generalists and specialists
III. Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
A. Morphologic Comparison. . . . . . . . . . . . . . 261 occur in virtually every ecosystem today, where
B. Fungal Spores . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 they colonize a wide variety of (micro-)habitats
C. Thin Sections and Acetate Peels . . . . . . . 263 and provide numerous functions ranging from
D. Plant Resins (Amber) . . . . . . . . . . . . . . . . . . 264 decomposing organic matter to immobilizing
IV. Major Fungal Lineages . . . . . . . . . . . . . . . . . . . . 265 nutrients (Cantrell et al. 2011). Considering
A. Chytridiomycota. . . . . . . . . . . . . . . . . . . . . . . . 265
B. Blastocladiomycota . . . . . . . . . . . . . . . . . . . . . 266 the remarkable diversity in types of fungi, the
C. Zygomycetous Fungi . . . . . . . . . . . . . . . . . . . 266 levels of interaction with other components of
C.1 So-Called Sporocarps . . . . . . . . . . . . . . 266 the biological and physical world, and their role
D. Glomeromycota. . . . . . . . . . . . . . . . . . . . . . . . . 267 as drivers of many processes in modern ecosys-
E. Basidiomycota . . . . . . . . . . . . . . . . . . . . . . . . . . 268 tems, it is surprising that fungi and fungal
F. Ascomycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
VI. Lichens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 activities have not been consistently studied in
VI. Enigmatic Fossils . . . . . . . . . . . . . . . . . . . . . . . . . . 271 the fossil record. In a very real sense, the study
A. Uncertain Affinities. . . . . . . . . . . . . . . . . . . . . 271 of fossil fungi has “fallen between the cracks”
B. Plant–Fungal Associations with Uncertain of geologic time because the activities of fungi
Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 (e.g., decomposition, parasitism) result in fos-
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 sil materials that are not especially appealing
and, therefore, not normally collected. This
has resulted in an underrepresentation of fossil
forms and an underappreciation of both their
biodiversity and their distribution in time and
space. In addition, fossil fungi continue to pres-
ent a dilemma to those interested in their geo-
logical history and biological features for
1
several reasons. One of these is the fact that,
Department of Ecology and Evolutionary Biology, University of
until relatively recently, there has not been a
Kansas, Lawrence, KS 66045-7534, USA; e-mail: tntaylor@ku.
edu; m.krings@lrz.uni-muenchen.de; etaylor@ku.edu concerted effort to systematically study fungi in
2
Natural History Museum and Biodiversity Institute, University the fossil record. A second continuing problem
of Kansas, Lawrence, KS 66045-7534, USA; e-mail: tntaylor@ku. centers on the “proprietary rights” of just who
edu; m.krings@lrz.uni-muenchen.de; etaylor@ku.edu should study fossil fungi. Paleontologists are
3
Department für Geo- und Umweltwissenschaften, Paläonto-
the most likely scientists to discover fungi or
logie und Geobiologie, Ludwig-Maximilians-Universität,
Richard-Wagner-Straße 10, Munich 80333, Germany; e-mail: their activities in the fossil record, but in most
m.krings@lrz.uni-muenchen.de instances, they either have no interest in these
4
Bayerische Staatssammlung für Paläontologie und Geologie, organisms or lack the scientific expertise to
Richard-Wagner-Straße 10, Munich 80333, Germany; e-mail: evaluate them. At the same time, the mycologi-
m.krings@lrz.uni-muenchen.de

260 T.N. Taylor et al.
cal community lacks access to these organisms, the scientific community of the day. In most
and thus there is little collaborative effort to cases, however, the presence of fungi was
date devoted to exploring questions relating to merely noted as a curiosity in early studies,
the fossils or the biodiversity of fungi in time although a few workers provided detailed
and space. descriptions of these life forms and discussed
Despite the preceding comments, there is how they might have affected host performance
already scattered historical evidence of fossil and ecosystem functioning in the past.
fungi that indicates an untapped wealth of Two series of historical contributions pro-
information and, more recently, a new empha- vide detailed information on fungi and other
sis on their paleodiversity and evolutionary microorganisms co-occurring with fossil
history that is beginning to emerge. The follow- plants. One of these is a series of papers by
ing sections provide examples of fossil fungi, as the French paleobotanist Bernard Renault
well as some enigmatic fossils that have been at (e.g., Renault and Bertrand 1885; Renault
one time or another considered to be fungal. 1894, 1895a, b, 1896a, 1903) on microorganisms
Although there is an increasing body of litera- associated with various Carboniferous plants
ture dealing with the importance of fungi in preserved in cherts from France. The renowned
paleoecology, sedimentology, coal geology, American botanist, paleontologist, and sociolo-
geobiology, and geobiochemistry (e.g., Stubble- gist Lester Ward called Renault’s studies “a
field and Taylor 1988; Gadd 2008; Hower et al. superb work on a very difficult, but at the
2009), we have focused the present review on same time very important subject” [quote in
the more biological dimensions of paleomycol- Andrews (1980)]. Renault’s observations were
ogy. Finally, we comment on some of the limit- summarized in the final section of Bassin
ing aspects of examining the record of the Houiller et Permien d’Autun et d’Epinac, Fasci-
activities of fossil fungi and suggest examples cule IV, Flore Fossile, Deuxième partie (1896b)
in which they represent a valuable data source and in his volume Sur Quelques Microorga-
that is unavailable using other scientific tools. nismes des Combustibles Fossiles (1900, atlas
1899). A similar approach was used later by
Robert Kidston and William H. Lang in their
work on the Lower Devonian Rhynie chert;
II. History of Fossil Fungi their studies appeared as a series of contribu-
tions (On Old Red Sandstone plants showing
Systematic analyses of fungi in the fossil record structure, from the Rhynie chert Bed, Aberdeen-
represent a relatively new avenue of research, shire) of the Royal Society of Edinburgh
despite the fact that fossil plants and animals (Kidston and Lang 1917, 1920a, b, 1921a, b).
have been studied for more than 250 years. In Part V of this series, Kidston and Lang
Nevertheless, many paleobotanists and paleo- (1921b) described numerous microorganisms
zoologists, especially during the nineteenth and from the Rhynie chert, including cyanobacteria,
early twentieth centuries, have occasionally algae, and a diverse assemblage of fungi and
made reference to or figured (what they funguslike organisms, many of which they
believed were) fungi or indications of fungal were able to document as being involved in
activity co-occurring with the plant and animal various levels of interactions with other organ-
fossils or sediment samples they were studying. isms. The studies of Renault and Kidston and
Even the most distinguished naturalists of the Lang, and perhaps a few others today, represent
day, including Charles Darwin, took notice of benchmarks in the analysis of the interrelation-
the presence of fungi associated with certain ships between microorganisms and land plants
plant fossils (Smith 1884). In fact, the earliest in fossil ecosystems.
compendium of fossil fungi was published in For the next 50 years, however, there was
1898 (Meschinelli 1898). Meschinelli’s book is a relatively little activity with fossil fungi [sur-
remarkable volume that is lavishly illustrated veyed in Tiffney and Barghoorn (1974) and
and demonstrated the interest in fossil fungi of Pirozynski (1976)]. In 1975, Pirozynski and
Fungal Diversity in the Fossil Record 261
Malloch published an influential paper in peeled or macerated from the rock; these
which they suggested that fungi formed a crit- often contain multiple epiphyllous fungi (e.g.,
ical component of the movement of plants Dilcher 1965; Daghlian 1978; Phipps and
onto the land. Their initial thesis postulated Rember 2004). While a number of Cenozoic
that some type of mutualistic relationship floras worldwide with cuticular preservation
between a fungus and a green alga provided have been extensively studied, most have not
the necessary physiological adaptations so that been analyzed for their epiphyllous fungi in any
this (new) organism could function in what systematic way. Epiphyllous fungi on fossil
must have been a very inhospitable environ- leaves can also serve as additional sources of
ment. Their hypothesis, together with an important information. One relates to the use of
increasing number of reports of Precambrian such leaf-borne fungi as proxy records for cer-
microbial life (Taylor et al. 2009), appears to tain climate variables (e.g., temperature limita-
have initiated a more general paleobiological tions) that can be used to help reconstruct
interest in evidence of microbial (including paleoclimate or to calibrate paleoclimatic mod-
fungal) activities from other, geologically els (Wells and Hill 1993; Phipps 2006; Singh and
younger, paleoecosystems (Taylor 1993). Chauhan 2008; Ding et al. 2011). A second use
may relate to the function of fungi in ancient
ecosystems (Tripathi 2009). For example, are
III. Techniques there specific types (species) of fossil fungi
that are consistently found on certain leaf
genera or species, and how do these relation-
A. Morphologic Comparison
ships relate to modern leaf/fungus associations?
No doubt the earliest technique used to study The answers to questions like these can be
fossil fungi simply involved the recognition and found in the extensive and robust data source
description of various spots and other struc- of compressed fossil leaves, a source that still
tures on the surface of compressed leaves of needs to be exploited.
angiosperms, especially those from the Ceno-
zoic. There are numerous descriptions of fungal
structure related to ascocarps and other repro- B. Fungal Spores
ductive structures in the literature (e.g., Unger
1850; Lesquereux 1877; Meschinelli 1898). The various types of spores, mycelia, and fruc-
These early reports relied on the so-called pic- tifications that are often recovered by acid
ture-matching technique with living fungi and digestion of sediments and rocks (e.g., Elsik
thus less accurately represented the taxonomy 1996; Kalgutkar and Jansonius 2000) represent
of the fossils than later studies. They did, how- another type of fungal fossil that is abundant
ever, appreciably contribute to the realization but is not a major source of information about
that various types of fungi were associated with the biology of ancient fungi. Historically, fungal
specific plant parts, as they are today, and that, spores (Fig. 10.1) and occasionally other dis-
in some instances, it appears that the same persed fungal remains have been used in
fungus was consistently present on the same stratigraphic studies based on palynomorph
type of plant or plant organ. On some speci- assemblages (e.g., Graham 1962) and have
mens it is possible to scrape off part of the been used to suggest extinction and recovery
fungus from the leaf surface and examine the events based on their presence or absence (e.g.,
hyphae and, in some cases, the spores or other Eshet et al. 1995; Vajda and McLoughlin 2004;
parts of the fossil fungal reproductive struc- Visscher et al. 2011). In a limited way, they have
tures. Compressed leaves, like those from the also been used to corroborate host–parasite
Eocene Geisel Valley in Saxony-Anhalt, Ger- interactions (e.g., Eocene angiosperm leaf
many, or the Miocene Clarkia beds in Idaho, type and epiphyllous fungus and spore type)
USA, have well-preserved cuticles that can be (Dilcher 1965).
Figs. 10.1–10.18 Representatives of fungal fossils Permian permineralization, Germany; bar¼10 mm.
(references in text). Fig. 10.1 Fungal spore; Lower Fig. 10.2 Aspergillus collembolorum, conidial head
One major problem in using fossil fungal spores as a replaced the organic matter of the cell walls.
taxonomic tool is their relative uniformity and lack of Such fossils occur in coal balls—concretions
ornamentation features (Sheffy and Dilcher 1971). This
is especially true of conidiospores; however, new imag-
of mineralized (calcium carbonate, pyrite)
ing systems may hold promise in this regard. In studies plant material (peat) of predominantly Carbon-
of modern fungal spores at the ultrastructural level the iferous and Permian age (Phillips et al. 1976).
focus has generally been on developmental features of On the other hand, in the preservational pro-
conidia (e.g., Mims et al. 1995), with little attention paid cess known as petrifaction, the intercellular
to features of the spore wall that might be useful in
systematics. Examination of surface ornamentation,
spaces and other voids are also filled with pre-
even at the nanometer scale, has generally been cipitated minerals, typically forms of silica
concerned with questions related to molecular interac- (chalcedony, quartz, opal), but the organic mat-
tions and cell-surface properties (e.g., Dufrêne et al. ter in the plant has been replaced by minerals as
1999). As far as we know, no studies have appeared well, so that very little or no organic matter
on the wall ultrastructure of any fossil fungal spores.
Whether this technique might yield additional informa-
remains. For example, the organisms in the
tion about the biological affinities or development of famous Early Devonian Rhynie chert paleoeco-
fossil fungal spores, as has been done for fossil pollen, system are petrified in silica.
remains unknown, but it may offer promise in the To examine the cells and tissue systems of
future. petrified fossils, it is necessary to prepare thin
sections (¼petrographic thin sections), in
which a piece of the fossil or the rock contain-
ing the fossil is cemented to a microscope slide
C. Thin Sections and Acetate Peels and then ground thin enough to be examined in
transmitted light. This technique was also used
The late 1800s witnessed a burst of activity in for early studies on permineralized plants in
paleobotany once structurally preserved (per- coal balls. While the primary focus of the early
mineralized) plant remains were discovered studies on coal balls was the plants, there were
associated with Carboniferous coals. Perminer- occasional reports of fungal remains in the
alizations represent a preservation type in matrix of plant debris and sometimes within
which minerals have precipitated in the cell plant tissue (e.g., Williamson 1878; Cash and
lumina and intercellular spaces but have not Hick 1879; Weiss 1904). As an alternative to the
⁄

Figs. 10.1–10.18 (continued) with chains of conidia; Devonian chert, Scotland; bar¼30 mm. Fig. 10.10 Acau-
Eocene amber, Baltic; bar¼25 mm (courtesy A.R. losporoid glomeromycotan spore; Lower Devonian
Schmidt). Fig. 10.3 Chytrid zoosporangia in outer wall chert, Scotland; bar¼100 mm. Fig. 10.11 Vesicles and
of spore; Lower Devonian chert, Scotland; bar¼20 mm. minute arbuscule-like structures (arrows) in inner cor-
Fig. 10.4 Chytrid zoosporangium in megaspore wall; tex of stigmarian appendage; Lower Pennsylvanian coal
arrows indicate rhizoidal system; Middle Mississippian ball, Great Britain; bar¼100 mm. Fig. 10.12 Hypha with
chert, France; bar¼20 mm. Fig. 10.5 Putative chytrid clamp connection; Middle Mississippian chert, France;
zoosporangia in solitary unicells; arrows indicate dis- bar¼10 mm. Fig. 10.13 Ganodermites libycus, longitu-
charge papillae; Middle Mississippian chert, France; dinal thick section (polished surface) through basidio-
bar¼10 mm. Fig. 10.6 Nothia aphylla prostrate axis, carp; lower Miocene permineralization, Libya; bar¼
showing host response to fungal attack in the form of 2 cm. Fig. 10.14 Septate conidium; middle Silurian
secondarily thickened cell walls; Lower Devonian chert, sandstone, Sweden; bar¼10 mm (from Sherwood-Pike
Scotland; bar¼0.5 mm. Fig. 10.7 Zygosporangium- and Gray 1985). Fig. 10.15 Tappania sp.; early Neopro-
apposed gametangia complex of Jimwhitea circumtecta terozoic shale, Canada; bar¼50 mm (courtesy N.J. But-
(Endogonaceae); Z mantled zygosporangium, MG terfield). Fig. 10.16 Winfrenatia reticulata, hyphal net
macrogametangium, mG microgametangium, MS enclosing cyanobacterial unicells; Lower Devonian
macrosuspensor, mS microsuspensor; Middle Triassic chert, Scotland; bar¼50 mm. Fig. 10.17 Cashhickia acu-
permineralized peat, Antarctica; bar¼20 mm. Fig. 10.8 minata in calamite rootlet, showing intracellular
So-called fungal sporocarp (type Sporocarpon sp.); hyphae arising from host cell walls; Upper Pennsylva-
Lower Pennsylvanian coal ball, Great Britain; bar¼ nian chert, France; bar¼30 mm. Fig. 10.18 Fungal pro-
100 mm. Fig. 10.9 Scutellosporites devonicus, germina- pagules in Sphenophyllum leaf; Upper Pennsylvanian
tion shield in near median longitudinal section; Lower chert, France; bar¼20 mm
expensive and time-consuming thin-section volatile terpenoid or phenolic secondary com-

technique, some early-twentieth-century scho- pounds. It may be produced and stored in
lars working on Carboniferous coal-ball fossils secretory canals/cavities or produced as the
started to use a liquid acetate mixture that was result of injury (Langenheim 2003). Fossil
poured onto acid-etched surfaces. Once the fungi preserved in amber can be easily observed
acetate mixture hardened, a peel of the surface and studied in detail using various microscopy
could be removed and examined with light techniques (Speranza et al. 2010). It is therefore
microscopy. A modification of this technique not surprising that fungi in amber were des-
was published in 1956 [Joy et al. (1956); see also cribed as early as the nineteenth and early
Galtier and Phillips (1999)] that affected how twentieth centuries (e.g., Goeppert and Berendt
coal-ball plants were studied and, at the same 1845; Caspary and Klebs 1907). Only recently,
time, may have decreased the number of dis- however, have these fossils received wider
coveries of fossil fungi in coal balls. In this scholarly attention. Today there are reports of
modification, preformed sheets of cellulose ace- representatives of many different groups of
tate were used to make peels. Using these fungi in amber because the translucent nature
sheets, the technique was easier to master and of the matrix makes it relatively easy to deter-
less time consuming and produced many peels mine even very delicate features useful in sys-
in the time it took for a single petrographic thin tematics, as well as those useful in determining
section or liquid peel to be made. As a result, interactions with other organisms. Some exam-
acetate peels became the technique of choice ples include basidiocarps (Hibbett et al. 1995,
in examining permineralized fossil plants. 1997a, 2003), a carnivorous soil fungus that
Although there are other reasons for the general traps nematodes (Schmidt et al. 2007), sooty
lack of information about fossil fungi (as noted molds (Rikkinen et al. 2003), a representative
earlier), it is now apparent that the classic, of the genus Aspergillus growing on a springtail
time-consuming thin-section technique repre- (Fig. 10.2) (Dörfelt and Schmidt 2005), and
sents the best method to study fossil fungi, evidence of animal parasitism by fungi (Sung
especially features that provide information et al. 2008). Because of the extraordinary pres-
about the interactions of fungi with other ervation potential that amber affords, it will
organisms (e.g., Krings et al. 2007a, b; Taylor continue to be an important avenue for
et al. 2011). As these studies have shown, the research on fossil fungi. Amber pieces usually
etching process used in the peel technique often are quite small, however, and thus rarely pro-
removes tiny organisms preserved in perminer- vide information on the ecological configura-
alized peat, including fungi. tion of the community in which the fungi (and
Transmitted light microscopy of acetate their host organisms) lived. Moreover, almost
peels and thin sections represent the primary all amber comes from Cretaceous and Cenozoic
methods currently being used in the analysis of strata, which is too geologically recent to record
fossil fungi, but there are others that are specific the origin or evolution of most major groups of
for various preservation types. For example, fungi. While amber has been reported from the
scanning electron microscopy (SEM), reflected Carboniferous (approximately 320 million
and transmitted light microscopy of plant years ago [Ma]) (Bray and Anderson 2009), it
cuticles, and other types of imaging systems has not been found to contain direct evidence
(Raman spectroscopy) are also in use. of fungi, with one possible exception (Smith
1898). Many woody plants from the Paleozoic
are known to have produced resins, however,
D. Plant Resins (Amber) and if amber of sufficient size and preservation
could be discovered, it might afford another
Amber is a type of woody seed-plant resin that source of information about very ancient
is a lipid soluble mixture of volatile and non- fungi.
IV. Major Fungal Lineages lycopsids), while others occur in unicells (uni-
cellular algae?) within the chert matrix; still
A. Chytridiomycota others reside on or within land-plant or fungal
spores (Fig. 10.4). Some of the chytrids are
Modern chytrids occur in diverse habitats from interpreted as endobiotic, with holocarpic thalli
the tropics to the Arctic and are found in almost that morphologically compare with zoosporan-
all forms of terrestrial and aquatic ecosystems, gia of the extant chytrid Olpidium (Fig. 10.5). In
so it is not surprising that they have also been some forms, there are distinct discharge pores
reported in the fossil record. Molecular clock or papillae, while others possess a stalk or an
estimates hypothesize that the chytrids are an apophysis that attaches the thallus to the
ancient group that inhabited the Earth at least substrate.
1.5 gigayears, or billion years, ago (Ga) or even Almost all of the chytridlike fossils that
earlier (Heckman et al. 2001). Unfortunately, have been reported to date are of uncertain
the body fossil record of this group in the Pre- affinity. In a few instances, however, some fos-
cambrian consists of only a few reports (e.g., sils of chytridlike organisms have been directly
Belova and Akhmedov 2006), and the interpre- referred to extant genera (e.g., Bradley 1967;
tation of these discoveries remains controver- Garcı́a Massini 2007) or to closely related
sial (Butterfield 2005). To date, the most forms (Daugherty 1941). In recent years, the
convincing chytrid fossils come from the Early approach has been to describe and illustrate
Devonian Rhynie chert ecosystem (Taylor et al. the diversity of chytridlike fossils in time and
2004a, b), which is dated at approximately space and to record the type of host and eco-
410 Ma (Pragian) (Fig. 10.3). The fossils occur system with which they are associated.
both within the silica matrix (Kidston and
Lang1921b) and associated with land plants One approach that has been informative relative to
(Illman 1984), charophytes (Taylor et al. fossil members of the Chytridiomycota is a focus on
1992a), degraded plant material (Boullard and the relationship between the chytrid and any existing
host response. Within the Rhynie chert are a variety of
Lemoigne 1971), and fungal spores (Hass et al. fungal spores, some of which are comparable to the
1994; Krings et al. 2009a). Some of these chy- asexual spores of extant members of the Glomeromy-
trids are so well preserved that they can be cota (Hass et al. 1994). Extending into the spore lumen
classified as holocarpic or eucarpic, mono- in many of these fossils are tapering structures termed
centric or polycentric, and with and without callosities, lignotubers, or papillae that represent a host
response to some invading organism. Many callosities
an operculum. Most of the Rhynie chert chytrid form as a response to microfungi, such as chytrids that
fossils represent structures interpreted as zoos- penetrate the spore wall. As this occurs the spore pro-
porangia because they are the same size and toplast in turn synthesizes new wall material around the
have the same morphology as modern forms. invading filament or hyphae. Other forms of Rhynie
At least one fossil from the Rhynie chert, how- chert microfungi with possible affinities to the Chytri-
diomycota are consistently found between particular
ever, has been interpreted as a chytrid zoospore wall layers of these large spores or completely occupy-
based on the presence of a single, posteriorly ing the lumen of the spore (Hass et al. 1994). Another
directed flagellum (Taylor et al. 1992b). host response to a parasitic chytrid attack occurs within
Within Carboniferous rocks are several the charophyte Palaeonitella cranii in the Rhynie chert
reports of chytridlike organisms associated (Taylor et al. 1992a). In this example, the parasitic
microfungus causes host cells to greatly expand as a
with pollen grains and spores (Millay and Tay- result of the infection, a process known as hypertrophy.
lor 1978), seeds (Oliver 1903), and other fungi What makes this host response so striking is the fact
(Krings et al. 2009b). In the Carboniferous of that a similar response occurs in some modern Chara
France are excellent examples of various types species (Karling 1928). In the fossil example, there are
of resting spores as well as different forms of several chytrids that have penetrated the host cell wall.
Other host responses with chytrids as the causative
zoosporangia (Krings et al. 2009b, c). Some of agents reported in certain Rhynie chert land plants
these are associated with (degraded) plant include hyperplasia, a pattern in which there is active
tissue (e.g., the wood or the periderm of cell division (increase in number of cells) associated
with the invasion of the parasite (Taylor et al. 2004a, b).
In addition, the Rhynie chert plant Nothia aphylla 1.4–1.2 Ga (Blair 2009); more conservative
responds effectively to fungal (chytrid?) attacks by estimates place the divergence at around
thickening of the cell walls that separate uninfected
from infected tissue and by controlled cell death in
800 Ma (Berbee and Taylor 2001). Neverthe-
infected areas of the tissue (Fig. 10.6) (Krings et al. less, documented fossil evidence of zygomy-
2007a). cetes is exceedingly rare. Several Precambrian
microfossils have been directly compared to
life history stages seen in modern zygomycetes
(e.g., Hermann and Podkovyrov 2006; Stane-
B. Blastocladiomycota vich et al. 2007), but none of these are con-
clusive. Perhaps the most persuasive pre-
This clade of core chytrids was included as an Mesozoic fossil interpreted as a member of
order of the Chytridiomycota until recently the zygomycetous fungi is Protoascon missour-
when it was elevated to the phylum level based iensis from the Carboniferous (Middle Penn-
on a life cycle with sporic meiosis and several sylvanian) of North America (Batra et al. 1964;
ultrastructural (James et al. 2006) and molecu- Baxter 1975). This fungus consists of a bulblike
lar features (Porter et al. 2011). One fossil that structure with appendages arising in a whorl
shows a number of features of this group is the from one end. The appendages form a basket-
Early Devonian microorganism Palaeoblasto- like structure around an ornamented sporan-
cladia milleri (Remy et al. 1994a). The fossil gium containing a single spore. Although
includes two types of thalli that are nearly iden- originally thought to be an ascomycete, Taylor
tical in morphology but that differ in the types et al. (2005a) reinterpreted P. missouriensis as
of reproductive structures they produce. One an azygo- or zygosporangium subtended by a
bears terminal zoosporangia and resting spor- suspensor forming appendages.
angia, whereas the other forms chains of 2–3 The record of fossil zygomycetes from the
gametangia. Based on a complement of features Mesozoic is equally scanty, but the few fossils
like those in certain extant Blastocladiomycota that have been attributed to this group of fungi
and the presence of a subgenus Euallomyces- are far more informative than the Paleozoic
type life cycle [alternating diploid sporophytic records. The most compelling Mesozoic fossil
and haploid gametophytic forms; Eucladiella of a zygomycetous fungus documented to date
type of Karling (1973)], it is hypothesized that comes from the Triassic of Antarctica and has
P. milleri had an alternation of generations with been named Jimwhitea circumtecta (Krings
sporic meiosis like that of some extant species et al. 2012). This fossil (Fig. 10.7) is interpreted
of Allomyces. Another interesting microfungus as a zygosporangium-apposed gametangium
from the Rhynie chert is Kryphiomyces catenu- complex that closely resembles the zygosporan-
latus, which occurs as an endobiotic mycelial gium–gametangium complexes seen in certain
thallus in a glomeromycotan spore (Krings extant species of Endogone. Other fossils of
et al. 2010a). The fossil consists of catenulate zygomycetous sexual reproductive structures
hyphae and terminal spherical reproductive from the Triassic of Antarctica include several
structures or propagules. Hyphal morphology intact sporocarps containing spores, in part
in K. catenulatus is reminiscent of that in cer- sheathed by a hyphal mantle, also suggested as
tain extant Hyphochytridiomycota, Chytridio- belonging to the Endogonales (White and
mycota, Blastocladiomycota (i.e., Gonapodya), Taylor 1989, 1991).
and even Ascomycota, but specific features that
can be used to assign the fossil to any modern
group are absent. C.1 So-Called Sporocarps
Some of the most puzzling microfossils in Carbonifer-
ous coal balls and cherts are spherical structures
C. Zygomycetous Fungi (approximately 0.1–1.5 mm in diameter) composed of
a central cavity surrounded by a complex, and in some
Molecular clock estimates suggest that the forms highly ornamented, investment (Fig. 10.8) (e.g.,
first zygomycetes appeared approximately Hutchinson 1955; Baxter 1960; Stubblefield et al. 1983;
Stubblefield and Taylor 1983; Taylor et al. 1994); similar mycetes based on spores dating to the Precam-
structures have also been found in permineralized peat brian (Pirozynski and Dalpé 1989) and
from the Triassic of Antarctica (e.g., Taylor and White
1989; White and Taylor 1991). These structures, which
Ordovician (Redecker et al. 2000, 2002); the
are today commonly termed sporocarps (but see Krings latter report includes some hyphae. None of
et al. 2011a), may occur singly, but there are many these reports, however, provides evidence of
specimens in which several individuals are clustered symbiotic associations, and there is concern
together. Sporocarps have been interpreted as fungal that at least some of the alleged fossils may
in origin based on the investment, which is constructed
of interlaced hyphae that form one to several distinct
represent modern contaminants. Geologically
layers. The number and configuration of the individual younger spores showing a suite of features like
investment layers, as well as surface ornamentation, those of modern Glomeromycota are common
may be highly variable and therefore have traditionally beginning in the Lower Devonian Rhynie chert
been used to distinguish between different sporocarp (Kidston and Lang 1921b), slightly younger
morphotypes, such as Dubiocarpon, Mycocarpon, Spor-
ocarpon, and Traquairia (Taylor et al. 2009).
Devonian rocks (Stubblefield and Banks 1983),
Many sporocarps contain one to several spherical and into the Carboniferous (e.g., Wagner and
structures in the cavity, which have led to their inter- Taylor 1981, 1982; Krings et al. 2011d), where
pretation as ascomycete cleistothecia (e.g., Stubblefield they occur in both the matrix and within the
and Taylor 1983). In this hypothesis, the larger internal tissues of various plants. Mesozoic records of
spherical structures would represent asci and the smal-
ler ones, ascospores. An alternative interpretation,
Glomeromycota include specimens found in
however, views the sporocarps as belonging to the the fossilized dung of herbivorous dinosaurs
zygomycetous fungi (Taylor and White 1989; White (Kar et al. 2004; Sharma et al. 2005).
and Taylor 1989). The large, inner sporelike body is
thought to represent the zygospore, while the sur- Some of the glomeromycotan spores from the Lower
rounding structure would be equivalent to the hyphal Devonian Rhynie chert provide details about certain
envelope or mantle seen in certain modern Endogo- features that are useful in identifying the mode of
nales. The smaller internal spheres reported in some spore germination, but they also provide characters
specimens are regarded as mycoparasites. Although that are useful in determining their systematic affi-
there is an increasing body of circumstantial evidence nities. Spores found in axes of Asteroxylon mackiei
to corroborate the hypothesis that at least some of the range up to 350 mm in diameter and possess a wall
so-called sporocarps represent zygomycetous repro- that consists of several distinct layers when examined
ductive structures (Krings et al. 2010b, 2011b, c), struc- in transmitted light. Associated with one spore wall
tural features confirming the zygomycetous affinity of layer is a round to oval, lobed structure that represents
these interesting fossils have not been documented to a germination shield, like those found in the modern
date. genus Scutellospora (Fig. 10.9) (Dotzler et al. 2006). In
modern forms, the germination shield produces one to
several germ tubes at maturity. Other large spores from
the Rhynie chert possess very complex walls and are
comparable to those found in modern members of the
D. Glomeromycota Acaulosporaceae in which the spore develops laterally
in the neck of a sporiferous saccule (Fig. 10.10) (Dotzler
This monophyletic group of fungi represents et al. 2008). These two studies demonstrate that even
one of the major drivers in modern ecosystems isolated spores can provide a wealth of information not
due to its role in global phosphorous and car- only about functional aspects of the spores but also
about the presence of structural and morphological
bon cycles. This function is accomplished in details useful in more clearly defining the systematic
approximately 80 % of land plants by means affinities of the spores, which can be used as markers
of a mutualistic symbiosis in the form of arbus- for minimal age dating in association with molecular
cular mycorrhizae (Schüßler and Walker 2011). data sets.
The Glomeromycota can be traced back at least
to the Early Devonian, where there is strong Although the presence of variously sized
evidence of a mycorrhizal system in place in spores, some with structural features like
both the free-living gametophyte and sporo- those of modern Glomeromycota, confirms
phyte phases of several early land plants the presence of the group in the Paleozoic, the
(Remy et al. 1994b; Taylor et al. 1995, 2005b). most convincing evidence of a biotrophic rela-
There have been putative reports of glomero- tionship with land plants is the presence of
arbuscules. The first unequivocal occurrence of enzyme systems for degradation. It is also pos-
these comes from both the sporophyte and sible, as is often the case in paleomycology, that
gametophyte phases of the land plant Aglao- the group has simply not been discovered to
phyton major (gametophyte¼Lyonophyton date. There is some support for this latter sug-
rhyniensis) from the Lower Devonian Rhynie gestion by the Middle Devonian, where there
chert. The fungus occurs in a narrow circum- are several examples of basidiomycete disease
ferential zone of cortical cells 2–3 layers thick symptoms in the wood of the progymnosperm
and consists of aseptate hyphae that give rise to Callixylon newberryi (Stubblefield et al. 1985).
intracellular branchlike structures that mor- Within the decaying wood are branched, sep-
phologically are identical to the modern physi- tate hyphae, some of which contain both in-
ological exchange structures in many extant tercalary and terminal spores. No clamp
plants. Arbuscule-like structures have also connections were found, but the secondary
been reported in other Paleozoic and Mesozoic xylem tracheids, decayed to varying degrees,
plant roots (Phipps and Taylor 1996; Strullu- show erosion troughs, cavities, and extensive
Derrien et al. 2009; Schwendemann et al. 2011), lysis of the walls, similar to symptoms caused
as well as in structures that functioned as root- by modern-day white-rot fungi. Another indi-
ing organs (Fig. 10.11) (Krings et al. 2011d). rect source of basidiomycete activity in the fos-
While there are older land plants from the Silu- sil record is patterns of secondary xylem decay
rian, to date none have been found that are in some Late Permian and Middle Triassic gym-
sufficiently well preserved to test the hypothesis nosperm woods from Antarctica. The most
advanced by Pirozynski and Malloch (1975) conspicuous of these occurs in the form of
that the earliest land plants were preadapted circular areas (pockets) that range up to several
to living in a terrestrial ecosystem because of millimeters in diameter and several centimeters
their biotrophic symbiotic relationship with a long that are completely devoid of cells. In the
fungal partner. adjacent wood are hyphae with clamp connec-
tions (Stubblefield and Taylor 1986). In addi-
tion, these woods show degradation of the cell
E. Basidiomycota walls in a sequential pattern, symptoms that are
anatomically identical to white rot and white
Surprisingly, the fossil record of basidiomy- pocket rot in several extant woody plants.
cetes is relatively poor, although today they To date, the oldest fossil evidence of a
are widespread and represent the primary clamp connection comes from the Middle Mis-
decay agents of cellulose and lignin. Even the sissippian (Visean) of central France (Krings
Lower Devonian Rhynie chert, which contains et al. 2010c). The fungus occurs in the cortical
several of the major groups of fungi (i.e., Chy- tissues of a small fern and consists of clamp-
tridiomycota, Blastocladiomycota, Glomero- bearing hyphae (Fig. 10.12) morphologically
mycota, Ascomycota), lacks any evidence of identical to those produced by many extant
fungi suggestive of being basidiomycetes. The basidiomycetes, as well as intercalary and ter-
lack of fossils of this group has led to the pro- minal swellings and structures resembling
posal of two possible reasons that basidiomy- chlamydospores. The presence of callosities in
cetous fungi were not present in this ecosystem: some host cells suggests that the plant was alive
there was an absence of lignin in the Early at the time the fungus invaded the tissues.
Devonian or degradation was accomplished by Other reports of late Paleozoic clamp-bearing
other fungi. Like so many fossil groups, the hyphae, interestingly also associated with
failure to find features like those in extant ferns, include Palaeancistrus martinii from
groups provides an immediate obstacle to the Upper Pennsylvanian of North America
interpreting the affinities of organisms that (Dennis 1970) and an unnamed form that
may have initially evolved a different set of thrives in the root mantle of Psaronius tree
morphological or reproductive characters but ferns from the Lower Permian of Germany
nevertheless still possess the appropriate (Barthel et al. 2010).
One basidiomycete group that has a more extensive of the land plant Asteroxylon mackiei and con-
fossil record are the polypores (surveyed in Fleisch- sists of perithecia approximately 400 mm in
mann et al. 2007). Although there have been some
reports of polypore basidiocarps from the Paleozoic
diameter. Each perithecium contains a short,
and early Mesozoic, many of these reports have since ostiolate neck; within the perithecium are
been discounted or reinterpreted as other organisms numerous asci intermixed with paraphyses.
(e.g., Pirozynski 1976; Hibbett et al. 1997b). In the Each ascus contains up to 16 uniseriate or
Cretaceous and Cenozoic there are far more reports of biseriate ascospores. What makes this fossil
polyporous fungi, and many of these can be closely
related to modern genera. The oldest bona fide record
even more interesting is the presence of acer-
of polypores comes from the Cretaceous of North vuli of thallic conidiophores producing cube-
America (Smith et al. 2004). Perhaps the most well- shaped arthrospores interspersed among the
preserved fossil polyporous basidiocarp is Ganoder- perithecia.
mites libycus from the Neogene of North Africa Mesozoic and Cenozoic rocks contain sev-
(Fig. 10.13) (Fleischmann et al. 2007). This perminer-
alized basidiocarp is stratified and shows pronounced
eral excellent examples of epiphyllous fungi
growth increments. The hymenium has equidistantly assigned to the Ascomycota. While most of
arranged pores that contain clavate basidia and ellip- these are associated with the leaves of angios-
soidal basidiospores, each with a two-layered ganoder- perms (e.g., Dilcher 1965; Smith 1980; Phipps
matoid wall. The extraordinary preservation of certain and Rember 2004), a few have been reported
features in the fossil, especially the unique basidios-
pores, makes it possible to place the fossil within the
on, or in association with, conifers and vascular
modern Ganodermataceae. cryptogams (e.g., Pons and Boureau 1977; Van
der Ham and Dortangs 2005; Shi et al. 2010;
Garcı́a Massini et al. 2012). Most of these
F. Ascomycota forms consist of isolated reproductive struc-
tures such as thyrothecia and pycnidia. Others
While the Ascomycota today constitute the consist of pseudoparenchymatous hyphae that
largest group of fungi, the fossil record does radiate from ascomata. Several epiphyllous
not demonstrate this level of diversity. To ascomycetes contain well-preserved hyphopo-
some extent, there has been an inherent bias dia that can be used to trace the life-history
based on the long-held assumption that the biology of the fungus. A wealth of information
group did not evolve until the Cretaceous remains to be obtained about the nutritional
(Pirozynski and Weresub 1979). Today, how- mode of fossil epiphyllous ascomycetes and
ever, there are scattered reports of ascomycete whether they represent generalist or specific
fossils dating back to the middle Silurian of types of parasites that can be correlated with
Sweden that have yielded hyphae and spores. distinct species of leaf fossils. Systematic data
The most often cited of these consists of chains on fossil epiphyllous fungi may also be useful as
of up to nine multiseptate spores as well as another proxy record of various climate para-
conidiogenous cells (phialides) (Fig. 10.14) meters through geologic time and space.
(Sherwood-Pike and Gray 1985). Because these From Cenozoic rocks there are also several
fossils were obtained in rock macerations, reports of excellent examples of fossil ascomy-
nothing is known about other stages of the life cetes involved in various types of intricate
history or the relationship with other organ- interactions with other organisms. For exam-
isms. Slightly younger (Early Devonian) speci- ple, from lower Eocene amber collected in
mens from Siberia attributed to the India, Beimforde et al. (2011) reported on ecto-
Ascomycota include asci and structures inter- mycorrhizal fungi that compare closely to an
preted as paraphyses that are regarded as evi- extant member of the Dothidiomycetes. Other
dence of the Microthyriales (Krassilov 1981). Eocene ascomycetes reported from perminera-
To date, the oldest structurally preserved lized specimens include mycoparasites (Currah
fossil attributed to the Ascomycota is Paleopyr- et al. 1998) and a single loculoascomycetous
enomycites devonicus, from the Lower Devo- ascoma (Mindell et al. 2007). An especially
nian Rhynie chert (Taylor et al. 2005c). This interesting case of fungal parasitism from the
fossil is preserved in the flattened appendages Eocene documents adaptive manipulation of
ants by a parasitic ascomycete in the form of Another Proterozoic fossil with a netlike organization
stereotypical death grip scars preserved in that has been interpreted to represent some level of
fungal organization is Tappania (Butterfield 2005;
angiosperm leaves from the famous Messel pit Nagovitsin 2009), an organism previously described as
in northern Germany (Hughes et al. 2011). an acritarch, a group of enigmatic fossil aquatic eukar-
yotes. In this fossil (Fig. 10.15), a series of filamentous
processes with cross walls forms a series of anasto-
moses surrounding a central vesicle. This multicellular
VI. Lichens level of organization has been used to suggest that
Tappania and a number of other acritarchs represent
putative fungi that fall somewhere between Ascomycota
It is surprising that the fossil record is not and zygomycetous fungi. Establishing the biological
replete with reports of lichens, because the affinities of these organisms, including whether they
structure of the thallus in many forms would are in fact fungal, however, will require more definitive
seem to have high preservational potential. evidence.
Some reasons for the lack of fossil lichens may
be the absence of a focus on these organisms, The direct physical association between
especially those that might have occurred early fungal hyphae and coccoid cells within a
in the terrestrialization of the Earth, or the three-dimensional netlike organization is also
failure to recognize examples of these sym- present in the Early Devonian thallus Winfre-
bioses in the fossil record. The enigmatic natia reticulata (Taylor et al. 1997; Karatygin
Early Devonian fossil Spongiophyton, histori- et al. 2009). The thallus is constructed of two
cally believed to represent a nonvascular plant distinct zones, a lower one of superimposed
form transitional between algae and land plants layers of parallel hyphae and an upper zone of
(Gensel et al. 1991), was interpreted as a lichen vertically oriented hyphae that are folded into
based on structural features seen in perminer- loops so as to create a series of ridges and
alized specimens (Stein et al. 1993). This inter- depressions. Extending into the depressions
pretation has been supported by evidence from are aseptate hyphae arranged in a loosely
carbon-isotope ratios [Jahren et al. 2003; but organized netlike structure (Fig. 10.16). Within
see Fletcher et al. (2004) for arguments against] the lacunae of the net are unicells up to 16 mm
and from ultrastructural analyses (Taylor et al. in diameter, each surrounded by a thin sheath.
2004b). There have been a few reports of lichens Cells at the base of the depression are generally
from the Precambrian to the Ordovician (e.g., small and solitary, while those that are more
Hallbauer and van Warmelo 1974; Retallack distal show increasing divisions resulting in
1994, 2009), but most of these have now been aggregations of up to 32 cells. The unicells of
discounted (Cloud 1976; Waggoner 1995) or the photobiont in W. reticulata show many
remain inconclusive. A structurally preserved similarities to certain cyanobacteria, but the
Precambrian fossil that is morphologically sim- absence of diagnostic features of the hyphae
ilar to a lichen comes from the Ediacaran (Neo- means that the affinities of the mycobiont in
proterozoic) upper Doushantuo Formation of W. reticulata remain unknown. Nevertheless,
South China (Yuan et al. 2005). This structure, the structural organization displayed in this
sometimes termed a biodictyon, consists of fossil, like the geologically earlier biodictyon
clusters of coccoid cells encased within a netlike in the Neoproterozoic, suggests that there was
arrangement of hyphae. It is not currently pos- physiological interaction between the two com-
sible to demonstrate the physiological interac- ponents, as is seen in modern cyanobiont
tions of the bionts in this association, the fact lichens.
that this Precambrian fossil shows a consistent, There have been several reports of Ceno-
physical relationship of coccoid cells, and the zoic lichen thalli preserved as impressions and
enclosing netlike mycelium provides support compressions in rocks (e.g., Peterson 2000) and
for the hypothesis that this fossil is indeed a amber (Rikkinen and Poinar 2008 and citations
lichen or lichenlike association. therein). An older specimen from the Middle
Jurassic of northeastern China possesses
numerous features that suggest an affinity with multiple specimens suggests that this fossil
the lichens (Wang et al. 2010). The thallus of may have had affinities with some basidiomy-
Daohugouthallus ciliiferus consists of a series of cete group (Hueber 2001); its heterotrophic
elongate primary axes from which extend lat- nutritional mode is also supported by isotopic
eral and terminal branches that repeatedly analysis (Boyce et al. 2007; Hobbie and Boyce
dichotomize. Extending from all branches are 2010). Prototaxites is constructed of three types
elongate structures that appear similar to cilia of septate and nonseptate tubes or hyphae, and
in modern lichens. in certain sections of the axes, there are what
Like the partners involved in mycorrhizae, are interpreted as growth increments of the
the physiological relationships within a lichen sporophore. Doliporelike septal structures
symbiosis based on fossils is currently impossi- have been reported, but they have not been
ble to prove unequivocally. Nevertheless, the thoroughly investigated in multiple specimens
fact that lichens are repeatedly regarded as or in different planes of a section (Schmid
ancient organisms based on sequence data 1976). A few structures are interpreted as
(e.g., Lutzoni et al. 2001), together with the clamps and sterigmata, but these remain equiv-
scattered reports of fossils, indicates that the ocal.
fossil record holds a potentially rich source of
information not only about the character states Another enigmatic organism that has been related to
present in ancient lichens, but also about the Prototaxites is Nematasketum (Burgess and Edwards
stages involved in the evolution of licheniza- 1988). It also consists of axially oriented tubes; the
larger ones often branch and have irregular thickenings
tion. on the internal wall, while the internal surface of the
narrower tube surface is sometimes smooth. On the
outside of Nematasketum are tissues interpreted as a
VI. Enigmatic Fossils rind (Edwards and Axe 2012). In regions interpreted as
sites of hyphal generation, Nematasketum exhibits dif-
ferentially thickened branching tubes, whereas the
There are numerous reports of fossil organisms hyphae are smooth in Prototaxites. Structures such as
that possess many of the characters of fungi, clamp connections, basidia, or septa have not been
and a number of these are known in great detail identified in any specimens to date. Although Nematas-
ketum is well preserved as a charcoalification and has
with regard to morphology or anatomy; how- been examined by SEM, the affinities of this organism
ever, for many of these, systematic assignment are still equivocal. Prototaxites and Nematasketum, as
to a specific group remains problematic. The well as some other enigmatic thalloid organisms from
problem in deciphering the affinities or nutri- the Silurian and Devonian known as nematophytes,
tional modes of these fossils has been due to may represent fungi, some type of symbiotic associa-
tion such as lichens (Selosse 2002), or life forms that
either the presence of distinguishing features, have no modern analogues. At present, these interest-
for example, clamp connections and basidia, or, ing fossils continue to defy attempts to place them
conversely, the absence of particular character- within a modern systematic framework (Taylor et al.
istics. 2010).
Another fossil structure that possesses a

A. Uncertain Affinities number of fungal features is the Carboniferous
taxon Palaeosclerotium pusillum (Rothwell
Most prominent among the enigmatic fossils 1972; Dennis 1976). This fossil appears to pos-
that have variously been attributed to the sess features of two major clades on modern
fungi is the late Silurian–Early Devonian organ- fungi. There are hyphae with what appear to be
ism Prototaxites. Despite the fact that some doliporelike septa that have clamp connections
Prototaxites specimens are large (some >1 m like those in modern basidiomycetes, but P.
in diameter) and anatomically preserved, the pusillum also has cleistothecia, perhaps with
affinities of this organism remain conjectural. asci, which are characteristics of the ascomy-
The most comprehensive study involving cetes. It is not yet resolved whether this fossil
represents an example of mycoparasitism or mycorrhizal relationships in extant plants

whether it may be a fungus that shares multiple based on structural (Brundrett 2009), molecu-
characters used to define modern groups lar (Wang and Qiu 2006), and functional
(McLaughlin 1976; Singer 1977). aspects (e.g., Bidartondo 2005). Examining the
actual type of physiological exchange mecha-
nism (e.g., Strullu-Derrien and Strullu 2007)
B. Plant–Fungal Associations with Uncertain will also be an important component of
Functions future studies involving the fossil record of
mycorrhizae.
There are a large number of fungi that colonize Despite the incredible amount of leaf bio-
roots today whose particular functions remain mass in the Carboniferous, it is surprising that
unknown. A fungus that occurs in the roots of a there are so few reports of fungi associated
Late Pennsylvanian calamite (Astromyelon) with leaves. Since these leaves were produced
presents a set of interesting questions with by ferns, other vascular cryptogams, and gym-
regard to its affinities, function, and nutritional nosperms (pteridosperms and cordaites), it has
mode (Taylor et al. 2012). This fungus, Cash- long been hypothesized that perhaps the pro-
hickia acuminata, consists of aseptate hyphae duction of secondary metabolites by these
that penetrate the outer periclinal and anticlinal plants restricted endophyte colonization. How-
(but not the inner periclinal) walls of the root ever, as is apparently the case with other fossil
cortical cells and extend approximately halfway fungi and the habitats they occupied, once
through each cell lumen (Fig. 10.17). Some of recognized, fungi associated with leaves are
the hyphae branch at right angles, and the tips rather common. For example, a small fern leaf
are sharply pointed. While the presence of cal- from the Upper Pennsylvanian of France con-
losities associated with some of the infected tains septate hyphae, some with prominent
cells suggests that the root was alive at the swellings, extending through the hypodermis
time of infection, the affinities of the fungus of the leaf (Krings et al. 2009d). Because there
and its nutritional mode remain unknown. is no observable host response, it is not possible
The presence of the pointed, aseptate hyphae to determine whether the host was alive at the
that only penetrate from one side of the cell and time of infection or in some stage of decay,
extend only about halfway into the lumen may thus suggesting the fungus was some type of
suggest that hyphal growth was perhaps influ- saprotroph.
enced by some nutritional gradient. In general,
these fossil hyphae appear similar to dark sep- A number of fungal remains that have recently been
tate hyphae (DSE), modern root endophytic described from Carboniferous leaf tissue consist of
hyphae that represent some conidial forms diverse assemblages of propagules in multiple leaf spe-
cimens of the same plant (Fig. 10.18) (e.g., Dotzler et al.
whose function and affinities remain poorly
2011; Krings et al. 2011c). These discoveries suggest
understood (Mandyam and Jumpponen 2005). that certain fungi were more widely distributed in
Historically fossil roots have generally not these tropical Paleozoic habitats, while others appear
been a major focus of terrestrial plant paleobot- to have had a rather limited host range. These differ-
any, even in those instances in which they were ences inevitably raise the question of whether this situ-
permineralized and showed extraordinary cell ation reflects generalists and specialists among the
fungi or is due merely to a current lack of resolution.
and tissue detail. In recent years, however, they Leaf endophytes today represent a significant percent-
have been examined in greater detail because age of overall fungal biodiversity, especially in tropical
they can serve as host to mycorrhizal fungi and habitats. We believe that their current underrepresen-
thus provide necessary information about pat- tation as fossils merely reflects a lack of attention to
terns in mycorrhizal development through time these fungi in the fossil record rather than some inher-
ent biological control in the past. An important contri-
(e.g., Strullu-Derrien et al. 2009; Krings et al. bution to paleomycology in the future will be to look at
2011d). Important work still to be done in such early leaves and leaflike organs to determine when the
studies includes a comprehensive analysis of phylloplane was first used as a fungal habitat.
VI. Conclusions Belova MY, Akhmedov AM (2006) Petsamomyces, a

new genus of organic-walled microfossils from
the coal-bearing deposits of the Early Proterozoic,
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171:9–18
11 Phylogenomics Enabling Genome-Based Mycology
JASON E. STAJICH1
CONTENTS A. Phylogenetic Reconstruction from Targeted

I. Application of Genomics to Molecular Genes
Phylogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
A. Phylogenetic Reconstruction from Targeted Amplification and sequencing of ribosomal
Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 DNA (rDNA) has been a widely successful tool
B. Genome-Based Phylogenetic for molecular phylogenetic identification of
Reconstruction. . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
C. Sequencing 1000 Fungal Genomes. . . . . . . 283
fungi (Bruns et al. 1992; White et al. 1990). The
II. Genomics Enabling Comparative Biology . 284 advent of polymerase chain reaction (PCR) and
A. Evolution of Gene Structures in the the development of universal primers for
Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 amplification of fungi opened avenues for phy-
B. Synteny and Gene Order Evolution . . . . . . 285 logenetic reconstruction from rDNA molecules,
C. Evolution of Gene Families . . . . . . . . . . . . . . 286
D. From Molecules to Morphology . . . . . . . . . 287
providing methods less prone to homoplasy and
III. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 often with greater resolution. Molecular-based
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 phylogenetic trees were shown to be more
robust and avoided the homoplasy that charac-
terized many morphological characters as a
result of convergent evolution (e.g., gills of
I. Application of Genomics to mushrooms, truffles) (Hibbett et al. 1997;
Molecular Phylogenetics McLaughlin et al. 1995; Moncalvo et al. 2002).
The individual loci within the rDNA provide
The application of molecular phylogenetics to needed variation in phylogenetic resolution: on
studies of fungal evolution ushered in an era of average, the 18S (small subunit—SSU) and 28S
more accurate methods for reconstructing the (large subunit—LSU) genes provided support
history and relationships of fungi. The advent for deep branches in the fungal tree while the
of high-throughput genome sequencing has internal transcribed spacers (ITS1 and ITS2)
even further improved the rate at which sys- were useful for species- and genus-level resolu-
tematics can resolve species relationships. The tion. However, as further fungal phylogenetic
application of genome sequences to phyloge- research was pursued, it emerged that rDNA
netics has also improved the accuracy of spe- genes had limitations in the resolution of phylo-
cies tree reconstructions and focused efforts on genetic relationships, and additional markers
obtaining samples and downstream data ana- were sought (Seifert et al. 1995). Multilocus
lyses rather than one-by-one efforts to obtain sequencing and combined analyses of markers
amplification from each sample. have mostly overcome problems in connection
with the limited phylogenetic resolution of
rDNA loci (James et al. 2006; Schoch et al.
1
2009). The development of additional loci
Department of Plant Pathology & Microbiology and Institute for
Integrative Genome Biology, University of California-Riverside,
organized by phylogenetic informativeness has
Riverside, CA 92521, USA; e-mail: jason.stajich@ucr.edu also shown improvements in species tree

280 J.E. Stajich
resolution from gene phylogenies (Townsend Genome sequencing involves a random

2007) so that loci can be chosen that are tuned sample of DNA and so provides roughly even
for the evolutionary distance to be resolved. The coverage of the genome. However, much of the
culmination of this work was a series of revi- genome evolves too rapidly or lacks correspon-
sions of the entire fungal phylogenetic tree, with dence across species for use in many phyloge-
200 species based on multiple markers (Hibbett netic analyses. Intergenic regions, introns, and
et al. 2007; James et al. 2006). transposable elements will be part of the sam-
However, locus-by-locus amplification has pled genome but are unlikely to be useful for
challenges and disadvantages and is a low- phylogenetic reconstruction for more than the
throughput operation. Optimization of PCR closest of species or for population genetic
conditions for some species is required due to analyses. A more targeted sample of only the
variation in sequence bias and the length of the gene regions can be obtained by sequencing
target loci and, when performed by either sam- the expressed transcripts of mRNA. This is
ple or locus, can be time consuming. In addi- achieved with protocols for so-called RNA-Seq
tion, locus-by-locus PCRs will work best if one that reverse-transcribe mRNA into cDNA using
is starting with a pure isolate of a species. This either random primers or at least one primer
can be difficult to obtain for some species that that is complementary to the poly-A tail found
are reticent to grow under laboratory condi- on eukaryotic mRNAs. The mRNA library must
tions or often impossible for obligate sym- also be normalized, or the majority of the
bionts. Finally, the resulting PCR product sequences obtained will originate from the
must be cloned and sequenced, which incurs most highly expressed genes, including mostly
reagent and sequencing costs for each sample ribosomal RNA, which will be only a limited
attempted and requires potential needs for and biased sample of the genes. To convert the
optimization of PCR primers for some clades short reads of RNA-Seq into usable long
of fungi. sequences for analyses, a tool such as Trinity
An alternative to locus-by-locus targeted (Grabherr et al. 2011) is well suited to construct
amplification is a random shotgun sequencing a de novo assembly of the reads. These assem-
of whole-genome DNA to generate a low- bled transcripts can then be compared to a
coverage (5–10) of the genome. With current preselected set of target markers and extraction
next-generation sequencing techniques it is and alignment of the sequences performed for
possible to obtain draft-level genome coverage phylogenetic analyses (Hittinger et al. 2010;
of a 40 Mb genome in 1/20 of an Illumina Hi- Rokas and Abbot 2009).
Seq lane. Using the paired-end approach would To best achieve uniformity from these
generate roughly 200 million reads that are sampling approaches, a set of conserved loci
100 bp long with current technology, or 40 must be developed to insure targeting of most
gigabases (4109). Splitting this among 20 sam- of the same loci across studies (Hittinger et al.
ples provides 2 gigabases (20 million bases) per 2010; Rokas and Abbot 2009). These loci should
genome, or 50 coverage of a 40 Mb haploid be single-copy or at least have paralogs that can
genome. This level of sequencing is not suffi- be easily distinguished from each other. They
cient to create a high-quality de novo assembly can be generated based on a reference set of
of genomes but should be sufficient for extract- fungal genomes representing the phylogenetic
ing a predefined set of gene markers (Lemmon diversity of the fungal kingdom. Comparative
and Lemmon 2012). Improvements to the next- analysis of these genomes using methods to
generation sequencing technologies, including cluster orthologous genes would generate a
upgrades to Illumina sequencing at 250 bp long compact set of loci that can be targeted. One
reads and the development of other sequencing method to accomplish this is to use the HAL
platforms (e.g., Ion Torrent, PacBio, Nano- pipeline to extract gene sets from the genomes
pore), will continue to lower the cost of these (Robbertse et al. 2006, 2011). This approach
approaches to data collection for phyloge- was used to develop a target set for the Fungal
nomics (Quail et al. 2012). Tree of Life II project and works by first
Phylogenomics Enabling Genome-Based Mycology 281
building a matrix of sequence similarities of all epoch (Townsend 2007). This approach enables
genes found in all the genomes and identifying a principled selection of markers for the species
the clusters that have only one copy per species. in a study that allows one to find an optimal set
Often some flexibility is allowed in the criteria, to resolve both deep and shallow nodes of the
which permits some species to be missing a phylogeny (Lopez-Giraldez and Townsend
gene to accommodate annotation errors or 2011; Townsend 2007; Townsend et al. 2012).
gene-loss events. The assumption is that these While the AFTOL project collected these mar-
losses or missing data are random. Another kers by PCR and sequencing of individual loci,
phylogenomic approach, implemented in the the availability of whole-genome sequencing
PhylomeDB project, constructs phylogenetic enables identification of the groups of ortho-
gene trees from the ortholog clusters (Gabaldon logs and their evaluation to identify a marker
2008; Huerta-Cepas et al. 2008; Huerta-Cepas set that is most phylogenetically informative for
et al. 2011). These gene trees can be tested the taxa selected.
against known phylogenies or, for consistent
species relationships, resolved in the different
clusters in order to select only a subset of B. Genome-Based Phylogenetic
markers that perform best for species tree Reconstruction
construction of the targeted organisms.
The dependence on PCR dictates the choice To fully utilize genome sequences in phyloge-
of markers for phylogenetic studies in which nomics requires the construction of species
genes can be amplified with common primers. trees from genome or transcriptome sequences
The identification of conserved genes, where a of a group of species. To produce these trees, a
single set of primers amplifies a very broad set of orthologous genes must be identified
taxonomic sample, was crucial for expansion from the set of genomes to be used in the
beyond rDNA loci. The protein-coding genes phylogenetic analysis. Genes are clustered into
Translation Elongation Factor 1 alpha (EF1a, homologous groups on the basis of their pro-
also called TEF1 in Saccharomyces cerevisiae) tein similarity. These clusters are further pro-
and RNA Polymerase II subunits 1 and 2 (RPB1 cessed using tools to evaluate the consistency of
and RPB2) have been developed as useful phy- the similarity, and in some cases the consis-
logenetic markers (Baldauf and Palmer 1993; tency of the phylogenetic signal, in order to
Liu et al. 1999). These three protein-coding best identify the subset of clusters of genes
genes were included in addition to rDNA as that are truly orthologous. To identify genes
the sampled loci for the Assembling the Fungal useful for phylogenetic analysis, simple criteria
Tree of Life (AFTOL) project. They demon- are applied such as selecting only orthologous
strated improved resolution of the fungal phy- clusters with one gene present per species in a
logenetic tree as markers with a variety of cluster. Other approaches accommodate clus-
evolutionary rates (James et al. 2006). Markers ters with paralogous genes by choosing only
with slower evolutionary rates resolve older one from a set of “in-paralogs,” genes dupli-
nodes in the phylogenetic tree, while those cated within a single species. Each set of puta-
with faster rates are more useful for younger tively orthologous sequences is aligned as a
nodes in the tree. The multilocus approach multiple alignment, and the resulting alignment
provided strong support for many previously is filtered for ambiguously aligned positions.
unresolved relationships in the fungal tree of This trimmed alignment is then analyzed
life. The evolutionary range of a marker to using phylogenetic tools that first choose an
resolve nodes of a particular age in a tree is its appropriate model of sequence evolution.
phylogenetic informativeness. In computing Finally, the trimmed alignment is subjected to
phylogenetic informativeness one calculates a variety of phylogenetic reconstruction meth-
the probability of a marker possessing the ods that include parsimony, distance-based
most appropriate evolutionary rate for a set of methods such as neighbor joining, maximum
nodes in a phylogenetic tree within a given likelihood, and Bayesian phylogenetic software.
282 J.E. Stajich
This operation can be performed in an auto- based primarily on sequence similarity (Chen
mated fashion following a series of logical steps et al. 2006; Fischer et al. 2011).
in a defined workflow. Several general work- Fungal species trees generated from fungal
flow systems have been developed for bioin- genomes give a similar perspective on the rela-
formatics, including Galaxy (Goecks et al. 2010) tionships inferred from only a few loci (Capella-
and Taverna (Hull et al. 2006; Oinn et al. 2004; Gutierrez et al. 2012; Fitzpatrick et al. 2006;
Tan et al. 2010) or combinations of both Marcet-Houben and Gabaldon 2009; Robbertse
(Abouelhoda et al. 2012). These systems are et al. 2006). The degree of resolution of the tree
typically Web-based tools designed for users can depend on whether there are conflicting
to interact with Web browser software. Ana- signals in the loci. Evaluation of supertrees
lyses can also be run within UNIX environ- from gene trees of multiple orthologs can be
ments of Linux or Mac OS X with a series of used to identify incongruences, since there will
steps executed as scripts that are run by be multiple topologies supported among the
the user. The improved standardization of gene trees (Creevey and McInerney 2005; Fitz-
these pipelines has reduced the amount of patrick et al. 2006; Haggerty et al. 2009). These
bioinformatics training required to adopt incongruences can be caused by horizontal
these approaches to new study systems. Further gene transfer, differential rates of evolution,
development of these workflow systems to and incomplete lineage sorting. It has also
support phylogenetic analyses and enable been shown that in some cases nearly all indi-
evolutionary model selection, hypothesis test- vidual gene trees of universally found orthologs
ing, and bootstrap analysis will simplify can have phylogenetic patterns that are differ-
accessto phylogenomics for inferring species ent from those of trees derived from concate-
relationships. nated alignments, indicating that testing the
To identify orthologous genes, groups of congruence of the phylogenetic signal is an
similar sequences must be identified (Fang important part of constructing species trees
et al. 2010). One approach uses clustering to from gene trees (Salichos and Rokas 2013).
build a network of genes based on sequence Work from the genomic data of S. cerevisiae
similarity computed from pairwise alignments and related species showed that for more than
using tools such as BLASTP and then clusters 1,070 orthologous families from genomes of 23
the network using Markov clustering (MCL) species, each had different topologies from that
(van Dongen 2000; van Dongen and Abreu- estimated from a concatenated alignment.
Goodger 2012), implemented first in Tribe- Phylogenomic approaches also use gene
MCL (Enright et al. 2002). Additional refine- families from similarity clusters to build can-
ments to the MCL approach for orthologous didate orthologous families but then use
genes was developed as OrthoMCL, which cor- phylogenetic trees to assess orthologous and
rects for paralogous gene families and has paralogous groups to provide detail on the his-
been shown to be one of the more accurate tory of gene families. One approach, Phylo-
and automated approaches (Chen et al. 2006, meDB, provides the historical perspective for
2007). A variation on this approach that simply each gene in a target genome by presenting a
tries to find single-copy genes suitable for phy- tree with the copies of orthologs and paralogs
logenetic analyses was developed in the pipe- for a selected set of genomes (Gabaldon 2008;
line HAL and has been successfully applied to Huerta-Cepas et al. 2008, 2011). The relation-
many fungal genome projects (Robbertse et al. ship of the genes within a family can also be
2006, 2011). Other fungal databases of gene assessed as to their relationship as paralogs (by
families include FUNYBASE, which provides a duplication) or as true orthologs (by specia-
presentation of multigene families as well as tion). This reconciliation of gene relationships
single-copy gene families across the fungi can be made through gene tree–species tree
(Marthey et al. 2008), and the OrthoMCL-DB reconciliation using software such as NOTUNG
database, which houses a collection of eukary- (Stolzer et al. 2012) or TreeBEST (Vilella et al.
otic and bacterial orthologous gene clusters, all 2009).
In some cases, genes’ history are not sim- example, one group of species inherits allele A
ply the same as the species history. This can be while others inherit allele B. This will make the
the result of events such as horizontal gene divergence between species seem older than it
transfer (HGT), where genes from one species actually is. This incomplete sorting can occur
are exchanged directly with another. There are with multiple alleles of a gene or sometimes
many documented examples of fungi accepting multiple duplicate copies of a gene, so that
bacterial genes (Hall and Dietrich 2007; Hall two lineages may each only have one copy of a
et al. 2005; Marcet-Houben and Gabaldon gene family, but they are different by paralogy
2010) as well as oomycete and fungal exchanges as ancient duplications that predate the species
(Richards et al. 2011). In many cases, the acqui- divergence. This can be a complicating factor
sition of these genes can be interpreted as a gain when these duplicates have acquired different
of function for a new biological process for the functions and display different rates of evolu-
species in its ecological niche. One example is tion, or when there are multiple species in a
the URA1 gene in Saccharomyces cerevisiae, lineage that could have fixed alternative ver-
which is required for uracil biosynthesis and sions of a duplicate gene, causing them to
is a HGT of a dihydroorotate dehydrogenase group by which allele was fixed, not by the
(DHOD) enzyme from Lactobacillus bacteria speciation.
(Gojkovic et al. 2004; Hall et al. 2005). The
confirmation of the gene’s history was revealed
through the gene tree, which had a pattern of C. Sequencing 1000 Fungal Genomes
fungal gene copies embedded within a large
bacterial phylogeny, indicating the fungal Questions on how to resolve the fungal tree of
copy was derived from a bacterial donor. The life, on a better understanding of the origins of
eukaryotic version of the DHOD gene is found novelty in enzyme families or morphology, and
in most fungi, but not in S. cerevisiae and Sac- on the evolution of genome structure can be
charomyces sensu stricto. Within the Saccharo- addressed by comparing genome sequences
myces sensu stricto clade, species contain the from fungi across the kingdom. Several
bacterial version of this gene, which can func- genome-sequencing projects have focused on
tion in anaerobic conditions. Acquisition of the a comprehensive sampling and genome seq-
DHOD gene by HGT was an important aspect of uencing of the Fungi, including projects housed
the evolution of a lifestyle that allows these at the U.S. Department of Energy’s Joint
yeasts to ferment alcohol. The comparison of Genome Institute (Martin et al. 2011) or the
gene trees of the copies of DHOD indicated that Broad Institute’s Fungal Genome Initiative
S. cerevisiae and relatives’ copies were embed- (Cuomo and Birren 2010). One approach, the
ded within the bacterial phylogeny, while the 1000 Fungal Genomes project (http://1000.fun-
eukaryotic version is found in all species out- galgenomes.org) or 1KFG, seeks to produce
side the clade. Examination of genomes of Klu- two genomes from each of the more than 500
veryomyces species also revealed copies of both families of fungi. A deep sampling of genomes
eukaryotic and eubacterial origins, confirming from across the fungal phylogeny will provide
that some present-day species have maintained an important resource for evolutionary studies
both copies. on how gene content changes as morphology or
Another scenario that can cause incongru- lifestyle changes and for phylogenetic resolu-
ence between a gene tree and the expected tion of the tree of life.
species tree (or the consensus among many Within kingdom Fungi there are diverse
but not all genes) is incomplete lineage sort- families of enzymes, secondary metabolite pro-
ing. This can occur when multiple alleles of a duction, and novel proteins to be discovered by
gene exist in a population and persist in the expanding the inventory of fungal genes. Other
formation of new species, but eventually the large-scale projects have used genome sequenc-
polymorphism sorts out as different fixed ing to explore phylogenomic diversity. The
alleles in the subsequent lineages where, for Genomic Encyclopedia of Bacteria and Archaea
284 J.E. Stajich
sequenced and analyzed 1,000 bacterial and that the shape and ultrastructure of organelles
archaeal genomes (Wu et al. 2009). The in zoosporic fungi are consistent with molecu-
researchers’ analysis revealed that the rate of lar phylogenetic trees, indicating that some
discovery of new protein families continued to morphological characters are robust enough
increase as new genomes were considered, and to use as phylogenetic characters (Barr 1980;
the curve representing family discovery did not Letcher et al. 2008). However, DNA sequences
indicate a saturation of the number of new and the proteins they encode can be some of the
families, even when all 1,000 were included. As most useful data for phylogenetics because they
hundreds of fungal genomes are sequenced and tend to change in a predictable fashion.
gene families compared, it will be important With the availability of genome sequencing,
to understand whether the same diversity is an inventory of genes in a species and compar-
exposed in Fungi. ison of gene content among species can com-
Phylogenetic studies will be able to take prise an alternative approach to studying
advantage of the complete genome sequences changes between species. The genes identified
produced through 1KFG and other efforts. The in a genome sequence can predict some aspects
availability of this deep sampling of genomes of lifestyle, such as the specializations of rusts
from Fungi should assist in providing better (Duplessis et al. 2011) or the potential to pro-
support for the backbone of the fungal tree. cess only some carbohydrates, and thus explain
For future projects that will apply low-coverage a specialized ecology (Eastwood et al. 2011) or
genome sequencing for phylogenetic studies, predict whether a species is sexual (Galagan
having a reference genome from a nearby et al. 2005), which can subsequently be tested
clade will enable better assembly of the data. (O’Gorman et al. 2009).
Furthermore, the extensive genome sampling Studies of how genomes have evolved can
will assist in building data sets of genes and provide more insight into an organism’s past
their conservation profile through the fungi, through what can be considered molecular
allowing for identification of the core fungal archaeology. These characters can include the
genes found in all species, and those specific evolution of how genes are encoded in the
to the phyla or classes. The origins and func- genome, chromosome structure and number,
tions of these gene groupings may provide and the copy number of gene family members.
additional insight into the biology and mechan- The specific gene content of clades of organ-
isms of morphological differences among isms can also provide some timing for when
groups of fungi. specific processes may have originated, back
to the most recent common ancestor of the
group. Dating approximately when genes or
groups of genes were gained or lost can help
II. Genomics Enabling Comparative establish when shifts in function and ecology
Biology may have occurred in fungal history.
Comparative biology in Fungi comprises the

study of the evolution of characters, particu- A. Evolution of Gene Structures in the Genome
larly with a focus on morphology, lifestyles,
and sexual reproduction (Lutzoni et al. 2001; The exon and intron structure of genes can
Schoch et al. 2009). Many fungal characteris- inform the relationships between organisms
tics, such as spore color (Hopple and Vilgalys because gene structures tend to be more similar
1999) and morphology (Hibbett 2004; Hibbett among orthologous genes (Irimia and Roy
2007; Hibbett and Binder 2002), have been 2008). A striking pattern in gene structure
shown to be evolutionarily labile and change was observed among the evolutionary clades
rapidly, making them homoplastic characters of Fungi. The Saccharomycotina yeasts (Can-
and uninformative for phylogenetic reconstruc- dida albicans, S. cerevisiae) and the Taphrino-
tion. Other phylogenetic analyses have shown mycotina fission yeast Schizosaccharomyces
pombe had on average one or no introns per the picoeukaryote Micromonas in thousands of
gene, while the filamentous ascomycetes and nearly identical copies within gene sequences.
basidiomycetes displayed a much broader The further discovery of intronerlike elements
range of intron distribution. This finding sug- in the Dothideomycete Cladosporium fulvum
gested that an ongoing process of intron change and five other fungi (van der Burgt et al. 2012)
had occurred during Fungal evolution and indicates that this mechanism might also con-
could have been driven either by widespread tribute to intron creation in fungi.
gains or series of losses in these yeasts. Several
studies examined the positions of the introns in
orthologous genes and found evidence that B. Synteny and Gene Order Evolution
introns were common in the ancestor of the
Fungi but that subsequent loss of introns had Beyond the conservation of gene structure
occurred, most extremely in the branches lead- among species, understanding how the ar-
ing to the Saccharomycotina and the branch rangement of genes along a chromosome
leading to S. pombe (Carmel et al. 2007, 2009; evolves can be important to picking out the
Nielsen et al. 2004; Stajich et al. 2007). This orthologous genes and understanding the
ongoing process of loss and gain, but predomi- mechanisms of chromosome evolution. A
nantly of loss, can be linked to ecology and significant event in the history of the Sacchar-
lifestyle changes of these fungi and the effective omycotina yeasts was the ancestral whole-
population sizes of the species (Lynch 2006; genome duplication (WGD). These fungi also
Lynch and Conery 2003). The transition from have high rates of duplication, providing a rich
a filamentous to yeast form may have altered environment for studies of these contributing
the effective population size, allowing for selec- factors to genome evolution (Dujon 2010;
tion to be more effective at purging the mildly Koszul et al. 2006). A careful curation of the
deleterious introns. Further studies of mechan- gene order in a collection of Saccharomyces
isms of intron changes suggest loss could occur species (Byrne and Wolfe 2005) provided a
through recombination or integration of map to determine the evolutionary mechan-
spliced mRNA into the genome (Fink 1987; isms by which the large number of duplicated
Stajich and Dietrich 2006). Intron gain has genes in S. cerevisiae were created. The fate of
also been posited to occur through several these duplicate genes can be examined in mul-
mechanisms, reviewed in Roy and Irimia tiple “experiments” by examining the different
(2009, 2012), that include recruitment of the patterns of gene loss among the duplicates
existing exonic sequence, tandem duplication, (Gordon et al. 2009) that ultimately lead to the
invasion during double-stranded break repair, formation of new species (Scannell et al. 2006,
organellar DNA insertion, and transposition of 2007). Large-scale duplication also occurs in
an intron by locus-specific targeting. An exam- other lineages of the Fungi, including the early
ple of duplication leading to intron gain was diverging Mucoromycotina, where at least one
observed in the Cryptococcus species (Sharpton WGD occurred (Ma et al. 2009).
et al. 2008), while locus-specific targeting was Additional studies of synteny have been
found in the accumulation of introns in Mag- useful in discovering clusters of genes that
naporthe oryzae (Nielsen et al. 2004). The intro- have moved. This includes the identification
nization of coding sequence and insertions of secondary metabolite clusters (Palmer and
were also observed to be major drivers in intron Keller 2010; Sanchez et al. 2012), for example,
changes in Fusarium and Cryptococcus species the aflatoxinlike gene cluster (Bradshaw et al.
(Croll and McDonald 2012). Recent discoveries 2013), the cyclosporin gene cluster (Bushley
of specific elements have shed additional light et al. 2013), fumonisin (Khaldi and Wolfe
on intron gains. A proliferating element called 2011), or the sterigmatocystin gene cluster
an introner, which resembles an intron with (Slot and Rokas 2011). Gene clusters of related
canonical splice sites and efficient removal function have also been demonstrated to be
from transcript, was found in the genome of transferred, and breaks in synteny make it
286 J.E. Stajich
easy to pick out the inserted regions. One gene families and how these sizes change
example is the transfer of nitrate assimilation among species can provide correlative evi-
genes that were transferred as a unit from a dence for adaptations. One approach is to
basidiomycete into the ancestor of the ascomy- count the number of copies of a gene family in
cete Trichoderma reesei (Slot and Hibbett two species and see whether they are signifi-
2007). cantly different. However, a more phylogenetic
The DAL (Wong and Wolfe 2005) and GAL approach would take into account the evolu-
clusters (Slot and Rokas 2010) present other tionary distance and the copy numbers across a
mechanisms of genome evolution where clus- clade of species (Hahn et al. 2005). One imple-
ters of genes migrated to a common location mentation, CAFE, estimates the rates of gene
from scattered locations throughout the Sac- gain and loss among families for each branch of
charomyces genomes. Syntenic comparisons of a phylogenetic tree (De Bie et al. 2006; Han et al.
these genomes permitted the conclusion that 2013). This allows for tests of shifts in rates of
the clusters were created, not transferred, duplication or loss that can be examined in the
because of the lack of shared gene order context of the evolution of other traits such as a
among the gene homologs in the related spe- shift from host-associated to saprotrophic life-
cies. styles or domestication. By identifying gene
Synteny is also often used to identify the families with unusual patterns of gene duplica-
mating type (MAT) region of fungi due to the tion or loss, inferences can be made about the
shared gene order that can be found through- relative importance and suggest the changes
out the Dikarya. For instance, the mating type that may be important to a lifestyle. In Fungi,
loci of Cryptococcus neoformans was first it appears that many of the changes involve
inferred through the identification of a lack gene families related to metabolism.
of shared gene order around the locus (Lenge- Many studies have taken this approach,
ler et al. 2002) in a comparison of two strains of identifying changes in carbohydrate-active
opposite mating types. Similarly for previously enzymes (CAZymes). CAZymes include carbo-
putatively asexual Aspergillus fumigatus, a pat- hydrate synthesis and cleaving enzymes, and
tern of gene order was used to find MAT genes their curation in the CAZy database has
between species (Galagan et al. 2005) and later provided a resource for studying the evolution
enabled confirmation of the capability for a of these families across Fungi (Cantarel et al.
sexual cycle (O’Gorman et al. 2009). 2009; Levasseur et al. 2013). Changes in family
An interesting pattern of gene order was composition have been interpreted in switches
discovered among the Dothideomycetes (Asco- in ecological association or in the preferred
mycota). Strict synteny is not maintained, and nutritional substrate. For example, a study
so genes on homologous chromosomes were among three species of Aspergillus was able to
not found in the same order when species relate some of the differences in enzyme poten-
were compared. However, the gene content of tial to their biological niche (Coutinho et al.
chromosomes, that is, the complement of genes 2009). Perhaps one of the most identifiable
found, was preserved, but the order was scram- associations of fungi is with wood-rotting basi-
bled. This property of gene content conserva- diomycetes. Brown rotters are unable to
tion but not gene order was dubbed degrade lignin and leave brown remains of a
mesosynteny (Hane et al. 2011) and presents a decomposed wood, while white rotters are able
curious but currently unexplained evolutionary to break down lignin, leaving white remains.
pattern that has only been observed in the Comparing the genome sequences of a broad
Dothideomycetes. sample of 31 genomes, including 7 brown rot-
ters and 9 white rotters, made it possible to
reconstruct the history and timing of the
C. Evolution of Gene Families evolution of rotting mechanisms among basi-
diomycetes (Floudas et al. 2012). This work
Phylogenomic approaches can also shed light revealed that expansion of lignin-degrading
on gene family evolution. Examining the size of enzymes occurred early in the evolution of the
Agaricomycotina and correlated with the sig- nutrition, while hemibiotrophs avoid host
nificant decrease in the deposition of organic detection in the early stages of infection and
carbon (coal) toward the end of the Permian. obtain nutrients from a living host. Com-
Furthermore, gene losses were found to accom- parison of the genomes of species with these
pany the transition to brown rot status, and differing lifestyles reveals in part a pattern of
brown rot evolved several times independently carbohydrate enzymes consistent with these
in these fungi. Work that investigated the dry predictions (de Wit et al. 2012). An analysis to
rot fungus Serpula lacrymans along with the compare genomes among the Dothediomycetes
brown rot fungus Postia placenta also showed (Ohm et al. 2012) also showed expansions and
that smaller gene families of oxidoreductases in contractions of CAZy families that tracked
these two separate lineages were likely caused with lifestyles. A comparison of two Colletotri-
by expansions in the white rot fungi, but that chum species found that C. higginsianum had
CAZY families were reduced in size in the an increased repertoire of enzymes for pectin
brown rotters (Eastwood et al. 2011). degradation, peptidases, and effectors, along
Approaches to studying pathogenic fungi with secondary metabolites, in comparison to
have also used gene family comparisons to the maize-infecting C. graminicola (O’Connell
understand what genomic changes supported et al. 2012). Overall, this work and others
the adaptations that led to virulence. A com- suggest that changes in copy number of some
parison of the amphibian pathogen Batracho- metabolic genes, especially those involved in
chytrium dendrobatidis with a relative that is carbohydrate metabolism, are correlated with
nonpathogenic identified pathogen-specific lifestyle changes and pathogenesis mode.
expansions of metallo-, serine, and aspartyl However, large-scale changes in gene
proteases (Joneson et al. 2011). The CAZy fam- families are not strictly related to metabolism.
ily CBM18 was also found to be greatly For example, the expansions of kinase and RAS
expanded only in the B. dendrobatidis patho- small GTPase families in the basidiomycete
gen, predicting some role for changes in how Laccaria bicolor suggest a major change in sig-
the cells sense or interact with chitin (Abram- naling pathways that may be related to changes
yan and Stajich 2012). Gene family contraction from saprotroph to symbiont (Rajashekar et al.
can occur as well. In the mammalian patho- 2009). Families of genes including P450, hydro-
genic fungi Coccidioides immitis and C. posa- phobins, laccases, and kinases are dramatically
dasii, several gene families related to the increased in the basidiomycetes (Hoegger et al.
degradation of plant material such as cellulases 2004; Ide et al. 2012; Plett et al. 2012; Stajich
and pectinases are absent. These families are et al. 2010). As more genomes are sequenced
missing in all the examined genomes in the from fungi, it will be important to see whether
Onygenales clade but are present in the the patterns of family changes continue to be
more distant phyto-saprotrophic Aspergilli good predictors of ecological niche and host-
and Eurotiales (Sharpton et al. 2009), while associated lifestyles.
expansions of protease families likely used for
keratin degradation were identified that may be
linked to the keratinophilic nature of these D. From Molecules to Morphology
fungi. Similar findings of an expanded ratio of
proteases to CAZy families were observed in the Much of the comparative biology of Fungi has
genomes of the Onygenales pathogens Paracoc- focused on how shapes change, such as the
cidioides lutzii and P. brasiliensis (Desjardins ultrastructure of organelles and fruiting body
et al. 2011). form (Barr 1980; Hibbett 2007; Hibbett and
Differences between hemibiotrophic and Binder 2002; Letcher et al. 2008; Velez et al.
necrotrophic plant pathogens could be related 2011). Changes in phenotype are caused by
to their repertoire of CAZy enzymes. The changes in the DNA, but we do not yet have
necrotrophic fungi do not need to avoid induc- the ability to predict which changes will im-
ing cell death since they rely on dead tissue for pact morphology. However, inferences and
288 J.E. Stajich
correlations in gene interactions can be drawn septum and septal pore cap, sterol composition,
as to the presence or absence of genes and the apical organization, and meiosporangium
observed morphological changes in a group of where meiotic spore products or apparatus for
organisms (Date and Marcotte 2003). For their delivery are formed. This collection of
example, a life stage with flagellated zoospores characters linked to the inventory of genes
is found in the Chytridiomycota and Blastocla- from genome sequences will provide even
diomyocta but is missing in the Dikarya. This more insight into the evolution of these char-
difference predicts the presence of flagellar acters. Research on the evolution of these and
genes only in the zoosporic fungi. Sequence other aspects of cell biology changes in Fungi
similarity searches confirm this pattern, and will be a rich area of future research.
flagellar genes are restricted to the early
diverging lineages but missing in species lack-
ing a flagellated life stage based on the analysis III. Conclusions
of several available zygomycete genomes from
the Mucoromycotina and Entomophthoromy- New technologies have improved the access to
cotina. genomic data for all biological systems. Molec-
A second example of changes in structure is ular phylogenetics has greatly benefited from
the evolution of the septal pore plugging pro- the reduced barrier to access to sequence data,
teins, which are necessary to plug septal pores and mycology has reaped these benefits to
that link adjacent cells in hyphae upon hyphal improve the ability to describe species indepen-
wounding. Without these proteins, hyphae dent of morphology or cultivability. The future
would leak cytoplasm after wounding, while of mycology with these genomics-enabled tools
intact septal plugs can protect the remainder may look different. One possibility is that
of the colony. Two different types of protein genome sequencing will become routine enough
perform these tasks in the filamentous basidio- that sample identification will proceed with a
mycetes and ascomycetes. In the Pezizomyco- whole-genome-sequencing approach and bioin-
tina (Ascomycota), the Woronin body is the formatics analysis of these data. A necessary
septal plug (Buller 1933; Voronin 1865) and is development will be streamlined tools for anal-
encoded by the hex gene (Jedd 2011; Jedd and ysis so that sequence data can be processed in a
Chua 2000). Isolation and sequencing of pro- standardized fashion. Studies in genome evolu-
teins responsible for the septal pore cap in tion and phylogenetics will certainly benefit
Agaricomycotina (Basidiomycota), an analo- from these approaches. However, cell biology
gous but nonhomologous structure to the Wor- and pathogenesis will be able to regularly use
onin body, identified a protein the authors later transcriptome sequencing to examine gene
named SPC18 (van Driel et al. 2008). However, expression in a routine manner, and sequencing
the sequences for these proteins are not similar to diagnose plant or animal infections can also
to each other, indicating that these identified be expected to be a primary means of classifying
components of septal plugs have independent unknown infections. The application of geno-
origins. mics methods will not remove the need to study
Further studies are needed to link observed and understand fungi from the microscopic to
changes in ultrastructure and morphology in the ecosystem level, but it should reduce the
the Fungi to the underlying molecular changes. time it takes to collect sequence data and
One effort to collect the descriptions of the improve the power and resolution of studies of
structure of fungal cellular components is fungal evolution.
being undertaken by the AFTOL project in the
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Biosystematic Index
A Arachnomycetales, 123, 128, 129

Acarosporaceae, 100 Archaeorhizomyces, 14
Aciculoconidium, 15 Arthonia, 145, 147, 152, 164–166
Acremonium, 189 A. endlicheri, 165
Acrospermales, 160 A. intexta, 147
Adelococcaceae, 131 A. muscigena, 152
Agaricomycetes, 181 Arthoniaceae, 152, 164–166
Agaricomycotina, 235, 287, 288 Arthoniales, 143, 148, 149, 152, 154, 163–166
Agaricus, 181 Arthophacopsis, 166
A. campestris, 181 Arthopyrenia, 156
Aglaophyton major, 268 Arthopyreniaceae, 152, 155
Agyriaceae, 108 Arthothelium, 164
Agyriales, 108 Arthrobotrys, 37, 40
Agyrium, 107, 109 Arthrodermataceae, 128, 133
Aigialaceae, 151, 156, 166 Ascobolaceae, 42, 43, 46, 48
Ajellomycetaceae, 128, 129, 133 Ascobolus, 44, 46
Albugo, 213 A. carbonarius, 46
Aleuriaceae, 48 Ascobotryozyma, 16, 19
Aliquandostipitaceae, 151, 158 Ascocorticiaceae, 72
Aliquandostipite, 157–158 Ascocoryne, 77
A. khaoyaiensis, 157 Ascodesmidiaceae, 46
Allomyces, 266 Ascodesmis, 42, 46, 48
Alternaria, 150, 156 A. nigricans, 244
Alyxoria, 165 Ascoidea, 14
Amanita, 216 Ascomycetes, 233, 235, 242, 249, 288
Ambrosiozyma, 8–9, 15 Ascomycota, 3, 232, 235, 237, 240, 247, 286
Amniculicolaceae, 151 Ascosphaera, 128, 129, 133
Amphisphaeriaceae, 67 Ascosphaeraceae, 128
Anamylopsoraceae, 103 Ascosphaerales, 121, 129
Angulospora, 40 Aspergillaceae, 124–126
Anisogramma anomala, 81 Aspergillus, 123–126, 132, 134–136, 264, 287
Anisomeridium, 153 A. alliaceus, 126
Anteaglonium, 157 A. collembolorum, 262
Antennulariaceae, 159 A. fischeri, 126
Apiosporina, 162 A. flavus, 125, 126, 134, 135
Arachnomyces, 129 A. fumigatus, 122, 126, 132, 134, 286
Arachnomycetaceae, 128, 129 A. lentulus, 126

296 Biosystematic Index
A. nidulans, 126, 134 Burenia, 14

A. niger, 125, 134, 135 Byssochlamys, 126, 127
A. ochraceus, 125, 134
A. oryzae, 126, 135, 136
C
A. parasiticus, 126, 134, 135
Cainiaceae, 67
A. sojae, 135, 136
Caliciaceae, 103
A. sydowii, 134
Caliciales, 131
A. tamarii, 135
Callixylon newberryi, 268
A. terreus, 125, 126, 134
Caloscyphaceae, 46
Aspergillus section Aspergillus, 122
Caloscypha fulgens, 42, 46
Aspergillus section Fumigati, 125
Calosphaeriales, 65
Asterinaceae, 147, 148, 161, 162, 166
Candelariaceae, 103
Asterinales, 161
Candida, 16
Asteroxylon mackiei, 267, 269
C. albicans, 6, 284
Astromyelon, 272
C. cylindracea, 4
Aulographina pinorum, 161
C. jeffriesii, 4–5
Aureobasidium pullulans, 160
C. oleophila, 6
Auriscalpium vulgare, 244
C. shehatae, 4
Capnodiaceae, 158, 159, 166
B Capnodiales, 152, 158, 159, 161, 167
Babjeviella, 16 Capronia pilosella, 121
Baeomycetaceae, 102 Cashhickia acuminata, 272
Balansia, 60 Catillariaceae, 108
Barnettozyma, 15 Catinella olivacea, 149
Barriopsis, 160 Celotheliaceae, 131
Basidiomycetes, 233, 249 Celotheliales, 131
Basidiomycota, 3, 231, 232, 235, 237, 247, 288 Celothelium, 131
Batrachochytrium dendrobatidis, 287 C. longisporum, 123
Baudoinia compniacensis, 160 Cenococcum geophilum, 149, 150, 157
Beauveria, 64 Cephaliophora, 44
Bionectriaceae, 63 Cephaloascus, 14
Bipolaris, 149, 188 Ceratocystis, 64
Blastobotrys, 16 Chaetomium, 60
Blastocladiomycota, 233, 240, 251, 288 Chaetosartorya, 125
Blastomyces, 129 Chaetosphaeriales, 66
B. dermatitidis, 129, 133 Chaetothyriaceae, 130, 159
Boletus, 216 Chaetothyriales, 121, 123, 130–132, 153, 159
Boliniales, 66 Chaetothyriomycetidae, 123, 130–132, 134, 136, 158
Botryosphaeria, 156, 160 Chaetothyrium, 123
Botryosphaeriaceae, 151, 155, 160 Chlorociboria, 75
Botryosphaeriales, 156, 160, 161 Chorioactidaceae, 42, 44, 46–47
Botryozyma, 16 Chrysosporium, 125, 128
Botrytis, 79 Chrysothrix, 165
B. cinerea, 79 Chrysotricacaceae, 164
Brachyphoris, 40 Chrysotrichaceae, 152, 164, 165
Brefeldiellaceae, 161 Chytridiales, 250
Brettanomyces, 15 Chytridiomycetes, 218, 250
Briancoppinsia, 149, 164 Chytridiomycota, 233, 240, 249, 251, 288
Brigantiaeaceae, 109 Cirrosporium, 137
Bulgariaceae, 72 C. novae-zelandiae, 123, 132
Biosystematic Index 297
Citeromyces, 16 Dactylellina, 40
Cladochytriales, 250 Dactylomyces, 126
Cladoniaceae, 105 Dactylospora, 131, 132
Cladosporium, 158 Daohugouthallus ciliiferus, 271
C. fulvum, 158 Davidiella, 158
Clathrospora heterospora var. simmonsii, 145 Davidiellaceae, 159
Clavicipitaceae, 63 Dawsophila, 152, 164
Clavispora, 15 Debaryomyces, 14
Clypeosphaeriaceae, 67 D. hansenii, 4
Coccidiascus, 16 Dekkera, 8, 15
Coccidioides, 122 Delitschiaceae, 156
C. immitis, 129, 133, 287 Dendrographa, 152, 165
C. posadasii, 133, 287 Dermateaceae, 72
Coccocarpiaceae, 107 Diaporthales, 65
Coccodiniaceae, 159 Diaporthe, 65
Coccotremataceae, 108 Diatrypaceae, 67
Cochliobolus, 149, 156, 188 Dibotryon, 162
Coelomycetes, 149 Dichlaenoideae, 127
Coenogoniaceae, 105 Dichotomomyces, 125
Collemataceae, 107 Dicranidion, 40
Colletotrichum, 60, 287 Diddensiella, 16
C. graminicola, 287 Didymella, 151, 167
C. higginsianum, 287 Didymellaceae, 156
Coniochaetales, 66 Dimidiographa, 165
Conocybe, 216 Diplodia, 160
Conotrema, 106 Dipodascopsis, 15
Coprinopsis, 216 Dipodascus, 14
Coprinus, 216 Discinaceae, 42, 47, 50
Cordyceps, 60 Disciotus, 47
Cordycipitaceae, 63 Discula destructiva, 65
Coronophorales, 63 Dolabra, 131
Cortinarius, 216 Dothidea, 144, 145, 150
Corynasca, 155 Dothideaceae, 150, 160
Coryneliales, 62, 123, 129, 130, 134 Dothideales, 144, 145, 153, 154, 158, 160, 163
Corynelia uberata, 122 Dothideomyceta, 143–145, 152–154, 157,
Corynespora cassiicola, 155 163, 167
Cryphonectria parasitica, 59 Dothideomycetes, 126, 129, 131, 286
Cryptococcus, 285 Dothideomycetidae, 144, 145, 151, 154, 158, 160
Cryptothecia, 147, 164 Dothidotthia, 156
Cucurbitariaceae, 156 Dothidotthiaceae, 156
Curvularia, 156 Dothiorella, 160
Cyberlindnera, 15 Drechslerella, 40
Cyniclomyces, 15 Dubiocarpon, 267
Cyphellophoraceae, 130 Dwayaangam, 40
Cystocoleus, 152, 158
Cyttaria, 70, 75
E
Cyttariales, 69–70
Elaphomyces, 122, 127, 128, 134
Elaphomycetaceae, 123, 127–128
D Eleutherascus, 42, 46
Dactylaria, 40 Elixiaceae, 110
Dactylella, 40 Elsinoaceae, 151, 160
Elsinoe Glomerellales, 63
E. australis, 160 Glomeromycota, 235, 240, 247
E. fawcettii, 160 Gloniopsis, 157
Emericella, 124–126 Glonium, 157
E. nidulans, 125 Gonapodya, 266
Endogone, 266 Graphidaceae, 97, 105
Endomyces, 14 Guignardia, 161
Engerulaceae, 161 Gyalectaceae, 105
Enterographa, 152, 165 Gymnoascaceae, 128, 129
Epibryaceae, 130
Epibryon, 134
Epichloë, 60 H
Eremascaceae, 128 Halosphaeriaceae, 64
Eremascus, 128, 129 Hanseniaspora, 15
Eremothecium, 5, 15 Helicogonium, 14
Eremothecium gossypii, 4 Helicoma, 162
Erysiphales, 62, 69–70, 235 Helicomyces, 162
Erythrodecton, 165 Helicoon, 40
Euallomyces, 266 Helicosporium, 162
Eucladiella, 266 Helotiaceae, 72
Eupenicillium, 124–126 Helotiales, 70
Eurotiales, 121, 123, 127–129, 131, 132, 135, 287 Helvellaceae, 41–42, 47, 48, 50
Eurotiomycetes, 122, 131, 134, 137, 158, 159, 167 Hemiphacidiaceae, 72
Eurotiomycetidae, 123, 130, 131, 136 Herpotrichiellaceae, 130
Eurotium, 122, 124–126, 132 Heydenia, 43
Histoplasma, 129
H. capsulatum, 122, 129, 133
F H. capsulatum var. duboisii, 133
Farlowiella, 147, 157 H. duboisii, 133
Fennellia, 126 Humariaceae, 48
Fulvophyton, 165 Hyalina, 40
Fusarium, 59 Hyalorbilia, 40, 41
Fuscideaceae, 110 Hyaloscyphaceae, 72
Fusicladium, 162 Hymenoscyphus, 76
Hyphomycetes, 149, 161
G Hyphopichia, 9, 14
Gaeumannomyces graminis, 65 Hypocrea, 63, 188
Galactomyces, 14 Hypocreaceae, 63
G. candidus, 5 Hypocreales, 63–64
Gamsylella, 40 Hypocreomycetidae, 62
Ganodermataceae, 269 Hypomyces, 60
Ganodermites libycus, 263, 269 Hyponectriaceae, 68
Geneaceae, 48 Hysteriaceae, 147, 155, 157
Geoglossaceae, 72 Hysteriales, 145, 147, 155, 157, 160
Geoglossomycetes, 77–78 Hysterium, 157
Geoglossum, 77 Hysterographium, 157
Geosmithia, 127
Geotrichum, 14
Glaziella aurantiaca, 47 I
Glaziellaceae, 47 Icmadophilaceae, 108
Glomerella, 63, 69 Ingaderia, 152
J Leotioaceae, 72
Jahnula, 157 Leotiomycetes, 57, 128
Jahnulales, 151, 155, 157, 158 Leptopeltidaceae, 161
Jimwhitea circumtecta, 263, 266 Leptosphaeria, 151, 156
Julella avicenniae, 155 L. maculans, 151
Leptosphaeriaceae, 156
Leptosphaerulina, 154
K Letrouitiaceae, 109
Kalapuya, 47 Leucoangium, 47
Kallistoskypha, 46 Lichenoconium, 153
Karstenellaceae, 47 Lindgomycetaceae, 151, 156
Karstenella vernalis, 47 Lipomyces, 4, 15
Kazachstania, 15 Lobariaceae, 107
Kellermania, 188 Lobulomycetales, 250
Kickxellomycotina, 232, 241 Loculoascomycetes, 144, 149, 150, 154, 158
Kloeckera, 15 Lodderomyces, 14
Kluyveromyces, 15, 283 Lophiaceae, 157
K. marxianus, 4 Lophiostomataceae, 156
Kodamaea, 15 Lophium mytilinum, 155
Koerberiaceae, 107 Loramycetaceae, 72
Komagataella, 16 Loxospora, 108
Koralionastetales, 62 Lulworthiales, 62
Kregervanrija, 15 Lyonophyton rhyniensis, 268
Kryphiomyces catenulatus, 266
Kuraishia, 16
Kurtzmaniella, 14 M
Macrorhabdus, 16
Magnaporthales, 65
L Magnaporthe, 187
Laboulbeniales, 62, 78 M. oryzae, 59, 285
Laboulbeniomycetes, 78 M. poae, 65
Laccaria bicolor, 287 Magnusiomyces, 14
Lachancea, 15 Malbranchea, 128
L. kluyveri, 9 Manglicolaceae, 158
Lachnocladiaceae, 233 Manglicola guatemalensis, 158
Lactarius, 216 Massalongiaceae, 107
Lalaria, 14 Massariaceae, 156, 157
Lamprospora, 42 Massarinaceae, 156
Lecanactis latebrarum, 165 Massarineae, 156
Lecanographa, 165 Mazosia, 152
Lecanographaceae, 164, 165 Medeolaria, 75
Lecanoraceae, 104 Medeolariales, 74
Lecanorales, 90, 91 Megalosporaceae, 109
Lecanoromycetes, 131, 143, 144, 152, 157, 166 Megasporaceae, 108
Lecideaceae, 105 Melanconis, 65
Lecophagus, 40 Melanommatales, 155, 157
Leiothecium, 125, 127 Melanospora, 63
Lentitheciaceae, 151, 156 Melanosporales, 64
Leotia, 75 Meliolales, 62, 68–69
Leotiaceae, 72 Metacoleroa, 162
Leotiales, 69 Metacordyceps, 64
Metarhizium, 64 Naumovozyma, 15
Metschnikowia, 15 Nectria, 63
M. fructicola, 6 Nectriaceae, 63
Meyerozyma, 14 Nematasketum, 271
M. guilliermondii, 4 Neocallimastigomycota, 240–241, 251
Microascales, 64–65 Neolecta, 14, 20
Micropeltidaceae, 161 Neomicrothyrium, 161
Micropeltis, 161 Neopetromyces, 126
Microsporidia, 192, 241 Neosartorya, 124–126
Microsporum, 128, 133 N. fumigata, 125
Microthyriaceae, 145, 147, 161, 162, 166 Nephromataceae, 107
Microthyriales, 161 Neurospora crassa, 60
Microthyrium microscopicum, 145, 147 Niessliaceae, 63
Millerozyma, 14 Nizziopsaceae, 129
Miltideaceae, 108 Nothia aphylla, 263, 266
Monacrosporium, 40 Nothojafnea thaxteri, 49
Monascaceae, 123, 127–128
Monascella, 42 O
Monascus, 125, 127 Ochrolechiaceae, 108
M. purpureus, 135, 136 Octospora, 42
Monoblastiaceae, 152, 153, 162, 163 Odontotremataceae, 105
Montagnulaceae, 156 Ogataea, 4, 15
Morchellaceae, 41–42, 47–48, 50 O. polymorpha, 4
Morosphaeriaceae, 151, 156, 166 Olpidium, 251, 265
Mucoromycotina, 241 Onygena, 122, 128
Muyocopron, 161 Onygenaceae, 128
Mycena, 216 Onygenales, 121, 123, 128–129, 131, 132, 287
Mycenella, 216 Oomycota, 216, 217, 219, 234
Mycocaliciales, 123, 131 Opegrapha, 147, 152, 165
Mycocaliciomycetidae, 123, 131, 134, 136 Opegraphaceae, 147, 152, 154, 164, 165
Mycocalicium, 123 Ophiocordycipitaceae, 63
Mycocarpon, 267 Ophioparmaceae, 110
Mycosphaerella, 159 Ophiostomatales, 65–66
Mycosphaerellaceae, 151, 159, 161 Ophiostoma ulmi, 59
Mycrothyriaceae, 161 Orbicula parietina, 43
Mycrothyrium microscopicum, 161 Orbilia, 37, 39, 40
Myeloconidaceae, 105 O. xanthostigma, 39
Myriangiaceae, 160 Orbiliaceae, 35–36, 40
Myriangiales, 144, 145, 158, 160 Orbiliales, 35–36
Mytilinidiaceae, 157 Orbiliomycetes, 35–38, 40, 50
Mytilinidiales, 145, 155, 157, 160 Ostreichnon, 157
Myxotrichaceae, 70, 128 Ostropales, 90
Myxozyma, 15 Otideaceae, 48
N P
Nadsonia, 16 Pachysolen, 16
Nakaseomyces, 15 P. tannophilus, 4
Nakazawaea, 16 Paecilomyces, 125–127
Nannizziopsiaceae, 128 Palaeancistrus martinii, 268
Palaeoblastocladia milleri, 266
Palaeonitella cranii, 265 Phoma, 153, 156, 165

Palaeosclerotium pusillum, 271 Phomopsis, 65
Paleopyrenomycites devonicus, 269 Phycopeltis, 152
Pannariaceae, 107 Phyllachorales, 62
Paracoccidioides, 133 Phyllosticta, 161
P. brasiliensis, 129, 133, 287 Phymatotrichopsis, 42, 44
P. lutzii, 287 P. ominivora, 42, 44
Paradoxomyces, 166 Physciaceae, 103
Paralecanographa, 165 Phytophthora, 216
Paramicrothyrium, 161 P. infestans, 206
Paraschismatomma, 165 Pichia, 15
Parmeliaceae, 104 P. kluyveri, 4
Parmulariaceae, 161, 166 Piedraiaceae, 158
Passalora fulva, 158 Piedraia hortae, 158
Patellariaceae, 157 Pinus, 162
Patellariales, 157, 160 Placopyrenium canellum, 123
Peltigeraceae, 107 Placynthiaceae, 107
Peltigerales, 91 Planistromellaceae, 188
Penicillium, 123–127, 134–136, 212 Plectocarpon, 166
P. camemberti, 135 Plectomycetes, 121, 127, 129, 136
P. chrysogenum, 122, 126, 136 Pleomassariaceae, 155
P. roquefortii, 135 Pleosporaceae, 156
subgenus Biverticillium, 124, 127 Pleosporales, 144, 145, 147, 151, 154–160,
Pentagenella, 152 162, 167
Perigrapha, 166 Pleosporineae, 150, 156
Pertusariaceae, 108 Pleosporomycetidae, 144, 145, 151, 154, 155,
Pestalotia, 67 157, 160, 162
Peterjamesia circumscripta, 165 Pneumocystis, 14, 21
Peterozyma, 16 P. carinii, 235
Petromyces, 126 Podocarpaceae, 134
Peziza, 48, 233 Podospora, 67
Pezizaceae, 41–44, 48 Polystomellaceae, 161
Pezizales, 37, 41 Porinaceae, 105
Pezizomycetes, 35–37, 41–43, 46–50 Porpidiaceae, 105
Pezizomycotina, 35, 126, 130, 232, 235, 237, 288 Postia placenta, 287
Phacidiaceae, 72 Priceomyces, 14
Phacographa, 165 Prosthemium, 155
Phacothecium, 166 Protoascon missouriensis, 266
Phaeomoniella, 131 Protomyces, 5, 14
Phaeosphaeriaceae, 153, 156 Protomycopsis, 14
Phaeotrichaceae, 146, 162 Protoventuria, 162
Phaeotrichum, 148 Psaronius, 268
Phaffomyces, 15 Pseudallescheria, 64
Phakopsora pachyrhizi, 212 Pseudocercospora, 159
Phaneromycetaceae, 105 Pseudorbilia, 40
Phialoascus, 14 Pseudotripoconidium, 40
Phialocephala, 75 Pseudotulostoma, 127, 128, 134
Phialophora, 65 P. volvata, 122, 127
Phialosimplex, 125 Psilopezia, 49
Phlyctidaceae, 105 Pucciniomycotina, 232, 235, 237
Phoebus, 166 Pyrenidium, 153
Pyrenomycetes, 59, 129, 130 Sarcosomataceae, 42, 44, 49

Pyrenophora, 151, 156 Sarrameana, 108
Pyrenula, 123, 154 Saturnispora, 15
P. aspistea, 154 Scheffersomyces, 14
P. concatervans, 123 S. stipitis, 4
P. pseudobufonia, 154 Schismatomma, 152, 165
Pyrenulaceae, 131 Schizoblastosporion, 16
Pyrenulales, 123, 131 Schizosaccharomyces, 13, 14
Pyricularia, 65, 187 S. pombe, 284
Pyronema, 44 Schizothyriaceae, 29, 32, 159, 161
Pyronemataceae, 41–43, 46, 48–50 Schwanniomyces, 14
Pythium, 216 Sclerococcum sphaerale, 131
Pyxidiophora, 78 Sclerophyton, 165
Pyxidiophorales, 78 S. cerebriforme, 165
S. muriforme, 165
Sclerospora graminicola, 213
R
Sclerotinia, 79
Racodium, 152, 158
S. sclerotiorum, 79
Ramalinaceae, 104
Sclerotiniaceae, 72
Ramularia, 159
Scutellospora, 267
Rasamsonia, 127
Scutellosporites devonicus, 263
Reichlingia, 149, 165
Septoria, 156, 167
Requiellaceae, 131
Serpula lacrymans, 287
Rhizaria, 231
Sordariales, 66–67
Rhizinaceae, 41–42, 44, 49
Sordariomycetes, 57, 126
Rhizina undulata, 42, 49
Sordariomycetidae, 62
Rhizocarpaceae, 108
Sparria, 165
Rhizophydiales, 250
Spathaspora, 14
Rhytidhysteron rufulum, 157
S. passalidarum, 4–5
Rhytisma, 79
Spathulospora, 68
Rhytismatales, 69–70
Spencermartinsia, 160
Roccella, 152, 163, 233
Spencermartinsiella, 16
R. galapagoensis, 166
Sphenophyllum, 263
Roccellaceae, 152, 164–166
Sphinctrinaceae, 131
Roccellographa, 165
Spilocaea, 162
Roccellographaceae, 165
Spongiophyton, 270
Roussoella, 156
Sporocarpon, 263, 267
Roussoellopsis, 156
Sporopachydermia, 16
Rutstroemiaceae, 72
Sporormiaceae, 147, 156
Sporostigma, 149
S Stachybotrys chartarum, 64
Saccharomyces, 4, 15 Starmera, 15
S. cerevisiae, 283 Starmerella, 4, 16
Saccharomycodes, 15 Stereocaulaceae, 105
Saccharomycopsis, 16 Stictidaceae, 105
Saccharomycotina, 3–27 Stictis, 106
Saccobolus, 46 Stigmina, 162
Sagenomella, 125, 127 Stirtonia, 147
Sagiolechiaceae, 105 Stomiopeltis, 161
Saitoella, 14, 17 S. versicolor, 161
Saprochaete, 14 Stramenopiles, 231
Sarcoscyphaceae, 42–44, 49 Straminipila, 217
Strigulaceae, 162, 163 T. magnatum, 49

Sugiyamaella, 16 T. melanosporum, 49
Sympoventuria, 162 Tuberaceae, 44, 47, 49–50
Sympoventuriaceae, 162 Tuberales, 44, 127
Tubeufiaceae, 155, 161–162
T Tylophorella, 149
Talaromyces, 124, 127 Tylophoron, 148, 164
T. marneffei, 134 Tyrannosorus pinicola, 162
Taphridium, 14
Taphrina, 5, 14
U
Taphrinales, 235
Umbilicariaceae, 110
Taphrinomycotina, 3–27
Uncinocarpus reesii, 129
Tappania, 263, 270
Uredo, 189
Teloschistaceae, 109
U. betae, 189
Teratosphaeriaceae, 158–160, 166
Urnula craterium, 42
Terfezia, 235
Ustilaginomycotina, 235
Tetrapisispora, 15
Tetraplosphaericeae, 156
Thecotheus, 46 V
Thelebolaceae, 70 Vahliellaceae, 107
Thelebolales, 70 Valsa, 65
Thelebolus, 70 Vanderwaltozyma, 15
Thelotremataceae, 97 Vararia, 233
Thermoascaceae, 123 Varicosporina, 64
Thermoascus, 126, 127 Venturia, 162
Thermomyces, 127 V. inaequalis, 162
Torula compniacensis, 160 Venturiaceae, 151, 155, 161, 162
Torulaspora, 4, 15 Venturiales, 162
Tothia, 161 Verrucariaceae, 131
Trapeliaceae, 109 Verrucariales, 123, 130–131
Trapeliales, 108 Verticillium, 63
Traquairia, 267 Vibrisseaceae, 72
Trematosphaeriaceae, 156 Vizellaceae, 161
Trentepohlia, 131, 152 Volkartia, 14
Trentepohliales, 152
Trichocoma, 127, 128
W
T. paradoxa, 122, 127
Wegea, 149
Trichocomaceae, 123, 124, 126–127
Wickerhamia, 14
Trichoderma, 60, 188
Wickerhamiella, 16
T. reesei, 286
Wickerhamomyces, 15
Trichodermataceae, 188
W. anomalus, 6
Trichomeriaceae, 130
W. canadensis, 9
Trichomonascus, 9, 16
Winfrenatia reticulata, 263, 270
Trichophyton, 128, 133
Trichosphaeriales, 62, 69
Tridentaria, 40 X
Trigonopsis, 16 Xeromyces, 125, 127
Trinacrium, 40 Xylaria, 60
Trypetheliaceae, 152, 154 Xylariaceae, 67
Trypetheliales, 162 Xylariales, 67
Tuber, 235 Xylariomycetidae, 62
Y Zopfiaceae, 147, 148, 156

Yamadazyma, 14 Zwackhia, 165
Yarrowia, 16 Zwackhiomyces, 153
Y. lipolytica, 4 Zygoascus, 16
Zygosaccharomyces, 4, 15
Zygotorulaspora, 15
Z
Zymoseptoria, 159
Zoopagomycotina, 241
Zopfia, 145, 148, 156
Subject Index
A Ascomata, 93
Access and benefit sharing (ABS), 215 Ascospores, 39, 43, 61, 97, 269
Acetate peels, 264 muriform, 163–165
Acronym, 207 Ascostroma, 147
Agar, 215 Ascus, asci, 39, 43, 61, 232, 233, 239
Agglutination, 9 bitunicate, 96, 147, 148, 150, 157
Amsterdam Declaration on Fungal dehiscence, 97
Nomenclature, 183 fissitunicate, 148, 150, 154
Anamorphs, 8, 21, 27–29, 31, 33, 37, inoperculate, 37
61, 183 operculum, 43
states, 44 prototunicate, 96
Ancient DNA (aDNA), 211 pseudoprototunicate, 148
length of, 214 rostrate, 148
Annotations, 211 semifissitunicate, 148
Antibiotics, 24 suboperculate, 43
Apical organization, 230, 232, 237, 248–249 unitunicate, 96
Apical ring, 61 Aspergillaceae
Apothecia asexual morphology, 125
lirellate, 94, 164 self-fertility, 125–126
perithecioid, 94 sexual morphology, 125–126
Apothecioid, 144, 145, 149, 159, 160 Atractosome, 232
Appressoria, 92 Author citations, 198
Ascogenous hyphae, 145, 148
Ascohymenial, 95, 144
Ascolocular, 95, 144 B
development, 144 Baking process, 4
Ascoma, 38–39, 42 Basidiospore, 232, 233
apothecial ascoma, 57 Basidium, 239
exothecia, 43 Basionym, 195
hypogeous, 41 Beverage, 4–5
hysteriaceous, 157 spoilage, 4–6
maculiform, 164 Biochemical characters, 229, 233–237,
mazaediate, 164 239, 251
perithecial ascoma, 57 Biocontrol, 5–6, 60
ptycothecia, 43 Biosafety, 215
stereothecia, 43 Biosecurity, 215
truffle, 43 Biotrophy, 68, 151

306 Subject Index
Brewing process, 4 Cryovials, 218

Bryophyte parasites, 42 Culture, 193
availability, 23–26
axenic, 214
C
collections, 26
Candidiasis, 6
ex-epitype, 195
Canopy fungi, 40
ex-holotype, 195
Carboniferous, 153, 260, 263–267, 272
ex-neotype, 195
Mississippian, 271
ex-type, 193, 195
Pennsylvanian, 271, 272
preservation, 216, 217, 220, 222
Carboniferous coal-ball, 264
purification, 24
Cell lysis, 213
Culture specimens, dried, 208
Cellular membranes, 220
preparing, 209
Cellular proteins, 220
storage of, 209–210
Cell wall, 234
Cystidia, 232, 237
Cenozoic rocks, 261, 264, 269
Centriole, 230, 232
Centrum, 60, 144, 149, 155, 157, 162
D
Character evolution, 237, 239
Devonian, 153, 167, 266–271
Character state, 230, 237–240, 250
Devonian Rhynie chert, 263, 265, 267, 268
Chemical fixation, 232, 242, 244, 245, 247, 248, 250
Diazonium Blue B (DBB), 233
Cherts, 260, 263, 265, 266
Dictyosomes, 232
Chestnut blight, 59
Differential scanning calorimetry (DSC), 220
Chlamydospores, 8
Dimethyl sulfoxide (DMSO), 218
Clamp connections (fossil), 263, 268, 271
Direct reference in new combination, 197
Cleistothecia, 148
Divergence time, 99
Cleistothecioid, 144
DNA
Coal-balls, 263, 264, 266
barcoding, 215
Coal geology, 260
degradation of, 214
Coccidioidomycosis, 133
quality
Coelomycete, 61
from herbarium specimens, 214
Collecting
DNA extraction, 207, 212–214
fungal specimen, 207
bead beating, 214
Collection management, 215
cell lysis, 213
Commensals, 152
clean area, 213
Conidia, 98
Dogwood anthracnose, 65
aquatic, 40
Dolipore, 231, 235
Conservation, 182
Drought tolerance, 220
Contamination
Dutch elm disease, 59
DNA, 212
of herbarium DNA samples, 212
Convention on Biological Diversity (CBD), 215 E
Coprophilic fungi, 156, 162 Ectomycorrhizae, 134
Cretaceous, 264, 269 Effectors, 151
Crozier, 61 Embedding, 243, 245–247, 251
Cryofixation, 229, 232, 244, 248, 249 Endophytes, 60, 150, 151, 153, 155, 160, 272
Cryogenic storage, 217–219 Endospores, 7–8
dynamic vapor phase, 218 Environmental sequences, 199
Cryopreservation, 217–219 Environmental study, 79–80
Cryoprotectants, 218, 220 Enzymatic degradation, 214
Subject Index 307
Eocene, 261, 263, 269 Geobiochemistry, 260

Epiphyllous fungi (fossil), 261, 269 Geobiology, 260
Epiphytes, 150, 155, 158 Germ slit, 67
Epitype, 209 Glass ampoules, 220, 221
Ethylene glycol, 218 Glass transition temperature, 220
Eurotiomycetes, 122, 131, 134, 137 Glomeromycotan spores (fossil), 267
animal pathogens, 132 Glycerol, 218
Coccidioidomycosis, 133 Golgi, 230
dermatophytes, 133 equivalents, 232
ectomycorrhizae, 134 Growth in culture, Orbiliomycetes, 41
extremophilism, 132
food contamination, 135
H
human pathogens, 134
Hamathecium, 144, 147, 162
industrial uses, 136
Hartig net, 42
lichens, 134
Haustoria, 92
metabolites, 135
Hemiangiocarpous development, 147
myrmecophytes, 135
Hemibiotrophic, 151, 156
onygenalean pathogens, 132–134
Herbarium cabinets, 209
plant pathogens, 134
Herbarium sheets, 208
Exciples, 164, 165
Herbarium specimens
Ex-type, 193, 195
annotations, 211
cross contamination, 212
F destructive sampling, 211
Flagellar apparatus, 230, 251 DNA extraction, 212–214
Flagellum, 230 labeling, 209
Food contamination, 135 mounting sheets, 208
Food spoilage, 4–6 paper packets, 208
Fossils, 100, 167 Holomorph, 183
record, 259, 260, 265, 268–273 Holotype, 209, 215
Freeze-dried material Homology, 237
reactivation, 221–222 Homonym, 181, 194
Freeze drying, 220 Host response, 263, 265, 272
dehydration, 220 Hyperparasites, 155, 161
primary drying, 220 Hyphal apex, 232, 248, 249
secondary drying, 220 Hyphal branching, 232
sublimation of ice, 220 Hyphomycete, 61
Freeze substitution, 244, 245 Hyphopodia, 161
Frozen storage, 25 Hysterothecia, 144
Fungal fossils, 261, 262 Hysterothecium, 157
Fungal Subcellular Ontology (FSO), 230,
240–242
I
Fungicolous fungi, 158
Ice-crystal dynamics, 217
Ice-crystals
G intracellular, 218
Genome, 78–79, 156, 160, 167 Identifier, 191, 194
Genome based phylogenetic reconstruction, Index Fungorum, 190
281–283 Index Herbariorum, 207, 209, 210
Genomics enabling comparative biology, Inoperculate discomycetes, 69
284–288 Insect infestations, 209
308 Subject Index
Insect pests, 212 Metabolism, 216

Interascal pseudoparenchyma, 144 Metabolites, 99
International Botanical Congress (IBC), 180 Methanol assimilating yeasts, 4
International Code, 149 Microbodies, 232, 237
International Code of Nomenclature (ICN) Microscala, 232
for algae, fungi and plants, 180–198 Microwave fixation, 246
International Commission on the Taxonomy Mineral oil, 216–217
of Fungi (ICTF), 187 Mites, 212, 216
International Mycological Congress infestations, 216
(IMC), 183 Mitochondrial cristae, 230
Isidia, 98 Mitosis, 232, 247, 250, 251
Motile cell, 230–233, 237, 239, 240, 251
Mounting sheets, 208
J
Multigene analyses, 13
Jurassic, 270–271
MycoBank, 191
Mycoparasitism, 153
K Mycorrhiza, 42
Karyogamy, 9
N
L Name
Labeling fungal specimens, 209 illegitimate, 181
Lactose, 220 invalid, 181
L-drying, 25 legitimate, 181
Lichenicolous fungi, 144, 147, 152, 153, 164, 166 new combination, 197
Lichenization, 100 registration, 190–191
Lichens, 91, 134, 153, 233 replacement, 197
combined classification, 150 sanctioned, 192–193, 195
crustose, 152, 162–165 superfluous, 181
fruticose, 152, 163, 165 Necrotrophy, 151
subfruticose, 165 Nematophyta, 271
Lifestyles, Lecanoromycetes, 91 Nematophytes, 271
Liquid nitrogen, 217 Nomenclature, 179
Liquid paraffin, 216 dual, 182
Long-term culture preservation, 216, 217, 220, original material, 195
222 Nomen Novum, 197
Lyophilization, 25, 220 Nuclear division, 40, 230, 232, 233, 237, 239,
cooling rates, 220 240, 247–248
glass transition temperature, 220
Lyoprotectants, 220
O
Lysine synthesis, 235
One Name Per Fungus, 182–189
Ontology, 240–242
M Operculate, 45
Marine species, 131–132 Original material, 195
Material transfer agreement (MTA), 215 Ostiole, 144, 145, 155
Meiosis, 9, 232, 247, 248
Melzer’s reagent, 233
Meristematic growth, 153 P
Mesosynteny, 167 Paleodiversity, 260
Metabolically active preservation, 215 Paleoecology, 260
Subject Index 309
Paleomycology, 268, 272, 273 effective, 181, 189–190

Paper packets, 208 electronic, 189–190
Paraphyses, 39–40, 43, 98, 232 ranks, 181
Paraphysoids, 144 valid, 181
Parasites, 49, 144, 150, 152, 155, 162 Pullulan, 160
species, 42, 150, 151, 156 Pyrenocarpous lichens, 162
Pathogens, 6, 132–134, 143–144, 150, 156, 159,
162
Q
Peptone, 220
Quality standards, 215
Periodic subculturing, 215
Quellkörper, 63
Periphyses, 144, 155, 158
Periphysoids, 144, 158
Perithecia, 94, 147, 150, 162 R
Permanent slide mounts, 208 Recombinant proteins, 4
Permian, 262, 263, 268 Red bread mold, 78
Photobionts, 91, 152, 165 Registration of fungal names, 190–191
Phylogeny, 61–62, 230–237, 249, 281–283 Rejection of fungal names, 182
analysis, 238–240 Replaced synonym, 195–197
and classification, 13–23 Requesting a specimen, 210
programs, 239 Residual moisture, 220
Phytosanitary regulations, 215 Rhynie chert, 260, 265–267, 269
Plant diseases, 5 Riboflavin, 4
Plant parasites, 49 Rice blast, 59
Plasmogamy, 9 Rock-inhabiting fungi (RIF), 146, 153, 158, 159
Plectenchyma, 144, 147 Roots, 268, 272
Plectomycetes
characteristics, 121
Polymerase chain reaction (PCR), 211 S
Polypore basidiocarps (fossil), 269 Saccharides, 220
Polyspory, 100 Safety cabinet, 218, 219, 222
Postmortem degradation, 214 Sanctioning, 182, 192, 195
Post-preservation survival, 218 Saprobes, 38, 42, 144, 150, 151, 156, 159, 161,
Powdery mildew, 76 163, 166
Precambrian, 261, 266, 270 dung, 42
Predation Saprotrophic nutritional mode, 81
nematode, 38 Sclerotia, 149, 150
Preservation Scutate wall of thyriothecia, 144
amber, 263, 264, 269, 270 Section staining, 246–247
Mississippian, 263 Sedimentology, 260
Pennsylvanian, 263, 268 Septal pore, 230–232, 235, 237, 240, 245
permineralizations, 262, 263 apparatus, 232, 237, 239
petrifaction, 263 cap, 232, 237
Preservation methods, 215 Septal structures, 40, 44, 46–49
Priority, 181, 188 Septate (true) hyphae, 7
Proterozoic (fossil), 263, 270 Septum, 230, 231, 239, 240
Protologue, 192 Sequencing 1000 fungal genomes, 283–284
Pseudohyphae, 7 Shipment of cultures, 26
Pseudoparaphyses, 144, 145, 151, 154, 156–158, Silurian, 263, 268, 269, 271
160, 162, 163 Skim milk, 220
Publication Sodium glutamate, 220
310 Subject Index
Soil Clone Group I (SCGI), 23 taxonomy, 126–127

Sooty mold, 158, 159, 167 Truffles, 49
Soredia, 98 Type, 181, 192
Special Committee on Electronic Publication, epitype, 196–197
189 fungal cultures, 193
Special Committee on Nomenclature of Fungi holotype, 193, 194
with a Pleomorphic Life Cycle, 183 isosyntype, 195
Spindle pole body (SPB), 230, 232, 239, isotype, 195
240, 247 lectotype, 193, 195
Spitzenkörper, 232, 249 neotype, 196
Spore body, 39 paratype, 195
Sporocarps (fossil), 263, 266–267 specimen, 209
Sporodochia, 164 syntype, 195
Sterol, 235, 239
Structural and Biocemical Database (SBD), 230,
U
237–240, 252
Ultrastructure, 61, 229, 248, 250
Subcellular characters, 230, 232, 249
Unique acronym, 209
Substrate, Lecanoromycetes, 91
Summer patch pathogen, 65
Symplechosome, 232 V
Synanamorphs, 61 Vapor phase, 217
Synonym Voucher specimens, 206
facultative, 193
heterotypic, 193
W
homotypic, 193
Wine making process, 4
nomenclatural, 193
Woronin bodies, 237
obligate, 193
replaced, 195–197
taxonomic, 193 X
Xylose fermentation, 236
T
Taxonomy, 179 Y
Teleomorph, 183 Yeast, 233, 235, 236, 247
Teliospores, 243, 248 asexual reproduction, 7
Thalli, Lecanoromycetes, 93 D1/D2 domains, rRNA gene, 12
Thermoascaceae distribution, 3–4
asexual morphology, 126 ecology, 3–4
sexual morphology, 126 fission, 7
Thin-sections, 263, 264 genotypic classification, 12–13
Thyriothecia, 144, 147, 161 industrial uses, 4–5
Thyriothecioid, 161 methanol assimilating, 4
Transport regulations, 215 occurrence, 3–4
Trapping mechanisms, 38 phenotypic characterization, 11–12
Trehalose, 220 purification of cultures, 24
Triassic, 266–268 septate (true) hyphae, 7
Trichocomaceae sexual reproduction, 9–11
asexual morphology, 127 Yeast budding
sexual morphology, 127 bipolar, 7
Subject Index 311
enteroblastic, 7 osmotic media, 24

holoblastic, 7 temperature, 23
multilateral, 7
Yeast culture Z
acidified media, 23 Zoospore, 230, 249, 250
isolation, 23–26 Zwischengruppe, 147
maintenance, 23–26 Zygomycete, 232, 247, 251
malt-peptone, 222 Zygosporic fungi, 232
membrane filters, 24

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Systematics and Evolution

I Growth, Differentiation and Sexuality

Professor Dr. David J. McLaughlin

ISBN 978-3-662-46010-8 ISBN 978-3-662-46011-5 (eBook)

Library of Congress Control Number: 2015936344

Springer Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer-Verlag GmbH Berlin Heidelberg is part of Springer Science+Business Media (www.springer.

Bochum, Germany KARL ESSER

Table 1 Taxonomic outline for Fungi and fungus-like organismsa

St. Paul, MN David J. McLaughlin

This is an exciting time to produce an overview of the systematics and evolution of

Mycota, Vol. I Mycota, Vol. VII

Mycota, Vol. VII Chapter 1, Vol. VII, Part A

St. Paul, MN DAVID J. MCLAUGHLIN

1 Saccharomycotina and Taphrinomycotina: The Yeasts and Yeastlike

2 Pezizomycotina: Pezizomycetes, Orbiliomycetes . . . . . . . . . . . . . . . . . . . . . . 35

3 Pezizomycotina: Sordariomycetes and Leotiomycetes . . . . . . . . . . . . . . . . 57

5 Pezizomycotina: Eurotiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

6 Pezizomycotina: Dothideomycetes and Arthoniomycetes . . . . . . . . . . . . 143

Nomenclature and Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

7 The Shifting Sands of Fungal Naming Under the ICN

8 The Role of Herbaria and Culture Collections . . . . . . . . . . . . . . . . . . . . . . . . 205

9 Subcellular Structure and Biochemical Characters

10 Fungal Diversity in the Fossil Record . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

11 Phylogenomics Enabling Genome-Based Mycology . . . . . . . . . . . . . . . . 279

Biosystematic Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

T.K. ARUN KUMAR

GERARD J.M. VERKLEY

CLETUS P. KURTZMAN1, JUNTA SUGIYAMA2

Systematics and Evolution, 2nd Edition

IV. Reproduction leads to the formation of multiple scars or

ellipsoidal, cylindrical, reniform, crescentic, V. Taxonomic Methods

Class Order Family Genus

Meyerozyma Kurtzman & M. Suzukid (T)

Class Order Family Genus

Nadsonia Sydow (T)

families is tentative for many of the taxa. The B. Classification

Fig. 1.4 (continued)

B. Maintenance bility. Lyophilized cultures are usually stored in

Systematics and Evolution, 2nd Edition

as O. luteorubella and O. xanthostigma, that addition to trapping nematodes, some hypho-

V. Reproduction ascus, the ectal excipulum, spore bodies, the

E. Septal Structures XI. Reproduction

Family Anamorphic form genus Teleomorphic genus References

Rhizinaceae Phymatotrichopsis None known Uppalapati et al. (2010)

12. Rhizinaceae In this family, ascomata are cupulate or urnu-

Webster J (1992) Anamorph-teleomorph relationships. Yang Y, Yang E, An Z, Liu X (2007) Evolution of

NING ZHANG1, ZHENG WANG2

CONTENTS VII. Culture and Maintenance . . . . . . . . . . . . . . . . . . 80

Systematics and Evolution, 2nd Edition

In addition to pathogens of plants, Sordar- of Nectriaceae and the Stachybotrys clade

3. Asci anamorphs also occur, most notably in the Glo-

5. Anamorphs C. Molecular Phylogeny

Sordariomycetes is an anamorph-rich class, Recent comprehensive taxonomic and phyloge-

Hypocreales, Melanosporales, and Microas- distinctions, and Wanderlei-Silva et al. (2003)

wide range of secondary metabolites and plant- e) Microascales

ciated with beetle dispersers, while related ana- f) Chaetosphaeriales