ZOJ 2024paratrichodorusallius-Complex Red

Zoological Journal of the Linnean Society, 2024, XX, 1–44
https://doi.org/10.1093/zoolinnean/zlad194
Advance access publication 24 January 2024
Original Article
Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlad194/7588341 by CSIC - Instituto De Ganaderia De Montana user on 29 January 2024
Original Article
Unravelling cryptic diversity in the Paratrichodorus allius-
group species complex to resolve eight new species of
the genus and new insights on the molecular phylogeny
(Nematoda: Trichodoridae)
Wilfrida Decraemer1,‡, Antonio Archidona-Yuste2,‡, Ilenia Clavero-Camacho2, Alessio Vovlas3,
Carolina Cantalapiedra-Navarrete2, Alba N Ruiz-Cuenca2,4, Pablo Castillo2,
Juan E Palomares-Rius2,*,
1
Ghent University, Department of Biology, Ledeganckstraat 35, B-9000 Ghent, Belgium
2
Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s/n, 14004 Córdoba, Campus de
Excelencia Internacional Agroalimentario, ceiA3, Spain
3
Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante (IPSP), v. G. Amendola, 122/D, 70126 Bari, Italy
4
Departament of Animal, Plant Biology and Ecology, Universidad de Jaén, Campus Las Lagunillas, Jaén, Spain
‡
These authors contributed equally to this work.
*
Corresponding author. Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s/n, 14004 Córdoba, Campus de
Excelencia Internacional Agroalimentario, ceiA3, Spain. E-mail: palomaresje@ias.csic.es
ABSTR ACT
The stubby-root nematode genus Paratrichodorus comprises species of plant polyphagous ectoparasitic nematodes distributed worldwide and
a few of them are plant virus-vectors (tobravirus). This is one of the most difficult nematode genera in terms of species identification, since it is
phenotypically conserved with closely related species and is highly sensitive to the specimen fixation and preservation processes. Using an inte-
grative and multidisciplinary taxonomy framework, we determined species boundaries among the Paratrichodorus populations from a P. allius
species-complex sampled in cultivated and natural ecosystems across Spain. We support the hypothesis that nematode diversity of the P. allius
species-complex remains underestimated and describe here a total of eight new species on the basis of detailed morphological and molecular
characterization of 16 Paratrichodorus populations: Paratrichodorus asidonaensis sp. nov., Paratrichodorus benalupensis sp. nov., Paratrichodorus
hinogerus sp. nov., Paratrichodorus litoralis sp. nov., Paratrichodorus paraallius sp. nov., Paratrichodorus rocianerus sp. nov., Paratrichodorus tarifaensis
sp. nov., and Paratrichodorus uliaensis sp. nov. This study emphasizes the importance of properly applying a broad taxonomic framework in plant-
parasitic nematodes. It is also relevant for plant health, since it correctly delimits species morphologically closed to the virus vector nematode, P.
allius, and gives proper molecular markers for its unequivocal identification.
Keywords: Bayesian inference; expansion domains of the large ribosomal subunit; internal transcriber spacer; multivariate morphometric
analysis; species delimitation; stubby root nematodes
INTRODUCTION plant-parasitic nematodes that reduce crop productivity by

Migratory root ectoparasitic nematodes of the family their impact on the plant root system (in some cases in sev-
Trichodoridae, particularly the genera Trichodorus Cobb 1913, eral crops), such as Nanidorus minor (Colbran 1956) Siddiqi
Nanidorus Siddiqi 1974, and Paratrichodorus Siddiqi 1974, cause 1974 and Paratrichodorus allius ( Jensen 1963) Siddiqi 1974
symptoms of stunted or stubby appearance of the root system (MacGowan 1983, Ingham et al. 1999, Hajihassani et al. 2018).
in infected plants in cultivated and wild plants and transmit Noteworthy, some species, such as Paratrichodorus allius, are
plant nepoviruses (Decraemer 1995, Decraemer and Geraert also demonstrated to transmit the tobravirus tobacco rattle
2013). Some species have been demonstrated to be important virus (TRV; Jensen and Allen 1964). This species is the most
Received 20 October 2023; revised 9 November 2023; accepted 30 November 2023

[Version of Record, first published online 24 January 2024 http://zoobank.org/urn:lsid:zoobank.org:pub:531FB687-C656-4F7F-8893-AA10FC8DC63A]
© The Author(s) 2024. Published by Oxford University Press on behalf of The Linnean Society of London. All rights reserved. For permissions, please e-mail: journals.permissions@
oup.com
2 • Decraemer et al.
important nematode vector of the corky ringspot strain (CRS) of two ventromedian precloacal supplements (F2), absence of
of TRV to potatoes in the western United States (Mojtahedi and ventromedian cervical papillae (D0), and nematode body shape
Santo 1999). This virus disease is widespread in Europe and it is straight or slightly and regularly curved (P1); while females have
one of the most important virus diseases in potato, especially in a didelphic genital system (D1), <75% of vulva position in re-
seed-production areas (Shumaker et al. 1984). TRV is a quaran- spect to body length (C1), and the position of vaginal sclerotized
tine pathogen in many countries (e.g. USA, Brazil, and Turkey) pieces widely separated (2.5–3.5 µm apart) (L3) (Decraemer
and their spread is controlled by seed-potato regulation in many and Baujard 1998). Therefore, molecular data, including from
countries of the European and Mediterranean Plant Protection topotypes (i.e. specimens of a known species collected at the
Organization (EPPO). Corky ringspot causes important brown locality from where it was originally described) of known spe-
rings on the surface and/or brown arcs or flecking of the inside cies are very important for reliable species identification. So far,
of infected potatoes. These symptoms make symptomatic tubers molecular data are available for only 30% of Paratrichodorus spe-
unmarketable, with rejection of a potato lot with the presence of cies. Molecular diversity and species delimitation are important
as few as 5% of tubers with corky ringspot symptoms [Fédération in order to design molecular tools useful for accurate species
Nationale des Producteurs de Plants de Pomme de Terre (France) identification, even in the absence of adult or adult specimens
2011]. Definitively, accurate identification of stubby root nema- with compromised morphology. Specifically, for P. allius, sev-
todes is crucial and with phytopathological consequences in eral molecular tools have been developed, such as conventional
field management and plant trade [plants or plant parts (tubers) and real-time polymerase chain reaction (PCR) using SYBR
infected with tobraviruses]. Identification to species level in Green and TaqMan probe (Huang et al. 2017a, b) and a one-step
Trichodoridae is often hampered by the impact of fixation on multiplex PCR for several Paratrichodorus (including P. allius)
morphological features and measurements, the need for spe- and Trichodorus species (Huang et al. 2019) using nematode
cimens in lateral view, overlapping morphometric data, and by and soil extractions. However, P. allius forms a species-complex
the co-occurrence of cryptic species (Decraemer and Subbotin with several closely related morphological species (see key in:
2021, Decraemer et al. 2021b). The challenge to species iden- Decraemer and Baujard 1998) and for this reason the diversity
tification is especially great for females of Paratrichodorus with and species delimitation is important in order to clarify which
several species lacking males or males being rare as is reflected species are important from a phytopathological or ecological
in only few (four) new species descriptions since the overview view. Phylogenetic analyses based on 18S rRNA, as well as on
paper by Decraemer and Baujard (1998) and others (Almeida D2-D3 expansion segments of 28S rRNA, group Paratrichodorus
et al. 2005, Decraemer et al. 2019). However, if available, male species in two main clades: a P. hispanus clade and a P. allius clade,
characters are very important to separate species in this genus. each containing about 50% of the known Paratrichodorus species
For species identification and determination of relationships (van Megen et al. 2009, Duarte et al. 2010, Decraemer et al. 2019,
based upon morphological similarities, the tabular identification Li et al. 2020, Subbotin et al. 2020). Both clades are supported
keys for males and females by Decraemer and Baujard (1998) by distinct morphological differences in the genital systems of
is commonly used. For Trichodoridae males, sorting on the pri- the species included. Species within the P. hispanus clade pos-
mary features F (number of ventromedian precloacal supple- sess a pore-like vulva in ventral view and large sperm cells with a
ments), feature D (number of ventromedian cervical papillae, large, sausage-shaped nucleus (few exceptions). Species of the P.
CP), and P (habitus), classifies all Paratrichodorus species, apart allius clade possess a longitudinal, slit-like vulva in ventral view
from three species [P. orrae Decraemer and Reay 1991 with ab- and very small sperm cells with small, rounded nucleus (few ex-
errant habitus and P. mirzai (Siddiqi 1960) Siddiqi 1974 and P. ceptions).
paramirzai Siddiqi 1991, both with 2 CP instead of 0 or 1] in Currently, nine out of 28 Paratrichodorus species, have been
species group 6 (Decraemer and Baujard 1998). Hereby, we have recorded from the Iberian Peninsula, i.e. P. allius, P. almadenensis,
to consider that since Decraemer and Baujard (1998), the genus P. anemones (Loof 1965) Siddiqi 1974, P. divergens, P. hispanus
Nanidorus Siddiqi (1974) has been generally accepted as a valid Roca and Arias 1986, P. pachydermus (Seinhorst 1954) Siddiqi
genus and includes seven species (Marais et al. 1996, Siddiqi 1974, P. porosus (Allen 1957) Siddiqi 1974, P. ramblensis, and
2002, Duarte et al. 2010), and for Paratrichodorus four new spe- P. teres (Hooper 1962) Siddiqi 1974 (López Pérez et al. 2001,
cies (P. almadenensis Decraemer et al. 2019, P. divergens Almeida Duarte et al. 2010, Decraemer et al. 2019); all, except P. allius,
et al. 2005, P. namibiensis Marais and Botha-Greeff 1997, and P. teres, and P. divergens, were found in cultivated and natural en-
P. ramblensis Decraemer et al. 2019) have been described since vironments in southern Spain. Together with P. divergens, they
then. For females, selection using prime features D (type of belong to the P. hispanus species group studied in Decraemer et
genital system) and C (vulva position), followed by features L al. (2019). The objectives of the current contribution are as fol-
(position of vaginal sclerotized pieces) and K (size of vaginal lows: (i) to study Paratrichodorus species of populations from
sclerotized pieces) places the species of Paratrichodorus in nine nematode surveys in cultivated and natural environments from
of the 12 subgroups of group 1; because of intraspecific vari- 2003 to 2016 in Spain belonging to the P. allius species-complex
ations in features L and K, some species were classified in up to clade based on a combined morphological and molecular ap-
five different subgroups. In addition, mode of sperm storage and proach; (ii) to describe and illustrate new species; and (iii) to
sperm structure were also considered as important diagnostic analyse relationships (genus/species) based on sequences of
features (Loof 1975, Decraemer 1989) and will be included in different genes and species delimitation support methods (tree-
the present contribution. Paratrichodorus allius species-complex based or/and non-tree-based). For comparative morphological
is characterized by a combination of three characters in males studies a population from southern Italy was also included.
New insights in Paratrichodorus allius-group • 3
M ATERIALS AND METHODS of 94°C for 30 s, an annealing temperature of 55°C for 45 s, and
72°C for 1 min, and one final cycle of 72°C for 10 min. PCR vol-
Nematode populations and morphological studies umes were adapted to 20 µL for each reaction, and primer con-
Nematode surveys were conducted in southern Spain during the centrations were as described in De Ley et al. (1999), Subbotin
2003–16 spring seasons in cultivated and natural environments, et al. (2005), and Holterman et al. (2006). We used 5x HOT
including grapevine (Vitis vinifera L.), unidentified grasses, FIREpol Blend Master Mix (Solis Biodyne, Tartu, Estonia)
stone pine (Pinus pinea L.), wild olive [Olea europaea subsp. in all PCR reactions. The PCR products were purified using
Silvestris (Mill.) Lehr), cork oak (Quercus suber L.), marram ExoSAP-IT (Affimetrix, USB products, Kandel, Germany) and
grass (Ammophila arenaria (L.) Link.], and cultivated olive used for direct sequencing in both directions with the corres-
[Olea europaea subsp. europaea (Mill.) Lehr], in southern Spain, ponding primers. The resulting products were run in a DNA
and one incidental sample was collected from marram grass in multicapillary sequencer (Model 3130XL Genetic Analyzer;
southern Italy (Table 1). Soil samples were collected from the Applied Biosystems, Foster City, CA, USA), using the BigDye
rhizosphere of host-plants with a hoe from the upper 50 cm of Terminator Sequencing Kit v.3.1 (Applied Bio-systems) at the
soil under four to five plants that were arbitrarily chosen in each Stab Vida sequencing facility (Caparica, Portugal). The se-
locality, and nematodes were extracted from 500 cm3 of soil by quence chromatograms of the three markers (18S, ITS, and
centrifugal flotation (Coolen 1979). Specimens were killed by D2-D3 expansion segments of 28S rRNA) were analysed using
gentle heat, fixed in a solution of 4% formaldehyde + 1% pro- DNASTAR LASERGENE SeqMan v.7.1.0. Basic local alignment
pionic acid and processed to pure glycerine using Seinhorst’s search tool (BLAST) at the National Center for Biotechnology
method (1966). Light micrographs from each nematode spe- Information (NCBI) was used to confirm the species identity of
cies were taken using a Leica DM6 compound microscope with the DNA sequences obtained in this study (Altschul et al. 1990).
a Leica DFC7000 T digital camera (Wetzlar, Germany) using The newly obtained sequences were deposited in the GenBank
mounted nematodes in glycerine. Nematodes were identified database under accession numbers indicated on Table 1 and the
at the species level, applying an integrative approach merging phylogenetic trees.
morphological analyses based on the tabular identification keys
for males and females (Decraemer and Baujard 1998), and mo- Species delimitation within Paratrichodorus allius
lecular analyses to achieve an efficient and accurate identifica- species-complex
tion (Decraemer et al. 2019, Subbotin et al. 2020). Two different approaches of species delimitation were used
to determine species boundaries within this P. allius species-
DNA extraction, PCR, and sequencing complex, including morphometric and molecular data. Only
For all the nematodes molecularly studied, DNA extraction was morphologically closely related species were included in the de-
based on single specimens and, more decisively, all the molecular limitation analyses.
markers studied of each individual originated from the same single Species delineation using morphometry was conducted with
DNA-extracted nematode in each PCR tube without any omission. principal component analysis (PCA) to estimate the degree of as-
Additionally, to avoid mistakes, in case of mixed Paratrichodorus sociation among some selected and morphological closely related
populations within the same soil sample (Table 1), single nema- populations within the P. allius species-complex (Legendre and
todes were provisionally deposited in a drop of 1 M NaCl containing Legendre 2012). PCA was performed on three populations be-
glass beads (to avoid nematode crushing/damaging specimens) to longing to two new species (Paratrichodorus uliaensis sp. nov.
ensure that specimens conformed with the unidentified popula- and Paratrichodorus benalupensis sp. nov.) using, separately,
tion. Nematode DNA was extracted from single individuals, and male and female specimens. Other taxa belonging to the P. allius
some PCR assays were conducted, as described by Subbotin et al. species-complex were not included in this analysis due to the un-
(2000). Briefly, an individual nematode was cut using a scalpel in availability of suitable and complete morphometric data (i.e. P.
a drop of PCR buffer (ThermoPol®, Biolabs, New England, USA) allius) and morphological non-similarity (i.e. Paratrichodorus
(20 µL) and 2 μL proteinase K (600 μg/mL) were added. The tubes paraallius sp. nov.). To do this, PCA was based on the main
were frozen at −80°C (15 min), then incubated at 65°C (1 h) and morphometric features used in the tabular identification keys
at 95°C (10 min) consecutively. Tubes were centrifuged (1 min, and based on importance in species diagnosis in literature for
16 000 × g) and kept at −20°C until used in PCR. The D2-D3 males and females (Loof 1975, Decraemer 1995, Decraemer and
expansion segments of 28S rRNA gene were amplified using the Baujard 1998) that included: (i) in both sexes: L (body length)
primers D2A (5ʹ-ACAAGTACCGTGAGGGAAAGTTG-3ʹ) and onchiostyle length; (ii) in females: V [(distance from anterior
and D3B (5ʹ-TCGGAAGGAACCAGCTACTA-3ʹ) (Nunn end to vulva/body length) × 100], the vagina shape (i.e. length
1992). The ITS1 region was amplified using forward primer vagina as % of width at midbody), vaginal sclerotized pieces
BL18 (5ʹ-CCCGTCGMTACTACCGATT-3ʹ) and re- length and distance between sclerotized pieces, the distance
verse primer 5818 (5ʹ-ACGARCCGAGTGATCCAC-3ʹ) from anterior end to secretory-excretory pore (EP), and the ratio
(Boutsika et al. 2004), and the partial 18S rRNA gene was a (body length/maximum body width); and (iii) in males: the
amplified as described by Holterman et al. (2006) using pri- distance from anterior end to ventromedian cervical papilla (CP),
mers 988F (5ʹ-CTCAAAGATTAAGCCATGC-3ʹ), 1912R and the spicule length. Prior to the statistical analysis, key diag-
(5ʹ-TTTACGGTCAGAACTAGGG-3ʹ), 1813F (5ʹ-CTGCGTG nostic characters were tested for collinearity (Zuur et al. 2010).
AGAGGTGAAAT-3ʹ), and 2646R (5ʹ-GCTACCTTGTTACG We used the collinearity test based on the values of the variance
ACTTTT-3ʹ) (Holterman et al. 2006). inflation factor (VIF) method that iteratively excludes numeric
The PCR cycling conditions for the 28S rRNA, ITS, and 18S covariates showing VIF values >10, as suggested by Montgomery
rRNA were as follows: 95°C for 15 min, followed by 35 cycles et al. (2012). In PCA, we used an orthogonal varimax rotation to
4
Table 1. Paratrichodorus samples used and sequenced in the present study.

Species Sample Location Host D2-D3 ITS 18S
code
• Decraemer et al.
Paratrichodorus M0156 Montemayor, Córdoba Vitis vinifera (grapevine) OR139109-OR139110

uliaensis sp. nov. (Spain)
Paratrichodorus M0157 La Rambla, Córdoba Vitis vinifera (grapevine) OR139111-OR139112 OR139998-OR140001 OR139140-OR139142
uliaensis sp. nov. (Spain)
Paratrichodorus Lomgr Benalup-Casas Viejas, Unidentified grasses OR139113-OR139114 OR140002-OR140004 OR139143
benalupensis sp. nov. Cádiz (Spain)
Paratrichodorus H0016 Hinojos, Huelva Pinus pinea (stone pine) OR139115-OR139116 OR140005 OR139145
hinogerus sp. nov. (Spain)
Paratrichodorus AR015 Sanlúcar de Barrameda, Olea europaea ssp. sylvestris OR139117-OR139118 OR139144
hinogerus sp. nov. Cádiz (Spain) (wild olive)
Paratrichodorus AR016 Sanlúcar de Barrameda, Olea europaea ssp. sylvestris OR139119-OR139120
hinogerus sp. nov. Cádiz (Spain) (wild olive)
Paratrichodorus H0156 Bonares, Huelva Quercus suber (cork oak) OR139121
hinogerus sp. nov. (Spain)
Paratrichodorus AR031 Tarifa, Cádiz (Spain) Olea europaea ssp. sylvestris OR139122 OR140006 OR139147
tarifaensis sp. nov. (wild olive)
Paratrichodorus NajQs Benalup-Casas Viejas, Quercus suber (cork oak) OR139123 OR139146
tarifaensis sp. nov. Cádiz (Spain)
Paratrichodorus AR004 Medina Sidonia, Cádiz Olea europaea ssp. sylvestris OR139124-OR139126 OR140007-OR140008
asidonaensis sp. nov. (Spain) (wild olive)
Paratrichodorus Lomac Benalup-Casas Viejas, Olea europaea ssp. sylvestris OR139127-OR139128
asidonaensis sp. nov. Cádiz (Spain) (wild olive)
Paratrichodorus H0117 Rociana del Condado, Vitis vinifera (grapevine) OR139129-OR139131 OR140009-OR1400011 OR139148-OR139149
rocianerus sp. nov. Huelva (Spain)
Paratrichodorus ADR01 Pantanaggianni, Ammophila arenaria OR139132-OR139133 OR140012 OR139150
litoralis sp. nov. Brindisi (Italy) (marram grass)
Paratrichodorus Culle Cullera, Valencia Ammophila arenaria OR139134 OR140013
litoralis sp. nov. (Spain) (marram grass)
Paratrichodorus AR003 Medina Sidonia, Cádiz Olea europaea ssp. sylvestris OR139135-OR139137 OR140014 OR139151
paraallius sp. nov. (Spain) (wild olive)
Paratrichodorus JAO44 Espiel, Córdoba Olea europaea ssp. europaea OR139138-OR139139 OR140015-OR140016
paraallius sp. nov. (Spain) (cultivated olive)
a
= not sequenced.
estimate the factor loadings where only components with sum of 2012) with the Akaike information criterion (AIC). The best-
squares (SS) loadings >1 were extracted (Legendre and Legendre fit model, the base frequency, the proportion of invariable sites,
2012). All data analyses were done with the R v.4.2.2 (R Core and the gamma distribution shape parameters and substitution
Team 2022; https://www.R-project.org). rates in the AIC were then used in MrBayes for the phylogen-
Species delineation was also based on molecular data etic analyses. The transversion model with invariable sites and
achieved by using species delimitation plugin (Masters et al. a gamma-shaped distribution (TVM+I+G) for the D2-D3 ex-
2011) from the program GENEIOUS PRIME v.2022.1.1. pansion segments of 28S rRNA, the one-parameter model with
(Geneious, Auckland, New Zealand), and was applied to com- invariable sites and gamma distribution model (TPM2uf+I+G)
pute intra- and interspecies variation by means of the Probability for the partial ITS rRNA gene, and the symmetrical model with
(P) Intra Distance (ID) liberal and the Rosenberg’s PAB value invariable sites and gamma distribution (SYM+I+G) for the
(The probability that species A represented by a sequences, in a partial 18S rRNA gene, were run with four chains for 4 × 106
clade of a + b sequences, will be reciprocally monophyletic with generations. The sampling for Markov chains was carried out
the remaining b sequences under the null model of random co- at intervals of 100 generations. For each analysis, two runs
alescence). Here, this approach was carried out on nematode were conducted. After discarding burn-in samples of 30% and
populations considering two sperate species-pair comparisons evaluating convergence, the remaining samples were retained for
based on morphologically similar species groups (P. uliaensis more in-depth analyses. The topologies were used to generate a
sp. nov. vs. P. benalupensis sp. nov., and P. paraallius sp. nov. vs. 50% majority-rule consensus tree. On each appropriate clade,
P. allius; please see species description below). The intra-/ posterior probabilities (PP) were given. FigTree software v.1.4.3
interspecies molecular variation was established by determining (Rambaut 2016) was used for visualizing trees from all analyses.
the ratio between the average genetic distance between speci-
mens within a species and the average genetic distance between
specimens belonging to sister-species (the average pairwise tree RESULTS
distance among members of a putative species/the average pair- Morphological characterization of the Paratrichodorus allius
wise tree distance between the members of one putative species, species-complex
and the members of the closest second putative species), if the Within the P. allius species group, the main morphological
ratio is less than 0.10, the probability of species identification is diagnostic features to differentiate species in males are, apart
high (Masters et al. 2011). The P ID (Liberal) value (Ross et al. from morphometrics, the number and position of CP and the
2008) represents the probability that a correct species identifi- number (total and in relation to retracted spicules) and position
cation would be made using best sequence alignment (BLAST), of precloacal supplements (SP) and, to a lesser extent, the ap-
closest genetic distance, or placement on a tree (falling within pearance of sperm in the testis; in females, the vaginal shape and
or being sister to a monophyletic species clade). Species with P length, the size, shape and position of the vaginal sclerotized
ID (Liberal) ≥0.93 were considered to be adequately delimited pieces in optical section, and sperm location are of main diag-
(Hamilton et al. 2014). The Rosenberg’s PAB represents the prob- nostic importance. Within the Paratrichodorus allius species-
ability that the monophyly of a group of sequences is the result complex, seven new Spanish species were found and compared
of random branching (Rosenberg 2007). with a new species from Italy. For all species, except one species
with unknown males, males were present either as common as
Phylogenetic analysis females or rare. We start with the description of the species with
D2-D3 expansion segments of 28S rRNA, ITS rRNA, and par- males with one CP followed by those without.
tial 18S rRNA gene sequences from the P. allius species-complex,
as well as different accessions belonging to the Trichodoridae Paratrichodorus species with males with a single
family that are available in GenBank, were used for phylogen- ventromedian cervical papilla
etic reconstruction. Outgroup taxa for each dataset were chosen
according to previously published data (Kumari and Subbotin Paratrichodorus uliaensis sp. nov. Decraemer et al.
2012, Decraemer et al. 2019, 2021a, Subbotin et al. 2020) that
included all the molecular variation in the analysed sequences (Figs 1–3A, B; Table 2)
(van Megen et al. 2009). Multiple sequence alignments of the Zoobank registration: urn:lsid:zoobank.org:act:7908CD13-
different genes were completed using the FFT-NS-2 algorithm BD88-45CE-BDEA-F24425B2590D
of MAFFT v.7.450 (Katoh et al. 2019). BioEdit program v.7.2.5
(Hall 1999) was used for sequence alignments’ visualization Type material: Holotype. Male extracted from soil samples
and manually edited and trimmed of the poorly aligned posi- collected from the rhizosphere of grapevine (Vitis vinifera) at
tions, using a light-filtering strategy (up to 20% of alignment Montemayor, Córdoba province, southern Spain (37°38ʹ11.7ʹʹ
positions), which has little impact on tree accuracy and may save N latitude, 004°42ʹ21.9ʹʹ W longitude) by J. Martín Barbarroja
some computation time as suggested by Tan et al. (2015), since and G. León Ropero, mounted in pure glycerine and deposited
methods for automated filtering of multiple sequence align- in the nematode collection of Ghent University, Ghent, Belgium
ments frequently worsen single-gene phylogenetic inference (slide number UGMD 104445).
(Tan et al. 2015). Phylogenetic analyses of the sequence datasets
were based on Bayesian inference (BI) using MrBayes 3.1.2 Paratypes: Male and female paratypes extracted from soil sam-
(Ronquist and Huelsenbeck 2003). The best-fit model of DNA ples collected from the rhizosphere of grapevine at Montemayor,
evolution was achieved using JModelTest v.2.1.7 (Darriba et al. Córdoba province, southern Spain, were deposited in the
Figure 1. Line drawings of Paratrichodorus uliaensis sp. nov.. Male. A, B, pharyngeal region. C, spicules. D, G, posterior body region with
copulatory apparatus. E, F, anterior part of testis. Female paratypes. H, I, pharyngeal region. J, K, reproductive system: posterior branch and
total system. L, N, vagina region. M, tail region. Holotype male, A, D; paratype male, F; paratype male, E. Specimens from La Rambla locality.
Scale bars = 20 µm.
Figure 2. Light micrographs of Paratrichodorus uliaensis sp. nov.. A–D, holotype male. A, pharyngeal region. B, total view. C, testis; D, tail region
with copulatory apparatus. Female paratypes. E, reproductive system. F, G, vagina region. Scale bars A, C-G = 10 µm; B = 100 µm.
following nematode collections: Ghent University, Ghent, rarely a combination of a short dorsal intestinal and a ventral
Belgium (slide numbers males and females paratype UGMD pharyngeal overlap; posterior ventrosublateral gland nuclei
104445- UGMD 104449) and one male and one female close to pharyngo-intestinal junction. Nerve ring surrounding
paratypes at the USDA Nematode Collection, Beltsville, MD, isthmus; secretory-excretory pore (SE-pore) at level of anterior
USA (collection number T-8015p). pharyngeal bulb when pharynx relaxed. A single ventromedian
CP at rather a short distance (10–22 µm) anterior to SE-pore.
Additional material examined: An additional population was col- Reproductive system monorchic, testis with short germinal
lected also in grapevine at La Rambla, Córdoba province, Spain zone (25 µm in holotype), and sperm with small oval-shaped
(37°37ʹ17.9ʹʹ N latitude, 4°42ʹ13.3ʹʹ W longitude). nucleus (2.5 × 1.5 µm), closely packed with fibrillar appear-
ance. Spicules medium-sized (average 44.5 µm), nearly straight,
Etymology: The species epithet refers to the roman name (Ulia) with marked capitulum, blade largely equally wide, but distally
of Montemayor, the type locality where the type specimens were tapered, transversely striated, except at extremities. Copulatory
collected. muscles hardly developed; capsule of suspensor muscles very
thin, around anterior half of spicule; gubernaculum with thick-
Description of male: Body more or less straight, cigar-shaped, ened distal part, parallel to spicules. Bursa narrow, rather short,
often with swollen cuticle (3.5–7.5 µm at midbody) upon fix- extending from head of retracted spicule to subterminally on
ation and anteriorly slightly tapered to a rounded lip region ap- tail. Three precloacal supplements, posterior two (SP1 and
pearing offset by the protruding double papillae (four cephalic SP2) opposite distal half of the retracted spicules with SP1 at
and two subdorsal and two subventral outer labial papillae). about equal distance from cloacal opening and from SP2; SP3
Onchiostyle rather small (average 33.5 µm long). Pharynx at little developed and non-protruding, located about 2.5 spicule
posterior isthmus either gradually widening to a posterior bulb lengths from the cloacal opening. A pair of well-developed
or bulb more or less marked, usually with a pronounced (8–37 postcloacal supplements close to the cloacal opening, the latter
µm) ventral overlap of the ventrosublateral pharyngeal glands, with a small protruding anterior lip flap (in ventral view visible
Figure 3. Line drawings for comparison of vagina in the Paratrichodorus allius species-complex group. A, B, P. uliaensis sp. nov.; C, D. P.
benalupensis sp. nov.; E, P. hinogerus sp. nov.; F, G, P. tarifaensis sp. nov.; H, P. asidonaensis sp. nov.; I, P. rocianerus sp. nov.; J, P. litoralis sp. nov.; K,
L, P. paraallius sp. nov.; M–P, P. allius based on Sturhan (1989); Q, P. allius, paratype; R, S, P. teres, paratypes.
as two flaps bordering the opening). Tail short, less than one secondary features. Digestive system with obvious ventral
anal body diameter long and rounded conical. A pair of caudal overlap of pharyngeal glands (15–53 µm). SE-pore at posterior
pores subterminally near tail tip; cuticle slightly thicker at tail tip level of isthmus or anterior end of pharyngeal bulb. Reproductive
(average 3.5 µm). system didelphic-amphidelphic with both branches about
equally developed, or one branch may be longer in presence of
Description of female: Body slightly ventrally curved; largely developed oocytes or egg; oviduct cells finely granular; sperm
similar to male apart from reproductive system and male cells, when present, spread along uterus, no spermathecae; vagina
Table 2. Morphometrics of Paratrichodorus uliaensis sp. nov. male and female paratype specimens and specimens from La Ramblaa.
Holotype Paratype Paratype La Rambla La Rambla
male males females males females
N 9 7 11 6
L 788 660–790 690–815 630–895 745–1000
708 ± 37 742 ± 43 748 ± 78 918 ± 97
Onchiostyle 36 30–37 34–37 33–38 34.5–41
33.5 ± 2.3 35.4 ± 31.2 36 ± 1.8 36.3 ± 2.5
Onchium 16 13–17.5 15–15.5 14–21.5 15–18
15.0 ± 1.5 15.3 ± 0.3 17.1 ± 2.5 16.3 ± 1.0
Pharyngostom 44 37–45 39–46 38.5–48 40–47
41.2 ± 2.3 42.3 ± 2.3 44.6 ± 3.5 44.0 ± 2.6
Pharynx 123 88–128 111–128 97–151 118–141
111 ± 12 115 ± 9 125 ± 18 132 ± 8
Anterior to guide ring 17 13–17 14–18 12.5–19 15–19
14 ± 1.9 16.2 ± 1.7 16.5 ± 2.5 17.2 ± 1.5
Anterior to nerve ring 52 44–57 45–53 49–63 49–55
49 ± 4.9 50 ± 3.2 53.3 ± 5.6 53.2 ± 3.7
Anterior to CP1/anterior to EP in female 83 72–96 87–134 62–101 74–116
80.7 ± 7.1 100 ± 18 81.1 ± 12 96.2 ± 18
Distance CP1-EP 11 10–22 9–26
13.9 ± 4.0 15.2 ± 7.6
mbd at cardia 30 27–37 24–38 26–39 30–35.5
32 ± 4.7 32 ± 4.9 31.1 ± 3.3 32.3 ± 2.1
mbd midbody 33 29–38 32–42 28–41 31–39
34.1 ± 5.9 35.7 ± 3.9 32.6 ± 3.6 33.8 ± 2.8
abd/♀: length anterior ovary 17 16–19 38–101 15.5–19 34–106
18 ± 2.1 80.3 ± 24.8 16.9 ± 1.4 75.7 ± 27.5
Spicule length/ ♀: length anterior branch 45 42–47 173–278 41–49 211–320
44.5 ± 1.6 231 ± 40 45.55 ± 2.6 259 ± 45
Gubernaculum/ ♀: length posterior ovary 16 10–15 56–113 9–16 34–109
11.8 ± 2.3 78.7 ± 19.8 11.9 ± 2.2 67.2 ± 24.4
Distance SP1-cloacal opening/♀ length posterior 12.5 9.5–12 153–279 6–13 139–276
branch 10.2 ± 0.9 211 ± 44 10.3 ± 1.8 197 ± 45
Distance SP1-SP2/ ♀ length vagina 9 10.5–14 7.5–9 10–16 7.5–9

12.1 ± 1.2 7.9 ± 0.7 13.0 ± 2.9 8.0 ± 0.6
Distance SP2-SP3/♀ vagina % cbw 104 65–120 20.7–23.7 76–115 23.1–24.2
95.3 ± 17.5 22.4 ± 1.5 87.3 ± 14.2 23.7 ± 0.5
Tail/♀G1 11 9–10 24.5–37.6 6.5–11 23.6–33.4
9.6 ± 0.4 31.0 ± 4.6 9.1 ± 1.53 28.3 ± 4.4
ab 23.88 14.5–25.0 17.6–22.5 16.2–26.8 24.0–28.4
20.8 ± 3.7 20.8 ± 1.9 22.6 ± 3.4 27.1 ± 2.0
b 6.41 5.4–7.38 5.5–7.3 4.8–7.7 5.5–7.6
6.4 ± 0.7 6.4 ± 0.7 6.3 ± 1.2 7.0 ± 0.9
c/♀V 71.64 69.6–79.6 48.4–60.0 66.5–110 52.56–59.4
73.4 ± 3.7 54.2 ± 4.2 81.3 ± 15.7 56.0 ± 2.6
T/♀G2 85.94 85.0–98.6 18.8–39.0 54.1–98.5 15.5–27.9
87.0 ± 6.2 28.6 ± 6.6 69.8 ± 18.8 21.7 ± 5.1
a
Measurements are in µm and in the form: range and mean ± SD.
b
a, body length/maximum body width; abw (anal body width); b, body length/pharyngeal length; c, body length/tail length; cbw (corresponding body width); CP (ventromedian
cervical papilla); CP1 (anterior ventromedian cervical papilla); G1, G2 (anterior, posterior genital branch length, respectively/body length) × 100; EP (secretory-excretory pore); L
(total body length); mbw (maximum body width); N (number of specimens studied); SP1, SP2 SP3 (posterior, second and third precloacal supplements, respectively); V (distance
from anterior end to vulva/body length) × 100; T (distance from cloacal aperture to anterior end of testis/body length) × 100.
more or less barrel-shaped, short [average 8 µm long or 22% of to oval sclerotized pieces, slightly obliquely oriented, tips
corresponding body diameter (cbd)]; pars refringens vaginae in 2.0–2.5 µm apart; vulva at about midbody (average 55%) and
optical section with minute (1.0–1.5 µm), rounded, triangular a longitudinal slit in ventral view. Sublateral body pores mostly
absent (observed in only three specimens from type popula- Type material: Holotype. Male extracted from soil samples col-
tion); could be obscured by swelling of body cuticle. lected from the rhizosphere of unidentified grasses at Benalup-
Casas Viejas, Cádiz province, southern Spain (36°18ʹ24.33ʹʹ N
Code within the tabular identification key (based on: Decraemer and latitude, 5°52ʹ52.38ʹʹ W longitude) by J. Martín Barbarroja and
Baujard 1998): For males: A 212 (average, min., max.), B11, C22, G. León Ropero, mounted in pure glycerine and deposited in the
D1, E0, F3, G2, H23, I2(4), J1, K2, L2, M27, N25, O1, P1. For nematode collection of Ghent University, Ghent, Belgium (slide
females: A212 (average, min., max.), B11, C11, D1, E23, F2 (4), number UGMD 104450).
G3, H53, I1, J13, K2, L3, M1, N1, O2, P25, Q4(1), R2, S1, T1.
Paratypes: Male and female paratypes extracted from soil sam-
Remarks: In three females, lateral body pores were observed ples collected from the rhizosphere of unidentified grasses at
(Q1), one postvulvar sublateral pore at nearly 2.5 vaginal body Benalup-Casas Viejas, Cádiz province, southern Spain, were de-
diameter from vulva, and one prevulvar sublateral body pore six posited in the following nematode collections: Ghent University,
body diameters anterior to vulva. Ghent, Belgium (slide numbers males and female paratype
UGMD 104450, UGMD 104453); and one male and one fe-
Diagnosis and relationships: Paratrichodorus uliaensis sp. nov. male paratypes at the USDA Nematode Collection, Beltsville,
is characterized in both sexes by the rather short onchiostyle MD, USA (collection number T-8016p).
(smaller than 40 µm) of 33.5 (30–37) µm in males and 35.4
(34–37) µm in females; in males by presence of a single CP, testis Etymology: The species epithet refers to Benalup-Casas Viejas,
with small sperm cells with small nucleus (2.5 × 1.5 µm), and the type locality where the type specimens were collected.
often fibrillar appearance, medium-sized spicules [44.5 (42–47)
µm] with marked capitulum and three precloacal supplements Description of male: Body nearly straight, cigar-shaped, with
with the posterior two within distal half of retracted spicules, swollen cuticle (average 4 µm at midbody) upon fixation; an-
and in females by the very short, less than 25% of corresponding teriorly clearly tapered to narrower, rounded, low-lip region
body diameter or 22.4% (20.7–23.7 %), barrel-shaped vagina appearing offset by the protruding double papillae (four ceph-
with minor midway indentation at level of constrictor muscles, alic and two subdorsal and two subventral outer labial papillae)
minute rounded triangular sclerotized pieces, oblique in orien- and swelling of body cuticle. Onchiostyle rather small (average
tation and clearly separated from one another, sublateral body 37 µm long); reserve onchium (14–17 µm) present in all
pores mostly not observed and vulva in ventral view a longitu- adults. Pharynx at posterior isthmus either gradually widening
dinal slit. Sperm spread along uteri. to a posterior bulb with a combined short ventral overlap of
Based upon the tabular identification keys for males ventrosublateral pharyngeal glands and short dorsal intestinal
(Decraemer and Baujard 1998), sorting on primary features F overlap; posterior ventrosublateral gland nuclei at some distance
(number of ventromedian precloacal supplements), feature D from dorsal gland nucleus. Nerve ring surrounding isthmus;
(number of CP), and P (habitus) the new species belongs to SE-pore opposite anterior pharyngeal bulb when pharynx re-
species group 6. Further sorting by feature N (type of sperm laxed. A single ventromedian CP at rather short distance (9–15
cells: small cell with small nucleus) grouped the new species µm) anterior to SE-pore. Reproductive system monorchic, testis
with P. catharinae Vermeulen and Heyns 1983, P. meyeri De with short germinal zone (34 µm in holotype) and sperm with
Waele and Kilian 1992, P. paraporosus Khan et al. 1989, and P. small, round to oval-shaped nucleus (2.0–2.5 µm × 1.5 µm),
tunisiensis (Siddiqi 1963) Siddiqi 1974. Paratrichodorus uliaensis closely packed, giving the vesicular seminalis a fibrillar appear-
sp. nov. can be distinguished from these four species by a shorter ance. Spicules medium-sized (average 48 µm), nearly straight,
onchiostyle (feature B), average less than or equal to 40 µm. with marked capitulum, blade largely equally wide, transversely
For females, sorting on primary features D and C, followed striated except anterior end with capitulum and in tapered
by L and K, resulted in a classification in subgroup 1-12 in distal end. Copulatory muscles hardly developed and obscure;
Decraemer and Baujard (1998). Further sorting on type of capsule of suspensor muscles very thin, around anterior half of
sperm cells (feature P) grouped the new species with the known spicule; gubernaculum with thickened distal part, parallel to
species P. acaudatus (Siddiqi 1960) Siddiqi 1974, P. allius, and spicules. Bursa narrow, extending largely along spicule region to
P. teres (subgroup 1-12) from which it can be distinguished by subterminally on tail. Three precloacal supplements, posterior
the shorter onchiostyle (<40 µm vs. >40µm). The new species two (SP1 and SP2) more or less at level of distal half of the re-
agrees with P. acaudatus in male being common but differs in tracted spicules with SP1 midway between cloacal opening and
vagina shape (quadrangular in P. acaudatus) and in male char- SP2; SP3 little developed and non-protruding, located about
acteristics such as absence of CP and much longer spicules in P. two spicule lengths from the cloacal opening. A pair of well-
acaudatus. Paratrichodorus uliaensis sp. nov. differs from both P. developed postcloacal supplements close to the cloacal opening;
allius and P. teres by males being common, from P. allius by lo- the anterior cloacal lip bifid with two thin-walled protrusions.
cation of sperm, i.e. only in the uteri and not in ovarial sac, and Tail short, less than one anal body diameter long and rounded
from P. teres in shape of vagina without widened midpart. conical. A pair of caudal pores subterminally near tail tip; cuticle
slightly thicker at tail tip.
Paratrichodorus benalupensis sp. nov. Decraemer et al.
Description of female: Body straight to slightly ventrally curved;
(Figs 3C, D, 4, 5; Table 3) largely similar to male, except for reproductive system and
Zoobank registration: urn:lsid:zoobank.org:act:FB05C936- male secondary features. Digestive system with a combined
0F15-4624-997A-9C54782EE924 dorsal intestinal and a ventral pharyngeal overlap; reserve
Figure 4. Line drawings of Paratrichodorus benalupensis sp. nov.. Male. A, pharyngeal region. B, C, E, tail region with copulatory apparatus. E, in
ventral view. D, testis, partim. Female paratype. F, pharyngeal region. G, vagina region. C, D, holotype. Scale bars = 20 µm.
onchium (12 µm) present in all females. Secretory-excretory Diagnosis and relationships: Paratrichodorus benalupensis sp. nov.
pore opposite anterior end of pharyngeal bulb. Reproductive is characterized by body medium-sized (average 900 µm in fe-
system didelphic-amphidelphic with both branches about male; 775 µm in male), onchiostyle medium-sized (average
equally developed, or one branch longer in the presence of de- 41 µm in female, 39 µm in male), and all adults with a reserve
veloped oocytes or egg; oviduct cells finely granular; sperm onchium. Male with a single CP, shortly anterior to SE pore;
cells, when present, spread along uteri, no spermathecae; va- straight spicules with marked capitulum, small sperm cells
gina more or less barrel-shaped with minor indentation at with clear nucleus closely packed giving fibrillar appearance
level of constrictor muscles, about 25% of corresponding body and thereby giving the vas deferens a striated appearance; three
width long; pars refringens vaginae in optical section with precloacal supplements, the posterior two more or less at level of
minute (1 µm) rounded triangular to oval sclerotized pieces, the posterior half of the retracted spicules; a pair of postcloacal
obliquely oriented, tips separated by 3 µm (average); vulva at supplements near cloacal opening; the latter with pronounced
about midbody (average 52%). No sublateral/subventral body bifid anterior lip. Females with vulva more or less barrel-shaped,
pores observed. about 25% of corresponding body width long; vaginal scler-
otized pieces in optical section minute (1 µm), rounded tri-
Code within the tabular identification key (based on: Decraemer angular to oval shaped, obliquely oriented, tips separated by 3
and Baujard 1998): For males: A 222, B1(2), C2, D1, E0, F3, µm (average). Sperm spread over uteri. No sublateral/subventral
G2, H3, I4, J1, K2, L2, M27, N5, O1, P1. For females: A222, body pores observed.
B12, C1, D1, E3, F4, G-, H33, I1, J1, K12, L3, M1, N1, O2, P2, Use of the tabular identification key for Paratrichodorus
Q4, R2, S1, T1. males (based on Decraemer and Baujard 1998) and sorting by
Figure 5. Light micrographs of Paratrichodorus benalupensis sp. nov.. A, anterior body region. D, testis with germinal zone. E, region pharyngo-
intestinal junction. F–H, posterior body region of male holotype with focus on spicule, gubernaculum and bursa respectively. Female paratype.
B, anterior body region. C, vagina region. Scale bars A–H = 10 µm.
primary feature D, followed by feature N (sperm cells), grouped feature N (occurrence of males) groups P. benalupensis sp. nov.
the P. benalupensis sp. nov. together with P. catharinae, P. meyeri, with P. acaudatus and P. tunisiensis, P. lobatus (Colbran 1965)
P. paraporosus, P. tunisiensis, and the previously described new Siddiqi 1974, and the previously described P. uliaensis sp. nov..
species P. uliaensis sp. nov.. It differs from the known species The new species differs from all these species by the presence
(except P. paraporosus) by the shorter onchiostyle ≤40 µm vs. of a reserve onchium in the adult and from all known species
>40 µm and from P. tunisiensis also in spicule shape [straight vs. by the shorter onchiostyle, a feature it has in common with the
more curved (type population, populations from Italy; Siddiqi previously described new species. So far, the presence of a re-
1963, Roca and Lamberti 1984, Tanha Maafi and Decraemer serve onchium in all specimens of a population has only been
2002)] and from P. paraporosus in the presence of only three SP used as a diagnostic feature in the genus Nanidorus to differen-
instead of four SP. Paratrichodorus benalupensis sp. nov. can be tiate Nanidorus minor and N. nanus (Allen 1957) Siddiqi 1974
distinguished from all four species by the presence of a reserve from the other species. In all other examples this criterion is con-
onchium in the adult. The presence of a reserve onchium in the sidered as accidental (see Discussion). Except for the presence of
adult is the main feature to distinguish it from P. uliaensis sp. nov.. a reserve onchium in the adult of P. uliaensis sp. nov., this species
For females of the Benalup sample, sorting on primary fea- is almost undistinguishable morphologically from P. benalupensis
tures D and C, followed by L and K (variable), resulted in a clas- sp. nov., being described here as separate new species, based on
sification in subgroups 1-11 or 1-12 in Decraemer and Baujard the additional support given by morphometrical and molecular
(1998); further sorting on feature P (type of sperm cells) and analyses (see below).
Table 3. Morphometrics of Paratrichodorus benalupensis sp. nov. male and female paratype specimensa.
Holotype male Paratype males Paratype females
N 4 4
L 800 610–882 841–986
775 ± 117 900 ± 62
Onchiostyle 37 36–42 39–42
39.3 ± 2.5 40.9 ± 1.3
Onchium 17 18–19 18.5–21.5
18.5 ± 0.5 19.8 ± 1.3
Pharyngostom 46 43–50 47–50
47.5 ± 3.3 48.8 ± 1.5
Pharynx 111 91–148 107–176
129 ± 27 147 ± 31
Anterior to guide ring 18 18.5–21 18–21
19.8 ± 1.8 19 ± 1.7
Anterior to nerve ring 94 45–56 52 (N = 1)
51.7 ± 5.9
Anterior to CP1/anterior to EP in female 94 52–93 101–106
78.5 ± 18.5 103 ± 4
Distance CP1-EP 15 9–13
11.5 ± 1.9
mbd at cardia 30 28–34 34–43
30.8 ± 2.5 38.4 ± 3.7
mbd midbody 33 33–34 38.5–46
33.3 ± 0.6 41.6 ± 3.5
abd/♀: length anterior ovary 18 17–27 63; 101 (2)
20.1 ± 4.6
Spicule length/ ♀: length anterior branch 49 48–49 191; 316 (2)
48.3 ± 0.6
Gubernaculum/ ♀: length posterior ovary 15 12–18.5 25–94
14.2 ± 3.8 64.5 ± 30.3
Distance SP1-cloacal opening/♀ length posterior branch 14 12–16 207–273
13.3 ± 1.9 233 ± 29
Distance SP1–SP2/ ♀ length vagina 18 12.5–18 7–8
15.6 ± 2.4 7.9 ± 0.6
Distance SP2–SP3/♀ vagina % cbw 108 72–124 22.6–26.6
107 ± 24.20 24.8 ± 1.8
Tail/♀G1 12 7.5–15 19.4; 35.1(2)
10.6 ± 3.2
ab 24.2 18.5–26.7 20.9–22.7
23.1 ± 3.4 21.6 ± 0.7
b 7.21 5.5–6.7 5.4–7.9
6.1 ± 0.6 6.1 ± 1.1
c/ ♀V 66.7 52.6–88.2 51.8–52.9
76.0 ± 15.9 52.4 ± 0.5
T/♀G2 66.0 58.7–63.4 23.8–30.3
61.2 ± 2.4 25.9 ± 3.0
a
b
Paratrichodorus hinogerus sp. nov. Decraemer et al. Type material: Holotype. A female was chosen as holotype be-
cause males were rare; only two males (one in dorso-ventral
(Figs 3E, 6, 7; Table 4) position) were found among 23 females and juveniles. Female
Zoobank registration: urn:lsid:zoobank.org:act:58A44816- extracted from soil samples collected from the rhizosphere of
941D-4441-8693-C448933A14E3 stone pine (Pinus pinea) at Hinojos, Huelva province, southern
Figure 6. Line drawings of Paratrichodorus hinogerus sp. nov.. Male paratype. A, pharyngeal region. B, posterior body region. C, reproductive
system and tail region. Female. D, pharyngeal region, paratype. E, vagina region paratype. G, reproductive system holotype. F, tail region,
holotype. Scale bars = 20 µm.
Spain (37°17ʹ42.2ʹʹ N latitude, 6°23ʹ46.0ʹʹ W longitude) by J. nematode collections: Ghent University, Ghent, Belgium (slide
Martín Barbarroja and G. León Ropero, mounted in pure gly- numbers male and females paratype UGMD 104451-104452);
cerine and deposited in the nematode collection of Ghent and one female paratype at the USDA Nematode Collection,
University, Ghent, Belgium (slide number UGMD 104451). Beltsville, MD, USA (collection number T-8017p).
Paratypes: Male and female paratypes extracted from soil sam- Additional material examined: Additional populations were
ples collected from the rhizosphere of stone pine at Hinojos, collected also in the rhizosphere of wild olive at Sanlúcar de
Huelva province, southern Spain, were deposited in the following Barrameda, Cádiz province, Spain (36°51ʹ20.0ʹʹN latitude,
Figure 7. Light micrographs of Paratrichodorus hinogerus sp. nov.. A–D, male paratype. A, total view. B, testis, germinal zone. C, tail region and
copulatory apparatus. D, bursa (arrow). E–I, females. E, total view paratype. F, pharyngeal region. G-H, vagina region. I, reproductive system in
holotype, arrow point to ovarial sac with sperm. Scale bars A, E = 100 µm; B–D, F–I = 20 µm.
6°19ʹ13.6ʹʹ W longitude), and the rhizosphere of cork oak widening in front of a more or less marked posterior bulb, with a
(Quercus suber) at Bonares, Huelva province, Spain (37°14ʹ11.1ʹʹ ventral overlap of the ventrosublateral pharyngeal glands as well
N latitude, 6°40ʹ34.7ʹʹ W longitude), unfortunately the speci- as a dorsal overlap by the intestine; posterior ventrosublateral
mens of this population were not in good conditions for light gland nuclei close to pharyngo-intestinal junction and far from
microscopic study, and were identified only molecularly. the dorsal gland nucleus situated at the anterior end of the bulb.
Nerve ring surrounding isthmus; SE-pore opposite anterior pha-
Etymology: The species epithet refers to the name of the inhabit- ryngeal bulb. Reproductive system didelphic-amphidelphic with
ants of Hinojos, the type locality where the type specimens were both branches more or less equally developed, with the posterior
collected. branch usually slightly shorter; influenced by difference in de-
velopment of ovarial sac with presence of sperm; oviduct cells
Description of female: Body medium-sized (average 730 µm), more finely granular. Vagina short (less than one-fifth of corresponding
or less straight cigar-shaped or slightly curved ventrad, often with body width), rounded with midway indentation and surrounded
swollen cuticle (4–7 µm at midbody) upon fixation; anteriorly by narrow constrictor muscles; pars refringens vaginae in optical
tapered to a narrower, rounded lip region with protruding double section with minute (1–1.5 µm) narrow triangular sclerotized
papillae (four cephalic and two subdorsal and two subventral pieces, slightly obliquely oriented, tips separated by 2–3 µm; vulva
outer labial papillae). Onchiostyle rather long (average 56 µm at about midbody (average 56%); a longitudinal slit in ventral view.
long). Pharynx with a short narrower part (isthmus), slightly No sublateral/subventral body pores. Tail minute, terminal cuticle
Table 4. Morphometrics of Paratrichodorus hinogerus sp. nov. male and female paratype specimens and specimens from Sanlúcar de
Barramedaa.
Holotype Paratype Paratype Sanlúcar de
female females male Barrameda females
N 8 1 3
L 796 625–810 725 850–1060
729 ± 70 981 ± 115
Onchiostyle 54 51–60 58 60–62
56.1 ± 2.5 61.3 ± 1.2
Onchium 26 25–30 30 28–32
28.0 ± 1.7 30.3 ± 2.1
Pharyngostom 62 60–67 61 69–71
64.3 ± 2.6 70.0 ± 1.0
Pharynx 130 124–148 142 158–173
136 ± 9 167 ± 8
Anterior to guide ring 33 24.5–28 27.5 26–28
25.6 ± 1.3 27.0 ± 1.0
Anterior to nerve ring 67 62–73 67 70–78
67.8 ± 3.8 73.0 ± 4.4
Anterior to CP1/anterior to EP in female 104 73–110 88
100 ± 14
Distance CP1-EP 22
mbd at cardia 34 35–48 34 43–46
39.6 ± 4.1 44.7 ± 1.5
mbd midbody 37 35–50 35 45–51
40.4 ± 4.7 48.0 ± 3.0
abd/♀: length anterior ovary 144 56–136 19 86–204
98 ± 27 150 ± 60
Spicule length/ ♀: length anterior branch 228 230–324 48 215–460
272 ± 38 353 ± 127
Gubernaculum/ ♀: length posterior ovary 87 79–120 11 87–145 104 ± 35
96 ± 17
Distance SP1-cloacal opening/♀ length posterior branch 207 153–306 15 175–355
232 ± 47 273 ± 93
Distance SP1–SP2/ ♀ length vagina 8.5 6.5–9.0 22 8.5–9.0
7.4 ± 0.8 8.7 ± 0.3
Distance SP2–SP3/♀ vagina % cbw 23 14.0–23.7 75 16.7–18.9
18.6 ± 3.3 18.1 ± 1.2
Tail/♀G1 37.1 31.1–40.2 11 25.2–43.5
36.7 ± 4.1 35.3 ± 9.3
ab 21.51 15.0–21.6 20.7 18.9–21.6
18.8 ± 2.7 20.4 ± 1.4
b 6.1 5.1–6.2 5.1 4.9–6.7
5.4 ± 0.4 5.9 ± 0.9
c/ ♀V 55.03 53.6–58.4 65.9 52.9–54.7
56.1 ± 3.3 53.7 ± 0.9
T/♀G2 26.0 21.1–37.9 57.10 16.3–34.4
31.1 ± 5.2 28.3 ± 10.3
a
b
slightly thicker; a pair of caudal pores present. The populations similar long onchiostyle (60–62 µm) but body on the higher range
from Sanlúcar de Barrameda also showed females with sperm (longer body) (average 980 µm for three females in sample code
at level ovarial sac, similar sclerotized pieces in size, shape, loca- AR016; 830 µm, in sample code AR015, one female) and slightly
tion, very short vagina (<20% of corresponding body diameter), more anterior vulva position with V% in AR016 of 53–54%.
Description of male: Largely similar to female, apart from repro- sperm cell) and by feature N (occurrence of males) groups the
ductive system, male secondary features and a somewhat longer new species with the known species P. allius and P. teres. As in
tail. Onchiostyle equally long and pharynx with clear ventral P. allius males are rare or absent in most populations of P. teres.
pharyngeal overlap (18 µm) and shorter (10 µm) dorsal overlap Paratrichodorus hinogerus sp. nov. also agrees with P. allius in the
by intestine. Secretory-excretory pore opposite anterior pha- presence of sperm in the ovarial sac vs. along the uteri in P. teres
ryngeal bulb but posterior to a single ventromedian cervical and in the narrow triangular, slightly oblique vaginal sclerotized
papilla; no lateral cervical papillae observed. Reproductive pieces and vagina barrel-shaped, slightly indented at level of
system monorchic, testis with very short germinal zone (12 µm) narrow constrictor muscles. In P. teres vaginal sclerotized pieces
and sperm with small, oval-shaped nucleus (2.0–2.5 × 1µm). are more oval or rounded triangular and the vagina mostly shows
Spicules medium-sized, slightly ventrally curved, hardly marked midway a widening (Fig. 3). Paratrichodorus hinogerus sp. nov.
equally wide capitulum; blade largely equally wide, tapering is also distinguished from these species by a longer onchiostyle
slightly anteriorly, more pronounced posteriorly and transversely (averages 56 µm vs. 42.5 µm and 45 µm, respectively).
striated, except for extremities. Copulatory muscles hardly de-
veloped, restricted to region of retracted spicules; capsule of Paratrichodorus species with males without ventromedian
suspensor muscles very thin, enclosing anterior half of spicule; cervical papillae
gubernaculum with thickened distal part, parallel to spicules. Paratrichodorus tarifaensis sp. nov. Decraemer et al.
Bursa narrow, extending from just posterior of spicule head to
subterminally on tail. Three precloacal supplements, posterior (Figs 3F, G, 8, 9; Table 5)
two (SP1 and SP2) opposite retracted spicules with SP1 at about Zoobank registration: urn:lsid:zoobank.org:act:B0C6E238-
equal distance from cloacal opening and from SP2; SP3 little de- 9592-411B-9AED-6C82D68A0CFF
veloped and non-protruding, located about 2.5 spicule lengths
from the cloacal opening. A pair of well-developed postcloacal Type material: Holotype. Male extracted from soil samples col-
supplements close to the cloacal opening, the latter with a small lected from the rhizosphere of wild olive (Olea europaea ssp.
protruding anterior lip flap (in ventral view visible as two flaps sylvestris) at Tarifa, Cádiz province, southern Spain (36°03ʹ49.5ʹʹ
bordering the opening). Tail short, less than one anal body N latitude, 5°40ʹ18.2ʹʹ W longitude) by J. Martín Barbarroja and
diameter long and rounded conical. A pair of posterior caudal G. León Ropero, mounted in pure glycerine and deposited in the
pores subterminally near tail tip. nematode collection of Ghent University, Ghent, Belgium (slide
number UGMD 104455).
Code within the tabular identification key (based on: Decraemer and
Baujard 1998): For males: A 222, B2, C2, D1, E0, F3, G2, H3, Paratypes: Male and female paratypes extracted from soil sam-
I4, J2, K23, L2, M27, N2, O1, P1. For females: A212 (average, ples collected from the rhizosphere of wild olive at Tarifa, Cádiz
min., max), B2(3), C1, D1, E3, F4, G3, H53, I1, J3, K2, L3, M1, province, southern Spain, were deposited in the following nema-
N2, O32, P2, Q4, R2, S1, T1. tode collections: Ghent University, Ghent, Belgium (slide num-
bers males and females paratype UGMD 104454-104458); and
Diagnosis and relationships: Paratrichodorus hinogerus sp. nov. is one male and one female paratypes at the USDA Nematode
characterized by a medium-sized body (average about 730 µm, Collection, Beltsville, MD, USA (collection number T-8018p).
type population) and rather long onchiostyle (56 µm average in
female, 58 µm in male) in both sexes. Females possess a very short Additional material examined: An additional population was
(less than one-fifth corresponding body width) rounded vagina collected also in the rhizosphere of cork oak (Quercus suber) at
with minute, narrow, triangular sclerotized pieces, obliquely Benalup-Casas Viejas, Cádiz province, Spain (36°17ʹ24.07ʹʹ N
orientation and clearly separated from one another. Reflexed latitude, 5°55ʹ57.05ʹʹ W longitude).
ovaries often with a long ovarial sac filled with sperm. No sub-
lateral/subventral body pores observed. Males rare compared to Etymology: The species epithet refers to Tarifa, the type locality
females and possess a single ventromedian cervical papilla, testis where the type specimens were collected.
with small sperm cells and small nucleus, medium-sized spicules
(48 µm) with slightly marked capitulum, blade finely striated Description of male: Body straight, cigar-shaped, often with
and three precloacal supplements with the posterior two spread swollen cuticle (3–7.5 µm at midbody) upon fixation; anteriorly
along the retracted spicules. tapered to low, rounded lip region appearing offset by the pro-
Based upon the tabular identification keys for males truding double papillae (four cephalic and two subdorsal and
(Decraemer and Baujard 1998), sorting of the data primarily two subventral outer labial papillae). Onchiostyle rather long
on character D (number of CP), followed by feature N (type (average 54 µm long). Pharynx at posterior isthmus gradually
of sperm cells), groups the new species with the known spe- widening to a posterior bulb, with a short to pronounced (5–48
cies P. catharinae, P. meyeri and P. tunisiensis and with former µm) ventral overlap of the ventrosublateral pharyngeal glands,
new species described P. uliaensis sp. nov. and P. benalupensis sp. rarely (one male) with a combination of a short dorsal overlap by
nov.. It differs from all these species by male being rare and by the intestine and a short ventral overlap; dorsal gland nucleus in
a longer onchiostyle (>55 µm average) and in addition from P. the anterior third of bulb, both pairs of ventrosublateral gland nu-
benalupensis sp. nov. by absence of reserve onchium. clei visible, the posterior pair located in the posterior third of the
For females, sorting on primary features D, C, and K and L bulb. Nerve ring surrounding isthmus close to the pharyngostom
of the tabular identification key followed by, feature P (type of base; SE-pore opposite anterior pharyngeal bulb. Ventromedian
Figure 8. Line drawings of Paratrichodorus tarifaensis sp. nov.. Males. A, E, D, pharyngeal region. B, C, E, F, tail region with copulatory
apparatus. F, ventral view. Females. G–H, pharyngeal region. I, reproductive system. J, vagina region. K, tail. Holotype (A, B), paratypes (C–K).
Scale bars = 20 µm.
Figure 9. Light micrographs of Paratrichodorus tarifaensis sp. nov.. Males. A, total view holotype. B, pharyngeal region holotype. C–E,
copulatory apparatus in holotype and paratypes. D, E, ventral view. F, testis, holotype. Female paratypes. G–J, vagina region. Scale bars A = 100
µm; B–E, G-J = 10 µm; F = 20 µm.
or lateral cervical papillae absent. Reproductive system spicules. Bursa narrow, extending usually from head of retracted
monorchic, sperm small with oval-shaped nucleus (3.0 × 1.5 spicule to subterminally on tail. Three precloacal supplements,
µm). Spicules medium-sized (average 53.5 µm), slightly ventrally posterior two (SP1 and SP2) opposite distal half or the retracted
curved, with marked capitulum, blade largely equally wide but spicules; SP3, little developed and non-protruding, located
slightly tapered towards an open distal tip; spicules ornamented about 2.5 spicule lengths from the cloacal opening. A pair of
with fine transverse striae, except at extremities. Exceptionally, well-developed postcloacal supplements adjacent to the cloacal
one male with different spicule length (52 µm for left spicule and opening, the latter with a small protruding anterior lip flap (in
56 µm for right spicule). Copulatory muscles hardly developed, ventral view visible as two flaps bordering the opening). Tail less
obscure; capsule of suspensor muscles thin, around anterior half than one anal body diameter long and broad conical. A pair of
of spicule; gubernaculum with thickened distal part parallel to posterior caudal pores subterminally near tail tip.
Table 5. Morphometrics of Paratrichodorus tarifaensis sp. nov. male and female paratype specimens and specimens from Benalup-Casas Viejasa.
Holotype Paratypes Benalup-Casas Viejas
male males females males females
N 9 9 3 3
L 975 610–980 655–1130 865–1180 836–1035
805 ± 123 870 ± 125 1048 ± 162 960 ± 89
Onchiostyle 56 50–59 50–57 49–56 46–53
54.2 ± 2.9 54.3 ± 2.2 51.7 ± 3.8 49.0 ± 2.9
Onchium 30 24–29 24–31 24.0–27.0 25–26 25.7 ± 0.5
26.6 ± 4.5 25.9 ± 7.3 25.0 ± 1.7
Pharyngostom 66 56–70 52–88 58–60 51–62
62.6 ± 4.6 64.7 ± 9.1 59.0 ± 1.41 58.0 ± 5.0
Pharynx 173 116–178 127–198 153–180 143–172
149 ± 24 158 ± 21 167 ± 19 157 ± 12
Ventral overlap pharynx 26 4.5–48 11–23 (N = 3) 11 (N = 1)
17.1 ± 14.5
Anterior to guide ring 26 23–26 21–27 21–27 18–24
24.8 ± 1.0 24.3 ± 1.9 24.0 ± 4.2 21.0 ± 2.5
Anterior to nerve ring 85 56–73 55–72 70–81 67, 72 (N = 2)
65.9 ± 6.6 67.6 ± 6.4 75.0 ± 7.8
Anterior to EP 119 76–152 83–121 115–130 104 (N = 1)
102 ± 24 107 ± 13 120 ± 9
mbd at cardia 32 32–45 33–41 31–34 31–39
36.6 ± 3.7 35.8 ± 2.5 32.0 ± 1.7 34.2 ± 3.5
mbd midbody 36 36.5–48 36–48 31–34 35–44
41.7 ± 3.8 40.7 ± 3.4 32.5 ± 1.5 38.3 ± 4.0
abd/♀: length anterior ovary 22 21–24 54–107 12–14 88–120
22.4 ± 0.9 83.8 ± 16.1 13 ± 1.4 103 ± 13
Spicule length/ ♀: length anterior branch 54 49–56 261–340 51–59 287–309
53.4 ± 2.0 275 ± 42 54.3 ± 4.2 299 ± 9
Gubernaculum/ ♀: length posterior ovary 14 12.5–14 65–107 11–14 88–120
13.3 ± 0.9 78.4 ± 17.0 12.7 ± 1.5 107 ± 14
Distance SP1-cloacal opening/♀ length posterior 7.5 9–15.5 139–320 15 279–349
branch 10.8 ± 2.1 239 ± 57 304 ± 32
Distance SP1–SP2/ ♀ length vagina 9 9–16 7.5–9 13–18 8–9

12.4 ± 2.9 8.6 ± 0.6 15.5 ± 3.5 8.5 ± 0.4
Distance SP2–SP3/♀ vagina % cbw 79 40–119 17–24.3 119–164 20.5–23.6
83 ± 24 21.02 ± 2.4 141 ± 32 22.3 ± 1.4
Tail/♀G1 18 10–22 25.4–38.1 11–19 27.7–35.9
14.1 ± 3.7 31.3 ± 4.2 14.7 ± 4.0 31.4 ± 3.4
ab 27.0 15.2–22.2 21.6–30.6 25.5–38.1 23.0–29.6
19.6 ± 3.0 21.6 ± 4.0 32.4 ± 6.4 25.3 ± 3.1
b 5.6 4.8–6.1 4.6–6.7 5.7–7.2 5.8–6.4
5.3 ± 0.8 5.5 ± 0.7 6.5 ± 0.7 6.1 ± 0.7
c/ ♀V 54.1 31.6–66.9 52.3–61.5 57.6–84.4 54.7–56.0
60.6 ± 12.1 56.55 ± 3.3 73.60 ± 14.1 55.48 ± 0.6
T/♀G2 53.9 42.2–98.4 19.8–36.3 55.4–68.5 27.5–34.6
70.1 ± 16.9 27.3 ± 5.7 60.0 ± 7.4 31.8 ± 3.1
a
b
Description of female: Body largely as in male. Reproductive small (1.0–1.5 µm), triangular, oblique to nearly parallel
system with small sperm spreading over the uteri; vagina to vaginal lumen in orientation and clearly separated
short with slight indentation; vaginal sclerotization pieces (2.0–3.0 µm).
Code within the tabular identification key (based on: Paratrichodorus asidonaensis sp. nov. Decraemer et al.
Decraemer and Baujard 1998): For males: A212 (average
(Figs 3H, 10, 11; Table 6)
min max), B22, C22, D0, E0, F3, G2, H3, I2(4), J2?, K2,
L2, M27, N2, O1, P1. For females: A213, B22, C1, D1, Zoobank registration: urn:lsid:zoobank.org:act:3BDC36BC-
E3, F2, G?, H35, I1, J1, K2, L3, M3, N1, O2, P2, Q4, R2, 3F6E-4C09-9CBC-1ABF082A8978
S1, T1.
Type material: Holotype. A female was chosen as holotype be-
Diagnosis and relationships: Paratrichodorus tarifaensis sp. nov. is cause males were rare; only two males (one dorso-ventral pos-
characterized by a similar proportion of males and females, with ition) were found among 23 females and juveniles. Female
a medium-sized body (average 805 µm in male, 870 µm in fe- extracted from soil samples collected from the rhizosphere of
male), medium-sized onchiostyle (average 54 µm), absence of wild olive (Olea europaea ssp. sylvestris) at Medina Sidonia, Cádiz
ventromedian and lateral cervical papillae, small sperm with oval province, southern Spain (36°19ʹ55.2ʹʹ N latitude, 5°51ʹ18.0ʹʹ
nucleus, pharynx mainly with overlapping ventrosublateral glands, W longitude) by J. Martín Barbarroja and G. León Ropero,
medium-sized spicule length (average 53 µm) with finely striated, mounted in pure glycerine and deposited in the nematode col-
slightly ventrally curved and marked capitulum, three SP of which lection of Ghent University, Ghent, Belgium (slide number
the posterior two opposite posterior half retracted spicule, tail UGMD 104459).
shorter than anal width, with a pair of postcloacal supplements
Paratypes: Female paratypes extracted from soil samples col-
near cloacal opening and nearly terminal pair of caudal pores.
lected from the rhizosphere of wild olive at Medina Sidonia,
Females with short vagina (21% cbd, average) with indentation
Cádiz, southern Spain, were deposited in the following nema-
and small triangular vaginal sclerotized pieces (1.5 µm) in optical
tode collections: Ghent University, Ghent, Belgium (slide
section, oblique to almost parallel with vaginal lumen and clearly
numbers male and females paratype UGMD 104459, UGMD
separated.
104460); and one female paratype at the USDA Nematode
Using the tabular identification keys for Paratrichodorus
Collection, Beltsville, MD, USA (collection number
males (Decraemer and Baujard 1998) with sorting of the data
T-8019p).
primarily on character D (number of CP), followed by char-
acter F (number of SP), groups P. tarifaensis sp. nov. with the Additional material examined: An additional population was
known species P. teres and P. rhodesiensis (Siddiqi and Brown collected also in the rhizosphere of wild olive at Benalup-
1965) Siddiqi 1974; three SP can exceptionally also occur in P. Casas Viejas, Cádiz province, Spain (36°18ʹ27.74ʹʹ N latitude,
allius (Siddiqi 1974), in P. lobatus (Decraemer and Reay 1991) 5°52ʹ56.40ʹʹ W longitude).
and in P. namibiensis. Paratrichodorus tarifaensis sp. nov. differs
from P. teres by male sperm with small nucleus but without Etymology: The species epithet refers to Asidona the visigothic
fibrillar appearance in testis and by male common instead of name of Medina Sidonia, the type locality where the type speci-
male rare in most populations of P. teres. It can be differenti- mens were collected.
ated from P. allius by male being common. The new species
differs from P. rhodesiensis by longer body average in type Description of female: Body short (average <550 µm), straight,
population 805 µm vs. 360–690 µm average 645 µm and from slightly tapered anteriorly to a low, rounded lip region with pro-
P. namibiensis by the shorter spicules (average 53 µm vs. 67 truding double papillae (four cephalic and two subdorsal and
µm) and sperm with small nucleus vs. threat-like without nu- subventral outer labial papillae), cuticle swollen (average 4 µm).
cleus in P. namibiensis. Paratrichodorus tarifaensis sp. nov. can Onchiostyle medium-sized (average 41 µm long). Pharynx at
show a similar pronounced ventral pharyngeal overlap as in posterior isthmus widened to a posterior bulb, usually with a
P. lobatus but differs in sperm without fibrillar appearance in short (5–15 µm) dorsal overlap by the intestine or a combined
male testis. short ventrosublateral overlap of pharyngeal glands (3; 15 µm)
Based on the tabular key for females (Decraemer and Baujard and dorsal intestinal overlap (5; 7 µm); dorsal gland nucleus
1998) sorting on primary features C and D, followed by features variable in position but mostly in the anterior third of the bulb.
K and L (size and position of vaginal sclerotized pieces in op- Nerve ring close to pharyngostom; SE-pore opposite posterior
tical section), groups the new species with P. acaudatus, P. allius isthmus or anterior pharyngeal bulb. Rectum nearly vertical,
and P. teres. Paratrichodorus tarifaensis sp. nov. differs from P. anus terminal.
acaudatus in the shorter onchiostyle (50–59 µm vs. 82–83 µm Reproductive system didelphic, amphidelphic, with ovaries
in type populations), in the terminal position of the anus, and reflexed; sperm cells small with oval nucleus (about 2 µm long),
in the shape of vaginal sclerotized pieces, triangular vs. quad- accumulated at the tip of the ovary flexure (i.e. at level ovarial
rangular. The new species differs from P. allius in sperm located sac). Vulva a short longitudinal slit (2 µm) in ventral view, lo-
in the uteri vs. sperm present in ovarial sacs and in the longer cated posterior midbody at about 60% of total body length from
onchiostyle (50–57 µm vs. 37–48 µm). The new species agrees anterior end. Vagina low, wide, rounded, half as long as wide and
with P. teres in presence of a pronounced ventral overlap of the occupying less than one-fifth of the corresponding body diam-
pharyngeal glands in some specimens but differs in the shape of eter; vaginal sclerotized pieces minute (1.0–1.5 µm), triangular,
vagina (with midway indentation instead of widening, common obliquely oriented and about 2.5 µm apart; vaginal constrictor
in P. teres), in shape of vaginal sclerotized pieces (shorter and muscles weakly developed. No (sub)lateral body pores ob-
oval in P. teres), and in the slightly longer onchiostyle (50–57 served. Tail almost non-existant due to terminal anus and a pair
µm vs. 42–50 µm). of terminal caudal pores.
Figure 10. Line drawings of Paratrichodorus asidonaensis sp. nov.. Females. A, total view holotype. B, pharyngeal region holotype. C,
reproductive system holotype. D, ovarial sac with sperm, anterior branch paratype. E, tail region holotype. F, vagina region, paratype. Male
paratype. G, pharyngeal region. H, posterior body region with reproductive system. Scale bars = 20 µm.
Figure 11. Light micrographs of Paratrichodorus asidonaensis sp. nov.. Male paratype. A, total view; B, testis germinal zone. Female holotype. D,
pharyngeal region. E, reproductive system. F, vagina region. G, tail region. Scale bars A = 100 µm; B, C, F, G = 10 µm; D, E = 20 µm.
Description of male: Apparently rare (only one specimen found). max), B21, C1, D1, E23, F34, G3, H5, I1, J1, K2, L33, M1, N2,
Body straight, cigar-shaped with swollen cuticle (5 µm) upon O3, P2, Q4, R2, S1, T1. For male: A100, B2, C1, D0, E0, F3, G2,
fixation. Digestive system with minute dorsal intestinal overlap H2, I3, J1, K2, L2, M27, N2, O1, P1.
(5 µm) of pharynx. No ventromedian or lateral cervical pa-
pillae; SE-pore opposite narrow pharyngeal region. Nerve ring Diagnosis and relationships: Paratrichodorus asidonaensis sp.
close to pharyngostom. Reproductive system monorchic, ger- nov. is small (<550 µm on average, type population). It is char-
minal zone 22-µm long, sperm cells small with oval-shaped acterized in female mainly by characteristics of the female re-
nucleus (2.5 × 1.5 µm). Spicules almost straight with slightly productive system with a very short (less than one-fifth of
knob-like marked capitulum, blade with fine transverse striae, corresponding body width), wide, rounded vagina, sclerotized
except at extremities, and slightly tapered towards a fine distal vaginal pieces obliquely orientated, well-separated, small (1–1.5
tip. Copulatory muscles hardly developed; capsule of suspensor µm), triangular in optical section, the vulva located at about 60%
muscles thin. Bursa narrow, extending from shortly anterior to of body length from anterior end with longitudinal slit-like shape
retracted spicule to mid-tail. Three precloacal supplements, pos- in ventral view and the unusual accumulation of sperm in the
terior two (SP1 and SP2) opposite distal half or spicules; SP3 ovarial sac at the flexure of ovaries. Males are rare and are char-
about three body widths anterior to cloacal opening and non- acterized by the absence of ventromedian and lateral cervical pa-
protruding. Tail with pair of well-developed postcloacal sup- pillae, vas deferens with small oval sperm, the presence of three
plements close to cloacal opening, the latter with a protruding ventromedian precloacal supplements, of which the posterior
anterior lip flap. A pair of posterior caudal pores subterminally, two are located within the posterior half of the medium-sized
i.e. posterior to the posterior precloacal supplements. Tail tip cu- (35 µm) retracted spicules; a narrow bursa extends from shortly
ticle 3.5 µm, hardly thickened. anterior to retracted spicules, till mid-tail (length less than one
anal body width).
Description of juveniles: One third-stage juvenile was observed. Based upon the tabular identification keys for females
Largely similar to female, except for adult characteristics; reserve (Decraemer and Baujard 1998), sorting on primary features D,
stylet 17 µm and genital primordium 32 µm long. C, followed by L and K, resulted in a classification of the new
species in subgroup 1-12. Further sorting on feature P (type of
Code within the tabular identification key (based on: Decraemer sperm cell), followed by feature O (location of sperm), groups P.
and Baujard 1998): For females: A100 i.e. A111 (average, min, asidonaensis sp. nov. with P. allius. Both species possess sperm in
Table 6. Morphometrics of Paratrichodorus asidonaensis sp. nov. male and female paratype specimens and specimens from Benalup-Casas
Viejasa.
Holotype Paratype Paratype Paratype Benalup-Casas
female female male juv. III Viejas female
N 5 1 1 9
L 530 480–565 475 398 570–835
515 ± 34 633 ± 79
Onchiostyle 41.5 37–43 42 40 41–43
40.9 ± 2.1 42.0 ± 0.9
Onchium 20 18.5–22 18 17 19–21.5
20.4 ± 1.2 20.3 ± 1.1
Pharyngostom 49 45–50 52 63 44.5–51
48.6 ± 2.0 49.1 ± 2.0
Pharynx 109 102–124 115 105 111–150
112 ± 8 122 ± 12
Anterior to guide ring 19 19–20 19 18 15–21
19.2 ± 0.4 18.6 ± 1.6
Anterior to nerve ring 50 51–52 53 63 47–77
51.8 ± 0.4 54.5 ± 10.2
Anterior to EP 74 67–83 74 64 68–105
73.6 ± 6.6 78.2 ± 10.1
mbd at cardia 30 29–30 28 25 27–33
29.5 ± 0.5 30.3 ± 1.7
mbd midbody 35 31–34 28 26 31–35.5
32.4 ± 1.0 33.2 ± 1.6
abd/♀: length anterior ovary 61 37–74 16 18–84
52.2 ± 12.3 45.7 ± 16.5
Spicule length/ ♀: length anterior branch 174 125–219 35 88–160
158 ± 33 150 ± 24
Gubernaculum/ ♀: length posterior ovary 40 43–63 21–85
51.8 ± 6.5 46.2 ± 17.2
Distance SP1-cloacal opening/♀ length posterior branch 133 110–181 80–154
143 ± 23 112 ± 22
Distance SP1–SP2/ ♀ length vagina 5.5 4.5–6 3.5–7.5
5.1 ± 0.6 5.6 ± 1.5
Distance SP2–SP3/♀ vagina % cbw 15.7 13.6–17.6 10.6–22.7
15.7 ± 1.6 16.8 ± 4.2
Tail/♀G1 32.8 25.0–38.8 15.4–30.4
30.5 ± 4.6 23.7 ± 4.8
ab 15.1 15.0–18.2 17.0 15.3 17.3–23.5
15.9 ± 1.2 19.1 ± 1.8
b 4.9 4.3–4.8 4.1 3.8 4.7–5.6
4.6 ± 0.2 5.2 ± 0.3
c/ ♀V 60.1 56.7–59.3 63.3 54.1–57.2
58.2 ± 1.2 56.2 ± 0.9
T/♀G2 25.1 22.9–33.3 51.7 14.0–24.2
27.7 ± 3.5 17.7 ± 6.5
a
b
a, body length/maximum body width; abw (anal body width); b, body length/pharyngeal length; c, body length/tail length; cbw (correspondingl body width); CP (ventromedian
ovarial sac, have a comparable onchiostyle length and males are was considered a senior synonym of P. tansaniensis Siddiqi 1974
rare. The new species can hardly be distinguished from P. allius by Sturhan (1989) because the main differential features, i.e. the
with a similar onchiostyle length (37–43 µm vs. 37–48 µm) but a slightly shorter body and onchiostyle length in P. tansaniensis,
somewhat shorter body length compared with the type popula- cannot be used because of the considerable impact of fixation
tion of P. allius (480–565 µm vs. 640–780 µm). However, P. allius (see Discussion). Considering that features K and L may show
some variation, we should also consider here P. rhodesiensis, al- Belgium (slide numbers males and females paratype UGMD
though, in general, feature L (distance between sclerotized 104463-104466); and one male and one female paratypes at the
pieces is smaller) and specimens also possess sperm at the level USDA Nematode Collection, Beltsville, MD, USA (collection
of the ovarial sac (Decraemer 1989). However, P. rhodesiensis number T-8020p).
shows more rounded sclerotized pieces.
For males, sorting of the data primarily on feature D (number Etymology: The species epithet refers to the name of the inhab-
of CP) and feature F (number of SP), places the new species itants of Rociana del Condado, the type locality where the type
in group 4 in Decraemer and Baujard (1998) together with P. specimens were collected.
rhodesiensis, P. teres, P. lobatus (2 SP), P. namibiensis (2 SP), and
previously described new species P. tarifaensis sp. nov.. The new Description of male: Body straight, cigar-shaped, often with
species most closely resembles P. rhodesiensis in the short body swollen cuticle (4.5–6.0 µm at midbody) upon fixation; an-
length [475 µm (N = 1) vs. 360–690 µm in type population teriorly tapered to rounded lip region appearing offset by the
of P. rhodesiensis] and in onchiostyle length (42 µm vs. 39–43 protruding double papillae (four cephalic and two subdorsal
µm) but differs in shorter spicule length (35 µm vs. 42–44 µm), and two subventral outer labial papillae). Onchiostyle rather
presence of dorsal intestinal overlap vs. ventrosublateral overlap short (average 39 µm long). Pharynx at posterior isthmus grad-
of pharyngeal glands. Paratrichodorus asidonaensis sp. nov. dif- ually widening to a posterior bulb, with a pronounced (24–27
fers from P. teres in slightly shorter body length [480–565µm µm) ventral overlap of the ventrosublateral pharyngeal glands;
vs. 720–860 µm in populations from The Netherlands and dorsal gland nucleus in the anterior third of bulb. Nerve ring
Belgium; see: Decraemer and De Waele (1981); no males in P. surrounding isthmus; SE-pore opposite posterior part pharyn-
teres type population] and spicule length (35 µm vs. 45–52 µm geal bulb. Ventromedian and lateral cervical papillae absent.
in P. flevensis population; P. flevensis syn. of P. teres), in presence of Reproductive system monorchic, testis with geminal zone 29 µm
dorsal intestinal overlap vs. ventrosublateral, or variable, marked in holotype; sperm small with oval-shaped nucleus, 5 by 3 µm
manubrium and sperm structure (non-fibrillar appearance in in size. Spicules medium-sized (average 45 µm), nearly straight,
testis). However, P. teres in the literature shows some variations with short widened capitulum, blade largely equally wide but
indicating that we are probably dealing with a species complex slightly tapered towards an open distal tip; spicules ornamented
(see general discussion). Paratrichodorus asidonaensis sp. nov. dif- with fine transverse striae, except at extremities. Copulatory
fers from P. lobatus and P. namibiensis apart from the common 3 muscles hardly developed, obscure; capsule of suspensor
SP vs. 2 SP and rarely 3 SP in P. lobatus and P. namibiensis, also in muscles thin, around anterior half of spicule; gubernaculum
shorter body, shorter spicules, and different pharyngo-intestinal with thickened distal part, parallel to spicules. Bursa narrow,
junction with anterior dorsal intestinal overlap vs. ventral pha- extending from head of retracted spicule to subterminal on
ryngeal overlap or a combined ventral and dorsal overlap re- tail. Three precloacal supplements, posterior two (SP1 and
spectively. In the sample from Benalup-Casas Viejas from the SP2) papilliform opposite distal half of retracted spicules; SP3,
rhizosphere wild olive, P. asidonaensis sp. nov. was found together little developed and non-protruding, located about 2.5 spicule
with female specimens from P. tarifaensis sp. nov., all possessing lengths from the cloacal opening. A pair of postcloacal supple-
a rather short onchiostyle and similar low, oval shape of vagina, ments shortly posterior to the cloacal opening, the latter with a
shape of vaginal sclerotized pieces in optical section and sperm small protruding anterior lip flap (in ventral view visible as two
in ovarial sac, but differing by males being rare in the former and flaps bordering the opening). Tail less than one anal body diam-
by its small body size and spicules, but well separated by mo- eter long (c’ average 0.64) and broad conical. A pair of posterior
lecular markers (see below), confirming another case of cryptic caudal pores subterminally near tail tip. Cuticle at tail tip not
diversity. thickened (2.0 µm).
Description of female: Body slightly longer than in male;

Paratrichodorus rocianerus sp. nov. Decraemer et al.
pharynx with a clear dorsal intestinal overlap (48 µm in one
(Figs 3I, 12, 13; Table 7) female) and with both an intestinal dorsal (45 µm) and a
Zoobank registration: urn:lsid:zoobank.org:act:8C8E441C- pronounced ventral overlap of pharyngeal glands (72 µm)
8A02-4028-93A6-1DA83FF06626 in another female. Reproductive system with small sperm all
along the uteri; vagina short (16% of cbd) with minor inden-
Type material: Holotype. Male extracted from soil samples tation at level of weak vaginal constrictor muscles; vaginal
collected from the rhizosphere of grapevine (Vitis vinifera) sclerotization pieces small (1–1.5 µm), oval, well separated
at Rociana del Condado, Huelva province, southern Spain (2.5–4.5 µm) and almost parallel to vaginal lumen in orien-
(37°19ʹ51.0ʹʹ N latitude, 6°35ʹ54.9ʹʹ W longitude) by J. Martín tation. A postvulvar lateral body pore observed at 111 µm
Barbarroja and G. León Ropero, mounted in pure glycerine posterior to vulva. Tail minute, caudal pores nearly terminal
and deposited in the nematode collection of Ghent University, on tail.
Ghent, Belgium (slide number UGMD 104462).
Code within the tabular identification key (based on: Decraemer
Paratypes: Male and female paratypes extracted from soil sam- and Baujard 1998): For male: A222 (average, min., max.), B12,
ples collected from the rhizosphere of grapevine at Rociana del C22, D0, E0, F3, G2, H4, I2, J1, K2, L2, M27, N2, O1, P1(3).
Condado, Huelva province, southern Spain, were deposited in For female: A223, B22, C1, D1, E4, F34, G?, H5, I1, J3, K2, L3,
the following nematode collections: Ghent University, Ghent, M3, N1, O2, P2, Q?, R21, S1, T1.
Figure 12. Line drawings of Paratrichodorus rocianerus sp. nov.. Holotype male. A, pharyngeal region. B, posterior body region. C, testis
germinal zone. Female paratype. E, pharyngeal region. F, anterior branch reproductive system. D, tail region. Scale bars = 20µm.
Figure 13. Light micrographs of Paratrichodorus rocianerus sp. nov.. Male holotype. A, total view. B, posterior body region with copulatory
apparatus. C, tail region with spicule. D, tail region showing bursa (arrow). Female paratypes. E, anterior body region. F, G, vagina region. H,
reproductive system. I. tail region. Scale bars A = 100 µm; B = 20 µm; C–G, I = 10 µm; H = 30 µm.
Diagnosis and relationships: Paratrichodorus rocianerus sp. nov. is P. rhodesiensis, P. lobatus (partim 3SP), and the previously de-
characterized by a medium-sized to large body (average 825 µm scribed new species P. tarifaensis sp. nov. and P. asidonaensis
in male; 975 µm in female), medium-sized onchiostyle (average sp. nov. Paratrichodorus rocianerus sp. nov. differs from P. teres
38.5 µm in male, 43 µm in female), absence of ventromedian by a different sex ratio (male as common as female vs. male ab-
and lateral cervical papillae in male, small sperm with oval nu- sent or rare in most populations of P. teres), by a slightly shorter
cleus, overlaps pharynx–intestinal junction variable, medium- onchiostyle (37–40 µm vs. >40 µm) and non-fibrillar sperm.
sized spicule length (average 45 µm) with finely striated, slightly It can be differentiated from P. lobatus by a slightly shorter
ventrally curved and short marked capitulum, three precloacal onchiostyle 37–40 µm vs. >40 µm, shorter spicules (44–46 µm
supplements, of which the posterior two opposite posterior vs. 53–59 µm) and from P. rhodesiensis by a slightly longer body
half retracted spicule, tail clearly shorter than anal width, with a (average 825 µm vs. 645 µm in type population) and smaller
pair of postcloacal supplements shortly posterior to the cloacal c’ value (<1 instead of >1 in P. rhodesiensis) and by the pro-
opening and nearly terminal pair of caudal pores. Females with nounced ventral overlap of the pharyngeal glands vs. a slight
short vagina (16% cbd) with minor indentation and small oval overlap; the single much smaller male specimen (360 µm)
vaginal sclerotized pieces (1.0–1.5 µm) in optical section, par- with 1 CP described in Baujard (1983) from Ivory Coast could
allel with vaginal lumen and clearly separated, sperm in uteri. represent a different species. It differs from P. asidonaensis
Based upon the tabular identification key for Paratrichodorus sp. nov. by males being as common as females, by longer body
males (Decraemer and Baujard 1998), sorting of the data and spicules, comparable with P. tarifaensis sp. nov., but differs
primarily on character D, followed by character F (number from the latter by a longer onchiostyle and more posterior pos-
SP) groups the new species with the known species P. teres, ition of EP-pore.
Table 7. Morphometrics of Paratrichodorus rocianerus sp. nov. male and female paratype specimensa.
N 4 6
L 985 755–890 775–1095
825 ± 52 975 ± 107
38.5 ± 1.1 43.1 ± 1.4
47.4 ± 1.3 53.2 ± 3.2
Pharynx 148 119–158 150–223
137 ± 16 182 ± 31
Anterior to guide ring 20 16; 20 (N = 2) 18; 21.5 (N = 2)
Anterior to nerve ring 51 59 (N = 1) 61–66 (N = 3)
Anterior to EP 116 94–170
125 ± 30
mbd at cardia 34.5 31–37 32–42
33.9 ± 2.6 37.2 ± 4.5
mbd midbody 35 32–40 33–48
37.5 ± 3.2 46.7 ± 5.1
abd/♀: length anterior ovary 19 18–22 109–145 (N = 3)
20.9 ± 1.7
Spicule length/ ♀: length anterior branch 46 44–46 288–456 (N = 3)
45.3 ± 0.8
Gubernaculum/ ♀: length posterior ovary 11 12–22 110–143 (N = 3)
15.4 ± 4.0
Distance SP1-cloacal opening/♀ length posterior 11 9.5–11.5 285–388 (N = 3)
branch 10.9 ± 0.8
Distance SP1–SP2/ ♀ length vagina 13 10–16 5.5–7

12.3 ± 2.3 6.1 ± 0.5
Distance SP2–SP3/♀ vagina % cbw 68 104–131 (N = 3) 15.0–16.7
16.0 ± 0.7
Tail/♀G1 15 12–14 30.7–41.6 (N = 3)
13.3 ± 0.8
ab 28.1 19.4–26.7 19.3–29.2
22.3 ± 2.9 24.3 ± 3.7
b 6.8 5.4–6.8 4.91–6.43 (N = 3)
6.2 ± 0.6
c/ ♀V 65.6 57.4–65.7 52.6–59.5
62.4 ± 3.1 56.8 ± 3.7
T/♀G2 56.7 44.5–64.1 30.4–38.1 (N = 3)
55.5 ± 7.4
a
b
Based upon the tabular identification keys for females anus vs. terminal and from both P. acaudatus and P. tarifaensis
(Decraemer and Baujard 1998), sorting on primary features sp. nov. in the shorter onchiostyle (41–45 µm vs. 82–83 µm
D and C, followed by L and K, resulted in a classification in and 50–57 µm, respectively) and in shape of vaginal sclerot-
subgroup 1-12 of group 1. Further sorting on feature P (type ized pieces (small oval vs. quadrangular and small triangular,
of sperm cell), feature O (location of sperm) and presence respectively). Females can hardly be distinguished from P.
of males joins the new species P.s rocianerus sp. nov. with P. benalupensis sp. nov. except for the absence of reserve stylet
acaudatus, and previously described species P. tarifaensis sp. vs. present. Features L and K appear to be variable (difficult to
nov. and P. benalupensis sp. nov.. Paratrichodorus rocianerus sp. describe precisely or illustrate because of their minute size in
nov. differs from P. acaudatus in the subterminal position of the optical section).
Paratrichodorus litoralis sp. nov. Decraemer et al. protruding. Tail short, less than one anal body diameter long,
conical. A pair of caudal pores subterminally near tail tip.
(Figs 3J, 14, 15; Table 8)
Zoobank registration: urn:lsid:zoobank.org:act:B7070089-B833- Description of female: Body similar in appearance and size as
43C5-B38C-CF4112497D1B in male, except for male reproductive system and features.
Onchiostyle equally long, with a reserve stylet in adults;
Type material: Holotype. Male extracted from soil samples pharyngo-intestinal junction with dorsal overlap 16–24 µm,
collected from the rhizosphere of marram grass (Ammophila rarely combined with a short ventral overlap. Reproductive
arenaria) at Pantanagianni, Brindisi province, southern Italy system with both genital branches about equally long and
(40°45ʹ01.27ʹʹ N latitude, 17°43ʹ13.65ʹʹ E longitude) by N. without spermatheca but small sperm with small nucleus spread
Vovlas, mounted in pure glycerine and deposited in the nema- over the uteri; vagina barrel-shaped with minor indentation at
tode collection of Ghent University, Ghent, Belgium (slide level weak constrictor muscles; vaginal sclerotization pieces very
number UGMD 104467). small (1.0 µm), rounded triangular, clearly apart (2.0–3.0 µm),
slightly oblique and vulva a longitudinal slit in ventral view. Tail
Paratypes: Male and female paratypes extracted from soil samples with caudal pores.
collected from the rhizosphere of marram grass at Pantanagianni,
Brindisi province, southern Italy, were deposited in the following
nematode collections: Ghent University, Ghent, Belgium (slide Code within the tabular identification key (based on: Decraemer
numbers males and females paratype UGMD 104467-104469, and Baujard 1998): For male: A212 (average, min., max.), B22,
UGMD 104461); and one male and one female paratypes at the C22, D0, E0, F3, G2, H2, I4, J2, K2, L2, M27, N2, O1, P1. For
USDA Nematode Collection, Beltsville, MD, USA (collection female: A222 (200), B23, C1, D1, E2, F3(4), G3, H3, I1, J1, K1,
number T-8021p). L3, M1, N1, O2, P2, Q-, R2, S1, T1.
Additional material examined: An additional population was Diagnosis and relationships: Paratrichodorus litoralis sp. nov. is
collected also in the rhizosphere of marram grass at Cullera, characterized by a medium-sized body (average 740 µm in male,
Valencia province, Spain (39°15ʹ64.7ʹʹ N latitude, 0°24ʹ08.2ʹʹ W 790 µm in female), rather long onchiostyle (average 57 µm in
longitude); unfortunately the specimens of this population were male, 60 µm in female), absence of ventromedian and lateral
not in good conditions for light microscopic study, and were cervical papillae in male, spicule length average 55 µm, three
identified only molecularly. precloacal supplements with the posterior two opposite the
distal two-thirds of the retracted spicules. Females with barrel-
Etymology: The species epithet 8s derived from the Latin shaped vagina, one-fifth of corresponding body width, and with
litoralis = pertaining to the seashore, and refers to the coastal en- minor indentation; minute vaginal sclerotized pieces (1 µm),
vironment where the type specimens were collected. slightly oblique and clearly separated (2.2–3 µm); sperm spread
over uteri. Both sexes with a reserve onchium in adults, dorsal
Description of male: Body nearly 750 µm long (average) straight, overlap by the intestine and caudal pores present.
cigar-shaped, with cuticle swollen upon fixation (5 µm at Based upon the tabular identification key for Paratrichodorus
midbody); anteriorly tapered to narrower, rounded, low-lip re- males (Decraemer and Baujard 1998), sorting of the data pri-
gion appearing offset by the protruding double papillae (four marily on character D (number of ventromedian cervical
cephalic and two subdorsal and two subventral outer labial pa- papillae), followed by character F (number of precloacal sup-
pillae) and swelling of body cuticle. Onchiostyle rather long plements) groups P. litoralis sp. nov. with the known species P.
(average 56 µm long); reserve onchium (21.5 µm average) allius, P. teres, and P. rhodesiensis, and with previously described
present in all adults. Pharynx at posterior isthmus gradually P. tarifaensis sp. nov. that shows similar measurements of body
widening to a posterior bulb and a short dorsal overlap by the length, onchiostyle, and spicule length. It also groups with P.
intestine. Nerve ring surrounding isthmus; SE-pore opposite lobatus and P. namibiensis (partim) with males with exception-
isthmus. Ventromedian or lateral cervical papillae absent. ally 3 SP. Paratrichodorus litoralis sp. nov. differs from all by the
Reproductive system monorchic, sperm small with oval-shaped presence of a reserve onchium in the adult, and from P. teres,
nucleus (3.0 × 1.5 µm). Spicules medium-sized (average 54.5 P. namibiensis, and P. lobatus in the non-fibrillar appearance
µm), slightly ventrally curved, with short slightly wider capit- of sperm vs. thread-like or fibrillar. The new species also dif-
ulum, blade largely equally wide but slightly tapered towards an fers from all known species by the longer onchiostyle (average
open distal tip; spicules ornamented with fine, transverse striae, >55 µm vs. <55 µm) and by the longer spicule length than in P.
except at extremities. Copulatory muscles hardly developed, allius, P. rhodesiensis, and P. teres (average >50µm vs. <50 µm) but
obscure; capsule of suspensor muscles thin, surrounding an- similar as in P. lobatus and shorter than in P. namibiensis (average
terior half of spicule; gubernaculum with thickened distal part 67 µm).
parallel to spicules. Bursa narrow, extending usually from head Based upon the tabular identification keys for females
of retracted spicule to subterminally on tail. Three precloacal (Decraemer and Baujard 1998), sorting on primary features D
supplements, posterior two (SP1 and SP2) opposite distal two- and C, followed by L and K, resulted in a classification in sub-
thirds of retracted spicules; anteriormost SP3, little developed group 1-12 of group 1. Further sorting on feature P (type of
and non-protruding, located about 2.5 spicule length anterior to sperm cell), feature O (location of sperm) and feature N (pres-
retracted spicules. A pair of well-developed postcloacal supple- ence of males) groups P. litoralis sp. nov. with known species
ments close to the cloacal opening; cloacal lips may be slightly P. acaudatus and P. allius. Paratrichodorus litoralis sp. nov. differs
Figure 14. Line drawings of Paratrichodorus litoralis sp. nov.. A, total view female paratype. B, pharyngeal region female paratype. C, tail region
and copulatory apparatus, male paratype. D, protruding spicule, paratype. E, posterior body region, holotype male. F, pharyngeal region
paratype male. G, female reproductive system, paratype. H, testis, germinal zone, paratype. Scale bars = 20 µm.
Figure 15. Light micrographs of Paratrichodorus litoralis sp. nov.. A, total view paratype female. B, anterior body region, arrow points to reserve
onchium, paratype female. C, vagina region, paratype. D, posterior genital branch, paratype female. E, anterior body region, arrow points to
reserve onchium, paratype male; tail region with copulatory apparatus, holotype male. Scale bars A = 100 µm; B, E = 20 µm; C, D = 10 µm.
from both by the presence of a reserve onchium in adult, a fea- and deposited in the nematode collection of Ghent University,
ture it has in common with P. benalupensis sp. nov.. It further dif- Ghent, Belgium (slide number UGMD 104471).
fers from P. allius by the longer onchiostyle (58–64 µm vs. 37–48
µm), the longer spicules (50–58 µm vs. 31 µm), the male–female Paratypes: Female paratypes extracted from soil samples col-
ratio, with males as common as females vs. males rare, and by lected from the rhizosphere of wild olive at Medina Sidonia,
the short dorsal overlap by the intestine vs. a ventral overlap in Cádiz province, southern Spain, were deposited in the following
P. allius. nematode collections: Ghent University, Ghent, Belgium (slide
numbers females paratype UGMD 104470-104471); and one
female paratype at the USDA Nematode Collection, Beltsville,
Paratrichodorus species without males but females with sperm MD, USA (collection number T-8022p).
at tip of ovary
Paratrichodorus paraallius sp. nov. Decraemer et al. Additional material examined: An additional population was
collected also in the rhizosphere of cultivated olive (Olea
(Figs 3K, L, 16, 17; Table 9) europaea ssp. europaea) at Espiel, Córdoba province, Spain
Zoobank registration: urn:lsid:zoobank.org:act:0FC78198- (38°06ʹ28.5ʹʹ N latitude, 5°04ʹ19.5ʹʹ W longitude); unfortu-
4409-4407-A1F1-1F63AC86050B nately the specimens of this population were not in good con-
ditions for light microscopic study, and were identified only
Type material: Holotype. Female extracted from soil samples col- molecularly.
lected from the rhizosphere of wild olive (Olea europaea subsp.
sylvestris) at Medina Sidonia, Cádiz province, southern Spain Etymology: The species epithet refers to Gr. prep. para, alongside
(36°19ʹ56.3ʹʹ N latitude, 5°51ʹ16.8ʹʹ W longitude) by J. Martín of, resembling; N.L. masc. n. allius, because of its close resem-
Barbarroja and G. León Ropero, mounted in pure glycerine blance to Paratrichodorus allius.
Table 8. Morphometrics of Paratrichodorus litoralis sp. nov. male and female paratype specimensa.
N 7 7
L 865 680–800 755–830
739 ± 43 788 ± 32
Onchium 29 24–29 27–30
26.4 ± 1.5 28.6 ± 0.9
56.9 ± 4.0 59.86 ± 2.0
68.6 ± 4.0 71.6 ± 2.3
Pharynx 155 148–161 135–171
154 ± 5 159 ± 11
Anterior to guide ring 26 24–30 26–30
26.2 ± 2.4 27.8 ± 1.3
Anterior to nerve ring 76 76; 86 (N = 2) 72–82
76.0 ± 4.7 77.4 ± 3.3
Anterior to EP 86-106
99 ± 7
mbd at cardia 44 41–43 38–54
41.7 ± 0.7 46.3 ± 4.6
mbd midbody 37–48 43–52
42.0 ± 4.3 47.3 ± 3.0
abd/♀: length anterior ovary 29 19–26 266–313
23.5 ± 2.5 290 ± 17
Spicule length/ ♀: length anterior branch 52 50–58 155–212
54.7 ± 2.5 195 ± 31
Gubernaculum/ ♀: length posterior ovary 14 9–18 38–75
13.1 ± 2.4 52.5 ± 14.2
Distance SP1-cloacal opening/♀ length posterior 14 10–16 133–220
branch 12.5 ± 2.2 165 ± 31
Distance SP1–SP2/ ♀ length vagina 17 11–16 9.5–11

14.2 ± 1.7 10.2 ± 0.6
Distance SP2–SP3/♀ vagina % cbw 72–96 19–23.3
81.3 ± 7.6 21.5 ± 1.7
Tail/♀G1 13 11.5–16 21.1–30.6
13.8 ± 1.8 24.8 ± 4.0
ab 19.7 16.4–19.2 14.5–18.6
18.5 ± 1.0 16.7 ± 1.2
b 5.6 4.5–5.2 4.5–5.9
5.0 ± 0.24 5.0 ± 0.5
c/ ♀V 66.5 48.1–57.1 49.92–56.0
54.7 ± 5.7 53.3 ± 1.8
T/♀G2 69.9 54.4–59.6 16.8–27.5
57.0 ± 2.1 21.9 ± 3.8
a
b
Description of female: Body medium-sized (average about 800 a pronounced dorsal overlap (11–23 µm) by the intestine,
µm), cigar-shaped, anteriorly tapered; cuticle non-swollen (2 with or without a short ventral overlap (average 6µm) of the
µm) at midbody; lip region rounded, with protruding double ventrosublateral pharyngeal glands; posterior ventrosublateral
papillae (four cephalic and two subdorsal and two subventral gland nuclei and dorsal gland nucleus well separated. Cardia
outer labial papillae). Onchiostyle medium-sized (average 47 obscure. Rectum nearly vertical with anus subterminal. Nerve
µm long). Pharynx with a short, narrower part (isthmus) and ring surrounding isthmus; SE-pore at level of posterior end
Figure 16. Line drawings of Paratrichodorus paraallius sp. nov.. Female. A, pharyngeal region, holotype. B, pharyngeal region, paratype. C,
anterior genital branch, paratype. D, tail region, holotype. E, vagina region, holotype. Scale bar = 10 µm.
isthmus or anterior part pharyngeal bulb. Reproductive system Male: Unknown.

didelphic-amphidelphic with both branches more or less equally
developed; oviduct cells finely granular, connection with re- Code within the tabular identification key (based on: Decraemer
flexed anterior end of ovary not clear but sperm observed near and Baujard 1998): For female: A222, B22, C1, D1, E23, F34,
tip reflexed ovary or sperm absent. Vagina short (average 20% G3, H2(8), I1, J9, K2, L3, M1, N3, O55, P2, Q4, R2, S1, T1.
of corresponding body width long), barrel-shaped to slight trap-
ezoidal with minor midway indentation at level of vaginal con- Diagnosis and relationships: Paratrichodorus paraallius sp. nov. is
strictor muscles; pars refringens vaginae in optical section with characterized by a body medium-sized (average about 800 µm),
short (1.5–2.0 µm) oval to rounded triangular sclerotized pieces, no males observed. Onchiostyle 47 µm (average) and without
nearly parallel to slightly obliquely oriented, about 2.5 µm apart reserve onchium in adult; pharyngo-intestinal junction with
(average); vulva situated on average 58% of body length from an- pronounced dorsal overlap by intestine with or without short
terior end and a longitudinal slit in ventral view. No sublateral/ ventral overlap by pharynx; SE pore near anterior end pharyn-
subventral body pores observed. Tail minute, terminal cuticle geal bulb; small oval sperm cells observed close to germinal
not thickened; a pair of terminal caudal pores present. zone reflexed ovary or absent; no sperm in uterus. Vagina short
Figure 17. Light micrographs of Paratrichodorus paraallius sp. nov.. Holotype female. A, total view. B, pharyngeal region. G, vagina region. H,
tail region. Paratype females. C, posterior pharyngeal region. D, ovary anterior genital branch, arrow indicates sperm near ovary tip. E-F. Vagina
region. Scale bars A = 100 µm; B–D = 20 µm; E–H = 10 µm.
(20% of corresponding body width), barrel-shaped to slightly P. paraallius sp. nov. is almost undistinguishable morphologic-
trapezoid with slight midway indentation at level of vaginal ally from P. allius, being described here as separate new species,
constrictor muscle; pars refringens vaginae in optical section based on the additional support given by molecular analyses (see
with short (1.5–2.0 µm) oval to rounded triangular sclerotized below).
pieces, nearly parallel to slightly obliquely oriented, 2.5 µm apart
(average). No sublateral or subventral body pores observed. Molecular characterization of the Paratrichodorus allius
Based upon the tabular identification keys for females species-complex
(Decraemer and Baujard 1998), sorting on primary features D Amplification of the D2-D3 expansion segments of 28S
and C, followed by L and K, resulted in a classification in sub- rRNA, ITS rRNA, and partial 18S rRNA gene from the new
group 1-12 of group 1. Further sorting on feature P (type of Paratrichodorus spp. yielded single fragments of approximately
sperm cell), feature N (presence of males), and feature O (lo- 900 bp, 1000 bp, and 1800, respectively, based on gel electro-
cation of sperm) groups P. paraallius sp. nov. with P. allius. The phoresis. Thirty-one sequences of D2-D3 expansion segments of
new species can hardly be differentiated based on morphological 28S rRNA gene (OR139109–OR139139), 19 sequences of ITS
features in female because of the variation in vaginal structures rRNA gene (OR139998–OR140016), and 12 sequences of 18S
but agrees in males being absent vs. absent or rare in P. allius and rRNA gene (OR139140–OR139151) were obtained for the first
in absence of sperm in the uteri but did not show accumulation time in this study (Table 1). No intraspecific variation was de-
of sperm in ovarial sac but in the ovary near the germinal zone, tected for D2-D3 expansion segments of 28S rRNA of P. uliaensis
a feature also observed in P. allius (Sturhan 1989). Definitely, sp. nov., and very low intraspecific variation for D2-D3 expansion
Table 9. Morphometrics of Paratrichodorus paraallius sp. nov. male was detected for other species ranging from 98.1% (19 bp, 2
and female paratype specimensa.
indels, differences) in P. litoralis sp. nov. to 99.9% (1 bp, 1 indel
Holotype female Paratype female differences) in P. asidonaensis sp. nov.. Finally, very low intraspe-
cific variation was detected for 18S rRNA of the P. allius species-
N 9 complex ranging from 98.6% (23 bp, 1 indel, differences) in P.
L 775 710–865 tarifaensis sp. nov. to 99.9% (1 bp, 1 indel differences) similar-
795 ± 30 ities in P. hinogerus sp. nov.. The closest species to P. uliaensis sp.
Onchiostyle 48 46–50 nov. for the D2-D3 expansion segments of 28S rRNA gene was
47.1 ± 1.3 P. benalupensis sp. nov. (OR139113–OR139114, 90.9% simi-
Pharyngostom 57 56–59 larity), followed by 90.3% similarity with P. allius (MG938549–
57.6 ± 0.5 MG93852, KX901757–KX901760). Paratrichodorus hinogerus
Pharynx 162 144–170 sp. nov. showed the highest similarity with P. tarifaensis sp.
162 ± 3.0 nov. (OR139122–OR139123, 92.1% similarity), followed
Anterior to guide ring 19 19–22 by Paratrichodorus sp. Zanjan (KY115134, 89.7% similarity).
21.1 ± 0.5 Paratrichodorus asidonaensis sp. nov. showed the highest simi-
Anterior to nerve ring 66 66–78 larity with P. rocianerus sp. nov. (OR139129–OR139131, 89.8%
70.1 ± 3.8 similarity), followed by P. allius (MG938549–MG93852,
Anterior to EP 104 97–113 88.2% similarity). Paratrichodorus litoralis sp. nov. showed
10.5 ± 5.3 the highest similarity with Paratrichodorus sp. Bostan Abad
mbd at cardia 36 33–36 (KY115136, 95.7% similarity), followed by P. paraallius sp. nov.
34.0 ± 1.2 (OR139115–OR139121, 89.9% similarity). Finally, P. paraallius
mbd midbody 39 31–39 sp. nov. showed the highest similarity with P. benalupensis sp. nov.
35.3 ± 2.9 (OR139113–OR139114, 93.9% similarity), followed by P. allius
length anterior ovary 87 36–130 (MG938549–MG93852, 90.5% similarity). All ITS sequences
76.8 ± 22.4 for the new P. allius species-complex described here were
length anterior branch 219 159–318 clearly different among them and from all ITS sequences from
247 ± 31 Paratrichodorus spp. in NCBI, except for one isolate of P. teres
length posterior ovary 79 61–123 from Washington State, USA (AM087125, 98,8% similarity),
91.9 ± 34.3 which should be considered conspecific with P. paraallius sp. nov.
length posterior branch 237 176–386 (see ITS phylogenetic tree). Finally, 18S sequences for the new P.
246 ± 63 allius species-complex described here showed some similarities
6.5 6.5–8.5 among them and with some sequences deposited in NCBI, par-
♀ length vagina
7.3 ± 0.7 ticularly with P. teres from The Netherlands (FJ040484, 90.6%
vagina % cbw 16.7 16.7–27.4 similarity).
20.9 ± 3.1
G1 28.2 22.0–37.9 Species delimitation using morphometry by principal com-
30.5 ± 3.7 ponent analysis
ab 19.9 19.2–25.1 In the PCA, the the first three components [sum of squares (SS)
22.6 ± 1.1 loadings >1] accounted for 72% (females) and 65% (males)
b 4.8 4.6–5.2 of the total variance in the morphometric characteristics of
4.9 ± 0.2 the P. uliaensis sp. nov., and P. benalupensis sp. nov. (Table 10).
V 56.9 55.5–58.3 Although no clear differences in morphology and morphomet-
57.6 ± 1.3 rics were observed between the two species (as mentioned in
G2 22.2 24.3–46.0 the species description section), it should be noted that when
30.8 ± 7.2 the PCA results were plotted, almost all specimens of either
males or females P. uliaensis sp. nov. and P. benalupensis sp. nov.
a
Measurements are in µm and in the form: range and mean ± SD. were separated from each other (Fig. 18). However, it is worth
b
a, body length/maximum body width; abw (anal body width); b, body length/
pharyngeal length; c, body length/tail length; cbw (corresponding body width); CP mentioning that there are some specimens of both species that
(ventromedian cervical papilla); CP1 (anterior ventromedian cervical papilla); G1, remain overlapping each other, which makes it difficult to sep-
G2 (anterior, posterior genital branch length, respectively/body length) × 100; EP
(secretory-excretory pore); L (total body length); mbw (maximum body width); arate them morphometrically. For both females and males, this
N (number of specimens studied); SP1, SP2 SP3 (posterior, second and third separation was mostly observed along the projection of the prin-
precloacal supplements, respectively); V (distance from anterior end to vulva/body
length) × 100; T (distance from cloacal aperture to anterior end of testis/body cipal component 1 (PC1; 34.9% and 65.1% of the total variance,
length) × 100. respectively; Fig. 18). Considering that the eigenvalues for each
character were used to identify the key morphological charac-
segments of 28S rRNA was detected for other species ranging ters for this species delimitation (Table 10), the PC1 was dom-
from 99.0% (1–7 bp, 1–5 indels, differences) in P. paraallius sp. inated by the length of vagina as percentage of width at midbody,
nov. to 99.9% (1 bp, 1 indel differences) in P. benalupensis sp. vaginal sclerotized pieces length, and distance between sclerot-
nov.. Similarly, no intraspecific variation was detected for ITS re- ized pieces with high correlations (eigenvalues = 0.50, 0.46 and
gion of P. uliaensis sp. nov., and very low intraspecific variation –0.45, respectively in females). And in males, PC1 was almost
Table 10. Eigenvector and SS loadings of the principal components Rosenberg’s PAB (Table 11). Analyses of species separation dem-
derived from nematode morphometric characters for Paratrichodorus onstrated that all morphologically closely related species were
uliaensis sp. nov. and P. benalupensis sp. nov. in bold characters to help clearly separated (P. uliaensis sp. nov., P. benalupensis sp. nov., P.
to separate both species. paraallius sp. nov., and P. allius from Oregon, USA). The intra-,
Character/ratioa PC1 PC2 PC3 interspecies molecular variation for D2-D3 expansion segments
of 28S rRNA and ITS genes of all four species was lower than
Females 0.10 (Table 11), suggesting that the probability of species iden-
Lb –0.28 0.30 –0.55 tification with both loci is high (Masters et al. 2011). Likewise,
Onchiostyle length –0.14 –0.57 –0.39 the P ID (Liberal) values for all four species and both loci were
Anterior to EP –0.28 0.07 0.36 ≥0.95, suggesting that species can be adequately separated (Ross
V 0.37 –0.11 –0.24 et al. 2008, Hamilton et al. 2014). All clade supports for the two
Length vagina as % mid body width –0.50 –0.09 –0.14 loci were well supported (PP = 1.00), and the Rosenberg’s PAB
Length of vaginal sclerotized pieces 0.46 –0.17 –0.41 values also support the monophyly of each species separately
Distance between sclerotized pieces –0.45 0.43 –0.08 (Rosenberg 2007).
a –0.15 0.59 –0.39
Phylogenetic relationships among the Paratrichodorus allius
SS loadings 1.67 1.36 1.19
species-complex within Trichodoridae
% of total variance 34.8 23.1 17.8
The phylogenetic relationships among the species in the
Cumulative % of total variance 34.9 558.0 75.7
family Trichodoridae inferred from analyses of the D2-D3 ex-
Males pansion segments of 28S rRNA, ITS rRNA, and partial 18S
Lb –0.51 0.51 –0.27 rRNA gene sequences using BI are given in Figures 19–21, re-
Onchiostyle length –0.49 –0.39 –0.68 spectively. The D2-D3 expansion segments of 28S rRNA gene
Anterior to CP –0.51 0.45 0.44 alignment (720 bp long) included 117 sequences and three
Spicules length –0.48 –0.62 0.52 outgroups species [Trypilina tamaki (GQ503060), Trypila
SS loadings 1.61 0.78 0.71 filicaudata (GQ503038), and Diphtherophora sp. PDL-2005
% of total variance 65.1 15.0 12.4 (DQ077790)]. The Bayesian 50% majority rule consensus
Cumulative % of total variance 65.1 80.1 92.6 tree inferred from D2-D3 expansion segments of 28S rRNA
alignment is given in Figure 19. BI of this marker revealed two
a
Based on two populations (considering means of each character/ratio) of major clades separating Paratrichodorus in a monophyletic
Paratrichodorus uliaensis sp. nov. (Table 2) and one population of P. benalupensis sp. nov.
(Table 3). 13 female and 20 male specimens of P. uliaensis sp. nov. from the paratype well-supported clade (PP = 1.00) from all other genera with
population and an additional population sample; four female and four male specimens available sequences, including Trichodorus, Nanidorus, and
of Paratrichodorus benalupensis sp. nov. from the type population.
b
Morphological and diagnostic characters according to Decraemer (1995) and Monotrichodorus in a low-supported clade (PP = 0.69) (Fig. 19).
Decraemer and Baujard (1998). Standardized canonical coefficients >0.4 are in bold The genus Trichodorus clustered in two moderately supported
font.
clades (PP = 0.96, PP = 0.92, respectively). The first clade com-
prises subclade I (PP = 0.87) encompassing 39 species from
equally dominated by all the characters considered in this study, several geographical origin and morphometric traits (Fig. 19);
with body, onchiostyle and spicule length, as well as the distance and subclade II (PP = 0.90), including four Nanidorus species
from anterior end to ventromedian CP with high correlations and three Trichodorus species (T. cedarus, T. japonicus, and T.
(eigenvalues = –0.51, –0.49, –0.51, and –0.48, respectively). nanjingensis) of Asian origin (Fig. 19). The second clade com-
In females, the range of these characters for the studied species prises subclade III (PP = 1.00), including 10 Trichodorus species
were 20.7–24.2 vs. 22.6–26.6 for length of vagina as % of width mostly from California, and subclade V (PP = 1.00), including
at midbody length, 1.0–1.5 vs. 0.8–1.5 µm for length of vaginal the three Monotrichodorus species sequenced up to date. The
sclerotized pieces; and 1.8–2.7 vs. 2.4–4.0 µm for the distance Paratrichodorus clade comprised subclade VI (PP = 1.00),
between sclerotized pieces between P. uliaensis sp. nov. and P. including the eight new species of P. allius species-complex de-
benalupensis sp. nov.. While for males, the ranges were 630–834 scribed herein, as well as six already described and undescribed
vs. 790–882, 30–39 vs. 37–50, 41–49 vs. 42–53, 77.5–110 vs. species counting P. allius, P. teres, Paratrichodorus sp. Arasbaran,
93–116µm for length of body, onchiostyle, and spicule, and Paratrichodorus sp. Zanjan, Paratrichodorus sp. Khalkhal, and
the distance from anterior end to ventromedian CP, respect- Paratrichodorus sp. Bostan Abad (Fig. 19). Finally, subclade IV
ively, between P. uliaensis sp. nov. and P. benalupensis sp. nov.. (PP = 0.97) comprised seven species of the P. hispanus species-
Nevertheless, it is emphasized that the separation observed group complex, including P. almadenensis, P. ramblensis, P. anem-
between both species was mostly due to the overall set of mor- ones, P. hispanus, P. pachydermus, P. porosus, and Paratrichodorus
phometric characteristics considered and not because of a key sp. CD3449I (Fig. 19). All the newly described species herein
character, given the similarity of the eigenvalues in the compo- were clearly separated from each other in well-supported
nents extracted from the PCA (Table 10). subclades (PP = 1.00): P. paraallius sp. nov. and P. benalupensis
sp. nov. clustered together in a well-supported clade (PP = 1.00),
Species separation based on ribosomal DNA and P. asidonaensis sp. nov. and P. tarifaensis sp. nov. clustered to-
Species separation by ribosomal DNA was based on four stat- gether in a moderately-supported clade (PP = 0.85), whereas P.
istical parameters: intra-/interspecies variation, P ID (Liberal) litoralis sp. nov., P. hinogerus sp. nov., P. uliaensis sp. nov., and P.
values, posterior probability of clades on Bayesian analyses, and rocianerus sp. nov. clustered in separate subclades (Fig. 19).
Figure 18. Principal component on morphometric characters to characterize Paratrichodorus uliaensis sp. nov. and P. benalupensis sp. nov..
Projections of P. allius species-complex on the plane of components 1 and 2 (A), 1 and 3 (B) for females, and components 1 and 2 (C), 1 and 3
(D) for males.
The ITS rRNA gene alignment (701 bp long) included 62 separated from each other in several non-supported subclades
sequences and two outgroups species [Hoplolaimus stephanus (Fig. 20).
(HQ678735) and Hoplolaimus sp. CB 2008 (EU515329)]. Finally, for the partial 18S rRNA gene, 88 sequences and
The Bayesian 50% majority rule consensus tree inferred from 1662 bp were included in the analyses, and this tree (Fig. 21)
ITS rRNA gene alignment is given in Figure 20. BI of this showed a similar topology to that of D2-D3 expansion segments
marker revealed two major clades, separating Paratrichodorus of 28S rRNA gene. Unfortunately, several trials for getting 18S
and Nanidorus in a well-supported clade (PP = 1.00) from rRNA of P. asidonaensis sp. nov. were unsuccessful. All other
Trichodorus (PP = 1.00). However, the relationships within the newly described species herein were clearly separated from each
clade of Paratrichodorus and Nanidorus were not well resolved other in well-supported subclades (PP = 1.00): P. paraallius
and need additional sequences for clarifying these relationships sp. nov. and P. benalupensis sp. nov. clustered together with P.
(Fig. 20). All the newly described species herein were clearly uliaensis sp. nov. in a well-supported clade (PP = 1.00), and P.
Table 11. Parameters evaluating Paratrichodorus allius species-complex delimitation based on two rRNA markers (D2-D3 expansion segments
of 28S rRNA, ITS) for four Paratrichodorus species of the species-complex.
Species Gene Intra/inter a Clade support b P ID (Liberal) c Rosenberg’s PAB d
Paratrichodorus uliaensis sp. nov. D2-D3 0.06 1.00e 0.97 (0.86,1.0) e 6.0 × 10–4
ITS 0.01 1.00 0.98 (0.87, 1.0) 4.8 × 10–3
Paratrichodorus benalupensis sp. nov. D2-D3 0.03 1.00 0.97 (0.81,1.0) 0.02
ITS 0.01 1.00 1.00 (0.86,1.0) 8.9 × 10–6
Paratrichodorus paraallius sp. nov. D2-D3 0.06 1.00 0.97 (0.87,1.0) 0.02
ITS 0.04 1.00 0.97 (0.86,1.0) 4.8 × 10–3
Paratrichodorus allius D2-D3 0.05 1.00 0.99 (0.93,1.0) 2.6 × 10–10
ITS 0.01 1.00 0.98 (0.88,1.0) 0.01
a
Intra-species variation relative to interspecies variation.
b
Clade support: posterior probabilities from Bayesian trees (Figs 19, 20).
c
The P ID (Liberal) value represents the probability (with the 95%confidence interval) for the prediction, of making a correct identification of an unknown specimen of the focal
species using DNA Barcoding (closest genetic distance). P ID (Liberal) values ≥0.93 considered to be delimited (Hamilton et al. 2014). Numbers in bold represent significant values.
d
Rosenberg’s PAB value is the probability that the monophyly of a group of sequences is the result of random branching.
e
Significant results are indicated in bold.
hinogerus sp. nov. and P. tarifaensis sp. nov. clustered together but P. paramirzai, with 2 CP in the male and with medium-sized
well separated from P. rocianerus sp. nov. clustering with P. teres, sperm cells with round nucleus and large sperm with sausage-
and P. litoralis sp. nov. clustering with Paratrichodorus sp. from shaped nucleus, respectively; no molecular data are available for
Iran (KY115107) (Fig. 21). these species; both species were reported from India and P. mirzai
was also recorded from Pakistan (Nasira and Maqbool 1994),
Japan (Edward and Misra 1970), and from Florida, but especially
DISCUSSION the latter identification, should be checked. Interestingly, the dif-
This study of Paratrichodorus species of the P. allius species- ference of sperm size and morphology within a single genus has
complex clearly illustrates the difficulty of differentiating species been studied in other nematode taxa, mainly in Caenorhabditis
based on morphology and morphometrics, and the necessity species where four repeated instances of convergent evolution
for molecular data. Most of the main diagnostic features used in of sperm ‘gigantism’ was observed (Vielle et al. 2016) and in
this species group, such as number of CP in the male, location gonochoristic or dioecious species in general make larger sperm;
of sperm in the female reproductive system, male/female ratio, self-fertilization reduce sperm competition. (Baldi et al. 2011). In
or, in both sexes, the presence of a reserve onchium in the adult a competitive environment, larger sperm have the advantage in
or a type of overlap at the pharyngo-intestinal junction, appear nematodes (LaMunyon and Ward 2002). Species within the P.
as convergent characters. Sperm morphology and vulva shape hispanus clade with large sperm are gonochoristic vs. the P. allius
seems to be maintained with the P. allius species-complex vs. P. clade with small sperm including gonochoristic and assumed
hispanus-group. However, even with these important difficul- automixis reproduction species (Sturhan 1989). Comparing
ties in morphology and morphometrics, this study presents an this trait at the phylogenetic level, based on D2-D3 expansion
important amount of Parartrichodorus new species, expanding segments of 28S rRNA, the genus Trichodorus appears paraphy-
their diversity, mainly in the Iberian Peninsula, including spe- letic; Nanidorus and Monotrichodorus form subclades among
cies in more than one country in the Mediterranean Basin (P. Trichodorus. Among the large clade of ‘Trichodorus’, including
litoralis sp. nov.). The use of molecular markers in an intregrative Monotrichodorus, only Nanidorus species show small sperm or
taxonomy (=combined morphology and molecular data) could non-functional thread-like sperm, and species reproduce by
resolve the species separation, giving unequivocal molecular autimixis vs. all other taxa, which are gonochoristic and possess
markers for species identification in the case of cryptic species large sperm cells, mostly with a large sausage-shaped or large
or in nematode stages with few morphological/morphometrical rounded nucleus (e.g. some USA species as T. elegans Allen 1957).
differences (juveniles or females). In the family Trichodoridae we could consider the ancestral form
In the male, easily visible diagnostic features, such as number as gonochoristic with large sperm cells. In the Paratrichodorus
of CP (rare variation), combined with the type of sperm, div- allius species-complex, automixis is a convergent feature. In
ides Paratrichodorus species largely in two main groups: (i) several nematode taxa, males produce copulatory plugs during
species with 1 CP, including the P. hispanus species group, char- copulation to impede females remating and in this way, sperm
acterized by large sperm with sausage-shaped sperm nucleus competition (Carta et al. 2009, Canales-Lazcano et al. 2019).
(except large, fusiform sperm in P. macrostylus or large sperm In most taxa, including Caenorhabditis, the copulatory plug is a
with large round nucleus in P. porosus) and four known species gelatinous mass that covers the vulva; more rarely, secretion is
of the P. allius species-complex with small sperm with round nu- found in the distal lumen vagina, as in Rhigonematoidea (Hunt
cleus (P. catharinae, P. meyeri, P. paraporosus, and P. tunisiensis) 2001); the evolutionary loss of copulatory plugs in self-fertilizing
and (ii) species without CP comprising the other species of populations suggest that copulatory plugs are costly to produce.
the P. allius species-complex with small sperm and small nu- In Trichodoridae, a copulatory plug has only been observed in
cleus. A third group (iii) contains only two species, P. mirzai and gonochoristic species with large sperm cells, such as Trichodorus
Figure 19. Phylogenetic relationships within Trichodoridae with the position of Paratrichodorus allius species-complex. Bayesian 50% majority
rule consensus tree as inferred from D2-D3 expansion segments of 28S rRNA gene sequence alignment under the transversion model with
invariable sites and a gamma-shaped distribution (TVM+I+G). Posterior probabilities of more than 0.70 are given for appropriate clades.
Newly obtained sequences in this are shown in bold. The scale bar indicates expected changes per site, and the boxes indicate the clade
association within P. allius species-complex.
species (Decraemer 2012) and species of the P. hispanus group two groups based on the presence of 1 CP (P. uliaensis sp. nov.,
(Decraemer et al. 2019). The copulatory plug does not cover the P. benalupensis sp. nov., and P. hinogerus sp. nov.) or absence of
vulva but is formed by male secretion hardening within the vagina CP (P. tarifaensis sp. nov., P. asidonaensis sp. nov., P. rocianerus
and results in more or less species-specific types in Trichodorus sp. nov., and P. litoralis sp. nov.). The phylogenetic tree based on
but with a uniform type in the few species of Paratrichodorus in D2-D3 expansion segments of 28S rRNA, showed that P. uliaensis
which a copulatory plug has been observed. Copulatory plugs sp. nov. and P. benalupensis sp. nov. belong to a clade including
do not prevent more than one mating because several plugs have P. paraallius sp. nov., a species without CP and also P. helapulensis
been observed in the uteri of trichodorids where they could pro- sp. nov. belongs to another clade including P. litoralis sp. nov., a
vide additional nutrients. In the P. allius species-complex with species without CP and an unknown species from Iran. This il-
small sperm, no copulatory plugs have been observed. lustrates that presence of CP is a convergent character. Within
During this study, the seven new species within the P. allius the P. allius species-complex, the species possessing 1 CP in
species-complex were morphologically easily separated into male P. uliaensis sp. nov. and P. benalupensis sp. nov. could only be
rule consensus tree as inferred from ITS rRNA gene sequence alignment under the one-parameter model with invariable sites and gamma
distribution model (TPM2uf+I+G). Posterior probabilities of more than 0.70 are given for appropriate clades. Newly obtained sequences
in this are shown in bold. The scale bar indicates expected changes per site, and the boxes indicate the clade association within P. allius
species-complex.
differentiated in males based on absence vs. presence of a reserve segments of 28S rRNA, illustrates that automixis is a convergent
stylet, a feature often considered as accidental. However, based feature, appearing more than once, e.g. in P. asidonaeensis sp. nov.,
on the additional support from ribosomal molecular markers P. rocianerus sp. nov., P. littoralis sp. nov., and P. paraallius sp. nov..
(28S rRNA, ITS, and partial 18S rRNA), as illustrated in the Females of the P. allius species-complex studied can hardly be
phylogenetic trees (Figs 19–21) and the separation by PCA ana- differentiated based on vaginal structures (shape/size vagina and
lysis (Fig. 18), both species were accepted and described as new shape/size/orientation of vaginal sclerotized pieces). Vaginal
species. We additionally support the usefulness of multivariate sclerotized pieces (optical section of a sclerotized ring) are very
morphometric analysis as a valuable tool in species delimitation small to inconspicuous. Therefore, coding of feature K based on
when morphologically and morphometrically undistinguishable size is tricky, the more so because shape (feature J) and orienta-
taxa appear within this nematode genus. However, we highlight tion (feature M) can also show some variation. Vagina shape can
that no single morphometric character can be used alone to dis- be influenced by the physiological condition of the specimen
criminate species here as has been seen in other nematode genera and by fixation. Consequently, other characters such as the loca-
(Archidona-Yuste et al. 2016). Among the four species without tion of sperm in the reproductive system should be investigated
CP, P. asidonaensis sp. nov. clusters with P. tarifaensis sp. nov., and as an additional tool in identification. For example, sperm accu-
P. rocianerus sp. nov. clusters with P. teres in a separate clade (Fig. mulation in an ovarial sac (P. hinogerus sp. nov. and P. asidonaensis
19). These four new species without CP were morphologically sp. nov.) or near ovary germinal zone (P. paraallius sp. nov.)
differentiated by morphometry (body length and onchiostyle points to P. allius and hermaphroditism (Decraemer and Chaves
length), also by the presence of males or gonochorism vs. 1989, Sturhan 1989, Decraemer 1995), while sperm in the uteri
automixis. The phylogenetic tree based on D2-D3 expansion (P. uliaensis sp. nov., P. benalupensis sp. nov., P. tarifaensis sp. nov.,
rule consensus tree as inferred from 18S rRNA gene sequence alignment under the symmetrical model with invariable sites and gamma
distribution (SYM+I+G). Posterior probabilities of more than 0.70 are given for appropriate clades. Newly obtained sequences in this are
shown in bold. The scale bar indicates expected changes per site, and the boxes indicate the clade association within Paratrichodorus allius
species-complex.
P. rocianerus sp. nov., and P. litoralis sp. nov.) points to similarities aff. sparsus morphotype 1 (Decraemer et al. 2008). So far, it has
with P. acaudatus, P. rhodesiensis, P. tunisiensis, or P. teres. Similarly been described and considered as a diagnostic feature only in
for males, morphometric data on body length and onchiostyle two Nanidorus species (N. minor and N. nanus) in which it was
length can further help in identification, as well as gender ratio observed in all specimens of several populations (Decraemer
or presence of a reserve onchium in the adult. The subdivision 1995). We observed a reserve onchium in all specimens of
of the P. allius species-complex studied in groups in males (1 CP two new species, P. benalupensis sp. nov. and P. litoralis sp. nov.,
or 0 CP) is not reflected in females. Ilieva-Makulee et al. (2017) which are not closely related. The feature of presence of a reserve
included shape and orientation of the rectum in females to dif- onchium is another convergent trait.
ferentiate P. teres specimens from Poland vs. Iranian specimens. Phylogenetic analyses based on 28S rDNA of Paratrichodorus
However, they found that the feature of the rectum (shape and species reveal two well-supported clades: (i) clade VI grouping
wall thickness) is of a restricted use and subjected to the fixation of the P. allius species-complex studied here, including sequences
process. from GenBank of P. teres (from Poland) (KU578055), P. allius
The criterion of the presence of a reserve onchium in adults (from USA), and other sequences of Paratrichodorus sp. from Iran
was not included in the tabular identification key because it is and (ii) a sister-clade IV grouping all species of the P. hispanus
often considered as an abnormal maintenance of a juvenile char- species group, including P. porosus. The species of the P. allius
acter; no attention is given to it, the more because the feature is clade all possess a longitudinal slit as vulva, and male and female
not always obvious. A reserve onchium in adults has been de- possess small sperm cells with small nuclei (with or without a fi-
scribed in P. porosus (Bird 1971, Decraemer and Cheng 1994), brillar appearance or rarely thread-like as in P. namibiensis). This
Trichodorus petrusalberti (Decraemer and Marais 1993), T. phylogenetic congruence with these clades is similar in partial
nanjingensis Liu and Cheng 1990 (Decraemer and Cheng 1994), 18S rRNA with 28S rRNA, while it is not clear with the ITS re-
and in all specimens of a population identified as Trichodorus gion. These features are typical for Paratrichodorus s.s. (Siddiqi
1974, 1980); in addition, the pharyngo-intestinal junction, al- León Ropero from IAS-CSIC, and Marjolein Couvreur (Ugent) for
though variable (Decraemer and De Waele 1981), often shows texcellent technical assistance.
a strong ventral overlap and males may be rare in some species/
populations. Species of the P. hispanus clade have females with CONFLICT OF IN TEREST
a pore-like vulva in ventral view, large sperm cells with a large
sausage-shaped nucleus or medium-sized cells with rounded nu- The authors declare that they are not aware of any conflict of interest.
cleus and, although variable, most species show a dorsal anterior
overlap of the pharynx by the intestine, never a pronounced ven-
FUNDING
tral overlap by the pharyngeal glands as in the P. allius species-
complex. All these features are characteristic for Atlantadorus This research was supported by grant P12-AGR 1486 from ‘Consejería
and support Atlantadorus as a valid genus, but this hypothesis de Economía, Innvovación y Ciencia’ from Junta de Andalucía, and
need to be confirmed with additional molecular studies on dif- Union Europea, Fondo Europeo de Desarrollo regional, ‘Una manera
ferent species/populations of this species group coincident de hacer Europa’, grant 219262 ArimNET_ERANET FP7 2012-2015
in a moderately support in D2-D3 expansion segments of 28S Project PESTOLIVE ‘Contribution of olive history for the manage-
ment of soilborne parasites in the Mediterranean basin’ from Instituto
rRNA (PP = 0.97) and a good support in partial 18S rRNA
Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA),
(PP = 1.00). and grant 201740E042, ‘Análisis de diversidad molecular, barcoding,
Several references in the literature and in GenBank of P. allius, y relaciones filogenéticas de nematodos fitoparásitos en cultivos
P. porosus, and P. teres probably refer to cryptic species. For ex- mediterráneos’ from Spanish National Research Council (CSIC).
ample: P. porosus was considered polyphyletic by Kumari and
Subbotin (2012); P. tansaniensis (junior synonym of P. allius)
from Italy was described based on morphology by Roca and DATA AVAIL ABILIT Y
Lamberti (1984) and probably belongs to another species All links and identifiers for our data are presented in the manuscript
(Sturhan 1989); and unpublished P. teres from Iran (KF550304) and in Tables 1–11. Additional data are available from the authors upon
is not coincident molecularly with published P. teres from Poland request.
(KU578055) (Ilieva-Makulec et al. 2017).
Sequences of nuclear rRNA and mtDNA genes have proven
to be a powerful tool for accurate molecular species identifica- REFERENCES
tion in Trichodoridae (Boutsika et al. 2004, Duarte et al. 2010, Allen MW. A review of the nematode genus Trichodorus with descrip-
Decraemer et al. 2013, Ilieva-Makulec et al. 2017, Pedram et al. tions of ten new species. Nematologica 1957;2:32–62. https://doi.
2017). In our case, the use of these molecular markers provides org/10.1163/187529257x00635
unequivocal nematode identification and only partial 18S rRNA Almeida MTM, Almeida Santos MSN, Oliveira Abrantes IM et al.
Paratrichodorus divergens sp. n., a new potential virus vector of to-
showed difficulties between some species, such as, P. paraallius bacco rattle virus and additional observations on P. hispanus Roca and
sp. nov. and P. teres (AM269896). Arias, 1986 from Portugal (Nematoda: Trichodoridae). Nematology
2005;7:343–61. https://doi.org/10.1163/156854105774355572
CONCLUSION Altschul SF, Gish W, Miller W et al. Basic local alignment search
tool. Journal of Molecular Biology 1990;215:403–10. https://doi.
In summary, the present study establishes the importance of org/10.1016/S0022-2836(05)80360-2
using integrative taxonomic identification for unravelling the Archidona-Yuste A, Navas-Cortés JA, Cantalapiedra-Navarrete C et al.
underestimated nematode biodiversity and highlights the time- Cryptic diversity and species delimitation in the Xiphinema americanum-
consuming aspect and difficulty of correct identification to the group complex (Nematoda: Longidoridae) as inferred from morpho-
species level within the P. allius species-complex, describing new metrics and molecular markers. Zoological Journal of the Linnean Society
2016;176:231–65. https://doi.org/10.1111/zoj.12316
species. Phylogenetic congruence of this species group is well- Baldi C, Viviano J, Ellis RE. A bias caused by ectopic development pro-
supported in the phylogenetic trees. This study also provides duces sexually dimorphic sperm in nematodes. Current Biology
molecular markers for resolving species delimitation in a cryptic 2011;21:1416–20. https://doi.org/10.1016/j.cub.2011.07.034
species complex or nematode stages (juveniles or females), and Baujard P. Observations sur les Trichodoridae Thome, 1935 (Ncmatoda)
for precise and unequivocal diagnosis of some species of the de l’Afrique de j’Ouest. Revue de Nématologie 1983;6:223–8.
genus Paratrichodorus to differentiate virus-vector species, such Bird GW. Digestive system of Trichodorus porosus. Journal of Nematology
1971;3:50–7.
as P. allius, with high accuracy. Boutsika K, Brown DJF, Philips MS et al. Molecular characterisa-
tion of the ribosomal DNA of Paratrichodorus macrostylus, P.
pachydermus, Trichodorus primitivus and T. similis (Nematoda:
ACKNOWLEDGE MENTS Trichodoridae). Nematology 2004;6:641–54. https://doi.
A. Archidona-Yuste is funded by the Ramón y Cajal program (RYC2021- org/10.1163/1568541042843432
Canales-Lazcano J, Contreras-Garduño J, Cordero C. Strategic ad-
031108-I), funded by MCIN/AEI/10.13039/501100011033, and
justment of copulatory plug size in a nematode. Current Zoology
UE ‘Next Generation EU/PRTR’. Spanish authors acknowledge grant 2019;65:571–7. https://doi.org/10.1093/cz/zoy067
PRE2019-090206 funded by MCIN and European Social Fund (ESF) Carta K, Handoo ZA, Hoberg EP et al. Evaluation of some vulval
‘Investing in your future’ for I. Clavero-Camacho. A.N. Ruiz-Cuenca is appendages in nematode taxonomy. Comparative Parasitology
a recipient of a postdoctoral grant for the requalification of the Spanish 2009;76:191–209.
University System 2021–2023 (modality ‘Margarita Salas’), and fi- Colbran RC. Studies of plant soil nematodes 1. Two new species from
nanced by Next Generation EU (NGEU) funding through the Spanish Queensland. Queensland Journal of Agricultural and Animal Science
Ministry of Universities. The authors thank J. Martín Barbarroja and G. 1956;13:123–6.
Coolen WA. Methods for extraction of Meloidogyne spp. and other nema- Duarte IM, de Almeida MTM, Brown DJF et al. Phylogenetic relation-
todes from roots and soil. In: Lamberti F, Taylor CE (eds), Root-knot ships based on SSU rDNA sequences, among the didelphic genera of
Nematodes (Meloidogyne species). Systematics, Biology, and Control. the family Trichodoridae from Portugal. Nematology 2010;12:171–
London, UK: Academic Press, 1979, 317–29. 80. https://doi.org/10.1163/156854109X461721
Darriba D, Taboada GL, Doallo R et al. jModelTest 2: more models, Edward JC, Misra SL. Two new species of Trichodorus from Uttar Pradesh,
new heuristics and parallel computing. Nature Methods 2012;9:772. India. Allahabad Farmer 1970;44:167–71.
https://doi.org/10.1038/nmeth.2109 Fédération Nationale des Producteurs de Plants de Pomme de Terre
Decraemer W. Identification of Trichodorids. In: Fortuner R (ed.), (France). A Practical Guide to Diseases, Pests and Disorders of The
Nematode Identification and Expert System Technology. NATO ASI Potato. France: FN3PT, Gnis and Arvalis, 2011, 196.
Series, Series A: Life Sciences 162. London: Plenum Press New York, Hajihassani A, Hamidi N, Dutta B et al. First Report of Stubby-Root
1989, 157–70. Nematode, Paratrichodorus minor, on Onion in Georgia, USA.
Decraemer W. The Family Trichodoridae: Stubby Root and Virus Vector Journal of Nematology 2018;50:453–5. https://doi.org/10.21307/
Nematodes. Dordrecht, Netherlands: Kluwer Academic Publishers, jofnem-2018-038
1995, 360. Hall TA. BioEdit: a user-friendly biological sequence alignment
Decraemer W. Tokens of love: possible diagnostic value of mating editor and analysis program for windows 95/98/NT. Nucleic Acids
plugs and refractive secretory uterine structures in Trichodorus Symposium Series 1999;41:95–8.
(Diphtherophorina: Trichodoridae). Nematology 2012;14:151–8. Hamilton CA, Hendrixson BE, Brewer MS et al. An evaluation of sam-
https://doi.org/10.1163/138855411x581703 pling effects on multiple DNA barcoding methods leads to an inte-
Decraemer W, Baujard P. A polytomous key for the identification of grative approach for delimiting species: A case study of the North
species of the family Trichodoridae Thorne, 1935 (Nematoda: American tarantula genus Aphonopelma (Araneae, Mygalomorphae,
Triplonchida). Fundamental and Applied Nematology 1998;21:37–62. Theraphosidae). Molecular Phylogenetics and Evolution 2014;71:79–
Decraemer W, Chaves E. Morphology of the reproductive system and 93. https://doi.org/10.1016/j.ympev.2013.11.007
structure of sperm cells in some species of the genus Paratrichodorus Holterman M, van den Wurff A, van den Elsen S et al. Phylum-wide ana-
with uncommon males (Nematoda: Diphtherophorina). Revue de lysis of SSU rDNA reveals deep phylogenetic relationships among
Nématologie 1989;11:405–9. nematodes and accelerated evolution toward crown clade. Molecular
Decraemer W, Cheng H. On the occurrence of an inner stylet in adult Biology and Evolution 2006;23:1792–800. https://doi.org/10.1093/
trichodorids and additional information on Trichodorus nanjingensis molbev/msl044
Liu and Cheng, 1990. Fundamental and Applied Nematology Hooper DJ. Three new species of Trichodorus (Nematoda: Dorylaimoidea)
1994;17:503–7. and observations on T. minor Colbran, 1956. Nematologica
Decraemer W, De Waele D. Taxonomic value of the position of oe- 1962;7:273–80. https://doi.org/10.1163/187529262x00567
sophageal gland nuclei and of oesophageal gland overlap in the Huang D, Yan G, Skantar A. Development of real-time and conventional
Trichodoridae (Diphtherophorina). Nematologica 1981;27:82–94. PCR assays for identifying stubby root nematode Paratrichodorus
https://doi.org/10.1163/187529281x00089 allius. Plant Disease 2017a;101:96–972. https://doi.org/10.1094/
Decraemer W, Geraert E. In: Perry RN, Moens M (eds), Plant Nematology, PDIS-10-16-1431-RE
2nd edn. Wallingford, UK: CAB International, 2013, 179–216. Huang D, Yan G, Gudmestad N et al. Quantification of Paratrichodorus
Decraemer W, Marais M. Two new species of Trichodorus from South allius in DNA extracted from soil using TaqMan probe and SYBR
Africa, with a note on T. petrusalberti (Nemata: Trichodoridae). Green real-time PCR assays. Nematology 2017b;19:987–1001.
Fundamental and Applied Nematology 1993;16:211–4. https://doi.org/10.1163/15685411-00003101
Decraemer W, Reay F. Trichodorid nematodes from Australia with de- Huang D, Yan G, Gudmestad N et al. Developing a one-step multiplex
scription of two new species from native vegetation. Australasian Plant PCR assay for rapid detection of four stubby-root nematode spe-
Pathology 1991;20:52–66. https://doi.org/10.1071/app9910052 cies, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus
Decraemer W, Subbotin SA. Molecular and morphological character- obtusus. Plant Disease 2019;103:404–10. https://doi.org/10.1094/
isation of Trichodorus species (Nematoda: Trichodoridae), with PDIS-06-18-0983-RE
description of T. pseudoaequalis n sp from California and other Hunt D. The African Carnoyidae (Nematoda: Rhigonematida). 1.
states of the USA. Nematology 2021;23:317–44. https://doi. Brumptaemilus sp n from Ghana with observations on copulatory
org/10.1163/15685411-bja10044 plugs and spermatophore development. Nematology 2001;3:313–23.
Decraemer W, Radivojević M, de la Peña E. Trichodoridae (Nematoda: https://doi.org/10.1163/156854101317020231
Triplonchida) from the Tara National Park, Serbia, and proposal of Ilieva-Makulec K, Rybarczyk-Mydłowska K, Winiszewska G et al.
Trichodorus pseudobursatus n sp. Nematology 2008;10:405–31. Morphological and molecular analysis of Paratrichodorus teres
Decraemer W, Palomares-Rius JE, Cantalapiedra-Navarrete C et al. Seven (Hooper 1962) (Nematoda: Trichodoridae): a groundwork for dis-
new species of Trichodorus (Diphtherophorina, Trichodoridae) from cussion on the phylogeny and pathogenicity of Paratrichodorus spe-
Spain, an apparent centre of speciation. Nematology 2013;15:57–100. cies. European Journal of Plant Pathology 2017;148:907–17. https://
https://doi.org/10.1163/156854112x645598 doi.org/10.1007/s10658-016-1145-x
Decraemer W, Cantalapiedra-Navarrete C, Archidona-Yuste A et al. Ingham RE, Hamm PB, McMorran JP et al. Manegement of Paratrichodorus
Integrative taxonomy unravels cryptic diversity in the Paratrichodorus allius Damage to Onion in the Columbia Basin of Oregon. Journal of
hispanus-group complex and resolves two new species of the genus and Nematology 1999;31:678–83.
the molecular phylogeny of the family (Nematoda: Trichodoridae). Jensen HJ. Trichodorus allius, a new species of stubby-root nematode from
Zoological Journal of the Linnean Society 2019;185:656–92. Oregon (Nemata: Dorylaimoidea). Proceedings of the Helminthological
Decraemer W, Kantor MR, Handoo ZA et al. Description of Trichodorus Society of Washington 1963;30:157–9.
marylandi n sp (Nematoda: Trichodoridae) from Maryland, USA. Jensen HJ, Allen TC. Trichodorus allius, a potential nematode vector of
Nematology 2021a;24:307–19. TRV. Phytopathology 1964;54:1434.
Decraemer W, Magnusson C, Bert W. Morphological and molecular char- Katoh K, Rozewicki J, Yamada KD. MAFFT online service: Multiple
acterisation of Trichodorus hellalae n sp (Nematoda: Trichodoridae) sequence alignment, interactive sequence choice and visualization.
from Norway. European Journal of Plant Pathology 2021b;161:91– Briefings in Bioinformatics 2019;20:1160–6. https://doi.org/10.1093/
105. https://doi.org/10.1007/s10658-021-02306-8 bib/bbx108
De Ley P, Felix MA, Frisse L et al. Molecular and morphological char- Kumari S, Subbotin SA. Molecular characterization and diag-
acterisation of two reproductively isolated species with mirror-image nostics of stubby root and virus vector nematodes of the
anatomy (Nematoda: Cephalobidae). Nematology 1999;1:591–612. family Trichodoridae (Nematoda: Triplonchida) using
ribosomal RNA genes. Plant Pathology 2012;61:1021–31. https:// Ross HA, Murugan S, Li WLS. Testing the reliability of genetic methods of
doi.org/10.1111/j.1365-3059.2012.02598.x species identification via simulation. Systematic Biology 2008;57:216–
LaMunyon CW, Ward S. Evolution of larger sperm in response to ex- 30. https://doi.org/10.1080/10635150802032990
perimentally increased sperm competition in Caenorhabditis elegans. Seinhorst JW. On Trichodorus pachydermus n sp (Nematoda: Enoplida).
Proceedings Biological Sciences 2002;269:1125–8. https://doi. Journal of Helminthology 1954;28:111–4. https://doi.org/10.1017/
org/10.1098/rspb.2002.1996 s0022149x00032727
Legendre P, Legendre L. Numerical Ecology. Amsterdam, The Netherlands: Seinhorst JW. Killing nematodes for taxonomic study with hot fa 4:1.
Elsevier, 2012, 990. Nematologica 1966;12:178.
Li X, Maria M, Cai R et al. Distribution of trichodorid species in main- Shumaker J, Weingartner C, Hensel D et al. Promising russet-skin po-
land China with description of Trichodorus hangzhouensis sp nov tato cultivars for north Florida. Soil and Crop Science Society of Florida
(Nematoda, Triplonchida). ZooKeys 2020;945:163–89. https://doi. 1984;43:166–9.
org/10.3897/zookeys.945.50424 Siddiqi MR. Two new species of the genus Trichodorus (Nematoda:
Loof PAA. Trichodorus anemones n. sp. with a note on T. teres Hooper, Dorylaimoidea) from India. Proceedings of the Helminthlogical Society
1962 (Nematoda: Enoplida). Verslagen en Mededelingen van de of Washington 1960;27:22–7.
Plantenziektenkundige Dienst te Wageningen 1965;142:132–6. Siddiqi MR. Trichodorus spp (Nematoda: Trichodoridae) from Tunisia
Loof PAA. Taxonomy of Trichodoridae. In: Lamberti F, Taylor CE, and Nicaragua. Nematologica 1963;9:69–85.
Seinhorst JW (eds), Nematode Vectors of Plant Viruses. New York, Siddiqi MR. Systematics of the genus Trichodorus Cobb, 1913 (Nematoda:
USA: Plenum Press Corporation, 1975, 103–27. Dorylaimida), with descriptions of three new species. Nematologica
López Pérez A, Arias A, Bello A. Trichodoridae (Nematoda: 1974;19:259–78.
Triplonchida) in Spain. Nematology 2001;3:403–9. https://doi. Siddiqi MR. On the generic status of Atlantadorus Siddiqi, 1974 and
org/10.1163/156854101753250728 Nanidorus Siddiqi, 1974 (Nematoda: Trichodoridae). Systematic
MacGowan JB. The stubby-root nematode Paratrichodorus christiei (Allen Parasitology 1980;1:151–2. https://doi.org/10.1007/bf00009861
1957) Siddiqi 1974. Fla. Dept. Agric. and Consumer Serv. Nemology Siddiqi MR. Ecuadorus equatorius gen. n., sp. n. and Nanidorus mexicanus
Circular 1983; No 97, 2. sp n (Nematoda: Trichodoridae). International Journal of Nematology
Marais M, Decraemer W, Quénéhervé P. Three Trichodoridae from 2002;12:197–202.
French Guiana and Martinique (Nemata: Diphtherophorina). Siddiqi MR, Brown KF. Trichodorus rhodesiensis and Amphidelus trichurus,
Nematropica 1996;26:121–8. two new nematode species from cultivated soils of Africa. Proceedings
Masters BC, Fan V, Ross HA. Species delimitation a Geneious of the Helminthological Society of Washington 1965;32:239–42.
plugin for the exploration of species boundaries. Molecular Sturhan D. On the species status of Paratrichodorus allius ( Jensen, 1963) and
Ecological Resources 2011;11:154–7. https://doi.org/10.1111/ P. tansaniensis Siddiqi, 1974 (Nematoda, Trichodoridae). Nematologica
j.1755-0998.2010.02896.x 1989;35:62–8. https://doi.org/10.1163/002825989x00052
Mojtahedi H, Santo GS. Ecology of Paratrichodorus allius and its relation- Subbotin SA, Waeyenberge L, Moens M. Identification of cyst forming
ship to the corky ring-spot disease of potato in the Pacific Northwest. nematodes of the genus Heterodera (Nematoda: Heteroderidae)
American Journal of Potato Research 1999;76:273–80. https://doi. based on the ribosomal DNA-RFLP. Nematology 2000;2:153–64.
org/10.1007/bf02853625 https://doi.org/10.1163/156854100509042
Montgomery DC, Peck EA, Vining GG. Introduction to Linear Regression Subbotin SA, Vovlas N, Crozzoli R et al. Phylogeny of Criconematina
Analysis: Hoboken, NJ, USA: Wiley, 2012. Siddiqi, 1980 (Nematoda: Tylenchida) based on morphology and
Nasira K, Maqbool MA. Two new species of Paratrichodorus Siddiqi, 1974 D2-D3 expansion segments of the 28S-rRNA gene sequences with
(Nematoda: Trichodoridae) with observation on P. mirzai (Siddiqi, application of a secondary structure model. Nematology 2005;7:927–
1960) Siddiqi, 1974 and P. renifer Siddiqi, 1974 from Pakistan. 44. https://doi.org/10.1163/156854105776186307
Fundamental and Applied Nematology 1994;17:323–32. Subbotin SA, Cid Del Prado Vera I, Inserra RI et al. Molecular character-
Nunn GB. Nematode molecular evolution: an investigation of evolu- isation of some stubby root nematodes (Nematoda: Trichodoridae)
tionary patterns among nematodes based upon DNA sequences. from the USA and other countries. Nematology 2020;22:39–52.
Ph.D. Dissertation, University of Nottingham, Nottingham, UK, https://doi.org/10.1163/15685411-00003279
1992. Tan G, Muffato M, Ledergerber C et al. Current methods for automated
Pedram M, Roshan-Bakhsh A, Pourjam E et al. Additional data filtering of multiple sequence alignments frequently worsen single-
on Iranian trichodorids (Triplonchida: Trichodoridae) and gene phylogenetic inference. Systematic Biology 2015;64:778–91.
first record of Trichodorus variabilis a rare species, Roca, https://doi.org/10.1093/sysbio/syv033
1998. Nematology 2017;19:121–9. https://doi.org/10.1163/ Tanha Maafi Z, Decraemer W. Description of Trichodorus gilanensis n.
15685411-00003036 sp. from a forest park in Iran and observations on Paratrichodorus
Rambaut A. FigTree v.1.4.3, A Graphical Viewer of Phylogenetic Trees, 2016. tunisiensis (Siddiqi, 1963) Siddiqi, 1974 (Nematoda:
http://tree.bio.ed.ac.uk/software/figtree/ (28 May 2023, date last Diphtherophorina). Nematology 2002;4:43–54. https://doi.
accessed). org/10.1163/156854102760082195
R_Core_Team. R: A Language and Environment for Statistical Computing. Van Megen H, Elsen SVD, Holterman M et al. A phylogenetic tree of
Vienna, Austria: R Foundation for Statistical Computing, 2022. nematodes based on about 1200 full-length small subunit ribo-
https://www.R-project.org/ (26 May 2023, date last accessed). somal DNA sequences. Nematology 2009;11:927–50. https://doi.
Roca F, Lamberti F. Trichodorids (Nematoda) from Italy. Nematologia org/10.1163/156854109X456862
mediterranea 1984;12:95–118. Vielle A, Callemeyn-Torre N, Gimond C et al. Convergent evolution of
Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic infer- sperm gigantism and the developmental origins of sperm size vari-
ence under mixed models. Bioinformatics 2003;19:1572–4. https:// ability in Caenorhabditis nematodes. Evolution 2016;70:2485–503.
doi.org/10.1093/bioinformatics/btg180 https://doi.org/10.1111/evo.13043
Rosenberg NA. Statistical tests for taxonomic distinctiveness from ob- Zuur AF, Ieno EN, Elphick CS. A protocol for data exploration to avoid
servations of monophyly. Evolution 2007;61:317–23. https://doi. common statistical problems. Methods in Ecology and Evolution
org/10.1111/j.1558-5646.2007.00023.x 2010;1:3–14. https://doi.org/10.1111/j.2041-210x.2009.00001.x

ZOJ 2024paratrichodorusallius-Complex Red

Uploaded by

Copyright:

Available Formats

ZOJ 2024paratrichodorusallius-Complex Red

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

ZOJ 2024paratrichodorusallius-Complex Red

Uploaded by

Copyright:

Available Formats

Zoological Journal of the Linnean Society, 2024, XX, 1–44

INTRODUCTION plant-parasitic nematodes that reduce crop productivity by

Received 20 October 2023; revised 9 November 2023; accepted 30 November 2023

Table 1. Paratrichodorus samples used and sequenced in the present study.

Paratrichodorus M0156 Montemayor, Córdoba Vitis vinifera (grapevine) OR139109-OR139110

Distance SP1-SP2/ ♀ length vagina 9 10.5–14 7.5–9 10–16 7.5–9

Distance SP1–SP2/ ♀ length vagina 9 9–16 7.5–9 13–18 8–9

Description of female: Body slightly longer than in male;

Distance SP1–SP2/ ♀ length vagina 13 10–16 5.5–7

Distance SP1–SP2/ ♀ length vagina 17 11–16 9.5–11

isthmus or anterior part pharyngeal bulb. Reproductive system Male: Unknown.

You might also like