Biological Sciencess

The orchid genus Calopogon R.Br. (Orchidaceae), native to eastern North America and the northern Caribbean, currently contains five species and up to three varieties. Using nuclear internal transcribed spacer (ITS) ribosomal DNA sequences, amplified fragment length polymorphisms (AFLPs), chloroplast DNA restriction fragments, and chromosome counts, we present a phylogenetic and taxonomic study of the genus. Calopogon multiflorus and C. pallidus are consistently sister species, but the relationships of C. barbatus, C. oklahomensis, and C. tuberosus are not as clear. In the ITS analysis C. oklahomensis is sister to C. barbatus, whereas it is sister to C. tuberosus in the plastid restriction fragment analysis. Furthermore, all species were found to have chromosome numbers of 2n ϭ 38 and 40, with the exception of the putatively hybrid-derived C. oklahomensis with 2n ϭ 114 and 120. The hexaploidy of the latter, plus the discrepancy in its position between the ITS and plastid restriction fragment trees, could suggest that it is of hybrid origin. However, the presence of unique morphological and molecular characters might indicate that it is either an ancient hybrid or not of hybrid derivation at all. Finally, using these molecular methods all taxa appear to generally be discrete groups, with the exception of C. tuberosus vars. latifolius and tuberosus, the former of which is best combined with the latter.

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements. angiosperm evolution ͉ molecular evolution A ngiosperms, the largest clade of land plants with Ͼ250,000 species, experienced rapid radiation soon after their first appearance in the fossil record (1). As a result, flowering plants exhibit incredible diversity in habit, morphology, anatomy, physiology, and reproductive biology. This variation has presented major challenges to biologists interested in the origin and evolution of these traits, and resolving these issues critically depends on having a well resolved and strongly supported phylogenetic framework. Over the past 20 years, numerous phylogenetic studies have used both morphological and molecular data to assess relationships among the major clades (reviewed in ref. 2), resulting in a widely accepted classification of angiosperms with 45 orders and 457 families (3).

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.

Geraniaceae plastid genomes are highly rearranged, and each of the four genera already sequenced in the family has a distinct genome organization. This study reports plastid genome sequences of six additional species, Francoa sonchifolia, Melianthus villosus,a n dViviania marifolia from Geraniales, and Pelargonium alternans, California macrophylla,a n d Hypseocharis bilobata from Geraniaceae. These genome sequences, combined with previously published species, provide sufficient taxon sampling to reconstruct the ancestral plastid genome organization of Geraniaceae and the rearrangements unique to each genus. The ancestral plastid genome of Geraniaceae has a 4 kb inversion and a reduced, Pelargonium-like small single copy region. Our ancestral genome reconstruction suggests that a few minor rearrangements occurred in the stem branch of Geraniaceae followed by independent rearrangements in each genus. The genomic comparison demonstrates that a series of inverted repeat boundary shifts and inversions played a major role in shaping genome organization in the family. The distribution of repeatsisstronglyassociatedwithbreakpointsintherearranged genomes, and the proportion and the number of large repeats (>20 bp and >60 bp) are significantly correlated with the degree of genome rearrangements. Increases in the degree of plastid genome rearrangements are correlated with the acceleration in nonsynonymous substitution rates (dN) but not with synonymous substitution rates (dS). Possible mechanisms that might contribute to this correlation, including DNA repair system and selection, are discussed.

Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants. Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant...

We have sequenced two complete chloroplast genomes in the Asteraceae, Helianthus annuus (sunflower), and Lactuca sativa (lettuce), which belong to the distantly related subfamilies, Asteroideae and Cichorioideae, respectively. The Helianthus chloroplast genome is 151 104 bp and the Lactuca genome is 152 772 bp long, which is within the usual size range for chloroplast genomes in flowering plants. When compared to tobacco, both genomes have two inversions: a large 22.8-kb inversion and a smaller 3.3-kb inversion nested within it. Pairwise sequence divergence across all genes, introns, and spacers in Helianthus and Lactuca has resulted in the discovery of new, fast-evolving DNA sequences for use in species-level phylogenetics, such as the trnY-rpoB, trnL-rpl32, and ndhC-trnV spacers. Analysis and categorization of shared repeats resulted in seven classes useful for future repeat studies: double tandem repeats, three or more tandem repeats, direct repeats dispersed in the genome, repeats found in reverse complement orientation, hairpin loops, runs of A's or T's in excess of 12 bp, and gene or tRNA similarity. Results from BLAST searches of our genomic sequence against expressed sequence tag (EST) databases for both genomes produced eight likely RNA edited sites (C ! U changes). These detailed analyses in Asteraceae contribute to a broader understanding of plastid evolution across flowering plants.

Asteraceae is the second largest family of plants, with over 20,000 species. For the past few decades, numerous phylogenetic studies have contributed to our understanding of the evolutionary relationships within this family, including comparisons of the fast evolving chloroplast gene, ndhF , rbcL, as well as non-coding DNA from the trnL intron plus the trnL-trnF intergenic spacer 4], matK [5], and, with lesser resolution, psbA-trnH [6]. This culminated in a study by Panero and Funk in 2002 [1] that used over 13,000 bp per taxon for the largest taxonomic revision of Asteraceae in over a hundred years. Still, some uncertainties remain, and it would be very useful to have more information on the relative rates of sequence evolution among various genes and on genome structure as a potential set of phylogenetic characters to help guide future phylogenetic structures.

The Chloroplast Genome Database (ChloroplastDB) is an interactive, web-based database for fully sequenced plastid genomes, containing genomic, protein, DNA and RNA sequences, gene locations, RNA-editing sites, putative protein families and alignments (http://chloroplast.cbio.psu.edu/). With recent technical advances, the rate of generating new organelle genomes has increased dramatically. However, the established ontology for chloroplast genes and gene features has not been uniformly applied to all chloroplast genomes available in the sequence databases. For example, annotations for some published genome sequences have not evolved with gene naming conventions. ChloroplastDB provides unified annotations, gene name search, BLAST and download functions for chloroplast encoded genes and genomic sequences. A user can retrieve all orthologous sequences with one search regardless of gene names in GenBank. This feature alone greatly facilitates comparative research on sequence evolution including changes in gene content, codon usage, gene structure and post-transcriptional modifications such as RNA editing. Orthologous protein sets are classified by TribeMCL and each set is assigned a standard gene name. Over the next few years, as the number of sequenced chloroplast genomes increases rapidly, the tools available in ChloroplastDB will allow researchers to easily identify and compile target data for comparative analysis of chloroplast genes and genomes.

Angiosperm systematics has progressed to the point where it is now expected that multiple, independent markers be used in phylogenetic studies. Universal primers for amplifying informative regions of the chloroplast genome are readily available, but in the faster-evolving nuclear genome it is challenging to discover priming sites that are conserved across distantly related taxa. With goals including the identification of informative markers in rosids, and perhaps other angiosperms, we screened 141 nuclear primer combinations for phylogenetic utility in two distinct groups of rosids at different taxonomic levels-Psiguria (Cucurbitaceae) and Geraniaceae. We discovered three phylogenetically informative regions in Psiguria and two in Geraniaceae, but none that were useful in both groups. Extending beyond rosids, we combined our findings with those of another recent effort testing these primer pairs in Asteraceae, Brassicaceae, and Orchidaceae. From this comparison, we identified 32 primer combinations that amplified regions in representative species of at least two of the five distantly related angiosperm families, giving some prior indication about phylogenetic usefulness of these markers in other flowering plants. This reduced set of primer pairs for amplifying low-copy nuclear markers along with a recommended experimental strategy provide a framework for identifying phylogenetically informative regions in angiosperms.

Evolution operates on whole genomes through direct rearrangements of genes, such as inversions, transpositions, and inverted transpositions, as well as through operations, such as duplications, losses, and transfers, that also affect the gene content of the genomes. Because these events are rare relative to nucleotide substitutions, gene order data offer the possibility of resolving ancient branches in the tree of life; the combination of gene order data with sequence data also has the potential to provide more robust phylogenetic reconstructions, since each can elucidate evolution at different time scales. Distance corrections greatly improve the accuracy of phylogeny reconstructions from DNA sequences, enabling distance-based methods to approach the accuracy of the more elaborate methods based on parsimony or likelihood at a fraction of the computational cost. This paper focuses on developing distance correction methods for phylogeny reconstruction from whole genomes. The main question we investigate is how to estimate evolutionary histories from whole genomes with equal gene content, and we present a technique, the empirically derived estimator (EDE), that we have developed for this purpose. We study the use of EDE on whole genomes with identical gene content, and we explore the accuracy of phylogenies inferred using EDE with the neighbor joining and minimum evolution methods under a wide range of model conditions. Our study shows that tree reconstruction under these two methods is much more accurate when based on EDE distances than when based on other distances previously suggested for whole genomes.

Evolution operates on whole genomes through mutations that change the order and strandedness of genes within the genomes. Thus analyses of gene-order data present new opportunities for discoveries about deep evolutionary events, provided that sufficiently accurate methods can be developed to reconstruct evolutionary trees. In this paper we present two new methods of character coding for parsimony-based analysis of genomic rearrangements: one called MPBE-2, and a new parsimony-based method which we call MPME (based on an encoding of Bryant), both variants of the MPBE method. We then conduct computer simulations to compare this class of methods to distance-based methods (NJ under various distance measures). Our empirical results show that two of our new methods return highly accurate estimates of the true tree, outperforming the other methods significantly, especially when close to saturation.

The Campanulaceae (the "hare bell" or "bellflower" family) is a derived angiosperm family comprised of about 600 species treated in 35 to 55 genera. Taxonomic treatments vary widely and little phylogenetic work has been done in the family. Gene order in the chloroplast genome usually varies little among vascular plants. However, chloroplast genomes of Campanulaceae represent an exception and phylogenetic analyses solely based on chloroplast rearrangement characters support a reasonably well-resolved tree. Chloroplast DNA physical maps were constructed for eighteen representatives of the family. So many gene order changes have occurred among the genomes that characterizing individual mutational events was not always possible. Therefore, we examined different, novel scoring methods to prepare data matrices for cladistic analysis. These approaches yielded largely congruent results but varied in amounts of resolution and homoplasy. The strongly supported nodes were c...

The breakpoint phylogeny is an optimization problem proposed by Blanchette et al. for reconstructing evolutionary trees from gene order data. These same authors also developed and implemented BPAnalysis [3], a heuristic method (based upon solving many instances of the travelling salesman problem) for estimating the breakpoint phylogeny. We present a new heuristic for this purpose; although not polynomial-time, our heuristic is much faster in practice than BPAnalysis. We present and discuss the results of experimentation on synthetic datasets and on the flowering plant family Campanulaceae with three methods: our new method, BPAnalysis, and the neighbor-joining method [25] using several distance estimation techniques. Our preliminary results indicate that, on datasets with slow evolutionary rates and large numbers of genes in comparison with the number of taxa (genomes), all methods recover quite accurate reconstructions of the true evolutionary history (although BPAnalysis is too sl...

The first heuristic for reconstructing phylogenetic trees from gene order data was introduced by Blanchette et al.. It sought to reconstruct the breakpoint phylogeny and was applied to a variety of datasets. We present a new heuristic for estimating the breakpoint phylogeny which, although not polynomial-time, is much faster in practice than BP-Analysis. We use this heuristic to conduct a phylogenetic analysis of chloroplast genomes in the flowering plant family Campanulaceae. We also present and discuss the results of experimentation on this real dataset with three methods: our new method, BPAnalysis, and the neighbor-joining method, using breakpoint distances, inversion distances, and inversion plus transposition distances.

The first heuristic for reconstructing phylogenetic trees from gene order data was introduced by Blanchette et al.. It sought to reconstruct the breakpoint phylogeny and was applied to a variety of datasets. We present a new heuristic for estimating the breakpoint phylogeny which, although not polynomial-time, is much faster in practice than BPAnalysis. We use this heuristic to conduct a phylogenetic analysis of chloroplast genomes in the flowering plant family Campanulaceae. We also present and discuss the results of experimentation on this real dataset with three methods: our new method, BPAnalysis, and the neighbor-joining method, using breakpoint distances, inversion distances, and inversion plus transposition distances. 1

The breakpoint phylogeny is an optimization problem proposed by Blanchette et al. for reconstructing evolutionary trees from gene order data. These same authors developed and implemented BPAnalysis [4], a heuristic method (based upon solving many instances of the Travelling Salesman Problem) for estimating the breakpoint phylogeny. We present a new heuristic for estimating the breakpoint phylogeny which, although not polynomial-time, is much faster in practice than BPAnalysis. We use this heuristic to conduct a phylogenetic analysis of the flowering plant family Campanulaceae. We also present and discuss the results of experimentation on this real dataset with three methods: our new method, BPAnalysis, and the neighbor-joining method [21], using breakpoint distances, inversion distances, and inversion plus transposition distances. Introduction Phylogenetic tree reconstruction is a major aspect of much biological research. This is a very difficult computational problem b...