Cryptic species diversity in a widespread neotropical tree genus: The case of Cedrela odorata.

PREMISE: Reconciling the use of taxonomy to partition morphological variation and describe genetic divergence within and among closely related species is a persistent challenge in phylogenetics. We reconstructed phylogenetic relationships among Cedrela odorata (Meliaceae) and five closely allied species to test the genetic basis for the current model of species delimitation in this economically valuable and threatened genus. METHODS: We prepared a nuclear species tree with the program SNPhylo and 16,000 single-nucleotide polymorphisms from 168 Cedrela specimens. Based on clades present and ancestral patterns ADMIXTURE, we designed nine species delimitation models and compared each model to current taxonomy with Bayes factor delimitation. Timing of major lineage divergences was estimated with the program SNAPP. RESULTS: The resulting analysis revealed that modern C. odorata evolved from two genetically distinct ancestral sources. All species delimitation models tested better fit the data than the model representing current taxonomic delimitation. Models with the greatest marginal likelihoods separated Mesoamerican C. odorata and South American C. odorata into two species and lumped C. angustifolia and C. montana as a single species. We estimated that Cedrela diversified in South America within the last 19 million years following one or more dispersal events from Mesoamerican lineages. CONCLUSIONS: Our analyses show that the present taxonomic understanding within the genus obscures divergent lineages in C. odorata due in part to morphological differentiation and taxonomic distinctions that are not predictably associated with genetic divergence. A more accurate application of taxonomy to C. odorata and related species may aid in its conservation, management, and restoration efforts.

Genomic resources for the Neotropical tree genus Cedrela (Meliaceae) and its relatives.

BACKGROUND: Tree species in the genus Cedrela P. Browne are threatened by timber overexploitation across the Neotropics. Genetic identification of processed timber can be used to supplement wood anatomy to assist in the taxonomic and source validation of protected species and populations of Cedrela. However, few genetic resources exist that enable both species and source identification of Cedrela timber products. We developed several 'omic resources including a leaf transcriptome, organelle genome (cpDNA), and diagnostic single nucleotide polymorphisms (SNPs) that may assist the classification of Cedrela specimens to species and geographic origin and enable future research on this widespread Neotropical tree genus. RESULTS: We designed hybridization capture probes to enrich for thousands of genes from both freshly preserved leaf tissue and from herbarium specimens across eight Meliaceae species. We first assembled a draft de novo transcriptome for C. odorata, and then identified putatively low-copy genes. Hybridization probes for 10,001 transcript models successfully enriched 9795 (98%) of these targets, and analysis of target capture efficiency showed that probes worked effectively for five Cedrela species, with each species showing similar mean on-target sequence yield and depth. The probes showed greater enrichment efficiency for Cedrela species relative to the other three distantly related Meliaceae species. We provide a set of candidate SNPs for species identification of four of the Cedrela species included in this analysis, and present draft chloroplast genomes for multiple individuals of eight species from four genera in the Meliaceae. CONCLUSIONS: Deforestation and illegal logging threaten forest biodiversity globally, and wood screening tools offer enforcement agencies new approaches to identify illegally harvested timber. The genomic resources described here provide the foundation required to develop genetic screening methods for Cedrela species identification and source validation. Due to their transferability across the genus and family as well as demonstrated applicability for both fresh leaves and herbarium specimens, the genomic resources described here provide additional tools for studies examining the ecology and evolutionary history of Cedrela and related species in the Meliaceae.

Evolution at the tips: Asclepias phylogenomics and new perspectives on leaf surfaces.

PREMISE OF THE STUDY: Leaf surface traits, such as trichome density and wax production, mediate important ecological processes such as anti-herbivory defense and water-use efficiency. We present a phylogenetic analysis of Asclepias plastomes as a framework for analyzing the evolution of trichome density and presence of epicuticular waxes. METHODS: We produced a maximum-likelihood phylogeny using plastomes of 103 species of Asclepias. We reconstructed ancestral states and used model comparisons in a likelihood framework to analyze character evolution across Asclepias. KEY RESULTS: We resolved the backbone of Asclepias, placing the Sonoran Desert clade and Incarnatae clade as successive sisters to the remaining species. We present novel findings about leaf surface evolution of Asclepias-the ancestor is reconstructed as waxless and sparsely hairy, a macroevolutionary optimal trichome density is supported, and the rate of evolution of trichome density has accelerated. CONCLUSIONS: Increased sampling and selection of best-fitting models of evolution provide more resolved and robust estimates of phylogeny and character evolution than obtained in previous studies. Evolutionary inferences are more sensitive to character coding than model selection.

A time and a place for everything: phylogenetic history and geography as joint predictors of oak plastome phylogeny.

Owing to high rates of introgressive hybridization, the plastid genome is poorly suited to fine-scale DNA barcoding and phylogenetic studies of the oak genus (Quercus, Fagaceae). At the tips of the oak plastome phylogeny, recent gene migration and reticulation generally cause topology to reflect geographic structure, while deeper branches reflect lineage divergence. In this study, we quantify the simple and partial effects of geographic proximity and nucleome-inferred phylogenetic history on oak plastome phylogeny at different evolutionary scales. Our study compares pairwise phylogenetic distances based on complete plastome sequences, pairwise phylogenetic distances from nuclear restriction site-associated DNA sequences (RADseq), and pairwise geographic distances for 34 individuals of the white oak clade representing 24 North American and Eurasian species. Within the North American white oak clade alone, phylogenetic history has essentially no effect on plastome variation, while geography explains 11%-21% of plastome phylogenetic variance. However, across multiple continents and clades, phylogeny predicts 30%-41% of plastome variation, geography 3%-41%. Tipwise attenuation of phylogenetic informativeness in the plastome means that in practical terms, plastome data has little use in solving phylogenetic questions, but can still be a useful barcoding or phylogenetic marker for resolving questions among major clades.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PREMISE OF THE STUDY: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. METHODS: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). KEY RESULTS: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. CONCLUSIONS: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Transcriptome characterization and polymorphism detection between subspecies of big sagebrush (Artemisia tridentata).

BACKGROUND: Big sagebrush (Artemisia tridentata) is one of the most widely distributed and ecologically important shrub species in western North America. This species serves as a critical habitat and food resource for many animals and invertebrates. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is a serious threat to big sagebrush ecosystem sustainability. Lack of genomic data has limited our understanding of the evolutionary history and ecological adaptation in this species. Here, we report on the sequencing of expressed sequence tags (ESTs) and detection of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in subspecies of big sagebrush. RESULTS: cDNA of A. tridentata sspp. tridentata and vaseyana were normalized and sequenced using the 454 GS FLX Titanium pyrosequencing technology. Assembly of the reads resulted in 20,357 contig consensus sequences in ssp. tridentata and 20,250 contigs in ssp. vaseyana. A BLASTx search against the non-redundant (NR) protein database using 29,541 consensus sequences obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e⁻¹5). A total of 20,952 SNPs and 119 polymorphic SSRs were detected between the two subspecies. SNPs were validated through various methods including sequence capture. Validation of SNPs in different individuals uncovered a high level of nucleotide variation in EST sequences. EST sequences of a third, tetraploid subspecies (ssp. wyomingensis) obtained by Illumina sequencing were mapped to the consensus sequences of the combined 454 EST assembly. Approximately one-third of the SNPs between sspp. tridentata and vaseyana identified in the combined assembly were also polymorphic within the two geographically distant ssp. wyomingensis samples. CONCLUSION: We have produced a large EST dataset for Artemisia tridentata, which contains a large sample of the big sagebrush leaf transcriptome. SNP mapping among the three subspecies suggest the origin of ssp. wyomingensis via mixed ancestry. A large number of SNP and SSR markers provide the foundation for future research to address questions in big sagebrush evolution, ecological genetics, and conservation using genomic approaches.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

BACKGROUND: Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. RESULTS: A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. CONCLUSIONS: The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.

Plastome Structural Evolution and Homoplastic Inversions in Neo-Astragalus (Fabaceae).

The plastid genomes of photosynthetic green plants have largely maintained conserved gene content and order as well as structure over hundreds of millions of years of evolution. Several plant lineages, however, have departed from this conservation and contain many plastome structural rearrangements, which have been associated with an abundance of repeated sequences both overall and near rearrangement endpoints. We sequenced the plastomes of 25 taxa of Astragalus L. (Fabaceae), a large genus in the inverted repeat-lacking clade of legumes, to gain a greater understanding of the connection between repeats and plastome inversions. We found plastome repeat structure has a strong phylogenetic signal among these closely related taxa mostly in the New World clade of Astragalus called Neo-Astragalus. Taxa without inversions also do not differ substantially in their overall repeat structure from four taxa each with one large-scale inversion. For two taxa with inversion endpoints between the same pairs of genes, differences in their exact endpoints indicate the inversions occurred independently. Our proposed mechanism for inversion formation suggests the short inverted repeats now found near the endpoints of the four inversions may be there as a result of these inversions rather than their cause. The longer inverted repeats now near endpoints may have allowed the inversions first mediated by shorter microhomologous sequences to propagate, something that should be considered in explaining how any plastome rearrangement becomes fixed regardless of the mechanism of initial formation.

eDNA as a tool for identifying freshwater species in sustainable forestry: A critical review and potential future applications.

Environmental DNA (eDNA) is an emerging biological monitoring tool that can aid in assessing the effects of forestry and forest manufacturing activities on biota. Monitoring taxa across broad spatial and temporal scales is necessary to ensure forest management and forest manufacturing activities meet their environmental goals of maintaining biodiversity. Our objectives are to describe potential applications of eDNA across the wood products supply chain extending from regenerating forests, harvesting, and wood transport, to manufacturing facilities, and to review the current state of the science in this context. To meet our second objective, we summarize the taxa examined with targeted (PCR, qPCR or ddPCR) or metagenomic eDNA methods (eDNA metabarcoding), evaluate how estimated species richness compares between traditional field sampling and eDNA metabarcoding approaches, and compare the geographical representation of prior eDNA studies in freshwater ecosystems to global wood baskets. Potential applications of eDNA include evaluating the effects of forestry and forest manufacturing activities on aquatic biota, delineating fish-bearing versus non fish-bearing reaches, evaluating effectiveness of constructed road crossings for freshwater organism passage, and determining the presence of at-risk species. Studies using targeted eDNA approaches focused on fish, amphibians, and invertebrates, while metagenomic studies focused on fish, invertebrates, and microorganisms. Rare, threatened, or endangered species received the least attention in targeted eDNA research, but are arguably of greatest interest to sustainable forestry and forest manufacturing that seek to preserve freshwater biodiversity. Ultimately, using eDNA methods will enable forestry and forest manufacturing managers to have data-driven prioritization for conservation actions for all freshwater species.

A draft genome and transcriptome of common milkweed (Asclepias syriaca) as resources for evolutionary, ecological, and molecular studies in milkweeds and Apocynaceae.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3,415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed 11 linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5ß-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae).

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

Quantitative analysis of transcript accumulation from genes duplicated by polyploidy using cDNA-SSCP.

Repeated rounds of polyploidy have been commonplace in the lineages leading to modern eukaryotic genomes, giving rise to widespread gene duplication. Genes duplicated by polyploidy, or "homoeologs," may continue to be expressed at equal levels following polyploidization or their expression may be dramatically altered. In this report, we describe how SSCP analysis of RT-PCR products can be used to evaluate the expression status (presence and relative quantity) of highly similar homoelogous gene pairs from an allotetraploid genome. This cDNA-SSCP approach was used to evaluate transcript abundance in "synthetic tetraploid" mRNA pools (i.e., mixtures of diploid products) and three natural homoeologous gene pairs expressed in tetraploid cotton (Gossypium hirsutum) ovules. Results from replicated tests show that cDNA-SSCP reliably separates duplicated transcripts with 99% sequence identity. Most significantly, the method yields quantitative estimates of transcript ratios in template pools that range from equimolar to approximately 100:1.

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

PREMISE OF THE STUDY: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • METHODS AND RESULTS: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • CONCLUSIONS: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Horizontal transfer of DNA from the mitochondrial to the plastid genome and its subsequent evolution in milkweeds (apocynaceae).

Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae]) and found evidence of intracellular HGT for a 2.4-kb segment of mitochondrial DNA to the rps2-rpoC2 intergenic spacer of the plastome. The transferred region contains an rpl2 pseudogene and is flanked by plastid sequence in the mitochondrial genome, including an rpoC2 pseudogene, which likely provided the mechanism for HGT back to the plastome through double-strand break repair involving homologous recombination. The plastome insertion is restricted to tribe Asclepiadeae of subfamily Asclepiadoideae, whereas the mitochondrial rpoC2 pseudogene is present throughout the subfamily, which confirms that the plastid to mitochondrial HGT event preceded the HGT to the plastome. Although the plastome insertion has been maintained in all lineages of Asclepiadoideae, it shows minimal evidence of transcription in A. syriaca and is likely nonfunctional. Furthermore, we found recent gene conversion of the mitochondrial rpoC2 pseudogene in Asclepias by the plastid gene, which reflects continued interaction of these genomes.

Adventures in the enormous: a 1.8 million clone BAC library for the 21.7 Gb genome of loblolly pine.

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu).

Development of novel chloroplast microsatellite markers to identify species in the Agrostis complex (Poaceae) and related genera.

We needed a reliable way to identify species and confirm potential interspecific and intergeneric hybrids in a landscape level study of gene flow from transgenic glyphosate-resistant Agrostis stolonifera (Poaceae) to compatible relatives. We developed 12 new polymorphic chloroplast microsatellite markers to aid in identifying species recipient of transgenic pollen both within the Agrostis complex and the related genera Polypogon.

Length polymorphism scanning is an efficient approach for revealing chloroplast DNA variation.

Phylogeographic and population genetic screens of chloroplast DNA (cpDNA) provide insights into seed-based gene flow in angiosperms, yet studies are frequently hampered by the low mutation rate of this genome. Detection methods for intraspecific variation can be either direct (DNA sequencing) or indirect (PCR-RFLP), although no single method incorporates the best features of both approaches. We show that screening universal chloroplast amp li cons for length polymorphism provides an accurate and efficient method for identifying cpDNA variation. By sequencing 4500 bp of cpDNA from 17 accessions of Purshia tridentata (bitterbrush), we detected 9 haplotypes, 8 of which were identifiable by unique multilocus length combinations resolvable by automated fragment analysis. In silico estimates of PCR-RFLP for these loci show that 5 haplotypes would be resolved by agarose electrophoresis. A survey of 4 intraspecific data sets from diverse angiosperms revealed that length variation in cpDNA amplicons is nearly ubiquitous, and 61 of 67 haplotypes identified by direct sequencing could be identified by screening length variation. Combined with automated fluorescent detection, length polymorphism screening of universal cpDNA regions offers a simple screen for intraspecific variation that can be used across angiosperms with minimal optimization, providing detection limits that rival direct sequencing at a fraction of the cost.

Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes.

Previous molecular phylogenetic studies have failed to resolve the branching order among the major cotton (Gossypium) lineages, and it has been unclear whether this reflects actual history (rapid radiation) or sampling properties of the genes evaluated. In this paper, we reconsider the phylogenetic relationships of diploid cotton genome groups using DNA sequences from 11 single-copy nuclear loci (10 293 base pairs [bp]), nuclear ribosomal DNA (695 bp), and four chloroplast loci (7370 bp). Results from individual loci and combined nuclear and chloroplast DNA partitions reveal that the cotton genome groups radiated in rapid succession following the formation of the genus. Maximum likelihood analysis of nuclear synonymous sites shows that this radiation occurred within a time span equivalent to 17% of the time since the separation of Gossypium from its nearest extant relatives in the genera Kokia and Gossypioides. Chloroplast and nuclear phylogenies differ significantly with respect to resolution of the basal divergence in the genus and to interrelationships among African cottons. This incongruence is due to limited character evolution in cpDNA and either previously unsuspected hybridization or unreliable phylogenetic performance of the cpDNA characters. This study highlights the necessity of using multiple, independent data sets for resolving phylogenetic relationships of rapidly diverged lineages.

Feast and famine in plant genomes.

Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a 'one-way ticket to genomic obesity', as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.

Rate variation among nuclear genes and the age of polyploidy in Gossypium.

Molecular evolutionary rate variation in Gossypium (cotton) was characterized using sequence data for 48 nuclear genes from both genomes of allotetraploid cotton, models of its diploid progenitors, and an outgroup. Substitution rates varied widely among the 48 genes, with silent and replacement substitution levels varying from 0.018 to 0.162 and from 0.000 to 0.073, respectively, in comparisons between orthologous Gossypium and outgroup sequences. However, about 90% of the genes had silent substitution rates spanning a more narrow threefold range. Because there was no evidence of rate heterogeneity among lineages for any gene and because rates were highly correlated in independent tests, evolutionary rate is inferred to be a property of each gene or its genetic milieu rather than the clade to which it belongs. Evidence from approximately 200,000 nucleotides (40,000 per genome) suggests that polyploidy in Gossypium led to a modest enhancement in rates of nucleotide substitution. Phylogenetic analysis for each gene yielded the topology expected from organismal history, indicating an absence of gene conversion or recombination among homoeologs subsequent to allopolyploid formation. Using the mean synonymous substitution rate calculated across the 48 genes, allopolyploid cotton is estimated to have formed circa 1.5 million years ago (MYA), after divergence of the diploid progenitors about 6.7 MYA.