Phylogenomics and the rise of the angiosperms.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5-7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

Target Enrichment and Extensive Population Sampling Help Untangle the Recent, Rapid Radiation of Oenothera Sect. Calylophus.

Oenothera sect. Calylophus is a North American group of 13 recognized taxa in the evening primrose family (Onagraceae) with an evolutionary history that may include independent origins of bee pollination, edaphic endemism, and permanent translocation heterozygosity. Like other groups that radiated relatively recently and rapidly, taxon boundaries within Oenothera sect. Calylophus have remained challenging to circumscribe. In this study, we used target enrichment, flanking noncoding regions, gene tree/species tree methods, tests for gene flow modified for target-enrichment data, and morphometric analysis to reconstruct phylogenetic hypotheses, evaluate current taxon circumscriptions, and examine character evolution in Oenothera sect. Calylophus. Because sect. Calylophus comprises a clade with a relatively restricted geographic range, we were able to extensively sample across the range of geographic, edaphic, and morphological diversity in the group. We found that the combination of exons and flanking noncoding regions led to improved support for species relationships. We reconstructed potential hybrid origins of some accessions and note that if processes such as hybridization are not taken into account, the number of inferred evolutionary transitions may be artificially inflated. We recovered strong evidence for multiple evolutionary origins of bee pollination from ancestral hawkmoth pollination, edaphic specialization on gypsum, and permanent translocation heterozygosity. This study applies newly emerging techniques alongside dense infraspecific sampling and morphological analyses to effectively reconstruct the recalcitrant history of a rapid radiation. [Gypsum endemism; Oenothera sect. Calylophus; Onagraceae; phylogenomics; pollinator shift; recent radiation; target enrichment.].

A Comprehensive Phylogenomic Platform for Exploring the Angiosperm Tree of Life.

The tree of life is the fundamental biological roadmap for navigating the evolution and properties of life on Earth, and yet remains largely unknown. Even angiosperms (flowering plants) are fraught with data gaps, despite their critical role in sustaining terrestrial life. Today, high-throughput sequencing promises to significantly deepen our understanding of evolutionary relationships. Here, we describe a comprehensive phylogenomic platform for exploring the angiosperm tree of life, comprising a set of open tools and data based on the 353 nuclear genes targeted by the universal Angiosperms353 sequence capture probes. The primary goals of this article are to (i) document our methods, (ii) describe our first data release, and (iii) present a novel open data portal, the Kew Tree of Life Explorer (https://treeoflife.kew.org). We aim to generate novel target sequence capture data for all genera of flowering plants, exploiting natural history collections such as herbarium specimens, and augment it with mined public data. Our first data release, described here, is the most extensive nuclear phylogenomic data set for angiosperms to date, comprising 3099 samples validated by DNA barcode and phylogenetic tests, representing all 64 orders, 404 families (96$\%$) and 2333 genera (17$\%$). A "first pass" angiosperm tree of life was inferred from the data, which totaled 824,878 sequences, 489,086,049 base pairs, and 532,260 alignment columns, for interactive presentation in the Kew Tree of Life Explorer. This species tree was generated using methods that were rigorous, yet tractable at our scale of operation. Despite limitations pertaining to taxon and gene sampling, gene recovery, models of sequence evolution and paralogy, the tree strongly supports existing taxonomy, while challenging numerous hypothesized relationships among orders and placing many genera for the first time. The validated data set, species tree and all intermediates are openly accessible via the Kew Tree of Life Explorer and will be updated as further data become available. This major milestone toward a complete tree of life for all flowering plant species opens doors to a highly integrated future for angiosperm phylogenomics through the systematic sequencing of standardized nuclear markers. Our approach has the potential to serve as a much-needed bridge between the growing movement to sequence the genomes of all life on Earth and the vast phylogenomic potential of the world's natural history collections. [Angiosperms; Angiosperms353; genomics; herbariomics; museomics; nuclear phylogenomics; open access; target sequence capture; tree of life.].

Green land: Multiple perspectives on green algal evolution and the earliest land plants.

Green plants, broadly defined as green algae and the land plants (together, Viridiplantae), constitute the primary eukaryotic lineage that successfully colonized Earth's emergent landscape. Members of various clades of green plants have independently made the transition from fully aquatic to subaerial habitats many times throughout Earth's history. The transition, from unicells or simple filaments to complex multicellular plant bodies with functionally differentiated tissues and organs, was accompanied by innovations built upon a genetic and phenotypic toolkit that have served aquatic green phototrophs successfully for at least a billion years. These innovations opened an enormous array of new, drier places to live on the planet and resulted in a huge diversity of land plants that have dominated terrestrial ecosystems over the past 500 million years. This review examines the greening of the land from several perspectives, from paleontology to phylogenomics, to water stress responses and the genetic toolkit shared by green algae and plants, to the genomic evolution of the sporophyte generation. We summarize advances on disparate fronts in elucidating this important event in the evolution of the biosphere and the lacunae in our understanding of it. We present the process not as a step-by-step advancement from primitive green cells to an inevitable success of embryophytes, but rather as a process of adaptations and exaptations that allowed multiple clades of green plants, with various combinations of morphological and physiological terrestrialized traits, to become diverse and successful inhabitants of the land habitats of Earth.

The uncharacterized gene EVE contributes to vessel element dimensions in Populus.

The radiation of angiosperms led to the emergence of the vast majority of today's plant species and all our major food crops. Their extraordinary diversification occurred in conjunction with the evolution of a more efficient vascular system for the transport of water, composed of vessel elements. The physical dimensions of these water-conducting specialized cells have played a critical role in angiosperm evolution; they determine resistance to water flow, influence photosynthesis rate, and contribute to plant stature. However, the genetic factors that determine their dimensions are unclear. Here we show that a previously uncharacterized gene, ENLARGED VESSEL ELEMENT (EVE), contributes to the dimensions of vessel elements in Populus, impacting hydraulic conductivity. Our data suggest that EVE is localized in the plasma membrane and is involved in potassium uptake of differentiating xylem cells during vessel development. In plants, EVE first emerged in streptophyte algae, but expanded dramatically among vessel-containing angiosperms. The phylogeny, structure and composition of EVE indicates that it may have been involved in an ancient horizontal gene-transfer event.

Differential gene expression associated with a floral scent polymorphism in the evening primrose Oenothera harringtonii (Onagraceae).

BACKGROUND: Plant volatiles play an important role in both plant-pollinator and plant-herbivore interactions. Intraspecific polymorphisms in volatile production are ubiquitous, but studies that explore underlying differential gene expression are rare. Oenothera harringtonii populations are polymorphic in floral emission of the monoterpene (R)-(-)-linalool; some plants emit (R)-(-)-linalool (linalool+ plants) while others do not (linalool- plants). However, the genes associated with differential production of this floral volatile in Oenothera are unknown. We used RNA-Seq to broadly characterize differential gene expression involved in (R)-(-)-linalool biosynthesis. To identify genes that may be associated with the polymorphism for this trait, we used RNA-Seq to compare gene expression in six different Oenothera harringtonii tissues from each of three linalool+ and linalool- plants. RESULTS: Three clusters of differentially expressed genes were enriched for terpene synthase activity: two were characterized by tissue-specific upregulation and one by upregulation only in plants with flowers that produce (R)-(-)-linalool. A molecular phylogeny of all terpene synthases identified two putative (R)-(-)-linalool synthase transcripts in Oenothera harringtonii, a single allele of which is found exclusively in linalool+ plants. CONCLUSIONS: By using a naturally occurring polymorphism and comparing different tissues, we were able to identify candidate genes putatively involved in the biosynthesis of (R)-(-)-linalool. Expression of these genes in linalool- plants, while low, suggests a regulatory polymorphism, rather than a population-specific loss-of-function allele. Additional terpene biosynthesis-related genes that are up-regulated in plants that emit (R)-(-)-linalool may be associated with herbivore defense, suggesting a potential economy of scale between plant reproduction and defense.

Paralogs and off-target sequences improve phylogenetic resolution in a densely-sampled study of the breadfruit genus (Artocarpus, Moraceae).

We present a 517-gene phylogenetic framework for the breadfruit genus Artocarpus (ca. 70 spp., Moraceae), making use of silica-dried leaves from recent fieldwork and herbarium specimens (some up to 106 years old) to achieve 96% taxon sampling. We explore issues relating to assembly, paralogous loci, partitions, and analysis method to reconstruct a phylogeny that is robust to variation in data and available tools. While codon partitioning did not result in any substantial topological differences, the inclusion of flanking non-coding sequence in analyses significantly increased the resolution of gene trees. We also found that increasing the size of datasets increased convergence between analysis methods but did not reduce gene tree conflict. We optimized the HybPiper targeted-enrichment sequence assembly pipeline for short sequences derived from degraded DNA extracted from museum specimens. While the subgenera of Artocarpus were monophyletic, revision is required at finer scales, particularly with respect to widespread species. We expect our results to provide a basis for further studies in Artocarpus and provide guidelines for future analyses of datasets based on target enrichment data, particularly those using sequences from both fresh and museum material, counseling careful attention to the potential of off-target sequences to improve resolution.

Incomplete reproductive isolation and low genetic differentiation despite floral divergence across varying geographic scales in Castilleja.

PREMISE: Divergence depends on the strength of selection and frequency of gene flow between taxa, while reproductive isolation relies on mating barriers and geographic distance. Less is known about how these processes interact at early stages of speciation. Here, we compared population-level differentiation in floral phenotype and genetic sequence variation among recently diverged Castilleja to explore patterns of diversification under different scenarios of reproductive isolation. METHODS: Using target enrichment enabled by the Angiosperms353 probe set, we assessed genetic distance among 50 populations of four Castilleja species. We investigated whether patterns of genetic divergence are explained by floral trait variation or geographic distance in two focal groups: the widespread C. sessiliflora and the more restricted C. purpurea species complex. RESULTS: We document that C. sessiliflora and the C. purpurea complex are characterized by high diversity in floral color across varying geographic scales. Despite phenotypic divergence, groups were not well supported in phylogenetic analyses, and little genetic differentiation was found across targeted Angiosperms353 loci. Nonetheless, a principal coordinate analysis of single nucleotide polymorphisms revealed differentiation within C. sessiliflora across floral morphs and geography and less differentiation among species of the C. purpurea complex. CONCLUSIONS: Patterns of genetic distance in C. sessiliflora suggest species cohesion maintained over long distances despite variation in floral traits. In the C. purpurea complex, divergence in floral color across narrow geographic clines may be driven by recent selection on floral color. These contrasting patterns of floral and genetic differentiation reveal that divergence can arise via multiple eco-evolutionary paths.

A Universal Probe Set for Targeted Sequencing of 353 Nuclear Genes from Any Flowering Plant Designed Using k-Medoids Clustering.

Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost of developing targeted sequencing approaches is associated with the generation of preliminary data needed for the identification of orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants). We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes identified by the One Thousand Plant Transcriptomes Initiative to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm group. To maximize the phylogenetic potential of the probes, while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, 5-15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order groups of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order groups, including the entire angiosperm clade itself.

Signal, Uncertainty, and Conflict in Phylogenomic Data for a Diverse Lineage of Microbial Eukaryotes (Diatoms, Bacillariophyta).

Diatoms (Bacillariophyta) are a species-rich group of eukaryotic microbes diverse in morphology, ecology, and metabolism. Previous reconstructions of the diatom phylogeny based on one or a few genes have resulted in inconsistent resolution or low support for critical nodes. We applied phylogenetic paralog pruning techniques to a data set of 94 diatom genomes and transcriptomes to infer perennially difficult species relationships, using concatenation and summary-coalescent methods to reconstruct species trees from data sets spanning a wide range of thresholds for taxon and column occupancy in gene alignments. Conflicts between gene and species trees decreased with both increasing taxon occupancy and bootstrap cutoffs applied to gene trees. Concordance between gene and species trees was lowest for short internodes and increased logarithmically with increasing edge length, suggesting that incomplete lineage sorting disproportionately affects species tree inference at short internodes, which are a common feature of the diatom phylogeny. Although species tree topologies were largely consistent across many data treatments, concatenation methods appeared to outperform summary-coalescent methods for sparse alignments. Our results underscore that approaches to species-tree inference based on few loci are likely to be misled by unrepresentative sampling of gene histories, particularly in lineages that may have diversified rapidly. In addition, phylogenomic studies of diatoms, and potentially other hyperdiverse groups, should maximize the number of gene trees with high taxon occupancy, though there is clearly a limit to how many of these genes will be available.

Bridging the micro- and macroevolutionary levels in phylogenomics: Hyb-Seq solves relationships from populations to species and above.

Reconstructing phylogenetic relationships at the micro- and macroevoutionary levels within the same tree is problematic because of the need to use different data types and analytical frameworks. We test the power of target enrichment to provide phylogenetic resolution based on DNA sequences from above species to within populations, using a large herbarium sampling and Euphorbia balsamifera (Euphorbiaceae) as a case study. Target enrichment with custom probes was combined with genome skimming (Hyb-Seq) to sequence 431 low-copy nuclear genes and partial plastome DNA. We used supermatrix, multispecies-coalescent approaches, and Bayesian dating to estimate phylogenetic relationships and divergence times. Euphorbia balsamifera, with a disjunct Rand Flora-type distribution at opposite sides of Africa, comprises three well-supported subspecies: western Sahelian sepium is sister to eastern African-southern Arabian adenensis and Macaronesian-southwest Moroccan balsamifera. Lineage divergence times support Late Miocene to Pleistocene diversification and climate-driven vicariance to explain the Rand Flora pattern. We show that probes designed using genomic resources from taxa not directly related to the focal group are effective in providing phylogenetic resolution at deep and shallow evolutionary levels. Low capture efficiency in herbarium samples increased the proportion of missing data but did not bias estimation of phylogenetic relationships or branch lengths.

Phylogenomics reveals an extensive history of genome duplication in diatoms (Bacillariophyta).

PREMISE OF THE STUDY: Diatoms are one of the most species-rich lineages of microbial eukaryotes. Similarities in clade age, species richness, and primary productivity motivate comparisons to angiosperms, whose genomes have been inordinately shaped by whole-genome duplication (WGD). WGDs have been linked to speciation, increased rates of lineage diversification, and identified as a principal driver of angiosperm evolution. We synthesized a large but scattered body of evidence that suggests polyploidy may be common in diatoms as well. METHODS: We used gene counts, gene trees, and distributions of synonymous divergence to carry out a phylogenomic analysis of WGD across a diverse set of 37 diatom species. KEY RESULTS: Several methods identified WGDs of varying age across diatoms. Determining the occurrence, exact number, and placement of events was greatly impacted by uncertainty in gene trees. WGDs inferred from synonymous divergence of paralogs varied depending on how redundancy in transcriptomes was assessed, gene families were assembled, and synonymous distances (Ks) were calculated. Our results highlighted a need for systematic evaluation of key methodological aspects of Ks-based approaches to WGD inference. Gene tree reconciliations supported allopolyploidy as the predominant mode of polyploid formation, with strong evidence for ancient allopolyploid events in the thalassiosiroid and pennate diatom clades. CONCLUSIONS: Our results suggest that WGD has played a major role in the evolution of diatom genomes. We outline challenges in reconstructing paleopolyploid events in diatoms that, together with these results, offer a framework for understanding the impact of genome duplication in a group that likely harbors substantial genomic diversity.

Allele phasing has minimal impact on phylogenetic reconstruction from targeted nuclear gene sequences in a case study of Artocarpus.

PREMISE OF THE STUDY: Untapped information about allele diversity within populations and individuals (i.e., heterozygosity) could improve phylogenetic resolution and accuracy. Many phylogenetic reconstructions ignore heterozygosity because it is difficult to assemble allele sequences and combine allele data across unlinked loci, and it is unclear how reconstruction methods accommodate variable sequences. We review the common methods of including heterozygosity in phylogenetic studies and present a novel method for assembling allele sequences from target-enriched Illumina sequencing libraries. METHODS: We performed supermatrix phylogeny reconstruction and species tree estimation of Artocarpus based on three methods of accounting for heterozygous sequences: a consensus method based on de novo sequence assembly, the use of ambiguity characters, and a novel method for incorporating read information to phase alleles. We characterize the extent to which highly heterozygous sequences impeded phylogeny reconstruction and determine whether the use of allele sequences improves phylogenetic resolution or decreases topological uncertainty. KEY RESULTS: We show here that it is possible to infer phased alleles from target-enriched Illumina libraries. We find that highly heterozygous sequences do not contribute disproportionately to poor phylogenetic resolution and that the use of allele sequences for phylogeny reconstruction does not have a clear effect on phylogenetic resolution or topological consistency. CONCLUSIONS: We provide a framework for inferring phased alleles from target enrichment data and for assessing the contribution of allelic diversity to phylogenetic reconstruction. In our data set, the impact of allele phasing on phylogeny is minimal compared to the impact of using phylogenetic reconstruction methods that account for gene tree incongruence.

A transcriptome screen for positive selection in domesticated breadfruit and its wild relatives (Artocarpus spp.).

PREMISE OF THE STUDY: Underutilized crops, such as breadfruit (Artocarpus altilis, Moraceae) have the potential to improve global food security. Humans have artificially selected many cultivars of breadfruit since its domestication began approximately 3500 years ago. The goal of this research was to identify transcriptomic signals of positive selection and to develop genomic resources that may facilitate the development of improved breadfruit cultivars in the future. METHODS: A reference transcriptome of breadfruit was assembled de novo and annotated. Twenty-four transcriptomes of breadfruit and its wild relatives were generated and analyzed to reveal signals of positive selection that may have resulted from local adaptation or natural selection. Emphasis was placed on MADS-box genes, which are important because they often regulate fruiting timing and structures, and on carotenoid biosynthesis genes, which can impact the nutritional quality of the fruit. KEY RESULTS: Over 1000 genes showed signals of positive selection, and these genes were enriched for localization to plastids. Nucleotide sites and individuals under positive selection were discovered in MADS-box genes and carotenoid biosynthesis genes, with several sites located in cofactor or DNA-binding domains. A McDonald-Kreitman test comparing wild to cultivated samples revealed selection in one of the carotenoid biosynthesis genes, abscisic acid 8'-hydroxylase 3. CONCLUSIONS: This research highlights some of the many genes that may have been intentionally or unintentionally selected for during the human-mediated dispersal of breadfruit and stresses the importance of conserving a varied germplasm collection. It has revealed candidate genes for further study and produced new genomic resources for breadfruit.

Ancestral polyploidy in seed plants and angiosperms.

Whole-genome duplication (WGD), or polyploidy, followed by gene loss and diploidization has long been recognized as an important evolutionary force in animals, fungi and other organisms, especially plants. The success of angiosperms has been attributed, in part, to innovations associated with gene or whole-genome duplications, but evidence for proposed ancient genome duplications pre-dating the divergence of monocots and eudicots remains equivocal in analyses of conserved gene order. Here we use comprehensive phylogenomic analyses of sequenced plant genomes and more than 12.6 million new expressed-sequence-tag sequences from phylogenetically pivotal lineages to elucidate two groups of ancient gene duplications-one in the common ancestor of extant seed plants and the other in the common ancestor of extant angiosperms. Gene duplication events were intensely concentrated around 319 and 192 million years ago, implicating two WGDs in ancestral lineages shortly before the diversification of extant seed plants and extant angiosperms, respectively. Significantly, these ancestral WGDs resulted in the diversification of regulatory genes important to seed and flower development, suggesting that they were involved in major innovations that ultimately contributed to the rise and eventual dominance of seed plants and angiosperms.

Phylotranscriptomic analysis of the origin and early diversification of land plants.

Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

A phylotranscriptomic analysis of gene family expansion and evolution in the largest order of pleurocarpous mosses (Hypnales, Bryophyta).

The pleurocarpous mosses (i.e., Hypnanae) are a species-rich group of land plants comprising about 6,000 species that share the development of female sex organs on short lateral branches, a derived trait within mosses. Many of the families within Hypnales, the largest order of pleurocarpous mosses, trace their origin to a rapid radiation less than 100 million years ago, just after the rise of the angiosperms. As a result, the phylogenetic resolution among families of Hypnales, necessary to test evolutionary hypotheses, has proven difficult using one or few loci. We present the first phylogenetic inference from high-throughput sequence data (transcriptome sequences) for pleurocarpous mosses. To test hypotheses of gene family evolution, we built a species tree of 21 pleurocarpous and six acrocarpous mosses using over one million sites from 659 orthologous genes. We used the species tree to investigate the genomic consequences of the shift to pleurocarpy and to identify whether patterns common to other plant radiations (gene family expansion, whole genome duplication, or changes in the molecular signatures of selection) could be observed. We found that roughly six percent of all gene families have expanded in the pleurocarpous mosses, relative to acrocarpous mosses. These gene families are enriched for several gene ontology (GO) terms, including interaction with other organisms. The increase in copy number coincident with the radiation of Hypnales suggests that a process such as whole genome duplication or a burst of small-scale duplications occurred during the diversification. In over 500 gene families we found evidence of a reduction in purifying selection. These gene families are enriched for several terms in the GO hierarchy related to "tRNA metabolic process." Our results reveal candidate genes and pathways that may be associated with the transition to pleurocarpy, illustrating the utility of phylotranscriptomics for the study of molecular evolution in non-model species.

Mechanisms of functional and physical genome reduction in photosynthetic and nonphotosynthetic parasitic plants of the broomrape family.

Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.

Examining the efficacy of a genotyping-by-sequencing technique for population genetic analysis of the mushroom Laccaria bicolor and evaluating whether a reference genome is necessary to assess homology.

Given the diversity and ecological importance of Fungi, there is a lack of population genetic research on these organisms. The reason for this can be explained in part by their cryptic nature and difficulty in identifying genets. In addition the difficulty (relative to plants and animals) in developing molecular markers for fungal population genetics contributes to the lack of research in this area. This study examines the ability of restriction-site associated DNA (RAD) sequencing to generate SNPs in Laccaria bicolor. Eighteen samples of morphologically identified L. bicolor from the United States and Europe were selected for this project. The RAD sequencing method produced anywhere from 290 000 to more than 3 000 000 reads. Mapping these reads to the genome of L. bicolor resulted in 84 000-940 000 unique reads from individual samples. Results indicate that incorporation of non-L. bicolor taxa into the analysis resulted in a precipitous drop in shared loci among samples, suggests the potential of these methods to identify cryptic species. F-statistics were easily calculated, although an observable "noise" was detected when using the "All Loci" treatment versus filtering loci to those present in at least 50% of the individuals. The data were analyzed with tests of Hardy-Weinburg equilibrium, population genetic statistics (FIS and FST), and population structure analysis using the program Structure. The results provide encouraging feedback regarding the potential utility of these methods and their data for population genetic analysis. We were unable to draw conclusions of life history of L. bicolor populations from this dataset, given the small sample size. The results of this study indicate the potential of these methods to address population genetics and general life history questions in the Agaricales. Further research is necessary to explore the specific application of these methods in the Agaricales or other fungal groups.