Benefits and Limits of Phasing Alleles for Network Inference of Allopolyploid Complexes.

Accurately reconstructing the reticulate histories of polyploids remains a central challenge for understanding plant evolution. Although phylogenetic networks can provide insights into relationships among polyploid lineages, inferring networks may be hindered by the complexities of homology determination in polyploid taxa. We use simulations to show that phasing alleles from allopolyploid individuals can improve phylogenetic network inference under the multispecies coalescent by obtaining the true network with fewer loci compared to haplotype consensus sequences or sequences with heterozygous bases represented as ambiguity codes. Phased allelic data can also improve divergence time estimates for networks, which is helpful for evaluating allopolyploid speciation hypotheses and proposing mechanisms of speciation. To achieve these outcomes in empirical data, we present a novel pipeline that leverages a recently developed phasing algorithm to reliably phase alleles from polyploids. This pipeline is especially appropriate for target enrichment data, where depth of coverage is typically high enough to phase entire loci. We provide an empirical example in the North American Dryopteris fern complex that demonstrates insights from phased data as well as the challenges of network inference. We establish that our pipeline (PATÉ: Phased Alleles from Target Enrichment data) is capable of recovering a high proportion of phased loci from both diploids and polyploids. These data may improve network estimates compared to using haplotype consensus assemblies by accurately inferring the direction of gene flow, but statistical non-identifiability of phylogenetic networks poses a barrier to inferring the evolutionary history of reticulate complexes.

Comprehensive phylogenomic time tree of bryophytes reveals deep relationships and uncovers gene incongruences in the last 500 million years of diversification.

PREMISE: Bryophytes form a major component of terrestrial plant biomass, structuring ecological communities in all biomes. Our understanding of the evolutionary history of hornworts, liverworts, and mosses has been significantly reshaped by inferences from molecular data, which have highlighted extensive homoplasy in various traits and repeated bursts of diversification. However, the timing of key events in the phylogeny, patterns, and processes of diversification across bryophytes remain unclear. METHODS: Using the GoFlag probe set, we sequenced 405 exons representing 228 nuclear genes for 531 species from 52 of the 54 orders of bryophytes. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed gene conflict, and estimated the timing of divergences based on 29 fossil calibrations. RESULTS: The phylogeny resolves many relationships across the bryophytes, enabling us to resurrect five liverwort orders and recognize three more and propose 10 new orders of mosses. Most orders originated in the Jurassic and diversified in the Cretaceous or later. The phylogenomic data also highlight topological conflict in parts of the tree, suggesting complex processes of diversification that cannot be adequately captured in a single gene-tree topology. CONCLUSIONS: We sampled hundreds of loci across a broad phylogenetic spectrum spanning at least 450 Ma of evolution; these data resolved many of the critical nodes of the diversification of bryophytes. The data also highlight the need to explore the mechanisms underlying the phylogenetic ambiguity at specific nodes. The phylogenomic data provide an expandable framework toward reconstructing a comprehensive phylogeny of this important group of plants.

GOgetter: A pipeline for summarizing and visualizing GO slim annotations for plant genetic data.

Premise: The functional annotation of genes is a crucial component of genomic analyses. A common way to summarize functional annotations is with hierarchical gene ontologies, such as the Gene Ontology (GO) Resource. GO includes information about the cellular location, molecular function(s), and products/processes that genes produce or are involved in. For a set of genes, summarizing GO annotations using pre-defined, higher-order terms (GO slims) is often desirable in order to characterize the overall function of the data set, and it is impractical to do this manually. Methods and Results: The GOgetter pipeline consists of bash and Python scripts. From an input FASTA file of nucleotide gene sequences, it outputs text and image files that list (1) the best hit for each input gene in a set of reference gene models, (2) all GO terms and annotations associated with those hits, and (3) a summary and visualization of GO slim categories for the data set. These output files can be queried further and analyzed statistically, depending on the downstream need(s). Conclusions: GO annotations are a widely used "universal language" for describing gene functions and products. GOgetter is a fast and easy-to-implement pipeline for obtaining, summarizing, and visualizing GO slim categories associated with a set of genes.

Functional traits and trait coordination change over the life of a leaf in a tropical fern species.

PREMISE: Plant ecological strategies are often defined by the integration of underlying traits related to resource acquisition, allocation, and growth. Correlations between key traits across diverse plants suggest that variation in plant ecological strategies is largely driven by a fast-slow continuum of plant economics. However, trait correlations may not be constant through the life of a leaf, and it is still poorly understood how trait function varies over time in long-lived leaves. METHODS: Here, we compared trait correlations related to resource acquisition and allocation across three different mature frond age cohorts in a tropical fern species, Saccoloma inaequale. RESULTS: Fronds exhibited high initial investments of nitrogen and carbon, but with declining return in photosynthetic capacity after the first year. In the youngest fronds, we found water-use efficiency to be significantly lower than in the oldest mature fronds due to increased transpiration rates. Our data suggest that middle-aged fronds are more efficient relative to younger, less water-use efficient fronds and that older fronds exhibit greater nitrogen investments without higher photosynthetic return. In addition, several trait correlations expected under the leaf economics spectrum (LES) do not hold within this species, and some trait correlations only appear in fronds of a specific developmental age. CONCLUSIONS: These findings contextualize the relationship between traits and leaf developmental age with those predicted to underlie plant ecological strategy and the LES and are among the first pieces of evidence for when relative physiological trait efficiency is maximized in a tropical fern species.

Deep vicariance and frequent transoceanic dispersal shape the evolutionary history of a globally distributed fern family.

PREMISE: The historical biogeography of ferns is typically expected to be dominated by long-distance dispersal due to their minuscule spores. However, few studies have inferred the historical biogeography of a large and widely distributed group of ferns to test this hypothesis. Our aims were to determine the extent to which long-distance dispersal vs. vicariance have shaped the history of the fern family Blechnaceae, to explore ecological correlates of dispersal and diversification, and to determine whether these patterns differ between the northern and southern hemispheres. METHODS: We used sequence data for three chloroplast loci to infer a time-calibrated phylogeny for 154 of 265 species of Blechnaceae, including representatives of all genera in the family. This tree was used to conduct ancestral range reconstruction and stochastic character mapping, estimate diversification rates, and identify ecological correlates of diversification. RESULTS: Blechnaceae originated in Eurasia and began diversifying in the late Cretaceous. A lineage comprising most extant diversity diversified principally in the austral Pacific region around the Paleocene-Eocene Thermal Maximum. Land connections that existed near the poles during periods of warm climates likely facilitated migration of several lineages, with subsequent climate-mediated vicariance shaping current distributions. Long-distance dispersal is frequent and asymmetrical, with New Zealand/Pacific Islands, Australia, and tropical America being major source areas. CONCLUSIONS: Ancient vicariance and extensive long-distance dispersal have shaped the history of Blechnaceae in both the northern and southern hemispheres. The exceptional diversity in austral regions appears to reflect rapid speciation in these areas; mechanisms underlying this evolutionary success remain uncertain.

Phylotranscriptomics Illuminates the Placement of Whole Genome Duplications and Gene Retention in Ferns.

Ferns are the second largest clade of vascular plants with over 10,000 species, yet the generation of genomic resources for the group has lagged behind other major clades of plants. Transcriptomic data have proven to be a powerful tool to assess phylogenetic relationships, using thousands of markers that are largely conserved across the genome, and without the need to sequence entire genomes. We assembled the largest nuclear phylogenetic dataset for ferns to date, including 2884 single-copy nuclear loci from 247 transcriptomes (242 ferns, five outgroups), and investigated phylogenetic relationships across the fern tree, the placement of whole genome duplications (WGDs), and gene retention patterns following WGDs. We generated a well-supported phylogeny of ferns and identified several regions of the fern phylogeny that demonstrate high levels of gene tree-species tree conflict, which largely correspond to areas of the phylogeny that have been difficult to resolve. Using a combination of approaches, we identified 27 WGDs across the phylogeny, including 18 large-scale events (involving more than one sampled taxon) and nine small-scale events (involving only one sampled taxon). Most inferred WGDs occur within single lineages (e.g., orders, families) rather than on the backbone of the phylogeny, although two inferred events are shared by leptosporangiate ferns (excluding Osmundales) and Polypodiales (excluding Lindsaeineae and Saccolomatineae), clades which correspond to the majority of fern diversity. We further examined how retained duplicates following WGDs compared across independent events and found that functions of retained genes were largely convergent, with processes involved in binding, responses to stimuli, and certain organelles over-represented in paralogs while processes involved in transport, organelles derived from endosymbiotic events, and signaling were under-represented. To date, our study is the most comprehensive investigation of the nuclear fern phylogeny, though several avenues for future research remain unexplored.

Get the shovel: morphological and evolutionary complexities of belowground organs in geophytes.

Herbaceous plants collectively known as geophytes, which regrow from belowground buds, are distributed around the globe and throughout the land plant tree of life. The geophytic habit is an evolutionarily and ecologically important growth form in plants, permitting novel life history strategies, enabling the occupation of more seasonal climates, mediating interactions between plants and their water and nutrient resources, and influencing macroevolutionary patterns by enabling differential diversification and adaptation. These taxa are excellent study systems for understanding how convergence on a similar growth habit (i.e., geophytism) can occur via different morphological and developmental mechanisms. Despite the importance of belowground organs for characterizing whole-plant morphological diversity, the morphology and evolution of these organs have been vastly understudied with most research focusing on only a few crop systems. Here, we clarify the terminology commonly used (and sometimes misused) to describe geophytes and their underground organs and highlight key evolutionary patterns of the belowground morphology of geophytic plants. Additionally, we advocate for increasing resources for geophyte research and implementing standardized ontological definitions of geophytic organs to improve our understanding of the factors controlling, promoting, and maintaining geophyte diversity.

A target enrichment probe set for resolving the flagellate land plant tree of life.

PREMISE: New sequencing technologies facilitate the generation of large-scale molecular data sets for constructing the plant tree of life. We describe a new probe set for target enrichment sequencing to generate nuclear sequence data to build phylogenetic trees with any flagellate land plants, including hornworts, liverworts, mosses, lycophytes, ferns, and all gymnosperms. METHODS: We leveraged existing transcriptome and genome sequence data to design the GoFlag 451 probes, a set of 56,989 probes for target enrichment sequencing of 451 exons that are found in 248 single-copy or low-copy nuclear genes across flagellate plant lineages. RESULTS: Our results indicate that target enrichment using the GoFlag451 probe set can provide large nuclear data sets that can be used to resolve relationships among both distantly and closely related taxa across the flagellate land plants. We also describe the GoFlag 408 probes, an optimized probe set covering 408 of the 451 exons from the GoFlag 451 probe set that is commercialized by RAPiD Genomics. CONCLUSIONS: A target enrichment approach using the new probe set provides a relatively low-cost solution to obtain large-scale nuclear sequence data for inferring phylogenetic relationships across flagellate land plants.

Insights into the evolutionary history and widespread occurrence of antheridiogen systems in ferns.

Sex expression of homosporous ferns is controlled by multiple factors, one being the antheridiogen system. Antheridiogens are pheromones released by sexually mature female fern gametophytes, turning nearby asexual gametophytes precociously male. Nevertheless, not all species respond. It is still unknown how many fern species use antheridiogens, how the antheridiogen system evolved, and whether it is affected by polyploidy and/or apomixis. We tested the response of 68 fern species to antheridiogens in cultivation. These results were combined with a comprehensive review of literature to form the largest dataset of antheridiogen interactions to date. Analyzed species also were coded as apomictic or sexual and diploid or polyploid. Our final dataset contains a total of 498 interactions involving 208 species (c. 2% of all ferns). About 65% of studied species respond to antheridiogen. Multiple antheridiogen types were delimited and their evolution is discussed. Antheridiogen responsiveness was not significantly affected by apomixis or polyploidy. Antheridiogens are widely used by ferns to direct sex expression. The antheridiogen system likely evolved multiple times and provides homosporous ferns with the benefits often associated with heterospory, such as increased rates of outcrossing. Despite expectations, antheridiogens may be beneficial to polyploids and apomicts.

The concluding chapter: recircumscription of Goodenia (Goodeniaceae) to include four allied genera with an updated infrageneric classification.

Close scrutiny of Goodenia (Goodeniaceae) and allied genera in the 'Core Goodeniaceae' over recent years has clarified our understanding of this captivating group. While expanded sampling, sequencing of multiple regions, and a genome skimming reinforced backbone clearly supported Goodenia s.l. as monophyletic and distinct from Scaevola and Coopernookia, there appears to be no synapomorphic characters that uniquely characterise this morphologically diverse clade. Within Goodenia s.l., there is strong support from nuclear, chloroplast and mitochondrial data for three major clades (Goodenia Clades A, B and C) and various subclades, which lead to earlier suggestions for the possible recognition of these as distinct genera. Through ongoing work, it has become evident that this is impractical, as conflict remains within the most recently diverged Clade C, likely due to recent radiation and incomplete lineage sorting. In light of this, it is proposed that a combination of morphological characters is used to circumscribe an expanded Goodenia that now includes Velleia, Verreauxia, Selliera and Pentaptilon, and an updated infrageneric classification is proposed to accommodate monophyletic subclades. A total of twenty-five new combinations, three reinstatements, and seven new names are published herein including Goodenia subg. Monochila sect. Monochila subsect. Infracta K.A.Sheph. subsect. nov. Also, a type is designated for Goodenia subg. Porphyranthus sect. Ebracteolatae (K.Krause) K.A.Sheph. comb. et stat. nov., and lectotypes or secondstep lectotypes are designated for a further three names.

The C-Fern (Ceratopteris richardii) genome: insights into plant genome evolution with the first partial homosporous fern genome assembly.

Ferns are notorious for possessing large genomes and numerous chromosomes. Despite decades of speculation, the processes underlying the expansive genomes of ferns are unclear, largely due to the absence of a sequenced homosporous fern genome. The lack of this crucial resource has not only hindered investigations of evolutionary processes responsible for the unusual genome characteristics of homosporous ferns, but also impeded synthesis of genome evolution across land plants. Here, we used the model fern species Ceratopteris richardii to address the processes (e.g., polyploidy, spread of repeat elements) by which the large genomes and high chromosome numbers typical of homosporous ferns may have evolved and have been maintained. We directly compared repeat compositions in species spanning the green plant tree of life and a diversity of genome sizes, as well as both short- and long-read-based assemblies of Ceratopteris. We found evidence consistent with a single ancient polyploidy event in the evolutionary history of Ceratopteris based on both genomic and cytogenetic data, and on repeat proportions similar to those found in large flowering plant genomes. This study provides a major stepping-stone in the understanding of land plant evolutionary genomics by providing the first homosporous fern reference genome, as well as insights into the processes underlying the formation of these massive genomes.

EyeChrom and CCDBcurator: Visualizing chromosome count data from plants.

PREMISE OF THE STUDY: Chromosome count data are available for hundreds of plant species and can be explored in text-only format at the Chromosome Counts Database (http://ccdb.tau.ac.il). CCDBcurator and EyeChrom are an R package and a web application, respectively, that first curate and then visualize these data graphically, so that intra- and interspecific variation of chromosome numbers can be easily summarized and displayed for a given genus. METHODS AND RESULTS: We developed R code to clean, summarize, and display in several formats the chromosome count data for a selected genus or set of species present in the Chromosome Counts Database. These data and figures can be exported for use in analyses, publications, or teaching. CONCLUSIONS: Chromosome count data are critical for a number of evolutionary studies in plant biology, and their importance is underscored by the increasing appreciation of the prevalence of polyploidy in land plants. CCDBcurator and EyeChrom provide a fast, easy, and reproducible means of cleaning, curating, and then visualizing the chromosome count data currently available for plants.

Community phylogeny of the globally critically imperiled pine rockland ecosystem.

PREMISE OF THE STUDY: Community phylogenetic methods incorporate information on evolutionary relationships into studies of organismal assemblages. We used a community phylogenetic framework to investigate relationships and biogeographic affinities and to calculate phylogenetic signal of endemism and invasiveness for the flora of the pine rocklands-a globally critically imperiled ecosystem with a significant portion of its distribution in South Florida, United States. METHODS: We reconstructed phylogenetic relationships of 538 vascular plant taxa, which represent 92.28% of the vascular flora of the pine rocklands. We estimated phylogenetic signal for endemism and invasiveness using phylogenetic generalized linear mixed models. We determined the native range for each species in the data set and calculated the total number of species sourced from each region and all possible combinations of these regions. KEY RESULTS: The pine rockland flora includes representatives of all major vascular plant lineages, and most species have native ranges in the New World. There was strong phylogenetic signal for endemism, but not for invasiveness. CONCLUSIONS: Community phylogenetics has high potential value for conservation planning, particularly for fragmented and endangered ecosystems like the pine rockland. Strong phylogenetic signal for endemic species in our data set, which also tend to be threatened or endangered, can help to identify species at risk, as well as fragments where those species occur, highlighting conservation priorities. Our results indicate, at least in the pine rockland ecosystem, no phylogenetic signal for invasive species, and thus other information must be used to predict the potential for invasiveness.

Employing hypothesis testing and data from multiple genomic compartments to resolve recalcitrant backbone nodes in Goodenia s.l. (Goodeniaceae).

Goodeniaceae is a primarily Australian flowering plant family with a complex taxonomy and evolutionary history. Previous phylogenetic analyses have successfully resolved the backbone topology of the largest clade in the family, Goodenia s.l., but have failed to clarify relationships within the species-rich and enigmatic Goodenia clade C, a prerequisite for taxonomic revision of the group. We used genome skimming to retrieve sequences for chloroplast, mitochondrial, and nuclear markers for 24 taxa representing Goodenia s.l., with a particular focus on Goodenia clade C. We performed extensive hypothesis tests to explore incongruence in clade C and evaluate statistical support for clades within this group, using datasets from all three genomic compartments. The mitochondrial dataset is comparable to the chloroplast dataset in providing resolution within Goodenia clade C, though backbone support values within this clade remain low. The hypothesis tests provided an additional, complementary means of evaluating support for clades. We propose that the major subclades of Goodenia clade C (C1-C3 + Verreauxia) are the result of a rapid radiation, and each represents a distinct lineage.

Community assembly of the ferns of Florida.

PREMISE OF THE STUDY: Many ecological and evolutionary processes shape the assembly of organisms into local communities from a regional pool of species. We analyzed phylogenetic and functional diversity to understand community assembly of the ferns of Florida at two spatial scales. METHODS: We built a phylogeny for 125 of the 141 species of ferns in Florida using five chloroplast markers. We calculated mean pairwise dissimilarity (MPD) and mean nearest taxon distance (MNTD) from phylogenetic distances and functional trait data for both spatial scales and compared the results to null models to assess significance. KEY RESULTS: Our results for over vs. underdispersion in functional and phylogenetic diversity differed depending on spatial scale and metric considered. At the county scale, MPD revealed evidence for phylogenetic overdispersion, while MNTD revealed phylogenetic and functional underdispersion, and at the conservation area scale, MPD revealed phylogenetic and functional underdispersion while MNTD revealed evidence only of functional underdispersion. CONCLUSIONS: Our results are consistent with environmental filtering playing a larger role at the smaller, conservation area scale. The smaller spatial units are likely composed of fewer local habitat types that are selecting for closely related species, with the larger-scale units more likely to be composed of multiple habitat types that bring together a larger pool of species from across the phylogeny. Several aspects of fern biology, including their unique physiology and water relations and the importance of the independent gametophyte stage of the life cycle, make ferns highly sensitive to local, microhabitat conditions.

The unexpected depths of genome-skimming data: A case study examining Goodeniaceae floral symmetry genes.

PREMISE OF THE STUDY: The use of genome skimming allows systematists to quickly generate large data sets, particularly of sequences in high abundance (e.g., plastomes); however, researchers may be overlooking data in low abundance that could be used for phylogenetic or evo-devo studies. Here, we present a bioinformatics approach that explores the low-abundance portion of genome-skimming next-generation sequencing libraries in the fan-flowered Goodeniaceae. METHODS: Twenty-four previously constructed Goodeniaceae genome-skimming Illumina libraries were examined for their utility in mining low-copy nuclear genes involved in floral symmetry, specifically the CYCLOIDEA (CYC)-like genes. De novo assemblies were generated using multiple assemblers, and BLAST searches were performed for CYC1, CYC2, and CYC3 genes. RESULTS: Overall Trinity, SOAPdenovo-Trans, and SOAPdenovo implementing lower k-mer values uncovered the most data, although no assembler consistently outperformed the others. Using SOAPdenovo-Trans across all 24 data sets, we recovered four CYC-like gene groups (CYC1, CYC2, CYC3A, and CYC3B) from a majority of the species. Alignments of the fragments included the entire coding sequence as well as upstream and downstream regions. DISCUSSION: Genome-skimming data sets can provide a significant source of low-copy nuclear gene sequence data that may be used for multiple downstream applications.

Differences in dehydration tolerance among populations of a gametophyte-only fern.

PREMISE OF THE STUDY: For many plant species, historical climatic conditions may have left lasting imprints that are detectable in contemporary populations. Additionally, if these historical conditions also prevented gene flow among populations, these populations may be differentiated with respect to one another and their contemporary environmental conditions. For the fern, Vittaria appalachiana, one theory is that historical conditions during the Pleistocene largely shaped both the distribution and lack of sporophyte production. Our goals-based on this theory-were to examine physiological differences among and within populations spanning the species' geographic range, and the contribution of historical climatic conditions to this differentiation. METHODS: We exposed explants from five populations to four drying treatments and examined differences in physiological response. Additionally, we examined the role of historical and current climatic conditions in driving the observed population differentiation. KEY RESULTS: Populations differ in their ability to tolerate varying levels of dehydration, displaying a pattern of countergradient selection. Exposure to historical and contemporary climatic conditions, specifically variation in temperature and precipitation regimes, resulted in population divergence observed among contemporary populations. CONCLUSIONS: Historical conditions have shaped not only the distribution of V. appalachiana, but also its current physiological limitations. Results from this study support the hypothesis that climatic conditions during the Pleistocene are responsible for the distribution of this species, and may be responsible for the observed differences in dehydration tolerance. Additionally, dehydration tolerance may be the driving factor for previously reported patterns of countergradient selection in this species.