Brad R. Ruhfel

Phylotranscriptomic analysis of the origin and early diversification of land plants

Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution. 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.

Phylogenetic patterns of species loss in Thoreau's woods are driven by climate change

Climate change has led to major changes in the phenology (the timing of seasonal activities, such as flowering) of some species but not others. The extent to which flowering-time response to temperature is shared among closely related species might have important consequences for community-wide patterns of species loss under rapid climate change. Henry David Thoreau initiated a dataset of the Concord, Massachusetts, flora that spans ≈150 years and provides information on changes in species abundance and flowering time. When these data are analyzed in a phylogenetic context, they indicate that change in abundance is strongly correlated with flowering-time response. Species that do not respond to temperature have decreased greatly in abundance, and include among others anemones and buttercups [Ranunculaceae pro parte (p.p.)], asters and campanulas (Asterales), bluets (Rubiaceae p.p.), bladderworts (Lentibulariaceae), dogwoods (Cornaceae), lilies (Liliales), mints (Lamiaceae p.p.), orchids (Orchidaceae), roses (Rosaceae p.p.), saxifrages (Saxifragales), and violets (Malpighiales). Because flowering-time response traits are shared among closely related species, our findings suggest that climate change has affected and will likely continue to shape the phylogenetically biased pattern of species loss in Thoreaus woods.

From algae to angiosperms–inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes

BackgroundNext-generation sequencing has provided a wealth of plastid genome sequence data from an increasingly diverse set of green plants (Viridiplantae). Although these data have helped resolve the phylogeny of numerous clades (e.g., green algae, angiosperms, and gymnosperms), their utility for inferring relationships across all green plants is uncertain. Viridiplantae originated 700-1500 million years ago and may comprise as many as 500,000 species. This clade represents a major source of photosynthetic carbon and contains an immense diversity of life forms, including some of the smallest and largest eukaryotes. Here we explore the limits and challenges of inferring a comprehensive green plant phylogeny from available complete or nearly complete plastid genome sequence data.ResultsWe assembled protein-coding sequence data for 78 genes from 360 diverse green plant taxa with complete or nearly complete plastid genome sequences available from GenBank. Phylogenetic analyses of the plastid data recovered well-supported backbone relationships and strong support for relationships that were not observed in previous analyses of major subclades within Viridiplantae. However, there also is evidence of systematic error in some analyses. In several instances we obtained strongly supported but conflicting topologies from analyses of nucleotides versus amino acid characters, and the considerable variation in GC content among lineages and within single genomes affected the phylogenetic placement of several taxa.ConclusionsAnalyses of the plastid sequence data recovered a strongly supported framework of relationships for green plants. This framework includes: i) the placement of Zygnematophyceace as sister to land plants (Embryophyta), ii) a clade of extant gymnosperms (Acrogymnospermae) with cycads + Ginkgo sister to remaining extant gymnosperms and with gnetophytes (Gnetophyta) sister to non-Pinaceae conifers (Gnecup trees), and iii) within the monilophyte clade (Monilophyta), Equisetales + Psilotales are sister to Marattiales + leptosporangiate ferns. Our analyses also highlight the challenges of using plastid genome sequences in deep-level phylogenomic analyses, and we provide suggestions for future analyses that will likely incorporate plastid genome sequence data for thousands of species. We particularly emphasize the importance of exploring the effects of different partitioning and character coding strategies.

Data access for the 1,000 Plants (1KP) project

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

Favorable climate change response explains non-native species' success in Thoreau's woods.

Invasive species have tremendous detrimental ecological and economic impacts. Climate change may exacerbate species invasions across communities if non-native species are better able to respond to climate changes than native species. Recent evidence indicates that species that respond to climate change by adjusting their phenology (i.e., the timing of seasonal activities, such as flowering) have historically increased in abundance. The extent to which non-native species success is similarly linked to a favorable climate change response, however, remains untested. We analyzed a dataset initiated by the conservationist Henry David Thoreau that documents the long-term phenological response of native and non-native plant species over the last 150 years from Concord, Massachusetts (USA). Our results demonstrate that non-native species, and invasive species in particular, have been far better able to respond to recent climate change by adjusting their flowering time. This demonstrates that climate change has likely played, and may continue to play, an important role in facilitating non-native species naturalization and invasion at the community level.

Phylogenomics and a posteriori data partitioning resolve the Cretaceous angiosperm radiation Malpighiales

The angiosperm order Malpighiales includes ∼16,000 species and constitutes up to 40% of the understory tree diversity in tropical rain forests. Despite remarkable progress in angiosperm systematics during the last 20 y, relationships within Malpighiales remain poorly resolved, possibly owing to its rapid rise during the mid-Cretaceous. Using phylogenomic approaches, including analyses of 82 plastid genes from 58 species, we identified 12 additional clades in Malpighiales and substantially increased resolution along the backbone. This greatly improved phylogeny revealed a dynamic history of shifts in net diversification rates across Malpighiales, with bursts of diversification noted in the Barbados cherries (Malpighiaceae), cocas (Erythroxylaceae), and passion flowers (Passifloraceae). We found that commonly used a priori approaches for partitioning concatenated data in maximum likelihood analyses, by gene or by codon position, performed poorly relative to the use of partitions identified a posteriori using a Bayesian mixture model. We also found better branch support in trees inferred from a taxon-rich, data-sparse matrix, which deeply sampled only the phylogenetically critical placeholders, than in trees inferred from a taxon-sparse matrix with little missing data. Although this matrix has more missing data, our a posteriori partitioning strategy reduced the possibility of producing multiple distinct but equally optimal topologies and increased phylogenetic decisiveness, compared with the strategy of partitioning by gene. These approaches are likely to help improve phylogenetic resolution in other poorly resolved major clades of angiosperms and to be more broadly useful in studies across the Tree of Life.

Phylogeny of the clusioid clade (Malpighiales): Evidence from the plastid and mitochondrial genomes

PREMISE OF THE STUDY The clusioid clade includes five families (i.e., Bonnetiaceae, Calophyllaceae, Clusiaceae s.s., Hypericaceae, and Podostemaceae) represented by 94 genera and ≈1900 species. Species in this clade form a conspicuous element of tropical forests worldwide and are important in horticulture, timber production, and pharmacology. We conducted a taxon-rich multigene phylogenetic analysis of the clusioids to clarify phylogenetic relationships in this clade. METHODS We analyzed plastid (matK, ndhF, and rbcL) and mitochondrial (matR) nucleotide sequence data using parsimony, maximum likelihood, and Bayesian inference. Our combined data set included 194 species representing all major clusioid subclades, plus numerous species spanning the taxonomic, morphological, and biogeographic breadth of the clusioid clade. KEY RESULTS Our results indicate that Tovomita (Clusiaceae s.s.), Harungana and Hypericum (Hypericaceae), and Ledermanniella s.s. and Zeylanidium (Podostemaceae) are not monophyletic. In addition, we place four genera that have not been included in any previous molecular study: Ceratolacis, Diamantina, and Griffithella (Podostemaceae), and Santomasia (Hypericaceae). Finally, our results indicate that Lianthus, Santomasia, Thornea, and Triadenum can be safely merged into Hypericum (Hypericaceae). CONCLUSIONS We present the first well-resolved, taxon-rich phylogeny of the clusioid clade. Taxon sampling and resolution within the clade are greatly improved compared to previous studies and provide a strong basis for improving the classification of the group. In addition, our phylogeny will form the foundation for our future work investigating the biogeography of tropical angiosperms that exhibit Gondwanan distributions.

Plastid phylogenomic analysis of green plants: A billion years of evolutionary history

PREMISE OF THE STUDY For the past one billion years, green plants (Viridiplantae) have dominated global ecosystems, yet many key branches in their evolutionary history remain poorly resolved. Using the largest analysis of Viridiplantae based on plastid genome sequences to date, we examined the phylogeny and implications for morphological evolution at key nodes. METHODS We analyzed amino acid sequences from protein-coding genes from complete (or nearly complete) plastomes for 1879 taxa, including representatives across all major clades of Viridiplantae. Much of the data used was derived from transcriptomes from the One Thousand Plants Project (1KP); other data were taken from GenBank. KEY RESULTS Our results largely agree with previous plastid-based analyses. Noteworthy results include (1) the position of Zygnematophyceae as sister to land plants (Embryophyta), (2) a bryophyte clade (hornworts, mosses + liverworts), (3) Equisetum + Psilotaceae as sister to Marattiales + leptosporangiate ferns, (4) cycads + Ginkgo as sister to the remaining extant gymnosperms, within which Gnetophyta are placed within conifers as sister to non-Pinaceae (Gne-Cup hypothesis), and (5) Amborella, followed by water lilies (Nymphaeales), as successive sisters to all other extant angiosperms. Within angiosperms, there is support for Mesangiospermae, a clade that comprises magnoliids, Chloranthales, monocots, Ceratophyllum, and eudicots. The placements of Ceratophyllum and Dilleniaceae remain problematic. Within Pentapetalae, two major clades (superasterids and superrosids) are recovered. CONCLUSIONS This plastid data set provides an important resource for elucidating morphological evolution, dating divergence times in Viridiplantae, comparisons with emerging nuclear phylogenies, and analyses of molecular evolutionary patterns and dynamics of the plastid genome.

Combined Morphological and Molecular Phylogeny of the Clusioid Clade (Malpighiales) and the Placement of the Ancient Rosid Macrofossil Paleoclusia

Premise of research. The clusioid clade is a member of the large rosid order Malpighiales and contains ∼1900 species in five families: Bonnetiaceae, Calophyllaceae, Clusiaceae sensu stricto (s.s.), Hypericaceae, and Podostemaceae. Despite recent efforts to clarify their phylogenetic relationships using molecular data, no such data are available for several critical taxa, including especially Hypericum ellipticifolium (previously recognized in Lianthus), Lebrunia, Neotatea, Thysanostemon, and the second-oldest rosid fossil (∼90 Ma), Paleoclusia chevalieri. Here, we (i) assess congruence between phylogenies inferred from morphological and molecular data, (ii) analyze morphological and molecular data simultaneously to place taxa lacking molecular data, and (iii) use ancestral state reconstructions (ASRs) to examine the evolution of traits that have been important for circumscribing clusioid taxa and to explore the placement of Paleoclusia. Methodology. We constructed a morphological data set including 69 characters and 81 clusioid species (or species groups). These data were analyzed individually and in combination with a previously published molecular data set of four genes (plastid matK, ndhF, and rbcL and mitochondrial matR) using parsimony, maximum likelihood (ML), and Bayesian inference. We used ML ASRs to infer the evolution of morphological characters. Pivotal results. Our phylogeny inferred from morphology alone was poorly supported but largely in agreement with molecular data. Moreover, our combined analyses were much better supported and largely confirm taxonomic hypotheses regarding relationships of extant taxa newly included here. The extinct Paleoclusia was placed as a member of stem group Clusiaceae s.s. or within crown group Clusiaceae s.s. as sister to one of its two major subclades. Conclusions. Despite poor overall bootstrap support for the placement of Paleoclusia, ancestral character state reconstructions are generally in agreement with our placements. Our recommendation is that Paleoclusia be treated as either a minimum stem group or a crown group age constraint of Clusiaceae s.s.

Digitization Workflows for Flat Sheets and Packets of Plants, Algae, and Fungi

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundations (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7–36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.