The link between ancient whole-genome duplications and cold adaptations in the Caryophyllaceae.

PREMISE: The Caryophyllaceae (the carnation family) have undergone multiple transitions into colder climates and convergence on cushion plant adaptation, indicating that they may provide a natural system for cold adaptation research. Previous research has suggested that putative ancient whole-genome duplications (WGDs) are correlated with niche shifts into colder climates across the Caryophyllales. Here, we explored the genomic changes potentially involved in one of these discovered shifts in the Caryophyllaceae. METHODS: We constructed a data set combining 26 newly generated transcriptomes with 45 published transcriptomes, including 11 cushion plant species across seven genera. With this data set, we inferred a dated phylogeny for the Caryophyllaceae and mapped ancient WGDs and gene duplications onto the phylogeny. We also examined functional groups enriched for gene duplications related to the climatic shift. RESULTS: The ASTRAL topology was mostly congruent with the current consensus of relationships within the family. We inferred 15 putative ancient WGDs in the family, including eight that have not been previously published. The oldest ancient WGD (ca. 64.4-56.7 million years ago), WGD1, was found to be associated with a shift into colder climates by previous research. Gene regions associated with ubiquitination were overrepresented in gene duplications retained after WGD1 and those convergently retained by cushion plants in Colobanthus and Eremogone, along with other functional annotations. CONCLUSIONS: Gene family expansions induced by ancient WGDs may have contributed to the shifts to cold climatic niches in the Caryophyllaceae. Transcriptomic data are crucial resources that help unravel heterogeneity in deep-time evolutionary patterns in plants.

Analysis of Paralogs in Target Enrichment Data Pinpoints Multiple Ancient Polyploidy Events in Alchemilla s.l. (Rosaceae).

Target enrichment is becoming increasingly popular for phylogenomic studies. Although baits for enrichment are typically designed to target single-copy genes, paralogs are often recovered with increased sequencing depth, sometimes from a significant proportion of loci, especially in groups experiencing whole-genome duplication (WGD) events. Common approaches for processing paralogs in target enrichment data sets include random selection, manual pruning, and mainly, the removal of entire genes that show any evidence of paralogy. These approaches are prone to errors in orthology inference or removing large numbers of genes. By removing entire genes, valuable information that could be used to detect and place WGD events is discarded. Here, we used an automated approach for orthology inference in a target enrichment data set of 68 species of Alchemilla s.l. (Rosaceae), a widely distributed clade of plants primarily from temperate climate regions. Previous molecular phylogenetic studies and chromosome numbers both suggested ancient WGDs in the group. However, both the phylogenetic location and putative parental lineages of these WGD events remain unknown. By taking paralogs into consideration and inferring orthologs from target enrichment data, we identified four nodes in the backbone of Alchemilla s.l. with an elevated proportion of gene duplication. Furthermore, using a gene-tree reconciliation approach, we established the autopolyploid origin of the entire Alchemilla s.l. and the nested allopolyploid origin of four major clades within the group. Here, we showed the utility of automated tree-based orthology inference methods, previously designed for genomic or transcriptomic data sets, to study complex scenarios of polyploidy and reticulate evolution from target enrichment data sets.[Alchemilla; allopolyploidy; autopolyploidy; gene tree discordance; orthology inference; paralogs; Rosaceae; target enrichment; whole genome duplication.].

Polyploid plants have faster rates of multivariate niche differentiation than their diploid relatives.

Polyploid speciation entails substantial and rapid postzygotic reproductive isolation of nascent species that are initially sympatric with one or both parents. Despite strong postzygotic isolation, ecological niche differentiation has long been thought to be important for polyploid success. Using biogeographic data from across vascular plants, we tested whether the climatic niches of polyploid species are more differentiated than their diploid relatives and if the climatic niches of polyploid species differentiated faster than those of related diploids. We found that polyploids are often more climatically differentiated from their diploid parents than the diploids are from each other. Consistent with this pattern, we estimated that polyploid species generally have higher rates of multivariate niche differentiation than their diploid relatives. In contrast to recent analyses, our results confirm that ecological niche differentiation is an important component of polyploid speciation and that niche differentiation is often significantly faster in polyploids.

Increasing phylogenetic stochasticity at high elevations on summits across a remote North American wilderness.

PREMISE: At the intersection of ecology and evolutionary biology, community phylogenetics can provide insights into overarching biodiversity patterns, particularly in remote and understudied ecosystems. To understand community assembly of the high alpine flora in the Sawtooth National Forest, USA, we analyzed phylogenetic structure within and between nine summit communities. METHODS: We used high-throughput sequencing to supplement existing data and infer a nearly completely sampled community phylogeny of the alpine vascular flora. We calculated mean nearest taxon distance (MNTD) and mean pairwise distance (MPD) to quantify phylogenetic divergence within summits, and assessed whether maximum elevation explains phylogenetic structure. To evaluate similarities between summits, we quantified phylogenetic turnover, taking into consideration microhabitats (talus vs. meadows). RESULTS: We found different patterns of community phylogenetic structure within the six most species-rich orders, but across all vascular plants phylogenetic structure was largely not different from random. There was a significant negative correlation between elevation and tree-wide phylogenetic diversity (MPD) within summits: overdispersion degraded as elevation increased. Between summits, we found high phylogenetic turnover driven by greater niche heterogeneity on summits with alpine meadows. CONCLUSIONS: Our results provide further evidence that stochastic processes may also play an important role in the assembly of vascular plant communities in high alpine habitats at regional scales. However, order-specific patterns suggest that adaptations are still important for assembly of specific sectors of the plant tree of life. Further studies quantifying functional diversity will be important in disentangling the interplay of eco-evolutionary processes that likely shape broad community phylogenetic patterns in extreme environments.

Impact of whole-genome duplication events on diversification rates in angiosperms.

PREMISE OF THE STUDY: Polyploidy or whole-genome duplication (WGD) pervades the evolutionary history of angiosperms. Despite extensive progress in our understanding of WGD, the role of these events in promoting diversification is still not well understood. We seek to clarify the possible association between WGD and diversification rates in flowering plants. METHODS: Using a previously published phylogeny spanning all land plants (31,749 tips) and WGD events inferred from analyses of the 1000 Plants (1KP) transcriptome data, we analyzed the association of WGDs and diversification rates following numerous WGD events across the angiosperms. We used a stepwise AIC approach (MEDUSA), a Bayesian mixture model approach (BAMM), and state-dependent diversification analyses (MuSSE) to investigate patterns of diversification. Sister-clade comparisons were used to investigate species richness after WGDs. KEY RESULTS: Based on the density of 1KP taxon sampling, 106 WGDs were unambiguously placed on the angiosperm phylogeny. We identified 334-530 shifts in diversification rates. We found that 61 WGD events were tightly linked to changes in diversification rates, and state-dependent diversification analyses indicated higher speciation rates for subsequent rounds of WGD. Additionally, 70 of 99 WGD events showed an increase in species richness compared to the sister clade. CONCLUSIONS: Forty-six of the 106 WGDs analyzed appear to be closely associated with upshifts in the rate of diversification in angiosperms. Shifts in diversification do not appear more likely than random within a four-node lag phase following a WGD; however, younger WGD events are more likely to be followed by an upshift in diversification than older WGD events.

Phylogenetic restriction of plant invasion in drought-stressed environments: Implications for insect-pollinated plant communities in water-limited ecosystems.

BACKGROUND: Plant-pollinator community diversity has been found to decrease under conditions of drought stress; however, research into the temporal dimensions of this phenomenon remains limited. In this study, we investigated the effect of seasonal drought on the temporal niche dynamics of entomophilous flowering plants in a water-limited ecosystem. We hypothesized that closely related native and exotic plants would tend to share similar life history and that peak flowering events would therefore coincide with phylogenetic clustering in plant communities based on expected phenological responses of plant functional types to limitations in soil moisture availability. LOCATION: Galiano Island, British Columbia, Canada. METHODS: Combining methods from pollinator research and phylogenetic community ecology, we tested the influence of environmental filtering over plant community phenology across gradients of landscape disturbance and soil moisture. Floral resource availability and community structure were quantified by counts of flowering shoots. We constructed a robust phylogeny to analyze spatial and temporal variation in phylogenetic patterns across the landscape, testing the significance of the observed patterns against a randomly generated community phylogeny. Phylogenetic metrics were then regressed against factors of disturbance and soil moisture availability. RESULTS: Critical seasonal fluctuations in floral resources coincided with significant phylogenetic clustering in plant communities, with decreasing plant diversity observed under conditions of increasing drought stress. Exotic plant species in the Asteraceae became increasingly pervasive across the landscape, occupying a late season temporal niche in drought-stressed environments. MAIN CONCLUSION: Results suggest that environmental filtering is the dominant assembly process structuring the temporal niche of plant communities in this water-limited ecosystem. Based on these results, and trends seen elsewhere, the overall diversity of plant-pollinator communities may be expected to decline with the increasing drought stress predicted under future climate scenarios.

Can functional genomic diversity provide novel insights into mechanisms of community assembly? A pilot study from an invaded alpine streambed.

An important focus of community ecology, including invasion biology, is to investigate functional trait diversity patterns to disentangle the effects of environmental and biotic interactions. However, a notable limitation is that studies usually rely on a small and easy-to-measure set of functional traits, which might not immediately reflect ongoing ecological responses to changing abiotic or biotic conditions, including those that occur at a molecular or physiological level. We explored the potential of using the diversity of expressed genes-functional genomic diversity (FGD)-to understand ecological dynamics of a recent and ongoing alpine invasion. We quantified FGD based on transcriptomic data measured for 26 plant species occurring along adjacent invaded and pristine streambeds. We used an RNA-seq approach to summarize the overall number of expressed transcripts and their annotations to functional categories, and contrasted this with functional trait diversity (FTD) measured from a suite of characters that have been traditionally considered in plant ecology. We found greater FGD and FTD in the invaded community, independent of differences in species richness. However, the magnitude of functional dispersion was greater from the perspective of FGD than from FTD. Comparing FGD between congeneric alien-native species pairs, we did not find many significant differences in the proportion of genes whose annotations matched functional categories. Still, native species with a greater relative abundance in the invaded community compared with the pristine tended to express a greater fraction of genes at significant levels in the invaded community, suggesting that changes in FGD may relate to shifts in community composition. Comparisons of diversity patterns from the community to the species level offer complementary insights into processes and mechanisms driving invasion dynamics. FGD has the potential to illuminate cryptic changes in ecological diversity, and we foresee promising avenues for future extensions across taxonomic levels and macro-ecosystems.

Pilot RNA-seq data from 24 species of vascular plants at Harvard Forest.

PREMISE: Large-scale projects such as the National Ecological Observatory Network (NEON) collect ecological data on entire biomes to track climate change. NEON provides an opportunity to launch community transcriptomic projects that ask integrative questions in ecology and evolution. We conducted a pilot study to investigate the challenges of collecting RNA-seq data from diverse plant communities. METHODS: We generated >650 Gbp of RNA-seq for 24 vascular plant species representing 12 genera and nine families at the Harvard Forest NEON site. Each species was sampled twice in 2016 (July and August). We assessed transcriptome quality and content with TransRate, BUSCO, and Gene Ontology annotations. RESULTS: Only modest differences in assembly quality were observed across multiple k-mers. On average, transcriptomes contained hits to >70% of loci in the BUSCO database. We found no significant difference in the number of assembled and annotated transcripts between diploid and polyploid transcriptomes. DISCUSSION: We provide new RNA-seq data sets for 24 species of vascular plants in Harvard Forest. Challenges associated with this type of study included recovery of high-quality RNA from diverse species and access to NEON sites for genomic sampling. Overcoming these challenges offers opportunities for large-scale studies at the intersection of ecology and genomics.

An empirical demonstration of using pentatricopeptide repeat (PPR) genes as plant phylogenetic tools: phylogeny of Verbenaceae and the Verbena complex.

The pentatricopeptide repeat (PPR) gene family, with hundreds of members in land plant genomes, has been recognized as a tremendous resource for plant phylogenetic studies based on publicly available genomic data from model organisms. However, whether this appealing nuclear gene marker system can be readily applied to non-model organisms remains questionable, particularly given the potential uncertainties in designing specific primers to only amplify the locus of interest from the sea of PPR genes. Here we demonstrate empirically the use of PPR genes in the family Verbenaceae and the Verbena complex. We also lay out a general scheme to design locus-specific primers to amplify and sequence PPR genes in non-model organisms. Intergeneric relationships within the family Verbenaceae were fully resolved with strong support. Relationships among the closely related genera within the Verbena complex and among some species groups within each genus were also well resolved, but resolution among very closely related species was limited. Our results suggest that PPR genes can be readily employed in non-model organisms. They may be best used to resolve relationships in a spectrum from among distantly related genera to among not-so-closely related congeneric species, but may have limited use among very closely related species.

TagSeq for gene expression in non-model plants: A pilot study at the Santa Rita Experimental Range NEON core site.

PREMISE: TagSeq is a cost-effective approach for gene expression studies requiring a large number of samples. To date, TagSeq studies in plants have been limited to those with a high-quality reference genome. We tested the suitability of reference transcriptomes for TagSeq in non-model plants, as part of a study of natural gene expression variation at the Santa Rita Experimental Range National Ecological Observatory Network (NEON) core site. METHODS: Tissue for TagSeq was sampled from multiple individuals of four species (Bouteloua aristidoides and Eragrostis lehmanniana [Poaceae], Tidestromia lanuginosa [Amaranthaceae], and Parkinsonia florida [Fabaceae]) at two locations on three dates (56 samples total). One sample per species was used to create a reference transcriptome via standard RNA-seq. TagSeq performance was assessed by recovery of reference loci, specificity of tag alignments, and variation among samples. RESULTS: A high fraction of tags aligned to each reference and mapped uniquely. Expression patterns were quantifiable for tens of thousands of loci, which revealed consistent spatial differentiation in expression for all species. DISCUSSION: TagSeq using de novo reference transcriptomes was an effective approach to quantifying gene expression in this study. Tags were highly locus specific and generated biologically informative profiles for four non-model plant species.

The pentatricopeptide repeat (PPR) gene family, a tremendous resource for plant phylogenetic studies.

* Despite the paramount importance of nuclear gene data in plant phylogenetics, the search for candidate loci is believed to be challenging and time-consuming. Here we report that the pentatricopeptide repeat (PPR) gene family, containing hundreds of members in plant genomes, holds tremendous potential as nuclear gene markers. * We compiled a list of 127 PPR loci that are all intronless and have a single orthologue in both rice (Oryza sativa) and Arabidopsis thaliana. The uncorrected p-distances were calculated for these loci between two Arabidopsis species and among three Poaceae genera. We also selected 13 loci to evaluate their phylogenetic utility in resolving relationships among six Poaceae genera and nine diploid Oryza species. * PPR genes have a rapid rate of evolution and can be best used at intergeneric and interspecific levels. Although with substantial amounts of missing data, almost all individual data sets from the 13 loci generate well-resolved gene trees. * With the unique combination of three characteristics (having a large number of loci with established orthology assessment, being intronless, and being rapidly evolving), the PPR genes have many advantages as phylogenetic markers (e.g. straightforward alignment, minimal effort in generating sequence data, and versatile utilities). We perceive that these loci will play an important role in plant phylogenetics.

For common community phylogenetic analyses, go ahead and use synthesis phylogenies.

Should we build our own phylogenetic trees based on gene sequence data, or can we simply use available synthesis phylogenies? This is a fundamental question that any study involving a phylogenetic framework must face at the beginning of the project. Building a phylogeny from gene sequence data (purpose-built phylogeny) requires more effort, expertise, and cost than subsetting an already available phylogeny (synthesis-based phylogeny). However, we still lack a comparison of how these two approaches to building phylogenetic trees influence common community phylogenetic analyses such as comparing community phylogenetic diversity and estimating trait phylogenetic signal. Here, we generated three purpose-built phylogenies and their corresponding synthesis-based trees (two from Phylomatic and one from the Open Tree of Life, OTL). We simulated 1,000 communities and 12,000 continuous traits along each purpose-built phylogeny. We then compared the effects of different trees on estimates of phylogenetic diversity (alpha and beta) and phylogenetic signal (Pagel's λ and Blomberg's K). Synthesis-based phylogenies generally yielded higher estimates of phylogenetic diversity when compared to purpose-built phylogenies. However, resulting measures of phylogenetic diversity from both types of phylogenies were highly correlated (Spearman's ρ  > 0.8 in most cases). Mean pairwise distance (both alpha and beta) is the index that is most robust to the differences in tree construction that we tested. Measures of phylogenetic diversity based on the OTL showed the highest correlation with measures based on the purpose-built phylogenies. Trait phylogenetic signal estimated with synthesis-based phylogenies, especially from the OTL, was also highly correlated with estimates of Blomberg's K or close to Pagel's λ from purpose-built phylogenies when traits were simulated under Brownian motion. For commonly employed community phylogenetic analyses, our results justify taking advantage of recently developed and continuously improving synthesis trees, especially the Open Tree of Life.

Leveraging contemporary species introductions to test phylogenetic hypotheses of trait evolution.

Plant trait evolution is a topic of interest across disciplines and scales. Phylogenetic studies are powerful for generating hypotheses about the mechanisms that have shaped plant traits and their evolution. Introduced plants are a rich source of data on contemporary trait evolution. Introductions could provide especially useful tests of a variety of evolutionary hypotheses because the environments selecting on evolving traits are still present. We review phylogenetic and contemporary studies of trait evolution and identify areas of overlap and areas for further integration. Emerging tools which can promote integration include broadly focused repositories of trait data, and comparative models of trait evolution that consider both intra and interspecific variation.

Riders in the sky (islands): using a mega-phylogenetic approach to understand plant species distribution and coexistence at the altitudinal limits of angiosperm plant life.

AIM: Plants occurring on high-alpine summits are generally expected to persist due to adaptations to extreme selective forces caused by the harshest climates where angiosperm life is known to thrive. We assessed the relative effects of this strong environmental filter and of other historical and stochastic factors driving plant community structure in very high-alpine conditions (up to 4,000m). LOCATION: European Alps, Écrins National Park, France. METHODS: Using species occurrence data collected from floristic surveys on 15 summits (2,791 m - 4,102 m a.s.l.) throughout the Écrins range, along with existing molecular sequence data obtained from GenBank, we used a mega-phylogenetic approach to evaluate the phylogenetic structure of high-alpine plant species assemblages. We used three nested species pools and two null models to address the importance of species-specific and species-neutral processes for driving coexistence. RESULTS: Compared to the entire species pool of the study region, alpine summits exhibited a strong signal of phylogenetic clustering. Restricting statistical sampling to environmentally and historically defined species pools reduced the significance of this pattern. However, we could not reject a model that explicitly incorporates neutral colonization and local extinction in shaping community structure for dominant plant orders. Between summits, phylogenetic turnover was generally lower than expected. Environmental drivers did not explain overall phylogenetic patterns, but we found significant geographic and climatic structure in phylogenetic diversity at finer taxonomic scales. MAIN CONCLUSIONS: Although we found evidence for strong phylogenetic clustering within alpine summits, we were not able to reject models of species-neutral processes to explain patterns of floristic diversity. Our results suggest that plant community structure in high-alpine regions can also be shaped by neutral processes, and not through the sole action of environmental selection as traditionally assumed for harsh and stressful environments.

An efficient field and laboratory workflow for plant phylotranscriptomic projects.

PREMISE OF THE STUDY: We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation. METHODS AND RESULTS: A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae. CONCLUSIONS: Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10-12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds.

A molecular phylogeny and classification of Verbenaceae.

PREMISE OF THE STUDY: Verbenaceae consist of trees, shrubs, lianas, and herbs distributed primarily in Latin America, where they occur in a wide array of ecosystems. A second center of diversity exists in Africa. Competing morphology-based classifications that rely on different traits conflict in significant ways. A broad phylogenetic study was undertaken to assess those classifications and to examine the historical geography of the family. • METHODS: Analysis of seven chloroplast DNA regions for 109 species, representing all genera except one monotypic genus, provide inference into evolutionary relationships in Verbenaceae. • KEY RESULTS: The phylogeny shows that none of the traditional classifications reflect phylogenetic relationships very well. Eight clades are recognized as tribes (Casselieae, Citharexyleae, Duranteae, Lantaneae, Neospartoneae trib. nov., Petreeae, Priveae, and Verbeneae). Two genera, Dipyrena and Rhaphithamnus, remain unplaced in these larger clades. Petreeae, which consist of Neotropical lianas, are sister to the rest of the family. Lantaneae and Verbeneae together form a derived clade that comprises approximately two-thirds of the species in Verbenaceae. • CONCLUSIONS: We present a new tribal classification, including one new tribe, Neospartoneae trib. nov., to accommodate three small genera of Argentine species (Diostea, Neosparton, and Lampaya). Phylogenetic inference suggests a South American origin for Verbenaceae, with approximately six colonization events having given rise to the Old World species.