A new phylogenetic data standard for computable clade definitions: the Phyloreference Exchange Format (Phyx).

To be computationally reproducible and efficient, integration of disparate data depends on shared entities whose matching meaning (semantics) can be computationally assessed. For biodiversity data one of the most prevalent shared entities for linking data records is the associated taxon concept. Unlike Linnaean taxon names, the traditional way in which taxon concepts are provided, phylogenetic definitions are native to phylogenetic trees and offer well-defined semantics that can be transformed into formal, computationally evaluable logic expressions. These attributes make them highly suitable for phylogeny-driven comparative biology by allowing computationally verifiable and reproducible integration of taxon-linked data against Tree of Life-scale phylogenies. To achieve this, the first step is transforming phylogenetic definitions from the natural language text in which they are published to a structured interoperable data format that maintains strong ties to semantics and lends itself well to sharing, reuse, and long-term archival. To this end, we developed the Phyloreference Exchange Format (Phyx), a JSON-LD-based text format encompassing rich metadata for all elements of a phylogenetic definition, and we created a supporting software library, phyx.js, to streamline computational management of such files. Together they form a foundation layer for digitizing and computing with phylogenetic definitions of clades.

Duplication and expression patterns of CYCLOIDEA-like genes in Campanulaceae.

BACKGROUND: CYCLOIDEA (CYC)-like transcription factors pattern floral symmetry in most angiosperms. In core eudicots, two duplications led to three clades of CYC-like genes: CYC1, CYC2, and CYC3, with orthologs of the CYC2 clade restricting expression dorsally in bilaterally symmetrical flowers. Limited data from CYC3 suggest that they also play a role in flower symmetry in some asterids. We examine the evolution of these genes in Campanulaceae, a group that contains broad transitions between radial and bilateral floral symmetry and 180° resupination (turning upside-down by twisting pedicle). RESULTS: We identify here all three paralogous CYC-like clades across Campanulaceae. Similar to other core eudicots, we show that CamCYC2 duplicated near the time of the divergence of the bilaterally symmetrical and resupinate Lobelioideae. However, in non-resupinate, bilaterally symmetrical Cyphioideae, CamCYC2 appears to have been lost and CamCYC3 duplicated, suggesting a novel genetic basis for bilateral symmetry in Cyphioideae. We additionally, utilized qRT-PCR to examine the correlation between CYC-like gene expression and shifts in flower morphology in four species of Lobelioideae. As expected, CamCYC2 gene expression was dorsoventrally restricted in bilateral symmetrical flowers. However, because Lobelioideae have resupinate flowers, both CamCYC2A and CamCYC2B are highly expressed in the finally positioned ventral petal lobes, corresponding to the adaxial side of the flower relative to meristem orientation. CONCLUSIONS: Our sequences across Campanulaceae of all three of these paralogous groups suggests that radially symmetrical Campanuloideae duplicated CYC1, Lobelioideae duplicated CYC2 and lost CYC3 early in their divergence, and that Cyphioideae lost CYC2 and duplicated CYC3. This suggests a dynamic pattern of duplication and loss of major floral patterning genes in this group and highlights the first case of a loss of CYC2 in a bilaterally symmetrical group. We illustrate here that CYC expression is conserved along the dorsoventral axis of the flower even as it turns upside-down, suggesting that at least late CYC expression is not regulated by extrinsic factors such as gravity. We additionally show that while the pattern of dorsoventral expression of each paralog remains the same, CamCYC2A is more dominant in species with shorter relative finally positioned dorsal lobes, and CamCYC2B is more dominant in species with long dorsal lobes.

Peeling back the layers: First phylogenomic insights into the Ledebouriinae (Scilloideae, Asparagaceae).

The Ledebouriinae (Scilloideae, Asparagaceae) are a widespread group of bulbous geophytes found predominantly throughout seasonal climates in sub-Saharan Africa, with a handful of taxa in Madagascar, the Middle East, India, and Sri Lanka. Phylogenetic relationships within the group have been historically difficult to elucidate. Here, we provide the first phylogenomic perspective into the Ledebouriinae. Using the Angiosperms353 targeted enrichment probe set, we consistently recovered four major clades (i.e., two Ledebouria clades, Drimiopsis, and Resnova). The two Ledebouria clades closely align with geography, either consisting almost entirely of sub-Saharan African taxa (Ledebouria Clade A), or East African and non-African taxa (Ledebouria Clade B). Our results suggest that the Ledebouriinae likely underwent a rapid radiation leading to rampant incomplete lineage sorting. We additionally find evidence for potential historical hybridization between Drimiopsis and a subclade within Ledebouria Clade A.

Evolution and biogeography of Memecylon.

PREMISE: The woody plant group Memecylon (Melastomataceae) is a large clade occupying diverse forest habitats in the Old World tropics and exhibiting high regional endemism. Its phylogenetic relationships have been previously studied using ribosomal DNA with extensive sampling from Africa and Madagascar. However, divergence times, biogeography, and character evolution of Memecylon remain uninvestigated. We present a phylogenomic analysis of Memecylon to provide a broad evolutionary perspective of this clade. METHODS: One hundred supercontigs of 67 Memecylon taxa were harvested from target enrichment. The data were subjected to coalescent and concatenated phylogenetic analyses. A timeline was provided for Memecylon evolution using fossils and secondary calibration. The calibrated Memecylon phylogeny was used to elucidate its biogeography and ancestral character states. RESULTS: Relationships recovered by the phylogenomic analyses are strongly supported in both maximum likelihood and coalescent-based species trees. Memecylon is inferred to have originated in Africa in the Eocene and subsequently dispersed predominantly eastward via long-distance dispersal (LDD), although a reverse dispersal from South Asia westward to the Seychelles was postulated. Morphological data exhibited high levels of homoplasy, but also showed that several vegetative and reproductive characters were phylogenetically informative. CONCLUSIONS: The current distribution of Memecylon appears to be the result of multiple ancestral LDD events. Our results demonstrate the importance of the combined effect of geographic and paleoclimatic factors in shaping the distribution of this group in the Old World tropics. Memecylon includes a number of evolutionarily derived morphological features that contribute to diversity within the clade.

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.

Niche dynamics of Memecylon in Sri Lanka: Distribution patterns, climate change effects, and conservation priorities.

Recent climate projections have shown that the distribution of organisms in island biotas is highly affected by climate change. Here, we present the result of the analysis of niche dynamics of a plant group, Memecylon, in Sri Lanka, an island, using species occurrences and climate data. We aim to determine which climate variables explain current distribution, model how climate change impacts the availability of suitable habitat for Memecylon, and determine conservation priority areas for Sri Lankan Memecylon. We used georeferenced occurrence data of Sri Lankan Memecylon to develop ecological niche models and assess both current and future potential distributions under six climate change scenarios in 2041-2060 and 2061-2080. We also overlaid land cover and protected area maps and performed a gap analysis to understand the impacts of land-cover changes on Memecylon distributions and propose new areas for conservation. Differences among suitable habitats of Memecylon were found to be related to patterns of endemism. Under varying future climate scenarios, endemic groups were predicted to experience habitat shifts, gains, or losses. The narrow endemic Memecylon restricted to the montane zone were predicted to be the most impacted by climate change. Projections also indicated that changes in species' habitats can be expected as early as 2041-2060. Gap analysis showed that while narrow endemic categories are considerably protected as demonstrated by their overlap with protected areas, more conservation efforts in Sri Lankan forests containing wide endemic and nonendemic Memecylon are needed. This research helped clarify general patterns of responses of Sri Lankan Memecylon to global climate change. Data from this study are useful for designing measures aimed at filling the gaps in forest conservation on this island.

A two-tier bioinformatic pipeline to develop probes for target capture of nuclear loci with applications in Melastomataceae.

PREMISE: Putatively single-copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference. However, suitable genomic resources are not available for many clades, including Melastomataceae. We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly related Memecylon and Tibouchina (Melastomataceae). METHODS: We present a two-tiered pipeline. First, we identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were then added (384 total loci). Second, using HybPiper, we retrieved 689 homologous template sequences for these loci using genome-skimming data from within the focal clades. RESULTS: We sequenced 193 loci common to Memecylon and Tibouchina. Probes designed from 56 template sequences successfully targeted sequences in both clades. Probes designed from genome-skimming data within a focal clade were more successful than probes designed from other sources. DISCUSSION: Our pipeline successfully identified and targeted SCN loci in Memecylon and Tibouchina, enabling phylogenomic studies in both clades and potentially across Melastomataceae. This pipeline could be easily applied to other clades with few genomic resources.

Tunicate bulb size variation in monocots explained by temperature and phenology.

Plant bulbs are modified shoot systems comprised of short internodes with apical bud(s) surrounded by layers of leaf bases. Bulb diameters can vary greatly, with overall bulb size playing a role in flower formation and resource allocation. Despite the importance of bulb size to the overall fitness of an individual, evolutionary and ecological aspects of this trait have been almost completely neglected. Examining over 2,500 herbarium vouchers for 115 selected species, we analyzed monocot tunicate bulb size within a phylogenetic context in order to investigate its evolutionary significance. We recorded two bulb diameter optima and observed that as bulb size increases taxa inhabit warmer areas with less temperature seasonality. Furthermore, we found that hysteranthous taxa, a habit where leaves emerge separately from flowers, exhibit overall larger bulbs potentially due to reliance upon belowground stored resources to flower rather than on current environmental inputs. This work highlights the importance of including the belowground portion of plants into ecological and evolutionary studies in order to gain a more complete understanding of the evolution of plant forms and functions.

Geophytism in monocots leads to higher rates of diversification.

Geophytes, plants with buds on underground structures, are found throughout the plant tree of life. These below ground structures allow plants to inhabit highly seasonal and disturbance-prone environments across ecosystems. Past researchers have hypothesised that the bulbous, cormous and tuberous habits promote diversification, but this had yet to be tested. Using a comprehensive monocot data set of almost 13 000 taxa, we investigated the effects of the geophytic habit on diversification using both state-dependent and state-independent models. We found that geophytes exhibit increased rates of diversification relative to nongeophytes. State-dependent analyses recovered higher yet similar rates of diversification for bulbous, cormous and tuberous taxa compared with rhizomatous and nongeophytic taxa. However, the state-independent model returned no difference in rates among the different traits. Geophytism shows higher rates of diversification relative to nongeophytes but we found little support for the hypothesis that the evolution of the bulb, corm or tuber appears to provide a diversification increase relative to rhizomatous and nongeophytic taxa. Our broad-scale analysis highlights the overall evolutionary importance of the geophytic habit (i.e. belowground bud placement). However, our results also suggest that belowground morphological diversity alone cannot explain this rate increase. In order to further test the evolutionary significance of these underground structures, future studies should consider these in combination with other biotic and abiotic factors.

Rates of niche and phenotype evolution lag behind diversification in a temperate radiation.

Environmental change can create opportunities for increased rates of lineage diversification, but continued species accumulation has been hypothesized to lead to slowdowns via competitive exclusion and niche partitioning. Such density-dependent models imply tight linkages between diversification and trait evolution, but there are plausible alternative models. Little is known about the association between diversification and key ecological and phenotypic traits at broad phylogenetic and spatial scales. Do trait evolutionary rates coincide with rates of diversification, are there lags among these rates, or is diversification niche-neutral? To address these questions, we combine a deeply sampled phylogeny for a major flowering plant clade-Saxifragales-with phenotype and niche data to examine temporal patterns of evolutionary rates. The considerable phenotypic and habitat diversity of Saxifragales is greatest in temperate biomes. Global expansion of these habitats since the mid-Miocene provided ecological opportunities that, with density-dependent adaptive radiation, should result in simultaneous rate increases for diversification, niche, and phenotype, followed by decreases with habitat saturation. Instead, we find that these rates have significantly different timings, with increases in diversification occurring at the mid-Miocene Climatic Optimum (∼15 Mya), followed by increases in niche and phenotypic evolutionary rates by ∼5 Mya; all rates increase exponentially to the present. We attribute this surprising lack of temporal coincidence to initial niche-neutral diversification followed by ecological and phenotypic divergence coincident with more extreme cold and dry habitats that proliferated into the Pleistocene. A lack of density-dependence contrasts with investigations of other cosmopolitan lineages, suggesting alternative patterns may be common in the diversification of temperate lineages.

The monocotyledonous underground: global climatic and phylogenetic patterns of geophyte diversity.

PREMISE: Geophytes-plants that typically possess a bulb, corm, tuber, and/or rhizome-have long captured the attention of hobbyists and researchers. However, despite the economic and evolutionary importance of these traits, the potential drivers of their morphological diversity remain unknown. Using a comprehensive phylogeny of monocots, we test for correlations between climate and geophyte growth form to better understand why we observe such a diversity of underground traits in geophytes. Understanding the evolutionary factors promoting independent origins of these potentially adaptive organs will lend insights into how plants adapt to environmental hardships. METHODS: Using a comprehensive phylogeny incorporated with global occurrence and climate data for the monocots, we investigated whether climatic patterns could explain differences between geophytes and non-geophytes, as well as differences among bulbous, cormous, tuberous, rhizomatous, and non-geophytic taxa. We used phylogenetically-informed ANOVAs, MANOVAs, and PCAs to test differences in climatic variables between the different growth forms. RESULTS: Geophytes inhabit cooler, drier, and more thermally variable climates compared to non-geophytes. Although some underground traits (i.e., bulb, corm, and tuber) appear to inhabit particular niches, a result supported by strong phylogenetic signal, our data has limited evidence for an overall role of climate in the evolution of these traits. However, temperature may be a driving force in rhizome evolution, as well as the evolution of taxa which we considered here as non-geophytic (e.g., trees, epiphytes, etc.). CONCLUSIONS: While precipitation patterns have played a role in the evolution of geophytism, our results suggest that temperature should be more strongly considered as a contributing factor promoting the evolution of belowground bud placement, specifically in rhizomatous and non-geophytic taxa. Bulbous, cormous, and tuberous taxa need closer examination of other mechanisms, such as anatomical constraints or genetic controls, in order to begin to understand the causes behind the evolution of their underground morphology.

A Phylogenomic Perspective on Evolution and Discordance in the Alpine-Arctic Plant Clade Micranthes (Saxifragaceae).

The increased availability of large phylogenomic datasets is often accompanied by difficulties in disentangling and harnessing the data. These difficulties may be enhanced for species resulting from reticulate evolution and/or rapid radiations producing large-scale discordance. As a result, there is a need for methods to investigate discordance, and in turn, use this conflict to inform and aid in downstream analyses. Therefore, we drew upon multiple analytical tools to investigate the evolution of Micranthes (Saxifragaceae), a clade of primarily arctic-alpine herbs impacted by reticulate and rapid radiations. To elucidate the evolution of Micranthes we sought near-complete taxon sampling with multiple accessions per species and assembled extensive nuclear (518 putatively single copy loci) and plastid (95 loci) datasets. In addition to a robust phylogeny for Micranthes, this research shows that genetic discordance presents a valuable opportunity to develop hypotheses about its underlying causes, such as hybridization, polyploidization, and range shifts. Specifically, we present a multi-step approach that incorporates multiple checks points for paralogy, including reciprocally blasting targeted genes against transcriptomes, running paralogy checks during the assembly step, and grouping genes into gene families to look for duplications. We demonstrate that a thorough assessment of discordance can be a source of evidence for evolutionary processes that were not adequately captured by a bifurcating tree model, and helped to clarify processes that have structured the evolution of Micranthes.

The future of cold-adapted plants in changing climates: Micranthes (Saxifragaceae) as a case study.

Research has shown species undergoing range contractions and/or northward and higher elevational movements as a result of changing climates. Here, we evaluate how the distribution of a group of cold-adapted plant species with similar evolutionary histories changes in response to warming climates. We selected 29 species of Micranthes (Saxifragaceae) representing the mountain and Arctic biomes of the Northern Hemisphere. For this analysis, 24,755 data points were input into ecological niche models to assess both present fundamental niches and predicted future ranges under climate change scenarios. Comparisons were made across the Northern Hemisphere between all cold-adapted Micranthes, including Arctic species, montane species, and species defined as narrow endemics. Under future climate change models, 72% of the species would occupy smaller geographical areas than at present. This loss of habitat is most pronounced in Arctic species in general, but is also prevalent in species restricted to higher elevations in mountains. Additionally, narrowly endemic species restricted to high elevations were more susceptible to habitat loss than those species found at lower elevations. Using a large dataset and modeling habitat suitability at a global scale, our results empirically model the threats to cold-adapted species as a result of warming climates. Although Arctic and alpine biomes share many underlying climate similarities, such as cold and short growing seasons, our results confirm that species in these climates have varied responses to climate change and that key abiotic variables differ between these two habitats.

Origins of East Asian Campanuloideae (Campanulaceae) diversity.

The Campanulaceae comprises approximately 2300 species that are distributed among five major lineages: Campanuloideae, Cyphioideae, Cyphocarpoideae, Lobelioideae, and Nemacladoideae. Of these, the Campanuloideae, a primarily Old World clade, has the largest diversity in East Asia. In this study, we reconstruct the phylogeny of East Asian Campanuloideae based on one nuclear gene (i.e., PPR70) and five plastid markers (i.e., atpB, matK, petD, rbcL, and trnL-trnF). We then use this phylogenetic framework to reconstruct the biogeographical history of the genus. Our molecular dataset includes 376 of the 1045 currently recognized species in the Campanuloideae. Of the 376 sampled species, 116 are from East Asia, representing ca. 60% of the East Asian Campanuloideae. Our PPR dataset included sequences for 156 accessions, representing 54 species, while our plastid dataset included sequences for 305 accessions, representing 354 species. Phylogenetic analyses recovered three large clades containing East Asian taxa: Campanulinae, Platycodinae, and Wahlenberginae. The historical assembly of Campanuloideae diversity in East Asia appears to have resulted from numerous, independent movements from Africa, Europe/W. Asia, and North America. Africa was inferred as the ancestral range for the Campanuloideae. Movement of the largest East Asian clade (Platycodinae) occurred at approximately 53.1 Ma (46.6-58.73 95% HPD) from Africa, with much of the current diversity found in East Asia having resulted from in situ diversification. Thirteen additional movements into East Asia, primarily from Europe/Western Asia, occurred subsequently. One dispersal event from western North America was also inferred. In contrast, only six movements out of East Asia were found. Our results suggest that East Asia has acted primarily as a sink for Campanuloideae diversity, with Europe, Western Asia, and Africa representing major source areas.

Pseudo-parallel patterns of disjunctions in an Arctic-alpine plant lineage.

Disjunct distributions have intrigued biologists for centuries. Investigating these biogeographic patterns provides insight into speciation and biodiversity at multiple spatial and phylogenetic scales. Some disjunctions have been intensively studied, yet others have been largely overlooked and remain poorly understood. Among the lesser-known disjunction patterns is that between the mountain ranges of western North America. Flora and fauna endemic to the mountains of this region provide important systems for investigating causes and results of disjunctions, given the relatively recent geological formation of this area and the intense climatic fluctuations that have occurred since its formation. In Micranthes (Saxifragaceae), which has high rates of montane endemism, two species, M. bryophora and M. tolmiei, show this biogeographical pattern. By reconstructing a time-calibrated phylogeny based on 518 low-copy nuclear markers and including multiple populations of each species from the Coast Ranges, Cascades, Sierra Nevada, and Rocky Mountains, this study provides a biogeographical and temporal framework for the evolution of Micranthes in western North America. Strongly supported east-west differentiated clades are recovered for M. bryophora and M. tolmiei in both maximum likelihood and coalescent-based species tree reconstructions. Biogeographic analysis suggests different patterns of dispersal for both taxa and the dating analyses recovered contrasting ages for each clade. Due to both the different geographic patterns and the timing of the initial diversification of each taxon corresponding to different geologic and climatic events, the disjunction patterns shown for these taxa are suggested to be an example of biogeographical pseudocongruence.

Naming diversity in an evolutionary context: Phylogenetic definitions of the Roucela clade (Campanulaceae/Campanuloideae) and the cryptic taxa within.

In recent times, evolution has become a central tenet of taxonomy, but nomenclature has consistently been decoupled from the tree-thinking process, often leading to significant issues in reconciling traditional (Linnaean) names with clades in the Tree of Life. Recent evolutionary studies on the Roucela clade, a group of endemic plants found in the Mediterranean Basin, motivated the establishment of phylogenetic concepts to formally anchor clade names on the Campanuloideae (Campanulaceae) tree. These concepts facilitate communication of clades that approximate traditionally defined groups, in addition to naming newly discovered cryptic diversity in a phylogenetic framework.

Embracing discordance: Phylogenomic analyses provide evidence for allopolyploidy leading to cryptic diversity in a Mediterranean Campanula (Campanulaceae) clade.

The Mediterranean Basin harbors a remarkable amount of biodiversity, a high proportion of which is endemic to this region. Here, we present an in-depth study of an angiosperm species complex, in which cryptic taxonomic diversity has been hypothesized. Specifically, we focus on four currently recognized species in the Roucela complex, a well-supported clade in the Campanulaceae/Campanuloideae: Campanula creutzburgii, C. drabifolia, C. erinus, and C. simulans. This study takes a phylogenomic approach, utilizing near-complete plastomes and 130 nuclear loci, to uncover cryptic diversity and test hypotheses regarding hybridization and polyploidy within this clade. Genome size estimates recovered tetraploid and octoploid lineages within the currently recognized, widespread species C. erinus, showing an east-west geographic pattern. Though genomic data clearly differentiate these two cytotypes, we failed to discern morphological differences. The formation of a cryptic octoploid lineage, distributed across the eastern Mediterranean, is hypothesized to be the result of an allopolyploid event in which one parental morphology is retained. The tetraploid C. erinus and C. creutzburgii (also a tetraploid) are implicated as parental lineages. Our results highlight the utility of target-enrichment approaches for obtaining genomic datasets for thorough assessments of species diversity and the importance of carefully considering gene-tree discordance within such datasets.

A global perspective on Campanulaceae: Biogeographic, genomic, and floral evolution.

PREMISE OF THE STUDY: The Campanulaceae are a diverse clade of flowering plants encompassing more than 2300 species in myriad habitats from tropical rainforests to arctic tundra. A robust, multigene phylogeny, including all major lineages, is presented to provide a broad, evolutionary perspective of this cosmopolitan clade. METHODS: We used a phylogenetic framework, in combination with divergence dating, ancestral range estimation, chromosome modeling, and morphological character reconstruction analyses to infer phylogenetic placement and timing of major biogeographic, genomic, and morphological changes in the history of the group and provide insights into the diversification of this clade across six continents. KEY RESULTS: Ancestral range estimation supports an out-of-Africa diversification following the Cretaceous-Tertiary extinction event. Chromosomal modeling, with corroboration from the distribution of synonymous substitutions among gene duplicates, provides evidence for as many as 20 genome-wide duplication events before large radiations. Morphological reconstructions support the hypothesis that switches in floral symmetry and anther dehiscence were important in the evolution of secondary pollen presentation mechanisms. CONCLUSIONS: This study provides a broad, phylogenetic perspective on the evolution of the Campanulaceae clade. The remarkable habitat diversity and cosmopolitan distribution of this lineage appears to be the result of a complex history of genome duplications and numerous long-distance dispersal events. We failed to find evidence for an ancestral polyploidy event for this clade, and our analyses indicate an ancestral base number of nine for the group. This study will serve as a framework for future studies in diverse areas of research in Campanulaceae.