Phylogenomics and the rise of the angiosperms.

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

The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

The phylogeny and global biogeography of Primulaceae based on high-throughput DNA sequence data.

The angiosperm family Primulaceae is morphologically diverse and distributed nearly worldwide. However, phylogenetic uncertainty has obstructed the identification of major morphological and biogeographic transitions within the clade. We used target capture sequencing with the Angiosperms353 probes, taxon-sampling encompassing nearly all genera of the family, tree-based sequence curation, and multiple phylogenetic approaches to investigate the major clades of Primulaceae and their relationship to other Ericales. We generated dated phylogenetic trees and conducted broad-scale biogeographic analyses as well as stochastic character mapping of growth habit. We show that Ardisia, a pantropical genus and the largest in the family, is not monophyletic, with at least 19 smaller genera nested within it. Neotropical members of Ardisia and several smaller genera form a clade, an ancestor of which arrived in the Neotropics and began diversifying about 20 Ma. This Neotropical clade is most closely related to Elingamita and Tapeinosperma, which are most diverse on islands of the Pacific. Both Androsace and Primula are non-monophyletic by the inclusion of smaller genera. Ancestral state reconstructions revealed that there have either been parallel transitions to an herbaceous habit in Primuloideae, Samolus, and at least three lineages of Myrsinoideae, or a common ancestor of nearly all Primulaceae was herbaceous. Our results provide a robust estimate of phylogenetic relationships across Primulaceae and show that a revised classification of Myrsinoideae and several other clades within the family is necessary to render all genera monophyletic.

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

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

Strategies of diaspore dispersal investment in Compositae: the case of the Andean highlands.

BACKGROUND AND AIMS: Understanding diaspore morphology and how much a species invests on dispersal appendages is key for improving our knowledge of dispersal in fragmented habitats. We investigate diaspore morphological traits in high-Andean Compositae and their main abiotic and biotic drivers and test whether they play a role in species distribution patterns across the naturally fragmented high-Andean grasslands. METHODS: We collected diaspore trait data for 125 Compositae species across 47 tropical high-Andean summits, focusing on achene length and pappus-to-achene length ratio, with the latter as a proxy of dispersal investment. We analysed the role of abiotic (temperature, elevation and latitude) and biotic factors (phylogenetic signal and differences between tribes) on diaspore traits and whether they are related to distribution patterns across the Andes, using phylogenomics, distribution modelling and community ecology analyses. KEY RESULTS: Seventy-five percent of the studied species show small achenes (length <3.3 mm) and 67% have high dispersal investment (pappus length at least two times the achene length). Dispersal investment increases with elevation, possibly to compensate for lower air density, and achene length increases towards the equator, where non-seasonal climate prevails. Diaspore traits show significant phylogenetic signal, and higher dispersal investment is observed in Gnaphalieae, Astereae and Senecioneae, which together represent 72% of our species. High-Andean-restricted species found across the tropical Andes have, on average, the pappus four times longer than the achene, a significantly higher dispersal investment than species present only in the northern Andes or only in the central Andes. CONCLUSIONS: Small achenes and high diaspore dispersal investment dominate among high-Andean Compositae, traits typical of mostly three tribes of African origin; but traits are also correlated with the environmental gradients within the high-Andean grasslands. Our results also suggest that diaspore dispersal investment is likely to shape species distribution patterns in naturally fragmented habitats.

Target sequence data shed new light on the infrafamilial classification of Araceae.

PREMISE: Recent phylogenetic studies of the Araceae have confirmed the position of the duckweeds nested within the aroids, and the monophyly of a clade containing all the unisexual flowered aroids plus the bisexual-flowered Calla palustris. The main objective of the present study was to better resolve the deep phylogenetic relationships among the main lineages within the family, particularly the relationships between the eight currently recognized subfamilies. We also aimed to confirm the phylogenetic position of the enigmatic genus Calla in relation to the long-debated evolutionary transition between bisexual and unisexual flowers in the family. METHODS: Nuclear DNA sequence data were generated for 128 species across 111 genera (78%) of Araceae using target sequence capture and the Angiosperms 353 universal probe set. RESULTS: The phylogenomic data confirmed the monophyly of the eight Araceae subfamilies, but the phylogenetic position of subfamily Lasioideae remains uncertain. The genus Calla is included in subfamily Aroideae, which has also been expanded to include Zamioculcadoideae. The tribe Aglaonemateae is newly defined to include the genera Aglaonema and Boycea. CONCLUSIONS: Our results strongly suggest that new research on African genera (Callopsis, Nephthytis, and Anubias) and Calla will be important for understanding the early evolution of the Aroideae. Also of particular interest are the phylogenetic positions of the isolated genera Montrichardia, Zantedeschia, and Anchomanes, which remain only moderately supported here.

Benefits of alignment quality-control processing steps and an Angiosperms353 phylogenomics pipeline applied to the Celastrales.

We examined the impact of successive alignment quality-control steps on downstream phylogenomic analyses. We applied a recently published phylogenomics pipeline that was developed for the Angiosperms353 target-sequence-capture probe set to the flowering plant order Celastrales. Our final dataset consists of 158 species, including at least one exemplar from all 109 currently recognized Celastrales genera. We performed nine quality-control steps and compared the inferred resolution, branch support, and topological congruence of the inferred gene and species trees with those generated after each of the first six steps. We describe and justify each of our quality-control steps, including manual masking, in detail so that they may be readily applied to other lineages. We found that highly supported clades could generally be relied upon even if stringent orthology and alignment quality-control measures had not been applied. But separate instances were identified, for both concatenation and coalescence, wherein a clade was highly supported before manual masking but then subsequently contradicted. These results are generally reassuring for broad-scale analyses that use phylogenomics pipelines, but also indicate that we cannot rely exclusively on these analyses to conclude how challenging phylogenetic problems are best resolved.

Evolving the structure: climatic and developmental constraints on the evolution of plant architecture. A case study in Euphorbia.

Plant architecture strongly influences ecological performance, yet its role in plant evolution has not been explored in depth. By testing both phylogenetic and environmental signals, it is possible to separate architectural traits into four categories: development constraints (phylogenetic signal only); convergences (environmental dependency only); key confluences to the environmental driver (both); unknown (neither). We analysed the evolutionary history of the genus Euphorbia, a model clade with both high architectural diversity and a wide environmental range. We conducted comparative analyses of 193 Euphorbia species world-wide using 73 architectural traits, a dated phylogeny, and climate data. We identified 14 architectural types in Euphorbia based on trait combinations. We found 22 traits and three types representing convergences under climate groups, 21 traits and four types showing phylogenetic signal but no relation to climate, and 16 traits and five types with both climate and phylogenetic signals. Major drivers of architectural trait evolution likely include water stress in deserts (selected for succulence, continuous branching), frost disturbance in temperate systems (selected for simple, prostrate, short-lived shoots) and light competition (selected for arborescence). Simple architectures allowed resilience to disturbance, and frequent transitions into new forms. Complex architectures with functional specialisation developed under stable climates but have low evolvability.

Combination of Sanger and target-enrichment markers supports revised generic delimitation in the problematic 'Urera clade' of the nettle family (Urticaceae).

Urera Gaudich, s.l. is a pantropical genus comprising c. 35 species of trees, shrubs, and vines. It has a long history of taxonomic uncertainty, and is repeatedly recovered as polyphyletic within a poorly resolved complex of genera in the Urticeae tribe of the nettle family (Urticaceae). To provide generic delimitations concordant with evolutionary history, we use increased taxonomic and genomic sampling to investigate phylogenetic relationships among Urera and associated genera. A cost-effective two-tier genome-sampling approach provides good phylogenetic resolution by using (i) a taxon-dense sample of Sanger sequence data from two barcoding regions to recover clades of putative generic rank, and (ii) a genome-dense sample of target-enrichment data for a subset of representative species from each well-supported clade to resolve relationships among them. The results confirm the polyphyly of Urera s.l. with respect to the morphologically distinct genera Obetia, Poikilospermum and Touchardia. Afrotropic members of Urera s.l. are recovered in a clade sister to the xerophytic African shrubs Obetia; and Hawaiian ones with Touchardia, also from Hawaii. Combined with distinctive morphological differences between Neotropical and African members of Urera s.l., these results lead us to resurrect the previously synonymised name Scepocarpus Wedd. for the latter. The new species epiphet Touchardia oahuensis T.Wells & A.K. Monro is offered as a replacement name for Touchardia glabra non H.St.John, and subgenera are created within Urera s.s. to account for the two morphologically distinct Neotropical clades. This new classification minimises taxonomic and nomenclatural disruption, while more accurately reflecting evolutionary relationships within the group.

Phylogenomics and biogeography of Cunoniaceae (Oxalidales) with complete generic sampling and taxonomic realignments.

PREMISE: Cunoniaceae are a family of shrubs and trees with 27 genera and ca. 335 species, mostly confined to tropical and wet temperate zones of the southern hemisphere. There are several known issues regarding generic limits, and the family also displays a number of intriguing long-range disjunctions. METHODS: We performed a phylogenomic study using the universal Angiosperms353 probe set for targeted sequence capture. We sampled 37 species covering all genera in the Cunoniaceae, and those in the three closely related families of the crown Oxalidales (Brunelliaceae, Cephalotaceae, and Elaeocarpaceae). We also performed analyses for molecular dating and ancestral area reconstruction. RESULTS: We recovered the topology (Cunoniaceae, (Cephalotaceae, (Brunelliaceae, Elaeocarpaceae))) and a well-resolved genus-level phylogeny of Cunoniaceae with strongly supported clades corresponding to all previously recognized tribes. As previously suspected, the genera Ackama and Weinmannia were recovered as paraphyletic. Australasia was inferred as the likely ancestral area for the family. CONCLUSIONS: The current distribution of Cunoniaceae is best explained by long-distance dispersal with a few possible cases of Australasian-American vicariance events. Extinctions may have been important in determining the mostly Oceanian distribution of this family while some genera in the tribe Cunonieae and in New Caledonia have undergone recent bursts of diversification. New generic diagnoses, 80 new combinations, and one new name are provided for a recircumscribed Ackama (including Spiraeopsis), a much smaller Weinmannia (mostly New World), and a resurrected Pterophylla to accommodate Old World taxa previously in Weinmannia.

Joining forces in Ochnaceae phylogenomics: a tale of two targeted sequencing probe kits.

PREMISE: Both universal and family-specific targeted sequencing probe kits are becoming widely used for reconstruction of phylogenetic relationships in angiosperms. Within the pantropical Ochnaceae, we show that with careful data filtering, universal kits are equally as capable in resolving intergeneric relationships as custom probe kits. Furthermore, we show the strength in combining data from both kits to mitigate bias and provide a more robust result to resolve evolutionary relationships. METHODS: We sampled 23 Ochnaceae genera and used targeted sequencing with two probe kits, the universal Angiosperms353 kit and a family-specific kit. We used maximum likelihood inference with a concatenated matrix of loci and multispecies-coalescence approaches to infer relationships in the family. We explored phylogenetic informativeness and the impact of missing data on resolution and tree support. RESULTS: For the Angiosperms353 data set, the concatenation approach provided results more congruent with those of the Ochnaceae-specific data set. Filtering missing data was most impactful on the Angiosperms353 data set, with a relaxed threshold being the optimum scenario. The Ochnaceae-specific data set resolved consistent topologies using both inference methods, and no major improvements were obtained after data filtering. Merging of data obtained with the two kits resulted in a well-supported phylogenetic tree. CONCLUSIONS: The Angiosperms353 data set improved upon data filtering, and missing data played an important role in phylogenetic reconstruction. The Angiosperms353 data set resolved the phylogenetic backbone of Ochnaceae as equally well as the family specific data set. All analyses indicated that both Sauvagesia L. and Campylospermum Tiegh. as currently circumscribed are polyphyletic and require revised delimitation.

An updated infra-familial classification of Sapindaceae based on targeted enrichment data.

PREMISE: The economically important, cosmopolitan soapberry family (Sapindaceae) comprises ca. 1900 species in 144 genera. Since the seminal work of Radlkofer, several authors have attempted to overcome challenges presented by the family's complex infra-familial classification. With the advent of molecular systematics, revisions of the various proposed groupings have provided significant momentum, but we still lack a formal classification system rooted in an evolutionary framework. METHODS: Nuclear DNA sequence data were generated for 123 genera (86%) of Sapindaceae using target sequence capture with the Angiosperms353 universal probe set. HybPiper was used to produce aligned DNA matrices. Phylogenetic inferences were obtained using coalescence-based and concatenated methods. The clades recovered are discussed in light of both benchmark studies to identify synapomorphies and distributional evidence to underpin an updated infra-familial classification. KEY RESULTS: Coalescence-based and concatenated phylogenetic trees had identical topologies and node support, except for the placement of Melicoccus bijugatus Jacq. Twenty-one clades were recovered, which serve as the basis for a revised infra-familial classification. CONCLUSIONS: Twenty tribes are recognized in four subfamilies: two tribes in Hippocastanoideae, two in Dodonaeoideae, and 16 in Sapindoideae (no tribes are recognized in the monotypic subfamily Xanthoceratoideae). Within Sapindoideae, six new tribes are described: Blomieae Buerki & Callm.; Guindilieae Buerki, Callm. & Acev.-Rodr.; Haplocoeleae Buerki & Callm.; Stadmanieae Buerki & Callm.; Tristiropsideae Buerki & Callm.; and Ungnadieae Buerki & Callm. This updated classification provides a backbone for further research and conservation efforts on this family.

A higher-level nuclear phylogenomic study of the carrot family (Apiaceae).

PREMISE: The carrot family (Apiaceae) comprises 466 genera, which include many well-known crops (e.g., aniseed, caraway, carrots, celery, coriander, cumin, dill, fennel, parsley, and parsnips). Higher-level phylogenetic relationships among subfamilies, tribes, and other major clades of Apiaceae are not fully resolved. This study aims to address this important knowledge gap. METHODS: Target sequence capture with the universal Angiosperms353 probe set was used to examine phylogenetic relationships in 234 genera of Apiaceae, representing all four currently recognized subfamilies (Apioideae, Azorelloideae, Mackinlayoideae, and Saniculoideae). Recovered nuclear genes were analyzed using both multispecies coalescent and concatenation approaches. RESULTS: We recovered hundreds of nuclear genes even from old and poor-quality herbarium specimens. Of particular note, we placed with strong support three incertae sedis genera (Platysace, Klotzchia, and Hermas); all three occupy isolated positions, with Platysace resolved as sister to all remaining Apiaceae. We placed nine genera (Apodicarpum, Bonannia, Grafia, Haplosciadium, Microsciadium, Physotrichia, Ptychotis, Tricholaser, Xatardia) that have never previously been included in any molecular phylogenetic study. CONCLUSIONS: We provide support for the maintenance of the four existing subfamilies of Apiaceae, while recognizing that Hermas, Klotzschia, and the Platysace clade may each need to be accommodated in additional subfamilies (pending improved sampling). The placement of the currently apioid genus Phlyctidocarpa can be accommodated by the expansion of subfamily Saniculoideae, although adequate morphological synapomorphies for this grouping are yet to be defined. This is the first phylogenetic study of the Apiaceae using high-throughput sequencing methods and represents an unprecedented evolutionary framework for the group.

Settling a family feud: a high-level phylogenomic framework for the Gentianales based on 353 nuclear genes and partial plastomes.

PREMISE: Comprising five families that vastly differ in species richness-ranging from Gelsemiaceae with 13 species to the Rubiaceae with 13,775 species-members of the Gentianales are often among the most species-rich and abundant plants in tropical forests. Despite considerable phylogenetic work within particular families and genera, several alternative topologies for family-level relationships within Gentianales have been presented in previous studies. METHODS: Here we present a phylogenomic analysis based on nuclear genes targeted by the Angiosperms353 probe set for approximately 150 species, representing all families and approximately 85% of the formally recognized tribes. We were able to retrieve partial plastomes from off-target reads for most taxa and infer phylogenetic trees for comparison with the nuclear-derived trees. RESULTS: We recovered high support for over 80% of all nodes. The plastid and nuclear data are largely in agreement, except for some weakly to moderately supported relationships. We discuss the implications of our results for the order's classification, highlighting points of increased support for previously uncertain relationships. Rubiaceae is sister to a clade comprising (Gentianaceae + Gelsemiaceae) + (Apocynaceae + Loganiaceae). CONCLUSIONS: The higher-level phylogenetic relationships within Gentianales are confidently resolved. In contrast to recent studies, our results support the division of Rubiaceae into two subfamilies: Cinchonoideae and Rubioideae. We do not formally recognize Coptosapelteae and Luculieae within any particular subfamily but treat them as incertae sedis. Our framework paves the way for further work on the phylogenetics, biogeography, morphological evolution, and macroecology of this important group of flowering plants.

Hundreds of nuclear and plastid loci yield novel insights into orchid relationships.

PREMISE: The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. METHODS: We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses. RESULTS: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. CONCLUSIONS: Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.

A nuclear phylogenomic study of the angiosperm order Myrtales, exploring the potential and limitations of the universal Angiosperms353 probe set.

PREMISE: To further advance the understanding of the species-rich, economically and ecologically important angiosperm order Myrtales in the rosid clade, comprising nine families, approximately 400 genera and almost 14,000 species occurring on all continents (except Antarctica), we tested the Angiosperms353 probe kit. METHODS: We combined high-throughput sequencing and target enrichment with the Angiosperms353 probe kit to evaluate a sample of 485 species across 305 genera (76% of all genera in the order). RESULTS: Results provide the most comprehensive phylogenetic hypothesis for the order to date. Relationships at all ranks, such as the relationship of the early-diverging families, often reflect previous studies, but gene conflict is evident, and relationships previously found to be uncertain often remain so. Technical considerations for processing HTS data are also discussed. CONCLUSIONS: High-throughput sequencing and the Angiosperms353 probe kit are powerful tools for phylogenomic analysis, but better understanding of the genetic data available is required to identify genes and gene trees that account for likely incomplete lineage sorting and/or hybridization events.

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

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

Spiny plants, mammal browsers, and the origin of African savannas.

Savannas first began to spread across Africa during the Miocene. A major hypothesis for explaining this vegetation change is the increase in C4 grasses, promoting fire. We investigated whether mammals could also have contributed to savanna expansion by using spinescence as a marker of mammal herbivory. Looking at the present distribution of 1,852 tree species, we established that spinescence is mainly associated with two functional types of mammals: large browsers and medium-sized mixed feeders. Using a dated phylogeny for the same tree species, we found that spinescence evolved at least 55 times. The diversification of spiny plants occurred long after the evolution of Afrotherian proboscideans and hyracoids. However, it is remarkably congruent with diversification of bovids, the lineage including the antelope that predominantly browse these plants today. Our findings suggest that herbivore-adapted savannas evolved several million years before fire-maintained savannas and probably, in different environmental conditions. Spiny savannas with abundant mammal herbivores occur in drier climates and on nutrient-rich soils, whereas fire-maintained savannas occur in wetter climates on nutrient-poor soils.

Diversification into novel habitats in the Africa clade of Dioscorea (Dioscoreaceae): erect habit and elephant's foot tubers.

BACKGROUND: Dioscorea is a widely distributed and highly diversified genus in tropical regions where it is represented by ten main clades, one of which diversified exclusively in Africa. In southern Africa it is characterised by a distinct group of species with a pachycaul or "elephant's foot" structure that is partially to fully exposed above the substrate. In contrast to African representatives of the genus from other clades, occurring mainly in forest or woodland, the pachycaul taxa and their southern African relatives occur in diverse habitats ranging from woodland to open vegetation. Here we investigate patterns of diversification in the African clade, time of transition from forest to more open habitat, and morphological traits associated with each habitat and evaluate if such transitions have led to modification of reproductive organs and mode of dispersal. RESULTS: The Africa clade originated in the Oligocene and comprises four subclades. The Dioscorea buchananii subclade (southeastern tropical Africa and South Africa) is sister to the East African subclade, which is respectively sister to the recently evolved sister South African (e. g., Cape and Pachycaul) subclades. The Cape and Pachycaul subclades diversified in the east of the Cape Peninsula in the mid Miocene, in an area with complex geomorphology and climate, where the fynbos, thicket, succulent karoo and forest biomes meet. CONCLUSIONS: Diversification out of forest is associated with major shifts in morphology of the perennial tuber (specifically an increase in size and orientation which presumably led them to become pachycaul) and rotation of stem (from twining to non-twining). The iconic elephant's foot morphology, observed in grasslands and thicket biomes, where its corky bark may offer protection against fire and herbivory, evolved since mid Miocene. A shift in pollination trait is observed within the forest, but entry into open habitat does not show association with reproductive morphology, except in the seed wing, which has switched from winged all round the seed margin to just at the base or at the apex of it, or has been even replaced by an elaiosome.

Savanna fire and the origins of the 'underground forests' of Africa.

The origin of fire-adapted lineages is a long-standing question in ecology. Although phylogeny can provide a significant contribution to the ongoing debate, its use has been precluded by the lack of comprehensive DNA data. Here, we focus on the 'underground trees' (=geoxyles) of southern Africa, one of the most distinctive growth forms characteristic of fire-prone savannas. We placed geoxyles within the most comprehensive dated phylogeny for the regional flora comprising over 1400 woody species. Using this phylogeny, we tested whether African geoxyles evolved concomitantly with those of the South American cerrado and used their phylogenetic position to date the appearance of humid savannas. We found multiple independent origins of the geoxyle life-form mostly from the Pliocene, a period consistent with the origin of cerrado, with the majority of divergences occurring within the last 2 million yr. When contrasted with their tree relatives, geoxyles occur in regions characterized by higher rainfall and greater fire frequency. Our results indicate that the geoxylic growth form may have evolved in response to the interactive effects of frequent fires and high precipitation. As such, geoxyles may be regarded as markers of fire-maintained savannas occurring in climates suitable for forests.