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.

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.].

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 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.

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.

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 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.

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.

Factors Affecting Targeted Sequencing of 353 Nuclear Genes From Herbarium Specimens Spanning the Diversity of Angiosperms.

The world's herbaria collectively house millions of diverse plant specimens, including endangered or extinct species and type specimens. Unlocking genetic data from the typically highly degraded DNA obtained from herbarium specimens was difficult until the arrival of high-throughput sequencing approaches, which can be applied to low quantities of severely fragmented DNA. Target enrichment involves using short molecular probes that hybridise and capture genomic regions of interest for high-throughput sequencing. In this study on herbariomics, we used this targeted sequencing approach and the Angiosperms353 universal probe set to recover up to 351 nuclear genes from 435 herbarium specimens that are up to 204 years old and span the breadth of angiosperm diversity. We show that on average 207 genes were successfully retrieved from herbarium specimens, although the mean number of genes retrieved and target enrichment efficiency is significantly higher for silica gel-dried specimens. Forty-seven target nuclear genes were recovered from a herbarium specimen of the critically endangered St Helena boxwood, Mellissia begoniifolia, collected in 1815. Herbarium specimens yield significantly less high-molecular-weight DNA than silica gel-dried specimens, and genomic DNA quality declines with sample age, which is negatively correlated with target enrichment efficiency. Climate, taxon-specific traits, and collection strategies additionally impact target sequence recovery. We also detected taxonomic bias in targeted sequencing outcomes for the 10 most numerous angiosperm families that were investigated in depth. We recommend that (1) for species distributed in wet tropical climates, silica gel-dried specimens should be used preferentially; (2) for species distributed in seasonally dry tropical climates, herbarium and silica gel-dried specimens yield similar results, and either collection can be used; (3) taxon-specific traits should be explored and established for effective optimisation of taxon-specific studies using herbarium specimens; (4) all herbarium sheets should, in future, be annotated with details of the preservation method used; (5) long-term storage of herbarium specimens should be in stable, low-humidity, and low-temperature environments; and (6) targeted sequencing with universal probes, such as Angiosperms353, should be investigated closely as a new approach for DNA barcoding that will ensure better exploitation of herbarium specimens than traditional Sanger sequencing approaches.

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.

Tackling Rapid Radiations With Targeted Sequencing.

In phylogenetic studies across angiosperms, at various taxonomic levels, polytomies have persisted despite efforts to resolve them by increasing sampling of taxa and loci. The large amount of genomic data now available and statistical tools to analyze them provide unprecedented power for phylogenetic inference. Targeted sequencing has emerged as a strong tool for estimating species trees in the face of rapid radiations, lineage sorting, and introgression. Evolutionary relationships in Cyperaceae have been studied mostly using Sanger sequencing until recently. Despite ample taxon sampling, relationships in many genera remain poorly understood, hampered by diversification rates that outpace mutation rates in the loci used. The C4 Cyperus clade of the genus Cyperus has been particularly difficult to resolve. Previous studies based on a limited set of markers resolved relationships among Cyperus species using the C3 photosynthetic pathway, but not among C4 Cyperus clade taxa. We test the ability of two targeted sequencing kits to resolve relationships in the C4 Cyperus clade, the universal Angiosperms-353 kit and a Cyperaceae-specific kit. Sequences of the targeted loci were recovered from data generated with both kits and used to investigate overlap in data between kits and relative efficiency of the general and custom approaches. The power to resolve shallow-level relationships was tested using a summary species tree method and a concatenated maximum likelihood approach. High resolution and support are obtained using both approaches, but high levels of missing data disproportionately impact the latter. Targeted sequencing provides new insights into the evolution of morphology in the C4 Cyperus clade, demonstrating for example that the former segregate genus Alinula is polyphyletic despite its seeming morphological integrity. An unexpected result is that the Cyperus margaritaceus-Cyperus niveus complex comprises a clade separate from and sister to the core C4 Cyperus clade. Our results demonstrate that data generated with a family-specific kit do not necessarily have more power than those obtained with a universal kit, but that data generated with different targeted sequencing kits can often be merged for downstream analyses. Moreover, our study contributes to the growing consensus that targeted sequencing data are a powerful tool in resolving rapid radiations.