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.

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.

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.

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.

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.

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.

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.