Artifactual Orthologs and the Need for Diligent Data Exploration in Complex Phylogenomic Datasets: A Museomic Case Study from the Andean Flora.

The Andes mountains of western South America are a globally important biodiversity hotspot, yet there is a paucity of resolved phylogenies for plant clades from this region. Filling an important gap in our understanding of the World's richest flora, we present the first phylogeny of Freziera (Pentaphylacaceae), an Andean-centered, cloud forest radiation. Our dataset was obtained via hybrid-enriched target sequence capture of Angiosperms353 universal loci for 50 of the ca. 75 spp., obtained almost entirely from herbarium specimens. We identify high phylogenomic complexity in Freziera, including the presence of data artifacts. Via by-eye observation of gene trees, detailed examination of warnings from recently improved assembly pipelines, and gene tree filtering, we identified that artifactual orthologs (i.e., the presence of only one copy of a multicopy gene due to differential assembly) were an important source of gene tree heterogeneity that had a negative impact on phylogenetic inference and support. These artifactual orthologs may be common in plant phylogenomic datasets, where multiple instances of genome duplication are common. After accounting for artifactual orthologs as source of gene tree error, we identified a significant, but nonspecific signal of introgression using Patterson's D and f4 statistics. Despite phylogenomic complexity, we were able to resolve Freziera into 9 well-supported subclades whose evolution has been shaped by multiple evolutionary processes, including incomplete lineage sorting, historical gene flow, and gene duplication. Our results highlight the complexities of plant phylogenomics, which are heightened in Andean radiations, and show the impact of filtering data processing artifacts and standard filtering approaches on phylogenetic inference.

Phylogenetic insights into the Salicaceae: The evolution of willows and beyond.

The Salicaceae includes approximately 54 genera and over 1,400 species with a cosmopolitan distribution. Members of the family are well-known for their diverse secondary plant metabolites, and they play crucial roles in tropical and temperate forest ecosystems. Phylogenetic reconstruction of the Salicaceae has been historically challenging due to the limitations of molecular markers and the extensive history of hybridization and polyploidy within the family. Our study employs whole-genome sequencing of 74 species to generate an extensive phylogeny of the Salicaceae. We generated two RAD-Seq enriched whole-genome sequence datasets and extracted two additional gene sets corresponding to the universal Angiosperms353 and Salicaceae-specific targeted-capture arrays. We reconstructed maximum likelihood-based molecular phylogenies using supermatrix and coalescent-based supertree approaches. Our fossil-calibrated phylogeny estimates that the Salicaceae originated around 128 million years ago and unravels the complex taxonomic relationships within the family. Our findings confirm the non-monophyly of the subgenus Salix s.l. and further support the merging of subgenera Chamaetia and Vetrix, both of which exhibit intricate patterns within and among different sections. Overall, our study not only enhances our understanding of the evolution of the Salicaceae, but also provides valuable insights into the complex relationships within the family.

Phylogenomics and morphology of Celmisiinae (Asteraceae: Astereae): Taxonomic and evolutionary implications.

The tribe Astereae (Asteraceae) includes 36 subtribes and 252 genera, and is distributed worldwide in temperate and tropical regions. One of the subtribes, Celmisiinae Saldivia, has been recently circumscribed to include six genera and ca. 160 species, and is restricted to eastern Australia, New Zealand, and New Guinea. The species show an impressive range of growth habit, from small herbs and ericoid subshrubs to medium-sized trees. They live in a wide range of habitats and are often dominant in subalpine and alpine vegetation. Despite the well-supported circumscription of Celmisiinae, uncertainties have remained about their internal relationships and classification at genus and species levels. This study exploited recent advances in high-throughput sequencing to build a robust multi-gene phylogeny for the subtribe Celmisiinae. The target enrichment Angiosperms353 bait set and the hybpiper-nf and paragone-nf pipelines were used to retrieve, infer, and assemble orthologous loci from 75 taxa representing all the main putative clades within the subtribe. Because of the diploidised ploidy level in Celmisiinae, as well as missing data in the assemblies, uncertainty remains surrounding the inference of orthology detection. However, based on a variety of gene-family sets, coalescent and concatenation-based phylogenetic reconstructions recovered similar topologies. Paralogy and missing data in the gene-families caused some problems, but the estimated phylogenies were well-supported and well-resolved. The phylogenomic evidence supported Celmisiinae and three main clades: the Pleurophyllum clade (Pleurophyllum, Macrolearia and Damnamenia), mostly in the New Zealand Subantarctic Islands, Celmisia of mainland New Zealand and Australia, and Shawia (including 'Olearia pro parte' and Pachystegia) of New Zealand, Australia and New Guinea. The results presented here add to the accumulating support for the Angiosperms353 bait set as an efficient method for documenting plant diversity.

Tangled webs and spider-flowers: Phylogenomics, biogeography, and seed morphology inform the evolutionary history of Cleomaceae.

PREMISE: Cleomaceae is an important model clade for studies of evolutionary processes including genome evolution, floral form diversification, and photosynthetic pathway evolution. Diversification and divergence patterns in Cleomaceae remain tangled as research has been restricted by its worldwide distribution, limited genetic sampling and species coverage, and a lack of definitive fossil calibration points. METHODS: We used target sequence capture and the Angiosperms353 probe set to perform a phylogenetic study of Cleomaceae. We estimated divergence times and biogeographic analyses to explore the origin and diversification of the family. Seed morphology across extant taxa was documented with multifocal image-stacking techniques and morphological characters were extracted, analyzed, and compared to fossil records. RESULTS: We recovered a well-supported and resolved phylogenetic tree of Cleomaceae generic relationships that includes 236 (~86%) species. We identified 11 principal clades and confidently placed Cleomella as sister to the rest of the family. Our analyses suggested that Cleomaceae and Brassicaceae diverged ~56 mya, and Cleomaceae began to diversify ~53 mya in the Palearctic and Africa. Multiple transatlantic disjunct distributions were identified. Seeds were imaged from 218 (~80%) species in the family and compared to all known fossil species. CONCLUSIONS: Our results represent the most comprehensive phylogenetic study of Cleomaceae to date. We identified transatlantic disjunctions and proposed explanations for these patterns, most likely either long-distance dispersals or contractions in latitudinal distributions caused by climate change over geological timescales. We found that seed morphology varied considerably but mostly mirrored generic relationships.

Easy353: A Tool to Get Angiosperms353 Genes for Phylogenomic Research.

The Angiosperms353 gene set (AGS) consists of a set of 353 universal low-copy nuclear genes that were selected by examining more than 600 angiosperm species. These genes can be used for phylogenetic studies and population genetics at multiple taxonomic scales. However, current pipelines are not able to recover Angiosperms353 genes efficiently and accurately from high-throughput sequences. Here, we developed Easy353, a reference-guided assembly tool to recover the AGS from high-throughput sequencing (HTS) data (including genome skimming, RNA-seq, and target enrichment). Easy353 is an open-source user-friendly assembler for diverse types of high-throughput data. It has a graphical user interface and a command-line interface that is compatible with all widely-used computer systems. Evaluations, based on both simulated and empirical data, suggest that Easy353 yields low rates of assembly errors.

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.

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.

Chloroplast Genomes and Phylogenetic Analysis of Three Carthamus (Asteraceae) Species.

The genus Carthamus Linnaeus, which belongs to the tribe Cardueae in the Asteraceae family, originated in the Mediterranean region and consists of approximately 20 species worldwide. Understanding the phylogeny of the Carthamus is crucial for the cultivation of C. tinctorius. Although chloroplast genomes are widely used for species identification and evolutionary studies, there have been limited investigations on the chloroplast genomes of Carthamus species. In this study, we assembled the chloroplast genomes of C. persicus, C. tinctorius × C. persicus, and C. lanatus and combined them with the five chloroplast genomes of C. tinctorius for comparative genomic analysis. The sizes of the chloroplast genomes of C. lanatus, C. persicus, and C. tinctorius × C. persicus were 152,602 bp, 153,177 bp, and 153,177 bp, respectively. Comparative analysis showed that the chloroplast genome structures of the four Carthamus species were highly conserved. Additionally, the phylogenomic analysis demonstrated that the plastid genome and angiosperms353 dataset significantly improved the phylogenetic support of Carthamus species. This analysis supported Carthamus as a monophyletic taxon and its internal division into the sect. Carthamus and sect. Atractylis. The Carthamus was closely related to Carduncellus, Femeniasia, Phonus, and Centaurea. In conclusion, this study not only expands our understanding of the cp genomes of Carthamus species but also provides support for more comprehensive phylogenetic studies of Carthamus.

Phylogenomics AND biogeography of Castanea (chestnut) and Hamamelis (witch-hazel) - Choosing between RAD-seq and Hyb-Seq approaches.

Hyb-Seq and RAD-seq are well-established high throughput sequencing technologies that have been increasingly used for plant phylogenomic studies. Each method has its own pros and cons. The choice between them is a practical issue for plant systematists studying the evolutionary histories of biodiversity of relatively recent origins. However, few studies have compared the congruence and conflict between results from the two methods within the same group of organisms in plants. In this study, we employed RAD-seq and Hyb-Seq of Angiosperms353 genes in phylogenomic and biogeographic studies of Hamamelis (the witch-hazels) and Castanea (chestnuts), two classic examples exhibiting the well-known eastern Asian (EA) -eastern North American (ENA) disjunct distribution, and compared them side by side. Our results showed congruences in phylogenetic inference and divergence time dating between the two data sets obtained through our customized procedures of library preparation and sequence trimming, although they differed in the number of loci and informative sites, the amount of missing data, and sampling within species. We provide recommendations regarding the selection of the two methods for phylogenomic study at generic level based on fund availability and sampling scale. If funds and time are not constrained, we recommend Hyb-Seq. If funds and time are somewhat limited and sampling is large, we recommend RAD-seq. However, we found greater conflict among gene trees from the RAD-seq data due to the short sequences per locus. Therefore, species tree building and network detecting with the RAD-seq data with short RAD-seq loci (e.g., <150 bp) should avoid using analytical methods relying on gene trees of individual locus, but using site-based methods such as SVDQuartets and D-statistic method. Our phylogenetic analyses of RAD-seq and Hyb-Seq data resulted in well-resolved species relationships. Analyses of the data using the D-statistic test and PhyloNet revealed ancient introgressions in both genera. Biogeographic analyses including fossil data using total evidence-based dated tree and DEC model applying specific inter-area dispersal probabilities revealed a complicated history for each genus, indicating multiple interareal dispersals and local extinctions within and outside areas of the taxa's modern ranges in both the Paleogene and Neogene. The study demonstrates the importance of including fossil taxa for a more accurate reconstruction of biogeographic histories of taxa to understand the EA and ENA floristic disjunction. Our results support a widespread ancestral range in EA-western North America (WNA) followed by early diversification in EA and expansion to North America (NA) and Europe for Castanea and a more widespread ancestral range in EA-ENA-WNA for Hamamelis. The origins of the modern EA-ENA disjunction in both genera were suggested to be the result of vicariance from widespread ancestors in Eurasia-ENA of the mid-Miocene and in EA-NA of the late Oligocene, respectively.

Incomplete lineage sorting and reticulate evolution mask species relationships in Brunelliaceae, an Andean family with rapid, recent diversification.

PREMISE: To date, phylogenetic relationships within the monogeneric Brunelliaceae have been based on morphological evidence, which does not provide sufficient phylogenetic resolution. Here we use target-enriched nuclear data to improve our understanding of phylogenetic relationships in the family. METHODS: We used the Angiosperms353 toolkit for targeted recovery of exonic regions and supercontigs (exons + introns) from low copy nuclear genes from 53 of 70 species in Brunellia, and several outgroup taxa. We removed loci that indicated biased inference of relationships and applied concatenated and coalescent methods to infer Brunellia phylogeny. We identified conflicts among gene trees that may reflect hybridization or incomplete lineage sorting events and assessed their impact on phylogenetic inference. Finally, we performed ancestral-state reconstructions of morphological traits and assessed the homology of character states used to define sections and subsections in Brunellia. RESULTS: Brunellia comprises two major clades and several subclades. Most of these clades/subclades do not correspond to previous infrageneric taxa. There is high topological incongruence among the subclades across analyses. CONCLUSIONS: Phylogenetic reconstructions point to rapid species diversification in Brunelliaceae, reflected in very short branches between successive species splits. The removal of putatively biased loci slightly improves phylogenetic support for individual clades. Reticulate evolution due to hybridization and/or incomplete lineage sorting likely both contribute to gene-tree discordance. Morphological characters used to define taxa in current classification schemes are homoplastic in the ancestral character-state reconstructions. While target enrichment data allows us to broaden our understanding of diversification in Brunellia, the relationships among subclades remain incompletely understood.

Phylogenomic discordance suggests polytomies along the backbone of the large genus Solanum.

PREMISE: Evolutionary studies require solid phylogenetic frameworks, but increased volumes of phylogenomic data have revealed incongruent topologies among gene trees in many organisms both between and within genomes. Some of these incongruences indicate polytomies that may remain impossible to resolve. Here we investigate the degree of gene-tree discordance in Solanum, one of the largest flowering plant genera that includes the cultivated potato, tomato, and eggplant, as well as 24 minor crop plants. METHODS: A densely sampled species-level phylogeny of Solanum is built using unpublished and publicly available Sanger sequences comprising 60% of all accepted species (742 spp.) and nine regions (ITS, waxy, and seven plastid markers). The robustness of this topology is tested by examining a full plastome dataset with 140 species and a nuclear target-capture dataset with 39 species of Solanum (Angiosperms353 probe set). RESULTS: While the taxonomic framework of Solanum remained stable, gene tree conflicts and discordance between phylogenetic trees generated from the target-capture and plastome datasets were observed. The latter correspond to regions with short internodal branches, and network analysis and polytomy tests suggest the backbone is composed of three polytomies found at different evolutionary depths. The strongest area of discordance, near the crown node of Solanum, could potentially represent a hard polytomy. CONCLUSIONS: We argue that incomplete lineage sorting due to rapid diversification is the most likely cause for these polytomies, and that embracing the uncertainty that underlies them is crucial to understand the evolution of large and rapidly radiating lineages.

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.

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.

Resolving species boundaries in a recent radiation with the Angiosperms353 probe set: the Lomatium packardiae/L. anomalum clade of the L. triternatum (Apiaceae) complex.

PREMISE: Speciation not associated with morphological shifts is challenging to detect unless molecular data are employed. Using Sanger-sequencing approaches, the Lomatium packardiae/L. anomalum subcomplex within the larger Lomatium triternatum complex could not be resolved. Therefore, we attempt to resolve these boundaries here. METHODS: The Angiosperms353 probe set was employed to resolve the ambiguity within Lomatium triternatum species complex using 48 accessions assigned to L. packardiae, L. anomalum, or L. triternatum. In addition to exon data, 54 nuclear introns were extracted and were complete for all samples. Three approaches were used to estimate evolutionary relationships and define species boundaries: STACEY, a Bayesian coalescent-based species tree analysis that takes incomplete lineage sorting into account; ASTRAL-III, another coalescent-based species tree analysis; and a concatenated approach using MrBayes. Climatic factors, morphological characters, and soil variables were measured and analyzed to provide additional support for recovered groups. RESULTS: The STACEY analysis recovered three major clades and seven subclades, all of which are geographically structured, and some correspond to previously named taxa. No other analysis had full agreement between recovered clades and other parameters. Climatic niche and leaflet width and length provide some predictive ability for the major clades. CONCLUSIONS: The results suggest that these groups are in the process of incipient speciation and incomplete lineage sorting has been a major barrier to resolving boundaries within this lineage previously. These results are hypothesized through sequencing of multiple loci and analyzing data using coalescent-based processes.

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.

A comprehensive phylogenomic study of the monocot order Commelinales, with a new classification of Commelinaceae.

PREMISE: Resolving relationships within order Commelinales has posed quite a challenge, as reflected in its unstable infra-familial classification. Thus, we investigated (1) relationships across families and genera of Commelinales; (2) phylogenetic placement of never-before sequenced genera; (3) how well off-target plastid data integrate with other plastid-based data sets; and (4) how the novel inferences coincide with the infra-familial classification. METHODS: We generated two large data sets (nuclear and plastome) by means of target sequence capture using the Angiosperms353 probe set, with additional sequences mined from publicly available transcriptomes and full plastomes. A third extended-plastid data set was considered, including all species with sequences in public repositories. Species trees were inferred under a multispecies coalescent framework from individual gene trees and also using maximum likelihood analyses from concatenated and partitioned data. RESULTS: The nuclear, plastome, and extended-plastid data sets include 52, 53, and 58 genera, respectively, and up to 290 species of Commelinales, representing the most comprehensive molecular sampling for the order to date, which includes seven never-before sequenced genera. CONCLUSIONS: We inferred robust phylogenies supporting the monophyly of Commelinales and its five constituent families, and we recovered the clades Pontederiaceae-Haemodoraceae and Hanguanaceae-Commelinaceae, as previously reported. The placement of Philydraceae remains contentious. Relationships within the two largest families, Commelinaceae and Haemodoraceae, are resolved. Based on the latter results, we confirm the subfamilial classification of Haemodoraceae and propose a new classification for Commelinaceae, which includes the synonymization of Tapheocarpa in Commelina.

Comprehending Cornales: phylogenetic reconstruction of the order using the Angiosperms353 probe set.

PREMISE: Cornales is an order of flowering plants containing ecologically and horticulturally important families, including Cornaceae (dogwoods) and Hydrangeaceae (hydrangeas), among others. While many relationships in Cornales are strongly supported by previous studies, some uncertainty remains with regards to the placement of Hydrostachyaceae and to relationships among families in Cornales and within Cornaceae. Here we analyzed hundreds of nuclear loci to test published phylogenetic hypotheses and estimated a robust species tree for Cornales. METHODS: Using the Angiosperms353 probe set and existing data sets, we generated phylogenomic data for 158 samples, representing all families in the Cornales, with intensive sampling in the Cornaceae. RESULTS: We curated an average of 312 genes per sample, constructed maximum likelihood gene trees, and inferred a species tree using the summary approach implemented in ASTRAL-III, a method statistically consistent with the multispecies coalescent model. CONCLUSIONS: The species tree we constructed generally shows high support values and a high degree of concordance among individual nuclear gene trees. Relationships among families are largely congruent with previous molecular studies, except for the placement of the nyssoids and the Grubbiaceae-Curtisiaceae clades. Furthermore, we were able to place Hydrostachyaceae within Cornales, and within Cornaceae, the monophyly of known morphogroups was well supported. However, patterns of gene tree discordance suggest potential ancient reticulation, gene flow, and/or ILS in the Hydrostachyaceae lineage and the early diversification of Cornus. Our findings reveal new insights into the diversification process across Cornales and demonstrate the utility of the Angiosperms353 probe set.

Phylogenomics of the pantropical Connaraceae: revised infrafamilial classification and the evolution of heterostyly.

Connaraceae is a pantropical family of about 200 species containing lianas and small trees with remarkably diverse floral polymorphisms, including distyly, tristyly, homostyly, and dioecy. To date, relationships within the family have not been investigated using a targeted molecular phylogenetic treatment, severely limiting systematic understanding and reconstruction of trait evolution. Accordingly, their last infrafamilial classification was based only on morphological data. Here, we used phylogenomic data obtained using the Angiosperms353 nuclear target sequence capture probes, sampling all tribes and almost all genera, entirely from herbarium specimens, to revise infrafamilial classification and investigate the evolution of heterostyly. The backbone of the resulting molecular phylogenetic tree is almost entirely resolved. Connaraceae consists of two clades, one containing only the African genus Manotes (4 or 5 species), which we newly recognize at the subfamily level. Vegetative and reproductive synapomorphies are proposed for Manotoideae. Within Connaroideae, Connareae is expanded to include the former Jollydoreae. The backbone of Cnestideae, which contains more than half of the Connaraceae species, remains incompletely resolved. Reconstructions of reproductive system evolution are presented that tentatively support tristyly as the ancestral state for the family, with multiple parallel losses, in agreement with previous hypotheses, plus possible re-gains. However, the great diversity of stylar polymorphisms and their phylogenetic lability preclude a definitive answer. Overall, this study reinforces the usefulness of herbarium phylogenomics, and unlocks the reproductive diversity of Connaraceae as a model system for the evolution of complex biological phenomena. Supplementary Information: The online version contains supplementary material available at 10.1007/s00606-024-01909-y.

Genomic incongruence accompanies the evolution of flower symmetry in Eudicots: a case study in the poppy family (Papaveraceae, Ranunculales).

Reconstructing evolutionary trajectories and transitions that have shaped floral diversity relies heavily on the phylogenetic framework on which traits are modelled. In this study, we focus on the angiosperm order Ranunculales, sister to all other eudicots, to unravel higher-level relationships, especially those tied to evolutionary transitions in flower symmetry within the family Papaveraceae. This family presents an astonishing array of floral diversity, with actinomorphic, disymmetric (two perpendicular symmetry axes), and zygomorphic flowers. We generated nuclear and plastid datasets using the Angiosperms353 universal probe set for target capture sequencing (of 353 single-copy nuclear ortholog genes), together with publicly available transcriptome and plastome data mined from open-access online repositories. We relied on the fossil record of the order Ranunculales to date our phylogenies and to establish a timeline of events. Our phylogenomic workflow shows that nuclear-plastid incongruence accompanies topological uncertainties in Ranunculales. A cocktail of incomplete lineage sorting, post-hybridization introgression, and extinction following rapid speciation most likely explain the observed knots in the topology. These knots coincide with major floral symmetry transitions and thus obscure the order of evolutionary events.