Multispecies Coalescent Approach Research Articles

Phylogenomics, Lineage Diversification Rates, and the Evolution of Diadromy in Clupeiformes (Anchovies, Herrings, Sardines, and Relatives).

Migration independently evolved numerous times in animals, with a myriad of ecological and evolutionary implications. In fishes, perhaps the most extreme form of migration is diadromy, the migration between marine and freshwater environments. Key and longstanding questions are: how many times has diadromy evolved in fishes, how frequently do diadromous clades give rise to non-diadromous species, and does diadromy influence lineage diversification rates? Many diadromous fishes have large geographic ranges with constituent populations that use isolated freshwater habitats. This may limit gene flow among some populations, increasing the likelihood of speciation in diadromous lineages relative to non-diadromous lineages. Alternatively, diadromy may reduce lineage diversification rates if migration is associated with enhanced dispersal capacity that facilitates gene flow within and between populations. Clupeiformes (herrings, sardines, shads and anchovies) is a model clade for testing hypotheses about the evolution of diadromy because it includes an exceptionally high proportion of diadromous species and several independent evolutionary origins of diadromy. However, relationships among major clupeiform lineages remain unresolved and existing phylogenies sparsely sampled diadromous species, limiting the resolution of phylogenetically-informed statistical analyses. We assembled a phylogenomic dataset and used multi-species coalescent and concatenation-based approaches to generate the most comprehensive, highly-resolved clupeiform phylogeny to date, clarifying associations among several major clades and identifying recalcitrant relationships needing further examination. We determined that variation in rates of sequence evolution (heterotachy) and base-composition (non-stationarity) had little impact on our results. Using this phylogeny, we characterized evolutionary patterns of diadromy and tested for differences in lineage diversification rates between diadromous, marine, and freshwater lineages. We identified thirteen transitions to diadromy, all during the Cenozoic Era (ten origins of anadromy, two origins of catadromy, and one origin of amphidromy), and seven losses of diadromy. Two diadromous lineages rapidly generated non-diadromous species, demonstrating that diadromy is not an evolutionary dead-end. We discovered considerably faster transition rates out of diadromy than to diadromy. The largest lineage diversification rate increase in Clupeiformes was associated with a transition to diadromy, but we uncovered little statistical support for categorically faster lineage diversification rates in diadromous versus non-diadromous fishes. We propose that diadromy may increase the potential for accelerated lineage diversification, particularly in species that migrate long distances. However, this potential may only be realized in certain biogeographic contexts, such as when diadromy allows access to ecosystems in which there is limited competition from incumbent species.

Major patterns in the introgression history of Heliconius butterflies

Gene flow between species, although usually deleterious, is an important evolutionary process that can facilitate adaptation and lead to species diversification. It also makes estimation of species relationships difficult. Here, we use the full-likelihood multispecies coalescent (MSC) approach to estimate species phylogeny and major introgression events in Heliconius butterflies from whole-genome sequence data. We obtain a robust estimate of species branching order among major clades in the genus, including the ‘melpomene-silvaniform’ group, which shows extensive historical and ongoing gene flow. We obtain chromosome-level estimates of key parameters in the species phylogeny, including species divergence times, present-day and ancestral population sizes, as well as the direction, timing, and intensity of gene flow. Our analysis leads to a phylogeny with introgression events that differ from those obtained in previous studies. We find that Heliconius aoede most likely represents the earliest-branching lineage of the genus and that ‘silvaniform’ species are paraphyletic within the melpomene-silvaniform group. Our phylogeny provides new, parsimonious histories for the origins of key traits in Heliconius, including pollen feeding and an inversion involved in wing pattern mimicry. Our results demonstrate the power and feasibility of the full-likelihood MSC approach for estimating species phylogeny and key population parameters despite extensive gene flow. The methods used here should be useful for analysis of other difficult species groups with high rates of introgression.

Major patterns in the introgression history of Heliconius butterflies.

Gene flow between species, although usually deleterious, is an important evolutionary process that can facilitate adaptation and lead to species diversification. It also makes estimation of species relationships difficult. Here, we use the full-likelihood multispecies coalescent (MSC) approach to estimate species phylogeny and major introgression events in Heliconius butterflies from whole-genome sequence data. We obtain a robust estimate of species branching order among major clades in the genus, including the 'melpomene-silvaniform' group, which shows extensive historical and ongoing gene flow. We obtain chromosome-level estimates of key parameters in the species phylogeny, including species divergence times, present-day and ancestral population sizes, as well as the direction, timing, and intensity of gene flow. Our analysis leads to a phylogeny with introgression events that differ from those obtained in previous studies. We find that Heliconius aoede most likely represents the earliest-branching lineage of the genus and that 'silvaniform' species are paraphyletic within the melpomene-silvaniform group. Our phylogeny provides new, parsimonious histories for the origins of key traits in Heliconius, including pollen feeding and an inversion involved in wing pattern mimicry. Our results demonstrate the power and feasibility of the full-likelihood MSC approach for estimating species phylogeny and key population parameters despite extensive gene flow. The methods used here should be useful for analysis of other difficult species groups with high rates of introgression.

Interspecific gene flow obscures phylogenetic relationships in an important insect pest species complex

As genomic data proliferates, the prevalence of post-speciation gene flow is making species boundaries and relationships increasingly ambiguous. Although current approaches inferring fully bifurcating phylogenies based on concatenated datasets provide simple and robust answers to many species relationships, they may be inaccurate because the models ignore inter-specific gene flow and incomplete lineage sorting. To examine the potential error resulting from ignoring gene flow, we generated both a RAD-seq and a 500 protein-coding loci highly multiplexed amplicon (HiMAP) dataset for a monophyletic group of 12 species defined as the Bactrocera dorsalis sensu lato clade. With some of the world's worst agricultural pests, the taxonomy of the B. dorsalis s.l. clade is important for trade and quarantines. However, taxonomic confusion confounds resolution due to intra- and interspecific phenotypic variation and convergence, mitochondrial introgression across half of the species, and viable hybrids. We compared the topological convergence of our datasets using concatenated phylogenetic and various multispecies coalescent approaches, some of which account for gene flow. All analyses agreed on species delimitation, but there was incongruence between species relationships. Under concatenation, both datasets suggest identical species relationships with mostly high statistical support. However, multispecies coalescent and multispecies network approaches suggest markedly different hypotheses and detected significant gene flow. We suggest that the network approaches are likely more accurate because gene flow violates the assumptions of the concatenated phylogenetic analyses, but the data-reductive requirements of network approaches resulted in reduced statistical support and could not unambiguously resolve gene flow directions. Our study highlights the importance of testing for gene flow, particularly with phylogenomic datasets, even when concatenated approaches receive high statistical support.

Transcriptome-based phylogenomic analysis reveals evidence of ancient hybridization events in the Mepraia genus (Hemiptera: Reduviidae: Triatominae)

IntroductionThe Mepraia genus is composed of three endemic species in Chile, namely Mepraia spinolai, Mepraia gajardoi, and Mepraia parapatrica, all capable of transmitting the causative agent of Chagas disease. Although species divergence within this genus is supported by morphological, molecular, and geological evidence, it has never been validated by genome-wide data.MethodsIn this sense, a pioneering phylogenomic study was conducted using transcriptome data of the three species, aiming to infer robust phylogenies and evaluate the impact of interspecific gene flow on the evolution of these insects. Our transcriptomes were used to robustly infer the phylogenetic relationships, employing multi-species coalescent and supermatrix approaches, as well as to investigate the occurrence of introgression.Results and discussionThe inferred phylogenies support the monophyly of the three described taxa and infer divergence times, although gene trees display high levels of discordance. Our results indicate widespread introgression among Mepraia lineages. Thus, our results support that Mepraia diversification occurred in a short period resulting in reticulate phylogenies and low genetic diversity. Additionally, we obtained evidence of introgression suggesting that ancient hybridization has had a major role in the evolution of the three Mepraia species.

Filtration of Gene Trees From 9,000 Exons, Introns, and UCEs Disentangles Conflicting Phylogenomic Relationships in Tree Frogs (Hylidae).

An emerging challenge in interpreting phylogenomic data sets is that concatenation and multi-species coalescent summary species tree approaches may produce conflicting results. Concatenation is problematic because it can strongly support an incorrect topology when incomplete lineage sorting (ILS) results in elevated gene-tree discordance. Conversely, summary species tree methods account for ILS to recover the correct topology, but these methods do not account for erroneous gene trees ("EGTs") resulting from gene tree estimation error (GTEE). Third, site-based and full-likelihood methods promise to alleviate GTEE as these methods use the sequence data from alignments. To understand the impact of GTEE on species tree estimation in Hylidae tree frogs, we use an expansive data set of ∼9,000 exons, introns, and ultra-conserved elements and initially found conflict between all three types of analytical methods. We filtered EGTs using alignment metrics that could lead to GTEE (length, parsimony-informative sites, and missing data) and found that removing shorter, less informative alignments reconciled the conflict between concatenation and summary species tree methods with increased gene concordance, with the filtered topologies matching expected results from past studies. Contrarily, site-based and full-likelihood methods were mixed where one method was consistent with past studies and the other varied markedly. Critical to other studies, these results suggest a widespread conflation of ILS and GTEE, where EGTs rather than ILS are driving discordance. Finally, we apply these recommendations to an R package named PhyloConfigR, which facilitates phylogenetic software setup, summarizes alignments, and provides tools for filtering alignments and gene trees.

Phylogenomics reveal extensive phylogenetic discordance due to incomplete lineage sorting following the rapid radiation of alpine butterflies (Papilionidae: Parnassius)

AbstractUnder rapid radiation, the earliest components of evolutionary divergence are often difficult to resolve, which were always driven by the characteristics of taxa and the limitations of alternative analytical methods. The origin and radiation of the alpine butterfly Parnassius, a high‐altitude mountainous insect group, can be attributed to the uplift of the Qinghai‐Tibet Plateau. Despite detailed phylogenetic analyses of the genus, deep phylogenetic relationships among the major subgenera remain recalcitrant. In this study, 102 individuals from 10 representative Parnassius species were sampled to resolve the phylogenetic relationships among subgenera based on nuclear and mitochondrial genome datasets. Gene‐tree/species‐tree conflicts were detected by concatenation and multispecies coalescent (MSC) approaches. We recovered a well‐supported species tree, despite these conflicts, and detected considerable phylogenetic discordance among genomic regions. The main explanation for the topological discordance among subgenera was extensive incomplete lineage sorting (ILS), whereas introgression events were not prominent. The origin and explosive radiation of Parnassius (i.e., rapid succession of speciation events) in the late Miocene associated with environmental events on the plateau led to short internal branches, thereby increasing ILS and topological conflicts, especially among closely related subgenera. Our results also suggested that MSC approaches (SNP and AFLP Package for Phylogenetic analysis [SNAPP] and SVDquartets) are accurate and superior to the concatenation approach; in particular, SVDquartets can explicitly accommodate gene‐tree/species‐tree conflicts caused by high ILS and demonstrate strong robustness. Finally, we explored the phylogenomic data by testing multiple sources of phylogenomic conflict to clarify the strengths and limitations of different approaches, while considering phylogenetic signal variation in mitochondrial loci. We anticipate that the phylogeny described here will be the backbone of future evolutionary studies of the genus and will provide insight into phylogenetic discordance due to rapid radiation.

Dispersal-limited Symbionts Exhibit Unexpectedly Wide Variation in Host Specificity.

A fundamental aspect of symbiotic relationships is host specificity, ranging from extreme specialists associated with only a single host species to generalists associated with many different species. Although symbionts with limited dispersal capabilities are expected to be host specialists, some are able to associate with multiple hosts. Understanding the micro- and macroevolutionary causes of variations in host specificity is often hindered by sampling biases and the limited power of traditional evolutionary markers. Here, we studied feather mites to address the barriers associated with estimates of host specificity for dispersal-limited symbionts. We sampled feather mites (Proctophyllodidae) from a nearly comprehensive set of North American breeding warblers (Parulidae) to study mite phylogenetic relationships and host-symbiont codiversification. We used pooled-sequencing (Pool-Seq) and short-read Illumina technology to interpret results derived from a traditional barcoding gene (cytochrome c oxidase subunit 1) versus 11 protein-coding mitochondrial genes using concatenated and multispecies coalescent approaches. Despite the statistically significant congruence between mite and host phylogenies, mite-host specificity varies widely, and host switching is common regardless of the genetic marker resolution (i.e., barcode versus multilocus). However, the multilocus approach was more effective than the single barcode in detecting the presence of a heterogeneous Pool-Seq sample. These results suggest that presumed symbiont dispersal capabilities are not always strong indicators of host specificity or of historical host-symbiont coevolutionary events. Comprehensive sampling at fine phylogenetic scales may help to better elucidate the microevolutionary filters that impact macroevolutionary processes regulating symbioses, particularly for dispersal-limited symbionts.

The use of Anchored Hybrid Enrichment data to resolve higher-level phylogenetic relationships: A proof-of-concept applied to Asterales (Eudicotyledoneae; Angiosperms)

Anchored Hybrid Enrichment (AHE) is a tool for capturing orthologous regions of the nuclear genome shared in low or single copy across lineages. Despite the increasing number of studies using this method, its usefulness to estimate relationships at deeper taxonomic levels in plants has not been fully explored. Here we present a proof of concept about the performance of nuclear loci obtained with AHE to infer phylogenetic relationships and explore the use of gene sampling schemes to estimate divergence times in Asterales. We recovered low-copy nuclear loci using the AHE method from herbarium material and silica-preserved samples. Maximum likelihood, Bayesian inference, and coalescence approaches were used to reconstruct phylogenomic relationships. Dating analyses were conducted under a multispecies coalescent approach by jointly inferring species tree and divergence times with random gene sampling schemes and multiple calibrations. We recovered 403 low-copy nuclear loci for 63 species representing nine out of eleven families of Asterales. Phylogenetic hypotheses were congruent among the applied methods and previously published results. Analyses with concatenated datasets were strongly supported, but coalescence-based analyses showed low support for the phylogenetic position of families Argophyllaceae and Alseuosmiaceae. Estimated family ages were congruent among gene sampling schemes, with the mean age for Asterales around 130 Myr. Our study documents the usefulness of AHE for resolving phylogenetic relationships at deep phylogenetic levels in Asterales. Observed phylogenetic inconsistencies were possibly due to the non-inclusion of families Phellinceae and Pentaphragmataceae. Random gene sampling schemes produced consistent age estimates with coalescence and species tree relaxed clock approaches.

Resurrection of Lethrinus scoparius Gilchrist amp; Thompson, 1908 (Perciformes: Lethrinidae), from South African waters.

Lethrinus nebulosus (Forsskl, 1775) is distributed throughout the Indian Ocean, Indonesia and the West-central Pacific. Given recent evidence suggesting two cryptic species of L. nebulosus in the southwestern Indian Ocean, we implemented a multispecies coalescent approach using nuclear and mitochondrial DNA loci and conducted Bayesian tests of species delimitation. One population extends from southern Mozambique to Kenya, and the other from northern KwaZulu-Natal (South Africa) southwards, with a zone of overlap between them. This study confirms that the South African population can be identified as a separate species, previously identified as Lethrinus scoparius Gilchrist Thompson, 1908, and can be distinguished from L. nebulosus by live and fresh colouration and with genetic data, but not consistently by morphometric or meristic parameters, other than in preorbital length cf. head length. Lethrinus scoparius is resurrected and redescribed herein.

New Insights Into the Relationships Within Subtribe Scorzonerinae (Cichorieae, Asteraceae) Using Hybrid Capture Phylogenomics (Hyb-Seq).

Subtribe Scorzonerinae (Cichorieae, Asteraceae) contains 12 main lineages and approximately 300 species. Relationships within the subtribe, either at inter- or intrageneric levels, were largely unresolved in phylogenetic studies to date, due to the lack of phylogenetic signal provided by traditional Sanger sequencing markers. In this study, we employed a phylogenomics approach (Hyb-Seq) that targets 1,061 nuclear-conserved ortholog loci designed for Asteraceae and obtained chloroplast coding regions as a by-product of off-target reads. Our objectives were to evaluate the potential of the Hyb-Seq approach in resolving the phylogenetic relationships across the subtribe at deep and shallow nodes, investigate the relationships of major lineages at inter- and intrageneric levels, and examine the impact of the different datasets and approaches on the robustness of phylogenetic inferences. We analyzed three nuclear datasets: exon only, excluding all potentially paralogous loci; exon only, including loci that were only potentially paralogous in 1–3 samples; exon plus intron regions (supercontigs); and the plastome CDS region. Phylogenetic relationships were reconstructed using both multispecies coalescent and concatenation (Maximum Likelihood and Bayesian analyses) approaches. Overall, our phylogenetic reconstructions recovered the same monophyletic major lineages found in previous studies and were successful in fully resolving the backbone phylogeny of the subtribe, while the internal resolution of the lineages was comparatively poor. The backbone topologies were largely congruent among all inferences, but some incongruent relationships were recovered between nuclear and plastome datasets, which are discussed and assumed to represent cases of cytonuclear discordance. Considering the newly resolved phylogenies, a new infrageneric classification of Scorzonera in its revised circumscription is proposed.

Gauging ages of tiger swallowtail butterflies using alternate SNP analyses

Divergence times underpin diverse evolutionary hypotheses, but conflicting age estimates across studies diminish the validity of such hypotheses. These conflicts have continued to grow as large genomics datasets become commonplace and analytical approaches proliferate. To provide more stable temporal intervals, age estimations should be interpreted in the context of both the type of data and analysis being used. Here, we use multispecies coalescent (MSC), concatenation-based, and categorical data transformation approaches on genome-wide SNP data to infer divergence ages within the Papilio glaucus group of tiger swallowtail butterflies in North America. While the SNP data supported previously recognized relationships within the group (P. multicaudata, ((P. eurymedon, P. rutulus), (P. appalachiensis, P. canadensis, P. glaucus))), estimated ages of divergence between the major lineages varied substantially among analyses. MSC produced wide credibility intervals particularly for deeper nodes, reflecting uncertainty in the coalescence times as a possible result of conflicting signal across gene trees. Concatenation, in contrast, gave narrower and more well-defined posterior distributions for the node ages; however, the higher precision of these time estimates is a likely artefact due to more simplistic underlying assumptions of this approach that do not account for conflict among gene trees. Transformed categorical data analysis gave the least precise and the most variable results, with its simple substitution model coupled with a relaxed clock tending to produce spurious results from large genome-wide datasets. While median node ages differed considerably between analyses (∼2 Mya between MSC and concatenation-based results), their corresponding credibility intervals nonetheless highlight common temporal patterns for deeper divergences in the group as well as finer-scale phylogeography. Age distributions across analyses support an origin of the group during the warm period of the early to mid-Pliocene. Late Pliocene climate aridification and cooling drove divergence between eastern and western groups that further diversified during the period of repeated Pleistocene glaciations. Our results provide a structured comparative assessment of divergence time estimates and evolutionary relationships in a well-studied group of butterflies, and support better understanding of analytical biases in divergence time estimation.

Using natural history to guide supervised machine learning for cryptic species delimitation with genetic data

The diversity of biological and ecological characteristics of organisms, and the underlying genetic patterns and processes of speciation, makes the development of universally applicable genetic species delimitation methods challenging. Many approaches, like those incorporating the multispecies coalescent, sometimes delimit populations and overestimate species numbers. This issue is exacerbated in taxa with inherently high population structure due to low dispersal ability, and in cryptic species resulting from nonecological speciation. These taxa present a conundrum when delimiting species: analyses rely heavily, if not entirely, on genetic data which over split species, while other lines of evidence lump. We showcase this conundrum in the harvester Theromaster brunneus, a low dispersal taxon with a wide geographic distribution and high potential for cryptic species. Integrating morphology, mitochondrial, and sub-genomic (double-digest RADSeq and ultraconserved elements) data, we find high discordance across analyses and data types in the number of inferred species, with further evidence that multispecies coalescent approaches over split. We demonstrate the power of a supervised machine learning approach in effectively delimiting cryptic species by creating a “custom” training data set derived from a well-studied lineage with similar biological characteristics as Theromaster. This novel approach uses known taxa with particular biological characteristics to inform unknown taxa with similar characteristics, using modern computational tools ideally suited for species delimitation. The approach also considers the natural history of organisms to make more biologically informed species delimitation decisions, and in principle is broadly applicable for taxa across the tree of life.

Phylogenetic Relationships Within the Hyper-Diverse Genus Eugenia (Myrtaceae: Myrteae) Based on Target Enrichment Sequencing.

Eugenia is one of the most taxonomically challenging lineages of flowering plants, in which morphological delimitation has changed over the last few years resulting from recent phylogenetic study based on molecular data. Efforts, until now, have been limited to Sanger sequencing of mostly plastid markers. These phylogenetic studies indicate 11 clades formalized as infrageneric groups. However, relationships among these clades are poorly supported at key nodes and inconsistent between studies, particularly along the backbone and within Eugenia sect. Umbellatae encompasses ca. 700 species. To resolve and better understand systematic discordance, 54 Eugenia taxa were subjected to phylogenomic Hyb-Seq using 353 low-copy nuclear genes. Twenty species trees based on coding and non-coding loci of nuclear and plastid datasets were recovered using coalescent and concatenated approaches. Concordant and conflicting topologies were assessed by comparing tree landscapes, topology tests, and gene and site concordance factors. The topologies are similar except between nuclear and plastid datasets. The coalescent trees better accommodate disparity in the intron dataset, which contains more parsimony informative sites, while concatenated trees recover more conservative topologies, as they have narrower distribution in the tree landscape. This suggests that highly supported phylogenetic relationships determined in previous studies do not necessarily indicate overwhelming concordant signal. Congruence must be interpreted carefully especially in concatenated datasets. Despite this, the congruence between the multi-species coalescent (MSC) approach and concatenated tree topologies found here is notable. Our analysis does not support Eugenia subg. Pseudeugenia or sect. Pilothecium, as currently circumscribed, suggesting necessary taxonomic reassessment. Five clades are further discussed within Eugenia sect. Umbellatae progress toward its division into workable clades. While targeted sequencing provides a massive quantity of data that improves phylogenetic resolution in Eugenia, uncertainty still remains in Eugenia sect. Umbellatae. The general pattern of higher site coefficient factor (CF) than gene CF in the backbone of Eugenia suggests stochastic error from limited signal. Tree landscapes in combination with concordance factor scores, as implemented here, provide a comprehensive approach that incorporates several phylogenetic hypotheses. We believe the protocols employed here will be of use for future investigations on the evolutionary history of Myrtaceae.

Phylogenomic analyses resolve relationships among garter snakes (Thamnophis: Natricinae: Colubridae) and elucidate biogeographic history and morphological evolution

Garter snakes (Thamnophis) are a successful group of natricines endemic to North America. They have become important natural models for ecological and evolutionary research, yet prior efforts to resolve phylogenetic relationships have resulted in conflicting topologies and weak support for certain relationships. Here, we use genomic data generated with a reduced representation double-digest RADseq approach to reassess evolutionary relationships across Thamnophis. We then use the resulting phylogeny to better understand how biogeography and feeding ecology have influenced lineage diversification and morphological evolution. We recovered highly congruent and strongly supported topologies from maximum likelihood and Bayesian analyses, but some discordance with a multispecies coalescent approach. All phylogenomic estimates split Thamnophis into two clades largely defined by northern and southern North American species. Divergence time estimates and biogeographic analyses indicate a mid-Miocene origin of Thamnophis in Mexico. In addition, historic vicariant events thought to explain biogeographic patterns in other lineages (e.g., Isthmus of Tehuantepec, Rocky Mountain Range, and Trans-Mexican Volcanic Belt) appear to have influenced patterns of diversification in Thamnophis as well. Analyses of morphological traits associated with feeding ecology showed moderate to strong phylogenetic signal. Nevertheless, phylogenetic ANOVA suggested significant differences in certain cranial morphologies between aquatic specialists and garter snakes that are terrestrial-aquatic generalists, independent of evolutionary history. Our new estimate of Thamnophis phylogeny yields an improved understanding of the biogeographic history and morphological evolution of garter snakes, and provides a robust framework for future research on these snakes.

Interspecific Gene Flow and Mitochondrial Genome Capture during the Radiation of Jamaican Anolis Lizards (Squamata; Iguanidae).

Gene flow and reticulation are increasingly recognized as important processes in the diversification of many taxonomic groups. With the increasing ease of collecting genomic data and the development of multispecies coalescent network approaches, such reticulations can be accounted for when inferring phylogeny and diversification. Caribbean Anolis lizards are a classic example of an adaptive radiation in which species have independently radiated on the islands of the Greater Antilles into the same ecomorph classes. Within the Jamaican radiation at least one species, Anolis opalinus, has been documented to be polyphyletic in its mitochondrial DNA, which could be the result of an ancient reticulation event or incomplete lineage sorting (ILS). Here, we generate mtDNA and genotyping-by-sequencing (GBS) data and implement gene tree, species tree, and multispecies coalescent network methods to infer the diversification of this group. Our mtDNA gene tree recovers the same relationships previously inferred for this group, which is strikingly different from the species tree inferred from our GBS data. Posterior predictive simulations suggest that our genomic data violate commonly adopted assumptions of the multispecies coalescent model (MSCM), so we use network approaches to infer phylogenetic relationships. The inferred network topology contains a reticulation event but does not explain the mtDNA polyphyly observed in this group; however, coalescent simulations suggest that the observed mtDNA topology is likely the result of past introgression. How common a signature of gene flow and reticulation is across the radiation of Anolis is unknown; however, the reticulation events that we demonstrate here may have allowed for adaptive evolution, as has been suggested in other, more recent, adaptive radiations. [Adaptive radiation; hybridization; introgression; multispecies network coalescent; posterior predictive simulation.].

Phylogeny, classification, and character evolution of tribe Citharexyleae (Verbenaceae).

As a family of Neotropical origin and primarily Neotropical distribution, the Verbenaceae are a good but understudied system with which to understand Neotropical evolution. Tribe Citharexyleae comprises three genera: Baillonia, Citharexylum-one of the largest genera in Verbenaceae-and Rehdera. A molecular phylogenetic approach was taken to resolve intergeneric relationships in Citharexyleae and infrageneric relationships in Citharexylum. The phylogeny is used to elucidate character evolution in a widespread, morphologically diverse Neotropical genus. Seven plastid regions, two nuclear ribosomal spacers, and six low-copy nuclear loci were analyzed for 64 species of Citharexyleae. Phylogenetic analyses were conducted using maximum likelihood, Bayesian inference, and multispecies coalescent approaches. Habit, presence or absence of thorns, inflorescence architecture, flower color, fruit color, and geography were examined to identify diagnostic character states for clades within Citharexylum. Rehdera is resolved as sister to Citharexylum, and Baillonia nested within Citharexylum. Two species, C. oleinum and C. tetramerum, are not closely related to tribe Citharexyleae, but may be related to members of tribe Duranteae instead. Seven clades within Citharexylum are inferred, each characterized by a combination of geography, fruit color and/or maturation, and inflorescence architecture. There is evidence of correlated evolution between habit, axillary inflorescences, and flower number per inflorescence. Shrubs with reduced inflorescences have evolved repeatedly. A subgeneric classification for Citharexylum is proposed. Although suites of associated traits are found, character morphology has been labile throughout Citharexylum's evolutionary history. Morphological diversity may be related to adaptation to differing mesic and xeric habitats.

Rare and widespread: integrating Bayesian MCMC approaches, Sanger sequencing and Hyb-Seq phylogenomics to reconstruct the origin of the enigmatic Rand Flora genus Camptoloma.

Genera that are widespread, with geographically discontinuous distributions and represented by few species, are intriguing. Is their achieved disjunct distribution recent or ancient in origin? Why are they species-poor? The Rand Flora is a continental-scale pattern in which closely related species appear codistributed in isolated regions over the continental margins of Africa. Genus Camptoloma (Scrophulariaceae) is the most notable example, comprising three species isolated from each other on the northwest, eastern, and southwest Africa. We employed Sanger sequencing of nuclear and plastid markers, together with genomic target sequencing of 2190 low-copy nuclear genes, to infer interspecies relationships and the position of Camptoloma within Scrophulariaceae by using supermatrix and multispecies-coalescent approaches. Lineage divergence times and ancestral ranges were inferred with Bayesian Markov chain Monte Carlo (MCMC) approaches. The population history was estimated with phylogeographic coalescent methods. Camptoloma rotundifolium, restricted to Southern Africa, was shown to be a sister species to the disjunct clade formed by C. canariense, endemic to the Canary Islands, and C. lyperiiflorum, distributed in the Horn of Africa-Southern Arabia. Camptoloma was inferred to be sister to the mostly South African tribes Teedieae and Buddlejeae. Stem divergence was dated in the Late Miocene, while the origin of the extant disjunction was inferred as Early Pliocene. The current disjunct distribution of Camptoloma across Africa was likely the result of fragmentation and extinction and/or population bottlenecking events associated with historical aridification cycles during the Neogene; the pattern of species divergence, from south to north, is consistent with the "climatic refugia" Rand Flora hypothesis.

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

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. 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. 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. 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.

Evaluating Species Delimitation Methods in Radiations: The Land Snail Albinaria cretensis Complex on Crete.

Delimiting species in radiations is notoriously difficult because of the small differences between the incipient species, the star-like tree with short branches between species, incomplete lineage sorting, and the possibility of introgression between several of the incipient species. Next-generation sequencing data may help to overcome some of these problems. We evaluated methods for species delimitation based on genome-wide markers in a land snail radiation on Crete. Species delimitation in the Albinaria cretensis group was based exclusively on shell characters until now and resulted in classifications distinguishing 3-9 species. We generated sequences of 4270 loci for 140 specimens of the A. cretensis group from 48 populations by double-digest restriction site-associated DNA sequencing. We evaluated three methods for species discovery. The multispecies coalescent approach implemented in the program Bayesian Phylogenetics and Phylogeography resulted in a drastic overestimating of the number of species, whereas Gaussian clustering resulted in an overlumping. Primary species hypotheses based on the maximum percentage of the genome of the individuals derived from ancestral populations as estimated with the program ADMIXTURE moderately overestimated the number of species, but this was the only approach that provided information about gene flow between groups. Two of the methods for species validation that we applied, BFD* and delimitR, resulted in an acceptance of almost all primary species hypotheses, even such based on arbitrary subdivisions of hypotheses based on ADMIXTURE. In contrast, secondary species hypotheses, resulting from an evaluation of primary species hypotheses based on ADMIXTURE with isolation by distance tests, approached the morphological classification, but also uncovered two cryptic species and indicated that some of the previously delimited units should be combined. Thus, we recommend this combination of approaches that provided more detailed insights in the distinctness of barriers between the taxa of a species complex and the spatial distribution of admixture between them than the other methods. The recognition and delimitation of undersampled species remained a major challenge. [ADMIXTURE; Clausiliidae; delimitR, Gaussian clustering; isolation-by-distance; land snails; multispecies coalescent; species delimitation.].