Multispecies Coalescent Model Research Articles

Machine learning approaches delimit cryptic taxa in a previously intractable species complex

Cryptic species are not diagnosable via morphological criteria, but can be detected through analysis of DNA sequences. A number of methods have been developed for identifying species based on genetic data; however, these methods are prone to over-splitting taxa with extreme population structure, such as dispersal-limited organisms. Machine learning methodologies have the potential to overcome this challenge. Here, we apply such approaches, using a large dataset generated through hybrid target enrichment of ultraconserved elements (UCEs). Our study taxon is the Aoraki denticulata species complex, a lineage of extremely low-dispersal arachnids endemic to the South Island of Aotearoa New Zealand. This group of mite harvesters has been the subject of previous species delimitation studies using smaller datasets generated through Sanger sequencing and analytical approaches that rely on multispecies coalescent models and barcoding gap discovery. Those analyses yielded a number of putative cryptic species that seems unrealistic and extreme, based on what we know about species’ geographic ranges and genetic diversity in non-cryptic mite harvesters. We find that machine learning approaches, on the other hand, identify cryptic species with geographic ranges that are similar to those seen in other morphologically diagnosable mite harvesters in Aotearoa New Zealand’s South Island. We performed both unsupervised and supervised machine learning analyses, the latter with training data drawn either from animals broadly (vagile and non-vagile) or from a custom training dataset from dispersal-limited harvesters. We conclude that applying machine learning approaches to the analysis of UCE-derived genetic data is an effective method for delimiting species in complexes of low-vagility cryptic species, and that the incorporation of training data from biologically relevant analogues can be critically informative.

Traditional multilocus phylogeny fails to fully resolve Palearctic ground squirrels (Spermophilus) relationships but reveals a new species endemic to West Siberia

Previous efforts to reconstruct evolutionary history of Palearctic ground squirrels within the genus Spermophilus have primarily relied on a single mitochondrial marker for phylogenetic data. In this study, we present the first phylogeny with comprehensive taxon sampling of Spermophilus via a conventional multilocus approach utilizing five mitochondrial and five nuclear markers. Through application of the multispecies coalescent model, we constructed a species tree revealing four distinct clades that diverged during the Late Miocene. These clades are 1) S. alaschanicus and S. dauricus from East Asia; 2) S. musicus and S. pygmaeus from East Europe and northwestern Central Asia; 3) the subgenus Colobotis found across Central Asia and its adjacent regions and encompassing S. brevicauda, S. erythrogenys, S. fulvus, S. major, S. pallidicauda, S. ralli, S. relictus, S. selevini, and S. vorontsovi sp. nov.; and 4) a Central/Eastern Europe and Asia Minor clade comprising S. citellus, S. taurensis, S. xanthoprymnus, S. suslicus, and S. odessanus. The latter clade lacked strong support owing to uncertainty of taxonomic placement of S. odessanus and S. suslicus. Resolving relationships within the subgenus Colobotis, which radiated rapidly, remains challenging likely because of incomplete lineage sorting and introgressive hybridization. Most of modern Spermophilus species diversified during the Early–Middle Pleistocene (2.2–1.0 million years ago). We propose a revised taxonomic classification for the genus Spermophilus by recognizing 18 species including a newly identified one (S. vorontsovi sp. nov.), which is found only in a limited area in the southeast of West Siberia. Employing genome-wide single-nucleotide polymorphism genotyping, we substantiated the role of the Ob River as a major barrier ensuring robust isolation of this taxon from S. erythrogenys. Despite its inherent limitations, the traditional multilocus approach remains a valuable tool for resolving relationships and can provide important insights into otherwise poorly understood groups. It is imperative to recognize that additional efforts are needed to definitively determine phylogenetic relationships between certain species of Palearctic ground squirrels.

Anomalous networks under the multispecies coalescent: theory and prevalence.

Reticulations in a phylogenetic network represent processes such as gene flow, admixture, recombination and hybrid speciation. Extending definitions from the tree setting, an anomalous network is one in which some unrooted tree topology displayed in the network appears in gene trees with a lower frequency than a tree not displayed in the network. We investigate anomalous networks under the Network Multispecies Coalescent Model with possible correlated inheritance at reticulations. Focusing on subsets of 4 taxa, we describe a new algorithm to calculate quartet concordance factors on networks of any level, faster than previous algorithms because of its focus on 4 taxa. We then study topological properties required for a 4-taxon network to be anomalous, uncovering the key role of [Formula: see text]-cycles: cycles of 3 edges parent to a sister group of 2 taxa. Under the model of common inheritance, that is, when each gene tree coalesces within a species tree displayed in the network, we prove that 4-taxon networks are never anomalous. Under independent and various levels of correlated inheritance, we use simulations under realistic parameters to quantify the prevalence of anomalous 4-taxon networks, finding that truly anomalous networks are rare. At the same time, however, we find a significant fraction of networks close enough to the anomaly zone to appear anomalous, when considering the quartet concordance factors observed from a few hundred genes. These apparent anomalies may challenge network inference methods.

Phylogenomic analysis reveals five independently evolved African forage grass clades in the genus Urochloa.

The grass genus Urochloa (Brachiaria) sensu lato includes forage crops that are important for beef and dairy industries in tropical and sub-tropical Africa, South America and Oceania/Australia. Economically important species include U. brizantha, U. decumbens, U. humidicola, U. mutica, U. arrecta, U. trichopus, U. mosambicensis and Megathyrsus maximus, all native to the African continent. Perennial growth habits, large, fast growing palatable leaves, intra- and interspecific morphological variability, apomictic reproductive systems and frequent polyploidy are widely shared within the genus. The combination of these traits probably favoured the selection for forage domestication and weediness, but trait emergence across Urochloa cannot be modelled, as a robust phylogenetic assessment of the genus has not been conducted. We aim to produce a phylogeny for Urochloa that includes all important forage species, and identify their closest wild relatives (crop wild relatives). Finally, we will use our phylogeny and available trait data to infer the ancestral states of important forage traits across Urochloa s.l. and model the evolution of forage syndromes across the genus. Using a target enrichment sequencing approach (Angiosperm 353), we inferred a species-level phylogeny for Urochloa s.l., encompassing 54 species (~40 % of the genus) and outgroups. Phylogenies were inferred using a multispecies coalescent model and maximum likelihood method. We determined the phylogenetic placement of agriculturally important species and identified their closest wild relatives, or crop wild relatives, based on well-supported monophyly. Further, we mapped key traits associated with Urochloa forage crops to the species tree and estimated ancestral states for forage traits along branch lengths for continuous traits and at ancestral nodes in discrete traits. Agricultural species belong to five independent clades, including U. brizantha and U. decumbens lying in a previously defined species complex. Crop wild relatives were identified for these clades supporting previous sub-generic groupings in Urochloa based on morphology. Using ancestral trait estimation models, we find that five morphological traits that correlate with forage potential (perennial growth habits, culm height, leaf size, a winged rachis and large seeds) independently evolved in forage clades. Urochloa s.l. is a highly diverse genus that contains numerous species with agricultural potential, including crop wild relatives that are currently underexploited. All forage species and their crop wild relatives naturally occur on the African continent and their conservation across their native distributions is essential. Genomic and phenotypic diversity in forage clade species and their wild relatives need to be better assessed both to develop conservation strategies and to exploit the diversity in the genus for improved sustainability in Urochloa cultivar production.

Ultrafast learning of four-node hybridization cycles in phylogenetic networks using algebraic invariants.

The abundance of gene flow in the Tree of Life challenges the notion that evolution can be represented with a fully bifurcating process which cannot capture important biological realities like hybridization, introgression, or horizontal gene transfer. Coalescent-based network methods are increasingly popular, yet not scalable for big data, because they need to perform a heuristic search in the space of networks as well as numerical optimization that can be NP-hard. Here, we introduce a novel method to reconstruct phylogenetic networks based on algebraic invariants. While there is a long tradition of using algebraic invariants in phylogenetics, our work is the first to define phylogenetic invariants on concordance factors (frequencies of four-taxon splits in the input gene trees) to identify level-1 phylogenetic networks under the multispecies coalescent model. Our novel hybrid detection methodology is optimization-free as it only requires the evaluation of polynomial equations, and as such, it bypasses the traversal of network space, yielding a computational speed at least 10 times faster than the fastest-to-date network methods. We illustrate our method's performance on simulated and real data from the genus Canis. We present an open-source publicly available Julia package PhyloDiamond.jl available at https://github.com/solislemuslab/PhyloDiamond.jl with broad applicability within the evolutionary community.

A comprehensive phylogenomic study unveils evolutionary patterns and challenges in the mitochondrial genomes of Carcharhiniformes: A focus on Triakidae

The complex evolutionary patterns in the mitochondrial genome (mitogenome) of the most species-rich shark order, the Carcharhiniformes (ground sharks) has led to challenges in the phylogenomic reconstruction of the families and genera belonging to the order, particularly the family Triakidae (houndsharks). The current state of Triakidae phylogeny remains controversial, with arguments for both monophyly and paraphyly within the family. We hypothesize that this variability is triggered by the selection of different a priori partitioning schemes to account for site and gene heterogeneity within the mitogenome. Here we used an extensive statistical framework to select the a priori partitioning scheme for inference of the mitochondrial phylogenomic relationships within Carcharhiniformes, tested site heterogeneous CAT + GTR + G4 models and incorporated the multi-species coalescent model (MSCM) into our analyses to account for the influence of gene tree discordance on species tree inference. We included five newly assembled houndshark mitogenomes to increase resolution of Triakidae. During the assembly procedure, we uncovered a 714 bp-duplication in the mitogenome of Galeorhinus galeus. Phylogenetic reconstruction confirmed monophyly within Triakidae and the existence of two distinct clades of the expanded Mustelus genus. The latter alludes to potential evolutionary reversal of reproductive mode from placental to aplacental, suggesting that reproductive mode has played a role in the trajectory of adaptive divergence. These new sequences have the potential to contribute to population genomic investigations, species phylogeography delineation, environmental DNA metabarcoding databases and, ultimately, improved conservation strategies for these ecologically and economically important species.

The genomic diversity of the Eliurus genus in northern Madagascar with a putative new species

Madagascar exhibits extraordinarily high level of species richness and endemism, while being severely threatened by habitat loss and fragmentation (HL&F). In front of these threats to biodiversity, conservation effort can be directed, for instance, in the documentation of species that are still unknown to science, or in investigating how species respond to HL&F. The tufted-tail rats genus (Eliurus spp.) is the most speciose genus of endemic rodents in Madagascar, with 13 described species, which occupy two major habitat types: dry or humid forests. The large species diversity and association to specific habitat types make the Eliurus genus a suitable model for investigating species adaptation to new environments, as well as response to HL&F (dry vs humid). In the present study, we investigated Eliurus spp. genomic diversity across northern Madagascar, a region covered by both dry and humid fragmented forests. From the mitochondrial DNA (mtDNA) and nuclear genomic (RAD-seq) data of 124 Eliurus individuals sampled in poorly studied forests of northern Madagascar, we identified an undescribed Eliurus taxon (Eliurus sp. nova). We tested the hypothesis of a new Eliurus species using several approaches: i) DNA barcoding; ii) phylogenetic inferences; iii) species delimitation tests based on the Multi-Species Coalescent (MSC) model, iv) genealogical divergence index (gdi); v) an ad-hoc test of isolation-by-distance within versus between sister-taxa, vi) comparisons of %GC content patterns and vii) morphological analyses. All analyses support the recognition of the undescribed lineage as a putative distinct species. In addition, we show that Eliurus myoxinus, a species known from the dry forests of western Madagascar, is, surprisingly, found mostly in humid forests in northern Madagascar. In conclusion, we discuss the implications of such findings in the context of Eliurus species evolution and diversification, and use the distribution of northern Eliurus species as a proxy for reconstructing past changes in forest cover and vegetation type in northern Madagascar.

The Species as a Reproductive Community Emerging From the Past

Biologists and philosophers of science have been unable to fully resolve the decades-long controversy as to what kind of unit of living biodiversity should receive the valued label “species”: reproductive communities (among sexual organisms), genealogical groups, or clusters of organisms that share traits. Among these choices, which represent a spectrum from process to history to observable outcome (respectively), the latter (more operationalist) concepts are not viable. Species of sexual organisms must embody or imply cohesive and integrating processes such as interbreeding and shared ecological pressures if they are to have sufficient power to bear the burden we give them: to predict or explain traits across the genome and among organisms. This commitment to cohesive process is needed whether biologists use species as taxonomic containers to synthesize data, as minimal phylogenetic units, or as actors in evolutionary diversification. These varied uses can be satisfied via a concept of reproductive community, but not the strict Biological Species Concept (BSC). Its two drawbacks are a focus on the contemporary and a restriction to intrinsic factors. Current reproductive compatibility may predict future matings, but it does not explain well the traits and genes that living organisms already have. The organisms alive today were shaped by isolating factors of the past, not those of the present, to whatever extent those differ. The most broadly-useful species concept must therefore see species retrospectively, as reproductive communities of the past. As well, the BSC’s exclusion of extrinsic factors renders each of its units incomplete in explanation and synthesis. Reproductive communities in nature were isolated not just by intrinsic (genetic) differences, but also by purely extrinsic (e.g., geographic) factors. Such reproductive communities were and are real, natural entities whose integrated and self-reinforcing cohesive processes constrained genealogical descent and aligned the distribution of many traits. This Retrospective Reproductive Community Concept (RRCC), formalized mathematically in multispecies coalescent models, justifies the traditional practice of taxonomists using morphological data to seek the echoes of past reproductive cohesion. However, which reproductive communities naturally deserve to be ranked as species, and which as demes or populations, is a vexing question. There is no natural, discrete and broadly informative species rank that applies universally, or perhaps even usually. To whatever extent species rank is justified, it is as justified for asexual organisms as for sexuals. The presence or absence of sex is just one example of the variability biologists confront. Because cohesive processes vary among clades, a useful and broadly-applicable species concept cannot specify detailed cohesive mechanisms. Nor can it perfectly align the named species of taxonomy with units of evolution, because the latter are not structured to match taxonomy’s partition of boxes. Taxonomic species should approximate, but can only approximate, evolutionary units. Settling on retrospection, letting go of a natural meaning for species rank, and accepting taxonomy as approximation allow biology to turn to the far more daunting task: listening to the natural world to understand the many interacting processes that built distinction and identity, that shaped the reproductive communities emerging out of the past into the present day.

Species delimitation with limited sampling: An example from rare trapdoor spider genus Cyclocosmia (Mygalomorphae, Halonoproctidae).

The outcome of species delimitation depends on many factors, including conceptual framework, study design, data availability, methodology employed and subjective decision making. Obtaining sufficient taxon sampling in endangered or rare taxa might be difficult, particularly when non-lethal tissue collection cannot be utilized. The need to avoid overexploitation of the natural populations may thus limit methodological framework available for downstream data analyses and bias the results. We test species boundaries in rare North American trapdoor spider genus Cyclocosmia Ausserer (1871) inhabiting the Southern Coastal Plain biodiversity hotspot with the use of genomic data and two multispecies coalescent model methods. We evaluate the performance of each methodology within a limited sampling framework. To mitigate the risk of species over splitting, common in taxa with highly structured populations, we subsequently implement a species validation step via genealogical diversification index (gdi), which accounts for both genetic isolation and gene flow. We delimited eight geographically restricted lineages within sampled North American Cyclocosmia, suggesting that major river drainages in the region are likely barriers to dispersal. Our results suggest that utilizing BPP in the species discovery step might be a good option for datasets comprising hundreds of loci, but fewer individuals, which may be a common scenario for rare taxa. However, we also show that such results should be validated via gdi, in order to avoid over splitting.

PhyloAcc-GT: A Bayesian method for inferring patterns of substitution rate shifts on targeted lineages accounting for gene tree discordance.

An important goal of evolutionary genomics is to identify genomic regions whose substitution rates differ among lineages. For example, genomic regions experiencing accelerated molecular evolution in some lineages may provide insight into links between genotype to phenotype. Several comparative genomics methods have been developed to identify genomic accelerations between species, including a Bayesian method called PhyloAcc, which models shifts in substitution rate in multiple target lineages on a phylogeny. However, few methods consider the possibility of discordance between the trees of individual loci and the species tree due to incomplete lineage sorting, which might cause false positives. Here we present PhyloAcc-GT, which extends PhyloAcc by modeling gene tree heterogeneity. Given a species tree, we adopt the multispecies coalescent model as the prior distribution of gene trees, use Markov chain Monte Carlo (MCMC) for inference, and design novel MCMC moves to sample gene trees efficiently. Through extensive simulations, we show that PhyloAcc-GT outperforms PhyloAcc and other methods in identifying target-lineage-specific accelerations and detecting complex patterns of rate shifts, and is robust to specification of population size parameters. PhyloAcc-GT is usually more conservative than PhyloAcc in calling convergent rate shifts because it identifies more accelerations on ancestral than on terminal branches. We apply PhyloAcc-GT to two examples of convergent evolution: flightlessness in ratites and marine mammal adaptations, and show that PhyloAcc-GT is a robust tool to identify shifts in substitution rate associated with specific target lineages while accounting for incomplete lineage sorting.

A reinvestigation of multiple independent evolution and Triassic-Jurassic origin of multicellular Volvocine algae.

The evolution of multicellular organisms is considered to be a major evolutionary transition, profoundly affecting the ecology and evolution of nearly all life on earth. The volvocine algae, a unique clade of chlorophytes with diverse cell morphology, provide an appealing model for investigating the evolution of multicellularity and development. However, the phylogenetic relationship and timescale of the volvocine algae are not fully resolved. Here, we use extensive taxon and gene sampling to reconstruct the phylogeny of the volvocine algae. Our results support that the colonial volvocine algae are not monophyletic group, and multicellularity independently evolve at least twice in the volvocine algae, once in Tetrabaenaceae and another in the Goniaceae + Volvocaceae. The simulation analyses suggest that incomplete lineage sorting is a major factor for the tree topology discrepancy, which imply that the multispecies coalescent model better fits the data used in this study. The coalescent-based species tree supports that the Goniaceae is monophyletic, and Crucicarteria is the earliest diverging lineage, followed by Hafniomonas and Radicarteria within the Volvocales. By considering the multiple uncertainties in divergence time estimation, the dating analyses indicate that the volvocine algae occurred during the Cryogenian to Ediacaran (696.6-551.1 Ma), and multicellularity in the volvocine algae originated from the Triassic to Jurassic. Our phylogeny and timeline provide an evolutionary framework for studying the evolution of key traits and the origin of multicellularity in the volvocine algae.

Potential Contribution of Ancient Introgression to the Evolution of a Derived Reproductive Strategy in Ricefishes.

Transitions from no parental care to extensive care are costly and involve major changes in life history, behavior, and morphology. Nevertheless, in Sulawesi ricefishes, pelvic brooding evolved from transfer brooding in two distantly related lineages within the genera Adrianichthys and Oryzias, respectively. Females of pelvic brooding species carry their eggs attached to their belly until the fry hatches. Despite their phylogenetic distance, both pelvic brooding lineages share a set of external morphological traits. A recent study found no direct gene flow between pelvic brooding lineages, suggesting independent evolution of the derived reproductive strategy. Convergent evolution can, however, also rely on repeated sorting of preexisting variation of an admixed ancestral population, especially when subjected to similar external selection pressures. We thus used a multispecies coalescent model and D-statistics to identify gene-tree-species-tree incongruencies, to evaluate the evolution of pelvic brooding with respect to interspecific gene flow not only between pelvic brooding lineages but also between pelvic brooding lineages and other Sulawesi ricefish lineages. We found a general network-like evolution in Sulawesi ricefishes, and as previously reported, we detected no gene flow between the pelvic brooding lineages. Instead, we found hybridization between the ancestor of pelvic brooding Oryzias and the common ancestor of the Oryzias species from the Lake Poso area. We further detected signs of introgression within the confidence interval of a quantitative trait locus associated with pelvic brooding in O. eversi. Our results hint toward a contribution of ancient standing genetic variation to the evolution of pelvic brooding in Oryzias.

Rearranging and completing the puzzle: Phylogenomic analysis of bark beetles Dendroctonus reveals new hypotheses about genus diversification

Studies carried out on bark beetles within Dendroctonus have been extensive and revealed diverse information in different areas of their natural history, taxonomy, evolution, and interactions, among others. Despite these efforts, phylogenetic hypotheses have remained obscured mainly due to limited information analyzed (taxonomic, gene sampling, or both) in studies focused on obtaining evolutionary hypotheses for this genus. With the aim of filling these gaps in the evolutionary history for Dendroctonus, we analyzed ∼1800 loci mapped to a reference genome obtained for 20 of the 21 species recognized to date, minimizing the impact of missing information and improving the assumption of orthology in a phylogenomic framework. We obtained congruent phylogenetic topologies from two phylogenomic inference strategies: loci concatenation (ML framework) and a multispecies coalescent model (MSC) through the analysis of site pattern frequencies (SNPs). Dendroctonus is composed of two major clades (A and B), each containing five and four subclades, respectively. According to our divergence dating analysis, the MRCA for Dendroctonus dates back to the early Eocene, while the MRCA for each major clade diverged in the mid-Eocene. Interestingly, most of the speciation events of extant species occurred during the Miocene, which could be correlated with the diversification of pine trees (Pinus). The MRCA for Dendroctonus inhabited large regions of North America, with all ancestors and descendants of clade A having diversified within this region. The Mexican Transition Zone is important in the diversification processes for the majority of clade A species. For clade B, we identified two important colonization events to the Old World from America: the first in the early Oligocene from the Arctic to Asia (via Beringia), and the second during the Miocene from the Arctic-Western-Alleghany region to Europe and Siberia (also via Beringia). Our genomic analyses also supported the existence of hidden structured lineages within the frontalis complex, and also that D. beckeri represent a lineage independent from D. valens, as previously suggested. The information presented here updates the knowledge concerning the diversification of a genus with remarkable ecological and economic importance.

Phylogenomic branch length estimation using quartets.

Branch lengths and topology of a species tree are essential in most downstream analyses, including estimation of diversification dates, characterization of selection, understanding adaptation, and comparative genomics. Modern phylogenomic analyses often use methods that account for the heterogeneity of evolutionary histories across the genome due to processes such as incomplete lineage sorting. However, these methods typically do not generate branch lengths in units that are usable by downstream applications, forcing phylogenomic analyses to resort to alternative shortcuts such as estimating branch lengths by concatenating gene alignments into a supermatrix. Yet, concatenation and other available approaches for estimating branch lengths fail to address heterogeneity across the genome. In this article, we derive expected values of gene tree branch lengths in substitution units under an extension of the multispecies coalescent (MSC) model that allows substitutions with varying rates across the species tree. We present CASTLES, a new technique for estimating branch lengths on the species tree from estimated gene trees that uses these expected values, and our study shows that CASTLES improves on the most accurate prior methods with respect to both speed and accuracy. CASTLES is available at https://github.com/ytabatabaee/CASTLES.

"Correcting" Gene Trees to be More Like Species Trees Frequently Increases Topological Error.

The evolutionary histories of individual loci in a genome can be estimated independently, but this approach is error-prone due to the limited amount of sequence data available for each gene, which has led to the development of a diverse array of gene tree error correction methods which reduce the distance to the species tree. We investigate the performance of two representatives of these methods: TRACTION and TreeFix. We found that gene tree error correction frequently increases the level of error in gene tree topologies by "correcting" them to be closer to the species tree, even when the true gene and species trees are discordant. We confirm that full Bayesian inference of the gene trees under the multispecies coalescent model is more accurate than independent inference. Future gene tree correction approaches and methods should incorporate an adequately realistic model of evolution instead of relying on oversimplified heuristics.

A taxonomic revision of the ecologically important Ochna holstii (Ochnaceae) complex using molecular and morphological data

Background and aims – Ochna holstii and its allies represent an ecologically important group in a variety of lowland to montane habitats in tropical eastern and southern Africa. Identifying and delimiting species within this group has proved challenging due to a lack of variation in morphological characters. We combine genomic-scale data of multiple accessions per species with morphological data to generate a taxonomic revision for the Ochna holstii complex using a multi-evidence approach. Material and methods – A total of 50 samples representing eight species were analysed using target enrichment and a custom bait kit. Phylogenetic analysis was conducted using the multi-species coalescent model and a concatenation maximum likelihood method, and gene tree discordance was investigated. Herbarium specimens of the O. holstii complex were studied in detail for informative morphological characters, supplemented where possible from field collections, and a full taxonomic revision is provided. Key results – Within Ochna sect. Schizanthera Clade I, our study confirms that all species are monophyletic and supported by morphological data with the exception of O. holstii, which is paraphyletic. A new species, O. mchanga, previously confused with O. polyneura, is described, and the placement of another recently described species, O. maguirei, is confirmed for the first time using molecular data. Conclusion – The widespread montane tree Ochna holstii is non-monophyletic in its current circumscription and likely gave rise to the more range-restricted O. oxyphylla and O. stolzii. We suggest that an integrated taxonomy approach, using both molecular and morphological data, is essential for deciphering difficult species relationships in Ochna.

Complex Patterns of Diversification in the Gray Zone of Speciation: Model-Based Approaches Applied to Patagonian Liolaemid Lizards (Squamata: Liolaemus kingii clade).

In this study we detangled the evolutionary history of the Patagonian lizard clade Liolaemus kingii, coupling dense geographic sampling and novel computational analytical approaches. We analyzed nuclear and mitochondrial data (restriction site-associated DNA sequencing and cytochrome b) to hypothesize and evaluate species limits, phylogenetic relationships, and demographic histories. We complemented these analyses with posterior predictive simulations to assess the fit of the genomic data to the multispecies coalescent model. We also employed a novel approach to time-calibrate a phylogenetic network. Our results show several instances of mito-nuclear discordance and consistent support for a reticulated history, supporting the view that the complex evolutionary history of the kingii clade is characterized by extensive gene flow and rapid diversification events. We discuss our findings in the contexts of the "gray zone" of speciation, phylogeographic patterns in the Patagonian region, and taxonomic outcomes. [Model adequacy; multispecies coalescent; multispecies network coalescent; phylogenomics; species delimitation.].

Genome-wide data reveal cryptic diversity and hybridization in a group of tree ferns

Discovery of cryptic diversity is essential to understanding both the process of speciation and the conservation of species. Determining species boundaries in fern lineages represents a major challenge due to lack of morphologically diagnostic characters and frequent hybridization. Genomic data has substantially enhanced our understanding of the speciation process, increased the resolution of species delimitation studies, and led to the discovery of cryptic diversity. Here, we employed restriction-site-associated DNA sequencing (RAD-seq) and integrated phylogenomic and population genomic analyses to investigate phylogenetic relationships and evolutionary history of 16 tree ferns with marginate scales (Cyatheaceae) from China and Vietnam. We conducted multiple species delimitation analyses using the multispecies coalescent (MSC) model and novel approaches based on genealogical divergence index (gdi) and isolation by distance (IBD). In addition, we inferred species trees using concatenation and several coalescent-based methods, and assessed hybridization patterns and rate of gene flow across the phylogeny. We obtained highly supported and generally congruent phylogenies inferred from concatenated and summary-coalescent methods, and the monophyly of all currently recognized species were strongly supported. Our results revealed substantial evidence of cryptic diversity in three widely distributed Gymnosphaera species, each of which was composite of two highly structure lineages that may correspond to cryptic species. We found that hybridization was fairly common between not only closely related species, but also distantly related species. Collectively, it appears that scaly tree ferns may contain cryptic diversity and hybridization has played an important role throughout the evolutionary history of this group.

Phylogenomic evolutionary insights in the fern family Gleicheniaceae

The pantropical fern family Gleicheniaceae comprises approximately 157 species. Seven genera are currently recognized in the family, although their monophyly is still uncertain due to low sampling in phylogenetic studies. We examined the monophyly of the genera through extended sampling, using the first phylogenomic inference of the family including data from both nuclear and plastid genomes. Seventy-six samples were sequenced (70 Gleicheniaceae species and six outgroups) using high throughput sequencing, including all seven currently recognized genera. Plastid and nuclear data were recovered and assembled; the nuclear data was phased to reduce paralogy as well as hybrid noise in the final recovered topology. Maximum likelihood trees were built for each locus, and a concatenated dataset was built for both datasets. A species tree based on a multispecies coalescent model was generated, and divergence time analyses performed. We here present the first genomic phylogenetic inferences concerning Gleicheniaceae, confirming the monophyly of most genera except Sticherus, which we recovered as paraphyletic. Although most of the extant genera of Gleicheniaceae originated during the Mesozoic, several genera show Neogene and even Quaternary diversifications, and our results suggest that reticulation and polyploidy may have played significant roles during this diversification. However, some genera, such as Rouxopteris and Stromatopteris, appear to represent evolutionary relicts.

Estimation of species divergence times in presence of cross-species gene flow.

Cross-species introgression can have significant impacts on phylogenomic reconstruction of species divergence events. Here, we used simulations to show how the presence of even a small amount of introgression can bias divergence time estimates when gene flow is ignored in the analysis. Using advances in analytical methods under the multispecies coalescent (MSC) model, we demonstrate that by accounting for incomplete lineage sorting and introgression using large phylogenomic data sets this problem can be avoided. The multispecies-coalescent-with-introgression (MSci) model is capable of accurately estimating both divergence times and ancestral effective population sizes, even when only a single diploid individual per species is sampled. We characterize some general expectations for biases in divergence time estimation under three different scenarios: 1) introgression between sister species, 2) introgression between non-sister species, and 3) introgression from an unsampled (i.e., ghost) outgroup lineage. We also conducted simulations under the isolation-with-migration (IM) model and found that the MSci model assuming episodic gene flow was able to accurately estimate species divergence times despite high levels of continuous gene flow. We estimated divergence times under the MSC and MSci models from two published empirical datasets with previous evidence of introgression, one of 372 target-enrichment loci from baobabs (Adansonia), and another of 1000 transcriptome loci from 14 species of the tomato relative, Jaltomata. The empirical analyses not only confirm our findings from simulations, demonstrating that the MSci model can reliably estimate divergence times but also show that divergence time estimation under the MSC can be robust to the presence of small amounts of introgression in empirical datasets with extensive taxon sampling. [divergence time; gene flow; hybridization; introgression; MSci model; multispecies coalescent].