Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Generalizing Bayesian phylogenetics to infer shared evolutionary events.

Many processes of biological diversification can simultaneously affect multiple evolutionary lineages. Examples include multiple members of a gene family diverging when a region of a chromosome is duplicated, multiple viral strains diverging at a "super-spreading" event, and a geological event fragmenting whole communities of species. It is difficult to test for patterns of shared divergences predicted by such processes because all phylogenetic methods assume that lineages diverge independently. We introduce a Bayesian phylogenetic approach to relax the assumption of independent, bifurcating divergences by expanding the space of topologies to include trees with shared and multifurcating divergences. This allows us to jointly infer phylogenetic relationships, divergence times, and patterns of divergences predicted by processes of diversification that affect multiple evolutionary lineages simultaneously or lead to more than two descendant lineages. Using simulations, we find that the method accurately infers shared and multifurcating divergence events when they occur and performs as well as current phylogenetic methods when divergences are independent and bifurcating. We apply our approach to genomic data from two genera of geckos from across the Philippines to test if past changes to the islands' landscape caused bursts of speciation. Unlike previous analyses restricted to only pairs of gecko populations, we find evidence for patterns of shared divergences. By generalizing the space of phylogenetic trees in a way that is independent from the likelihood model, our approach opens many avenues for future research into processes of diversification across the life sciences.

A set of principles and practical suggestions for equitable fieldwork in biology.

Field biology is an area of research that involves working directly with living organisms in situ through a practice known as "fieldwork." Conducting fieldwork often requires complex logistical planning within multiregional or multinational teams, interacting with local communities at field sites, and collaborative research led by one or a few of the core team members. However, existing power imbalances stemming from geopolitical history, discrimination, and professional position, among other factors, perpetuate inequities when conducting these research endeavors. After reflecting on our own research programs, we propose four general principles to guide equitable, inclusive, ethical, and safe practices in field biology: be collaborative, be respectful, be legal, and be safe. Although many biologists already structure their field programs around these principles or similar values, executing equitable research practices can prove challenging and requires careful consideration, especially by those in positions with relatively greater privilege. Based on experiences and input from a diverse group of global collaborators, we provide suggestions for action-oriented approaches to make field biology more equitable, with particular attention to how those with greater privilege can contribute. While we acknowledge that not all suggestions will be applicable to every institution or program, we hope that they will generate discussions and provide a baseline for training in proactive, equitable fieldwork practices.

Species Delimitation, Phylogenomics, and Biogeography of Sulawesi Flying Lizards: A Diversification History Complicated by Ancient Hybridization, Cryptic Species, and Arrested Speciation.

The biota of Sulawesi is noted for its high degree of endemism and for its substantial levels of in situ biological diversification. While the island's long period of isolation and dynamic tectonic history have been implicated as drivers of the regional diversification, this has rarely been tested in the context of an explicit geological framework. Here, we provide a tectonically informed biogeographical framework that we use to explore the diversification history of Sulawesi flying lizards (the Draco lineatus Group), a radiation that is endemic to Sulawesi and its surrounding islands. We employ a framework for inferring cryptic speciation that involves phylogeographic and genetic clustering analyses as a means of identifying potential species followed by population demographic assessment of divergence-timing and rates of bi-directional migration as means of confirming lineage independence (and thus species status). Using this approach, phylogenetic and population genetic analyses of mitochondrial sequence data obtained for 613 samples, a 50-SNP data set for 370 samples, and a 1249-locus exon-capture data set for 106 samples indicate that the current taxonomy substantially understates the true number of Sulawesi Draco species, that both cryptic and arrested speciations have taken place, and that ancient hybridization confounds phylogenetic analyses that do not explicitly account for reticulation. The Draco lineatus Group appears to comprise 15 species-9 on Sulawesi proper and 6 on peripheral islands. The common ancestor of this group colonized Sulawesi ~11 Ma when proto-Sulawesi was likely composed of two ancestral islands, and began to radiate ~6 Ma as new islands formed and were colonized via overwater dispersal. The enlargement and amalgamation of many of these proto-islands into modern Sulawesi, especially during the past 3 Ma, set in motion dynamic species interactions as once-isolated lineages came into secondary contact, some of which resulted in lineage merger, and others surviving to the present. [Genomics; Indonesia; introgression; mitochondria; phylogenetics; phylogeography; population genetics; reptiles.].

Ultraconserved elements-based phylogenomic systematics of the snake superfamily Elapoidea, with the description of a new Afro-Asian family.

The highly diverse snake superfamily Elapoidea is considered to be a classic example of ancient, rapid radiation. Such radiations are challenging to fully resolve phylogenetically, with the highly diverse Elapoidea a case in point. Previous attempts at inferring a phylogeny of elapoids produced highly incongruent estimates of their evolutionary relationships, often with very low statistical support. We sought to resolve this situation by sequencing over 4,500 ultraconserved element loci from multiple representatives of every elapoid family/subfamily level taxon and inferring their phylogenetic relationships with multiple methods. Concatenation and multispecies coalescent based species trees yielded largely congruent and well-supported topologies. Hypotheses of a hard polytomy were not retained for any deep branches. Our phylogenies recovered Cyclocoridae and Elapidae as diverging early within Elapoidea. The Afro-Malagasy radiation of elapoid snakes, classified as multiple subfamilies of an inclusive Lamprophiidae by some earlier authors, was found to be monophyletic in all analyses. The genus Micrelaps was consistently recovered as sister to Lamprophiidae. We establish a new family, Micrelapidae fam. nov., for Micrelaps and assign Brachyophis to this family based on cranial osteological synapomorphy. We estimate that Elapoidea originated in the early Eocene and rapidly diversified into all the major lineages during this epoch. Ecological opportunities presented by the post-Cretaceous-Paleogene mass extinction event may have promoted the explosive radiation of elapoid snakes.

Gene Flow Increases Phylogenetic Structure and Inflates Cryptic Species Estimations: A Case Study on Widespread Philippine Puddle Frogs (Occidozyga laevis).

In cryptic amphibian complexes, there is a growing trend to equate high levels of genetic structure with hidden cryptic species diversity. Typically, phylogenetic structure and distance-based approaches are used to demonstrate the distinctness of clades and justify the recognition of new cryptic species. However, this approach does not account for gene flow, spatial, and environmental processes that can obfuscate phylogenetic inference and bias species delimitation. As a case study, we sequenced genome-wide exons and introns to evince the processes that underlie the diversification of Philippine Puddle Frogs-a group that is widespread, phenotypically conserved, and exhibits high levels of geographically based genetic structure. We showed that widely adopted tree- and distance-based approaches inferred up to 20 species, compared to genomic analyses that inferred an optimal number of five distinct genetic groups. Using a suite of clustering, admixture, and phylogenetic network analyses, we demonstrate extensive admixture among the five groups and elucidate two specific ways in which gene flow can cause overestimations of species diversity: 1) admixed populations can be inferred as distinct lineages characterized by long branches in phylograms; and 2) admixed lineages can appear to be genetically divergent, even from their parental populations when simple measures of genetic distance are used. We demonstrate that the relationship between mitochondrial and genome-wide nuclear $p$-distances is decoupled in admixed clades, leading to erroneous estimates of genetic distances and, consequently, species diversity. Additionally, genetic distance was also biased by spatial and environmental processes. Overall, we showed that high levels of genetic diversity in Philippine Puddle Frogs predominantly comprise metapopulation lineages that arose through complex patterns of admixture, isolation-by-distance, and isolation-by-environment as opposed to species divergence. Our findings suggest that speciation may not be the major process underlying the high levels of hidden diversity observed in many taxonomic groups and that widely adopted tree- and distance-based methods overestimate species diversity in the presence of gene flow. [Cryptic species; gene flow; introgression; isolation-by-distance; isolation-by-environment; phylogenetic network; species delimitation.].

Cryptic diversity of a widespread global pathogen reveals expanded threats to amphibian conservation.

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Phylogenetics of mud snakes (Squamata: Serpentes: Homalopsidae): A paradox of both undescribed diversity and taxonomic inflation.

Mud snakes (Serpentes: Homalopsidae) are a family of 55 described, mainly aquatic, species primarily distributed throughout mainland Southeast Asia and the Indo-Australian Archipelago. Although they have been the focus of prior research, the basic relationships amongst genera and species remain poorly known. We used a combined mitochondrial and nuclear gene dataset to infer their phylogenetic relationships, using the highest levels of taxon and geographic sampling for any homalopsid phylogeny to date (62% generic and 62% species coverage; 140 individuals). Our results recover two reciprocally monophyletic groups: the fangless Brachyorrhos and its sister clade comprised of all rear-fanged homalopsids. Most genera and interspecific relationships were monophyletic and strongly supported, but intergeneric relationships and intraspecific population structure lack support. We find evidence of both undescribed diversity as well as cases of taxonomic inflation within several species. Tree-based species delimitation approaches (mPTP) support potential new candidate species as distinct from their conspecifics and also suggest that many named taxa may not be distinct species. Divergence date estimation and lineage-through-time analyses indicate lower levels of speciation in the Eocene, with a subsequent burst in diversification in the Miocene. Homalopsids may have diversified most rapidly during the Pliocene and Pleistocene, possibly in relation to tectonic shifts and sea-level fluctuations that took place in Sundaland and the Sahul Shelf. Our analyses provide new insights on homalopsid taxonomy, a baseline phylogeny for the family, and further biogeographic implications demonstrating how dynamic tectonics and Quaternary sea level changes may have shaped a widespread, diverse family of snakes.

Unexpectedly high levels of lineage diversity in Sundaland puddle frogs (Dicroglossidae: Occidozyga Kuhl and van Hasselt, 1822).

One of the most urgent contemporary tasks for taxonomists and evolutionary biologists is to estimate the number of species on earth. Recording alpha diversity is crucial for protecting biodiversity, especially in areas of elevated species richness, which coincide geographically with increased anthropogenic environmental pressures - the world's so-called biodiversity hotspots. Although the distribution of Puddle frogs of the genus Occidozyga in South and Southeast Asia includes five biodiversity hotspots, the available data on phylogeny, species diversity, and biogeography are surprisingly patchy. Samples analyzed in this study were collected throughout Southeast Asia, with a primary focus on Sundaland and the Philippines. A mitochondrial gene region comprising ~ 2000 bp of 12S and 16S rRNA with intervening tRNA Valine and three nuclear loci (BDNF, NTF3, POMC) were analyzed to obtain a robust, time-calibrated phylogenetic hypothesis. We found a surprisingly high level of genetic diversity within Occidozyga, based on uncorrected p-distance values corroborated by species delimitation analyses. This extensive genetic diversity revealed 29 evolutionary lineages, defined by the > 5% uncorrected p-distance criterion for the 16S rRNA gene, suggesting that species diversity in this clade of phenotypically homogeneous forms probably has been underestimated. The comparison with results of other anuran groups leads to the assumption that anuran species diversity could still be substantially underestimated in Southeast Asia in general. Many genetically divergent lineages of frogs are phenotypically similar, indicating a tendency towards extensive morphological conservatism. We present a biogeographic reconstruction of the colonization of Sundaland and nearby islands which, together with our temporal framework, suggests that lineage diversification centered on the landmasses of the northern Sunda Shelf. This remarkably genetically structured group of amphibians could represent an exceptional case for future studies of geographical structure and diversification in a widespread anuran clade spanning some of the most pronounced geographical barriers on the planet (e.g., Wallace's Line). Studies considering gene flow, morphology, ecological and bioacoustic data are needed to answer these questions and to test whether observed diversity of Puddle frog lineages warrants taxonomic recognition.

Target-capture phylogenomics provide insights on gene and species tree discordances in Old World treefrogs (Anura: Rhacophoridae).

Genome-scale data have greatly facilitated the resolution of recalcitrant nodes that Sanger-based datasets have been unable to resolve. However, phylogenomic studies continue to use traditional methods such as bootstrapping to estimate branch support; and high bootstrap values are still interpreted as providing strong support for the correct topology. Furthermore, relatively little attention has been given to assessing discordances between gene and species trees, and the underlying processes that produce phylogenetic conflict. We generated novel genomic datasets to characterize and determine the causes of discordance in Old World treefrogs (Family: Rhacophoridae)-a group that is fraught with conflicting and poorly supported topologies among major clades. Additionally, a suite of data filtering strategies and analytical methods were applied to assess their impact on phylogenetic inference. We showed that incomplete lineage sorting was detected at all nodes that exhibited high levels of discordance. Those nodes were also associated with extremely short internal branches. We also clearly demonstrate that bootstrap values do not reflect uncertainty or confidence for the correct topology and, hence, should not be used as a measure of branch support in phylogenomic datasets. Overall, we showed that phylogenetic discordances in Old World treefrogs resulted from incomplete lineage sorting and that species tree inference can be improved using a multi-faceted, total-evidence approach, which uses the most amount of data and considers results from different analytical methods and datasets.

Gene flow creates a mirage of cryptic species in a Southeast Asian spotted stream frog complex.

Most new cryptic species are described using conventional tree- and distance-based species delimitation methods (SDMs), which rely on phylogenetic arrangements and measures of genetic divergence. However, although numerous factors such as population structure and gene flow are known to confound phylogenetic inference and species delimitation, the influence of these processes is not frequently evaluated. Using large numbers of exons, introns, and ultraconserved elements obtained using the FrogCap sequence-capture protocol, we compared conventional SDMs with more robust genomic analyses that assess population structure and gene flow to characterize species boundaries in a Southeast Asian frog complex (Pulchrana picturata). Our results showed that gene flow and introgression can produce phylogenetic patterns and levels of divergence that resemble distinct species (up to 10% divergence in mitochondrial DNA). Hybrid populations were inferred as independent (singleton) clades that were highly divergent from adjacent populations (7%-10%) and unusually similar (<3%) to allopatric populations. Such anomalous patterns are not uncommon in Southeast Asian amphibians, which brings into question whether the high levels of cryptic diversity observed in other amphibian groups reflect distinct cryptic species-or, instead, highly admixed and structured metapopulation lineages. Our results also provide an alternative explanation to the conundrum of divergent (sometimes nonsister) sympatric lineages-a pattern that has been celebrated as indicative of true cryptic speciation. Based on these findings, we recommend that species delimitation of continuously distributed "cryptic" groups should not rely solely on conventional SDMs, but should necessarily examine population structure and gene flow to avoid taxonomic inflation.

Larger, unfiltered datasets are more effective at resolving phylogenetic conflict: Introns, exons, and UCEs resolve ambiguities in Golden-backed frogs (Anura: Ranidae; genus Hylarana).

Using FrogCap, a recently-developed sequence-capture protocol, we obtained >12,000 highly informative exons, introns, and ultraconserved elements (UCEs), which we used to illustrate variation in evolutionary histories of these classes of markers, and to resolve long-standing systematic problems in Southeast Asian Golden-backed frogs of the genus-complex Hylarana. We also performed a comprehensive suite of analyses to assess the relative performance of different genetic markers, data filtering strategies, tree inference methods, and different measures of branch support. To reduce gene tree estimation error, we filtered the data using different thresholds of taxon completeness (missing data) and parsimony informative sites (PIS). We then estimated species trees using concatenated datasets and Maximum Likelihood (IQ-TREE) in addition to summary (ASTRAL-III), distance-based (ASTRID), and site-based (SVDQuartets) multispecies coalescent methods. Topological congruence and branch support were examined using traditional bootstrap, local posterior probabilities, gene concordance factors, quartet frequencies, and quartet scores. Our results did not yield a single concordant topology. Instead, introns, exons, and UCEs clearly possessed different phylogenetic signals, resulting in conflicting, yet strongly-supported phylogenetic estimates. However, a combined analysis comprising the most informative introns, exons, and UCEs converged on a similar topology across all analyses, with the exception of SVDQuartets. Bootstrap values were consistently high despite high levels of incongruence and high proportions of gene trees supporting conflicting topologies. Although low bootstrap values did indicate low heuristic support, high bootstrap support did not necessarily reflect congruence or support for the correct topology. This study reiterates findings of some previous studies, which demonstrated that traditional bootstrap values can produce positively misleading measures of support in large phylogenomic datasets. We also showed a remarkably strong positive relationship between branch length and topological congruence across all datasets, implying that very short internodes remain a challenge to resolve, even with orders of magnitude more data than ever before. Overall, our results demonstrate that more data from unfiltered or combined datasets produced superior results. Although data filtering reduced gene tree incongruence, decreased amounts of data also biased phylogenetic estimation. A point of diminishing returns was evident, at which higher congruence (from more stringent filtering) at the expense of amount of data led to topological error as assessed by comparison to more complete datasets across different genomic markers. Additionally, we showed that applying a parameter-rich model to a partitioned analysis of concatenated data produces better results compared to unpartitioned, or even partitioned analysis using model selection. Despite some lingering uncertainties, a combined analysis of our genomic data and sequences supplemented from GenBank (on the basis of a few gene regions) revealed highly supported novel systematic arrangements. Based on these new findings, we transfer Amnirana nicobariensis into the genus Indosylvirana; and I. milleti and Hylarana celebensis to the genus Papurana. We also provisionally place H. attigua in the genus Papurana pending verification from positively identified (voucher substantiated) samples.

Multilocus phylogeny of Bornean Bent-Toed geckos (Gekkonidae: Cyrtodactylus) reveals hidden diversity, taxonomic disarray, and novel biogeographic patterns.

The gekkonid genus Cyrtodactylus is a highly diverse group of lizards (280 + species), which covers an expansive geographic range. Although this genus has been the focus of many taxonomic and molecular systematic studies, species on the Southeast Asian island of Borneo have remained understudied, leading to an unclear evolutionary history with cascading effects on taxonomy and biogeographic inferences. We assembled the most comprehensive multilocus Bornean dataset (one mitochondrial and three nuclear loci) that included 129 novel sequences and representatives from each known Cyrtodactylus species on the island to validate taxonomic status, assess species diversity, and elucidate biogeographic patterns. Our results uncovered a high proportion of cryptic diversity and revealed numerous taxonomic complications, especially within the C. consobrinus, C. malayanus, and C. pubisulcus groups. Comparisons of pairwise genetic distances and a preliminary species delimitation analysis using the Automatic Barcode Gap Discovery (ABGD) method demonstrated that some wide-ranging species on Borneo likely comprise multiple distinct and deeply divergent lineages, each with more restricted distributional ranges. We also tested the prevailing biogeographic hypothesis of a single invasion from Borneo into the Philippines. Our analyses revealed that Philippine taxa were not monophyletic, but were likely derived from multiple separate invasions into the geopolitical areas comprising the Philippines. Although our investigation of Bornean Cyrtodactylus is the most comprehensive to-date, it highlights the need for expanded taxonomic sampling and suggests that our knowledge of the evolutionary history, systematics, and biogeography of Bornean Cyrtodactylus is far from complete.

Parachute geckos free fall into synonymy: Gekko phylogeny, and a new subgeneric classification, inferred from thousands of ultraconserved elements.

Recent phylogenetic studies of gekkonid lizards have revealed unexpected, widespread paraphyly and polyphyly among genera, unclear generic boundaries, and a tendency towards the nesting of taxa exhibiting specialized, apomorphic morphologies within geographically widespread "generalist" clades. This is especially true in Australasia, where monophyly of Gekko proper has been questioned with respect to phenotypically ornate flap-legged geckos of the genus Luperosaurus, the Philippine false geckos of the genus Pseudogekko, and even the elaborately "derived" parachute geckos of the genus Ptychozoon. Here we employ sequence capture targeting 5060 ultraconserved elements (UCEs) to infer phylogenomic relationships among 42 representative ingroup gekkonine lizard taxa. We analyze multiple datasets of varying degrees of completeness (10, 50, 75, 95, and 100 percent complete with 4715, 4051, 3376, 2366, and 772 UCEs, respectively) using concatenated maximum likelihood and multispecies coalescent methods. Our sampling scheme addresses four persistent systematic questions in this group: (1) Are Luperosaurus and Ptychozoon monophyletic, and are any of these named species truly nested within Gekko? (2) Are prior phylogenetic estimates of Sulawesi's L. iskandari as the sister taxon to Melanesian G. vittatus supported by our genome-scale dataset? (3) Is the high-elevation L. gulat of Palawan Island correctly placed within Gekko? (4) And, finally, where do the enigmatic taxa P. rhacophorus and L. browni fall in a higher-level gekkonid phylogeny? We resolve these issues; confirm with strong support some previously inferred findings (placement of Ptychozoon taxa within Gekko; the sister taxon relationship between L. iskandari and G. vittatus); resolve the systematic position of unplaced taxa (L. gulat, and L. browni); and transfer L. iskandari, L. gulat, L. browni, and all members of the genus Ptychozoon to the genus Gekko. Our unexpected and novel systematic inference of the placement of Ptychozoon rhacophorus suggests that this species is not grouped with Ptychozoon or even Luperosaurus (as previously expected) but may, in fact, be most closely related to several Indochinese species of Gekko. With our resolved and strongly supported phylogeny, we present a new classification emphasizing the most inclusive, original generic name (Gekko) for these ~60 taxa, arranged into seven subgenera.

Lizards of the lost arcs: mid-Cenozoic diversification, persistence and ecological marginalization in the West Pacific.

Regions with complex geological histories often have diverse and highly endemic biotas, yet inferring the ecological and historical processes shaping this relationship remains challenging. Here, in the context of the taxon cycle model of insular community assembly, we investigate patterns of lineage diversity and habitat usage in a newly characterized vertebrate radiation centred upon the world's most geologically complex insular region: island arcs spanning from the Philippines to Fiji. On island arcs taxa are ecologically widespread, and provide evidence to support one key prediction of the taxon cycle, specifically that interior habitats (lowland rainforests, montane habitats) are home to a greater number of older or relictual lineages than are peripheral habitats (coastal and open forests). On continental fringes, however, the clade shows a disjunct distribution away from lowland rainforest, occurring in coastal, open or montane habitats. These results are consistent with a role for biotic interactions in shaping disjunct distributions (a central tenant of the taxon cycle), but we find this pattern most strongly on continental fringes not islands. Our results also suggest that peripheral habitats on islands, and especially island arcs, may be important for persistence and diversification, not just dispersal and colonization. Finally, new phylogenetic evidence for subaerial island archipelagos (with an associated biota) east of present-day Wallace's Line since the Oligocene has important implications for understanding long-term biotic interchange and assembly across Asia and Australia.

Discovery of an old, archipelago-wide, endemic radiation of Philippine snakes.

The extraordinarily rich land vertebrate biodiversity of the Philippines includes at least 112 species of terrestrial snakes (74% of which are endemic to the archipelago) in 41 genera (12% endemic). Endemic Philippine snake genera include Cyclocorus (two species), Hemibungarus (three species), Hologerrhum (two species), Oxyrhabdium (two species), and Myersophis (monotypic). Although Hemibungarus and Oxyrhabdium have been included in previous species-level phylogenies, the affinities of the other three Philippine endemic genera are completely unknown. We generated novel DNA sequences for six species from four genera and analyzed these in conjunction with data from earlier studies to infer a phylogeny for the group containing Colubridae, Elapoidea (Elapidae + Lamprophiidae), and Homalopsidae. We present a novel phylogenetic result that strongly supports the existence of an entirely endemic Philippine radiation of elapoid snakes that originated 35-25 million years ago. We provide a revised, phylogeny-based classification to accommodate the new clade, transfer Cyclocorus, Hologerrhum, and Myersophis to Lamprophiidae, and provide the first estimate of the evolutionary relationships among these genera and the related Oxyrhabdium, setting the stage for future investigation of this entirely endemic, novel Philippine elapoid radiation.

Comprehensive multi-locus phylogeny of Old World tree frogs (Anura: Rhacophoridae) reveals taxonomic uncertainties and potential cases of over- and underestimation of species diversity.

The family Rhacophoridae is one of the most diverse amphibian families in Asia, for which taxonomic understanding is rapidly-expanding, with new species being described steadily, and at increasingly finer genetic resolution. Distance-based methods frequently have been used to justify or at least to bolster the recognition of new species, particularly in complexes of "cryptic" species where obvious morphological differentiation does not accompany speciation. However, there is no universally-accepted threshold to distinguish intra- from interspecific genetic divergence. Moreover, indiscriminant use of divergence thresholds to delimit species can result in over- or underestimation of species diversity. To explore the range of variation in application of divergence scales, and to provide a family-wide assessment of species-level diversity in Old-World treefrogs (family Rhacophoridae), we assembled the most comprehensive multi-locus phylogeny to date, including all 18 genera and approximately 247 described species (∼60% coverage). We then used the Automatic Barcode Gap Discovery (ABGD) method to obtain different species-delimitation schemes over a range of prior intraspecific divergence limits to assess the consistency of divergence thresholds used to demarcate current species boundaries. The species-rich phylogeny was able to identify a number of taxonomic errors, namely the incorrect generic placement of Chiromantis inexpectatus, which we now move to the genus Feihyla, and the specific identity of Rhacophorus bipunctatus from Peninsular Malaysia, which we tentatively reassign to R. rhodopus. The ABGD analysis demonstrated overlap between intra- and interspecific divergence limits: genetic thresholds used in some studies to synonymize taxa have frequently been used in other studies to justify the recognition of new species. This analysis also highlighted numerous groups that could potentially be split or lumped, which we earmark for future examination. Our large-scale and en bloc approach to species-level phylogenetic systematics contributes to the resolution of taxonomic uncertainties, reveals possible new species, and identifies numerous groups that require critical examination. Overall, we demonstrate that the taxonomy and evolutionary history of Old-World tree frogs are far from resolved, stable or adequately characterized at the level of genus, species, and/or population.

A comprehensive phylogeny of the genus Kurixalus (Rhacophoridae, Anura) sheds light on the geographical range evolution of frilled swamp treefrogs.

Currently, the genus Kurixalus comprises 14 species distributed in Southern, Southeast and East Asia. Because of their relatively low dispersal capability and intolerance of seawater, this group is ideal for the study of terrestrial range evolution, especially that portion of its range that extends into the island archipelagos of Southern Asia. We assembled a large dataset of mitochondrial and nuclear genes, and estimated phylogeny by maximum likelihood and Bayesian methods, and we explored the history of each species via divergence-time estimation based on fossil-calibrations. A variety of ancestral-area reconstruction strategies were employed to estimate past changes of the species' geographical range, and to evaluate the impact of different abiotic barriers on range evolution. We found that frilled swamp treefrogs probably originated in Taiwan or South Vietnam in the Oligocene. Alternatively, the lineage leading to Kurixalus appendiculatus strongly supports a hypothesis of terrestrial connection between the Indian and Asian continents in the Oligocene. The outcome of both our divergence-time estimates and ancestral-area reconstruction suggests that the divergence between species from Indochina and Taiwan can probably be attributed to the opening of the South China Sea, approximately 33 million years ago. We could not find evidence for dispersal between mainland China and Taiwan Island. Formation of both Mekong and Red River valleys did not have any impact on Kurixalus species diversification. However, coincidence in timing of climate change and availability of plausible dispersal routes from the Oligocene to the middle Miocene, plausibly implied that Kurixalus diversification in Asia resulted from contemporaneous, climate-induced environmental upheaval (Late Oligocene Warming at 29 Ma; Mi-1 glaciation since 24.4-21.5 Ma; Mid-Miocene Climatic Optimum at 14 Ma), which alternatively opened and closed dispersal routes.

Large-scale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae).

Southeast Asia and southern China (SEA-SC) harbor a highly diverse and endemic flora and fauna that is under increasing threat. An understanding of the biogeographical history and drivers of this diversity is lacking, especially in some of the most diverse and threatened groups. The Asian leaf-litter frog genus Leptolalax Dubois 1980 is a forest-dependent genus distributed throughout SEA-SC, making it an ideal study group to examine specific biogeographic hypotheses. In addition, the diversity of this genus remains poorly understood, and the phylogenetic relationships among species of Leptolalax and closely related Leptobrachella Smith 1928 remain unclear. Herein, we evaluate species-level diversity based on 48 of the 53 described species from throughout the distribution of Leptolalax. Molecular analyses reveal many undescribed species, mostly in southern China and Indochina. Our well-resolved phylogeny based on multiple nuclear DNA markers shows that Leptolalax is not monophyletic with respect to Leptobrachella and, thus, we assign the former to being a junior synonym of the latter. Similarly, analyses reject monophyly of the two subgenera of Leptolalax. The diversification pattern of the group is complex, involving a high degree of sympatry and prevalence of microendemic species. Northern Sundaland (Borneo) and eastern Indochina (Vietnam) appear to have played pivotal roles as geographical centers of diversification, and paleoclimatic changes and tectonic movements seem to have driven the major divergence of clades. Analyses fail to reject an "upstream" colonization hypothesis, and, thus, the genus appears to have originated in Sundaland and then colonized mainland Asia. Our results reveal that both vicariance and dispersal are responsible for current distribution patterns in the genus.

Species delimitation with gene flow: A methodological comparison and population genomics approach to elucidate cryptic species boundaries in Malaysian Torrent Frogs.

Accurately delimiting species boundaries is a nontrivial undertaking that can have significant effects on downstream inferences. We compared the efficacy of commonly used species delimitation methods (SDMs) and a population genomics approach based on genomewide single-nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genomewide SNPs to identify putative species boundaries by implementing noncoalescent and coalescent-based SDMs (mPTP, iBPP, BFD*). We then tested the validity of putative boundaries by estimating spatiotemporal gene flow (fastsimcoal2, ABBA-BABA) to assess the extent of genetic isolation among putative species. Our results show that the A. larutensis complex runs the gamut of the speciation continuum from highly divergent, genetically isolated lineages (mean Fst  = 0.9) to differentiating populations involving recent gene flow (mean Fst  = 0.05; Nm  > 5). As expected, SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. This study does not discount the utility of SDMs but highlights the danger of violating model assumptions and the importance of carefully considering methods that appropriately fit the diversification history of a particular system.