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Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.

The gekkonid genus Cyrtodactylus is the third largest vertebrate genus on the planet with well over 300 species that range across at least eight biogeographic regions from South Asia to Melanesia. The ecological and morphological plasticity within the genus, has contributed to its ability to disperse across ephemeral seaways, river systems, basins, land bridges, and mountain ranges—followed by in situ diversification within specific geographic areas. Ancestral ranges were reconstructed on a mitochondrial phylogeny with 346 described and undescribed species from which it was inferred that Cyrtodactylus evolved in a proto-Himalaya region during the early Eocene. From there, it dispersed to what is currently Indoburma and Indochina during the mid-Eocene—the latter becoming the first major center of origin for the remainder of the genus that seeded dispersals to the Indian subcontinent, Papua, and Sundaland. Sundaland became a second major center of radiation during the Oligocene and gave rise to a large number of species that radiated further within Sundaland and dispersed to Wallacea, the Philippines, and back to Indochina. One Papuan lineage dispersed west to recolonize and radiate in Sundaland. Currently, Indochina and Sundaland still harbor the vast majority of species of Cyrtodactylus .

Accurate species delimitation and description are necessary to guide effective conservation of imperiled species, and this synergy is maximized when multiple data sources are used to delimit species. We illustrate this point by examining Drymarchon couperi (Eastern Indigo Snake), a large, federally-protected species in North America that was recently divided into two species based on gene sequence data from three loci and heuristic morphological assessment. Here, we re-evaluate the two-species hypothesis for D. couperi by evaluating both population genetic and gene sequence data. Our analyses of 14 microsatellite markers revealed 6-8 genetic population clusters with significant admixture, particularly across the contact zone between the two hypothesized species. Phylogenetic analyses of gene sequence data with maximum-likelihood methods suggested discordance between mitochondrial and nuclear markers and provided phylogenetic support for one species rather than two. For these reasons, we place Drymarchon kolpobasileus into synonymy with D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA are driven by high dispersal of males relative to females. We advocate for species delimitation exercises that evaluate admixture and gene flow in addition to phylogenetic analyses, particularly when the latter reveal monophyletic lineages. This is particularly important for taxa, such as squamates, that exhibit strong sex-biased dispersal. Problems associated with over-delimitation of species richness can become particularly acute for threatened and endangered species, because of high costs to conservation when taxonomy demands protection of more individual species than are supported by accumulating data.

Phylogenetic analyses based on 1459 base pairs of the mitochondrial gene ND2 and its flanking tRNAs indicate that Cyrtodactylus consobrinus from the type locality in Sarawak, East Malaysia (Borneo) and C. consobrinus from Peninsular Malaysia are not conspecific. Both populations as well as C. hutan from East Malaysia form a strongly supported monophyletic group even though their relationships to one another remain unresolved. Cyrtodactylus consobrinus from peninsular Malaysia is described herein as the new species C. peninsularis sp. nov. whose type locality is Gunung Belumut, Johor State. Cyrtodactylus peninsularis sp. nov. is diagnosable from all other species in the malayanus group by having statistically different morphospatial positions in multiple factor analyses (MFA) based on size-corrected morphometric and meristic characters. ANOVA analyses of these characters recovered significantly different mean values between C. peninsularis sp. nov. and varying combinations of all other malayanus group species across several size-corrected morphometric and meristic characters. Genetic variation within C. peninsularis sp. nov. is geographically structured across six well supported monophyletic mitochondrial lineages bearing an uncorrected pairwise sequence divergence ranging from 0.97–4.5%. Despite its well supported phylogeographic structure, PCAs and ANOVAs recovered statistically weak morphological separation among the lineages and as such, all are considered conspecific pending a genomic analysis. The phylogeographic structure within the forest-dwelling C. peninsularis sp. nov. is quite similar to that of the stream-adapted ranid frog genus Amolops and less so to that of the microhabitat specialists of the C. pulchellus group and the forest generalist C. quadrivirgatus , all of whom are sympatric across Peninsular Malaysia.

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.

Understanding the processes that underpin adaptive evolutionary shifts within major taxonomic groups has long been a research directive among many evolutionary biologists. Such phenomena are best studied in large monophyletic groups that occupy a broad range of habitats where repeated exposure to novel ecological opportunities has happened independently over time in different lineages. The gekkonid genus Cyrtodactylus is just such a lineage with approximately 300 species that range from South Asia to Melanesia and occupy a vast array of habitats. Ancestral state reconstructions using a stochastic character mapping analysis of nine different habitat preferences were employed across a phylogeny composed of 76% of the known species of Cyrtodactylus. This was done in order to ascertain which habitat preference is the ancestral condition and from that condition, the transition frequency to more derived habitat preferences. The results indicate that a general habitat preference is the ancestral condition for Cyrtodactylus and the frequency of transitioning from a general habitat preference to anything more specialized occurs approximately four times more often than the reverse. Species showing extreme morphological and/or ecological specializations generally do not give rise to species bearing other habitat preferences. The evolution of different habitat preferences is generally restricted to clades that tend to occur in specific geographic regions. The largest radiations in the genus occur in rocky habitats (granite and karst), indicating that the transition from a general habitat preference to a granite or karst‐dwelling life style may be ecologically uncomplicated. Two large, unrelated clades of karst‐associated species are centered in northern Indochina and the largest clade of granite‐associated species occurs on the Thai‐Malay Peninsula. Smaller, independent radiations of clades bearing other habitat preferences occur throughout the tree and across the broad distribution of the genus. With the exception of a general habitat preference, the data show that karst‐associated species far out‐number all others (29.6% vs. 0.4%–10.2%, respectively) and the common reference to karstic regions as “imperiled arcs of biodiversity” is not only misleading but potentially dangerous. Karstic regions are not simply refugia harboring the remnants of local biodiversity but are foci of speciation that continue to generate the most speciose, independent, radiations across the genus. Unfortunately, karstic landscapes are some of the most imperiled and least protected habitats on the planet and these data continue to underscore the urgent need for their conservation. The gekkonid genus is an ecologically labile lineage. Species with a general habitat preference have independently given rise to at least eight other distinct more specialized habitat preferences.

The exploration of unsurveyed areas in southern Thailand discovered two new karst-adapted species, Cyrtodactylus sungaiupe sp. nov. and Cyrtodactylus wangkhramensis sp. nov. , from Thung Wa and La-ngu Districts, Satun Province, respectively. These new species are members of the C. pulchellus group that occur along the Thai-Malay Peninsula. The new species can be distinguished from all other congeners by their key morphological characters and genetic divergence. Morphologically, Cyrtodactylus sungaiupe sp. nov. and Cyrtodactylus wangkhramensis sp. nov. can be diagnosed from other members by having a combination of differences in body size; degree of dorsal tuberculation; absence of tubercles on ventral surfaces; number of ventral scales, paravertebral tubercles and femoroprecloacal pores in males only; deep precloacal groove only in males; absence of a scattered pattern of white dorsal tubercles; number of dark body bands; and the extent of caudal tubercles on an original tail. Although the two species are sister taxa and have nearly identical morphologies, they are considered to be different species, based on a relatively high uncorrected pairwise genetic divergence of the mitochondrial ND2 gene (6.59–6.89%), statistically significant univariate and multivariate morphological differences (PERMANOVA and ANOVA) and diagnostic characteristics of caudal tuberculation on the original tail. Moreover, Cyrtodactylus sungaiupe sp. nov. and Cyrtodactylus wangkhramensis sp. nov. are currently restricted to their karstic type localities which may serve as a geographic barrier to dispersal and gene flow.

The taxonomy and phylogeny of the Cyrtodactylus pulchellus complex along the Thai-Malay Peninsular region has been the focus of many recent studies and has resulted in the recognition of 17 species. However, the majority of these studies were focused on Peninsular and insular Malaysia where there were specimens and genetic vouchers. The taxonomic status and phylogenetic relationships of the Thai species in this complex remain unresolved, due to the lack of genetic material of some species, especially C. phuketensis and C. macrotuberculatus from Thai populations. In this study, we investigated the phylogenetic relationship between C. phuketensis and its closely related species C. macrotuberculatus , using both morphometric and molecular data. Phylogenetic analyses of mitochondrial NADH dehydrogenase subunit 2 (ND2) gene revealed that C. phuketensis is embedded within a C. macrotuberculatus clade with 1.45–4.20% (mean 2.63%) uncorrected pairwise sequence divergences. Morphological comparisons showed nearly identical measurements of C. phuketensis and C. macrotuberculatus and overlapping ranges in meristic characters. Based on these data, C. phuketensis is considered to be a variant of C. macrotuberculatus , thus rendering C. phuketensis a junior synonym of C. macrotuberculatus .

Karstic landscapes are immense reservoirs of biodiversity and range-restricted endemism. Nowhere is this more evident than in the world’s third-largest vertebrate genus Cyrtodactylus (Gekkonidae) which contains well over 300 species. A stochastic character mapping analysis of 10 different habitat preferences across a phylogeny containing 344 described and undescribed species recovered a karst habitat preference occurring in 25.0% of the species, whereas that of the other eight specific habitat preferences occurred in only 0.2–11.0% of the species. The tenth category—general habitat preference—occurred in 38.7% of the species and was the ancestral habitat preference for Cyrtodactylus and the ultimate origin of all other habitat preferences. This study echoes the results of a previous study illustrating that karstic landscapes are generators of species diversity within Cyrtodactylus and not simply “imperiled arks of biodiversity” serving as refugia for relics. Unfortunately, the immense financial returns of mineral extraction to developing nations largely outweighs concerns for biodiversity conservation, leaving approximately 99% of karstic landscapes with no legal protection. This study continues to underscore the urgent need for their appropriate management and conservation. Additionally, this analysis supports the monophyly of the recently proposed 31 species groups and adds one additional species group.

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.