Integrative methods reveal multiple drivers of diversification in rice paddy snakes.

Divergence dating analyses in systematics provide a framework to develop and test biogeographic hypotheses regarding speciation. However, as molecular datasets grow from multilocus to genomic, sample sizes decrease due to computational burdens, and the testing of fine-scale biogeographic hypotheses becomes difficult. In this study, we use coalescent demographic models to investigate the diversification of poorly known rice paddy snakes from Southeast Asia (Homalopsidae: Hypsiscopus), which have conflicting dates of origin based on previous studies. We use coalescent modeling to test the hypothesis that Hypsiscopus diversified 2.5 mya during the Khorat Plateau uplift in Thailand. Additionally, we use ecological niche analyses to identify potential differences in the niche space of the two most widely distributed species in the past and present. Our results suggest Hypsiscopus diversified ~ 2.4 mya, supporting that the Khorat Plateau may have initiated the diversification of rice paddy snakes. We also find significant niche differentiation and shifts between species of Hypsiscopus, indicating that environmental differences may have sustained differentiation of this genus after the Khorat Plateau uplift. Our study expands on the diversification history of snakes in Southeast Asia, and highlights how results from smaller multilocus datasets can be useful in developing and testing biogeographic hypotheses alongside genomic datasets.

The natural and human-mediated expansion of a human-commensal lizard into the fringes of Southeast Asia.

BACKGROUND: Human-commensal species often display deep ancestral genetic structure within their native range and founder-effects and/or evidence of multiple introductions and admixture in newly established areas. We investigated the phylogeography of Eutropis multifasciata, an abundant human-commensal scincid lizard that occurs across Southeast Asia, to determine the extent of its native range and to assess the sources and signatures of human introduction outside of the native range. We sequenced over 350 samples of E. multifasciata for the mitochondrial ND2 gene and reanalyzed a previous RADseq population genetic dataset in a phylogenetic framework. RESULTS: Nuclear and mitochondrial trees are concordant and show that E. multifasciata has retained high levels of genetic structure across Southeast Asia despite being frequently moved by humans. Lineage boundaries in the native range roughly correspond to several major biogeographic barriers, including Wallace's Line and the Isthmus of Kra. Islands at the outer fringe of the range show evidence of founder-effects and multiple introductions. CONCLUSIONS: Most of enormous range of E. multifasciata across Southeast Asia is native and it only displays signs of human-introduction or recent expansion along the eastern and northern fringe of its range. There were at least three events of human-introductions to Taiwan and offshore islands, and several oceanic islands in eastern Indonesia show a similar pattern. In Myanmar and Hainan, there is a founder-effect consistent with post-warming expansion after the last glacial maxima or human introduction.

A new species of terrestrially-nesting fanged frog (Anura: Dicroglossidae) from Sulawesi Island, Indonesia.

Herein, we describe a new species of terrestrially-nesting fanged frog from Sulawesi Island, Indonesia. Though male nest attendance and terrestrial egg deposition is known in one other Sulawesi fanged frog (Limnonectes arathooni), the new species exhibits a derived reproductive mode unique to the Sulawesi assemblage; male frogs guard one or more clutches of eggs festooned to leaves or mossy boulders one to two meters above small slow-moving streams, trickles, or seeps. This island endemic has thus far been collected at three sites on Sulawesi: one in the Central Core of the island, and two on the Southwest Peninsula-south of the Tempe Depression (a major biogeographical boundary). The new Limnonectes has the smallest adult body size among its Sulawesi congeners-with a maximum snout-vent length of about 30 millimeters. Beyond its unique reproductive behavior and body size, the species is further diagnosed on the basis of advertisement call and genetic distance from sympatric fanged frogs. The discovery and description of the new species highlights the remarkable reproductive trait diversity that characterizes the Sulawesi fanged frog assemblage despite that most species in this radiation have yet to be formally described.

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.

Their fates intertwined: diversification patterns of the Asian gliding vertebrates may have been forged by dipterocarp trees.

The repeated evolution of gliding in diverse Asian vertebrate lineages is hypothesized to have been triggered by the dominance of tall dipterocarp trees in the tropical forests of Southeast Asia. These dipterocarp forests have acted as both centres of diversification and climatic refugia for gliding vertebrates, and support most of their extant diversity. We predict similarities in the diversification patterns of dipterocarp trees and gliding vertebrates, and specifically test whether episodic diversification events such as rate shifts and/or mass extinctions were temporally congruent in these groups. We analysed diversification patterns in reconstructed timetrees of Asian dipterocarps, the most speciose gliding vertebrates from different classes (Draco lizards, gliding frogs and Pteromyini squirrels) and compared them with similar-sized clades of non-gliding relatives (Diploderma lizards, Philautus frogs and Callosciurinae squirrels) from Southeast Asia. We found significant declines in net-diversification rates of dipterocarps and the gliding vertebrates during the Pliocene-Pleistocene, but not in the non-gliding groups. We conclude that the homogeneity and temporal coincidence of these rate declines point to a viable ecological correlation between dipterocarps and the gliding vertebrates. Further, we suggest that while the diversification decay in dipterocarps was precipitated by post-Miocene aridification of Asia, the crises in the gliding vertebrates were induced by both events concomitantly.

Elevational surveys of Sulawesi herpetofauna 1: Gunung Galang, Gunung Dako Nature Reserve.

The Indonesian island of Sulawesi has a unique geology and geography, which have produced an astoundingly diverse and endemic flora and fauna and a fascinating biogeographic history. Much biodiversity research has focused on the regional endemism in the island's Central Core and on its four peninsulas, but the biodiversity of the island's many upland regions is still poorly understood for most taxa, including amphibians and reptiles. Here, we report the first of several planned full-mountain checklists from a series of herpetological surveys of Sulawesi's mountains conducted by our team. In more than 3 weeks of work on Gunung Galang, a 2,254 m peak west of the city of Tolitoli, Sulawesi Tengah Province, on Sulawesi's Northern Peninsula, we recovered nearly fifty species of reptiles and amphibians, more than a dozen of which are either new to science or known but undescribed. The incompleteness of our sampling suggests that many more species remain to be discovered on and around this mountain.

Bewildering biogeography: Waves of dispersal and diversification across southern Wallacea by bent-toed geckos (genus: Cyrtodactylus).

Bent-toed Geckos, genus Cyrtodactylus, are one of the most diverse terrestrial vertebrate groups, and their range extends from South Asia into Australo-Papua and adjacent Pacific islands. Given the generally high faunal endemism on Wallacean islands, it is rather paradoxical that the diversity in these geckos appears to be so low (21 species in Wallacea, 15 in the Philippines) compared with continental shelf assemblages (>300 species on Sunda + Sahul Shelves + adjacent islands). To determine whether this shortfall was real or an artifact of historical undersampling, we analyzed mitochondrial DNA sequences of hundreds of southern Wallacean samples (Lesser Sundas + southern Maluku). After screening to guide sample selection for target capture data collection, we obtained a 1150-locus genomic dataset (1,476,505 bp) for 119 samples of southern Wallacean and closely related lineages. The results suggest that species diversity of Cyrtodactylus in southern Wallacea is vastly underestimated, with phylogenomic and clustering analyses suggesting as many as 25 candidate species, in contrast to the 8 currently described. Gene exchange between adjacent candidate species is absent or minimal across the archipelago with only one case of > 0.5 migrants per generation. Biogeographical analysis suggests that the hitherto unrecognized diversity is the result of at least three independent dispersals from Sulawesi or its offshore islands into southern Wallacea between 6 and 14 Ma, with one invasion producing small-bodied geckos and the other two or three producing larger-bodied geckos. The smaller-bodied laevigatus group appears to be able to coexist with members of either larger-bodied clade, but we have yet to find members of the two larger-bodied clades occurring in sympatry, suggesting that ecological partitioning or competitive exclusion may be shaping individual island assemblages.

Hummingbird ingestion of low-concentration ethanol within artificial nectar.

Both frugivores and nectarivores are potentially exposed to dietary ethanol produced by fermentative yeasts which metabolize sugars. Some nectarivorous mammals exhibit a preference for low-concentration ethanol solutions compared to controls of comparable caloric content, but behavioural responses to ethanol by nectar-feeding birds are unknown. We investigated dietary preference by Anna's Hummingbirds (Calypte anna) for ethanol-enhanced sucrose solutions. Via repeated binary-choice experiments, three adult male hummingbirds were exposed to sucrose solutions containing 0%, 1% or 2% ethanol; rates of volitional nectar consumption were measured over a 3 h interval. Hummingbirds did not discriminate between 0% and 1% ethanol solutions, but exhibited significantly reduced rates of consumption of a 2% ethanol solution. Opportunistic measurements of ethanol concentrations within hummingbird feeders registered values peaking at about 0.05%. Ethanol at low concentrations (i.e. up to 1%) is not aversive to Anna's Hummingbirds and may be characteristic of both natural and anthropogenic nectars upon which they feed. Given high daily amounts of nectar consumption by hummingbirds, chronic physiological exposure to ethanol can thus be substantial, although naturally occurring concentrations within floral nectar are unknown.

Reference genome of an iconic lizard in western North America, Blainville's horned lizard Phrynosoma blainvillii.

Genome assemblies are increasingly being used to identify adaptive genetic variation that can help prioritize the population management of protected species. This approach may be particularly relevant to species like Blainville's horned lizard, Phrynosoma blainvillii, due to its specialized diet on noxious harvester ants, numerous adaptative traits for avoiding predation (e.g. cranial horns, dorsoventrally compressed body, cryptic coloration, and blood squirting from the orbital sinuses), and status as Species of Special Concern in California. Rangewide decline since the early 20th century, the basis of its conservation status, has been driven mainly by habitat conversion, over-collecting, and invasion of a non-native ant that displaces its native ant prey base. Here, we report on a scaffold-level genome assembly for P. blainvillii as part of the California Conservation Genomics Project (CCGP), produced using Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology. The de novo assembly has 78 scaffolds, a total length of ~2.21 Gb, a scaffold N50 length of ~352 Mb, and BUSCO score of 97.4%. This is the second species of Phrynosoma for which a reference genome has been assembled and represents a considerable improvement in terms of contiguity and completeness. Combined with the landscape genomics data being compiled by the CCGP, this assembly will help strategize efforts to maintain and/or restore local genetic diversity, where interventions like genetic rescue, translocation, and strategic land preservation may be the only means by which P. blainvillii and other low-vagility species can survive in the fragmented habitats of California.

Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes.

BACKGROUND: Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods. RESULTS: With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80-90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes. CONCLUSIONS: This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Hummingbird blood traits track oxygen availability across space and time.

Predictable trait variation across environments suggests shared adaptive responses via repeated genetic evolution, phenotypic plasticity or both. Matching of trait-environment associations at phylogenetic and individual scales implies consistency between these processes. Alternatively, mismatch implies that evolutionary divergence has changed the rules of trait-environment covariation. Here we tested whether species adaptation alters elevational variation in blood traits. We measured blood for 1217 Andean hummingbirds of 77 species across a 4600-m elevational gradient. Unexpectedly, elevational variation in haemoglobin concentration ([Hb]) was scale independent, suggesting that physics of gas exchange, rather than species differences, determines responses to changing oxygen pressure. However, mechanisms of [Hb] adjustment did show signals of species adaptation: Species at either low or high elevations adjusted cell size, whereas species at mid-elevations adjusted cell number. This elevational variation in red blood cell number versus size suggests that genetic adaptation to high altitude has changed how these traits respond to shifts in oxygen availability.

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.].

Geography and elevation as drivers of cloacal microbiome assemblages of a passerine bird distributed across Sulawesi, Indonesia.

BACKGROUND: Empirical field studies allow us to view how ecological and environmental processes shape the biodiversity of our planet, but collecting samples in situ creates inherent challenges. The majority of empirical vertebrate gut microbiome research compares multiple host species against abiotic and biotic factors, increasing the potential for confounding environmental variables. To minimize these confounding factors, we focus on a single species of passerine bird found throughout the geologically complex island of Sulawesi, Indonesia. We assessed the effects of two environmental factors, geographic Areas of Endemism (AOEs) and elevation, as well as host sex on the gut microbiota assemblages of the Sulawesi Babbler, Pellorneum celebense, from three different mountains across the island. Using cloacal swabs, high-throughput-amplicon sequencing, and multiple statistical models, we identified the core microbiome and determined the signal of these three factors on microbial composition. RESULTS: The five most prevalent bacterial phyla within the gut microbiome of P. celebense were Proteobacteria (32.6%), Actinobacteria (25.2%), Firmicutes (22.1%), Bacteroidetes (8.7%), and Plantomycetes (2.6%). These results are similar to those identified in prior studies of passeriform microbiomes. Overall, microbiota diversity decreased as elevation increased, irrespective of sex or AOE. A single ASV of Clostridium was enriched in higher elevation samples, while lower elevation samples were enriched with the genera Perlucidibaca (Family Moraxellaceae), Lachnoclostridium (Family Lachnospiraceae), and an unidentified species in the Family Pseudonocardiaceae. CONCLUSIONS: While the core microbiota families recovered here are consistent with other passerine studies, the decreases in diversity as elevation increases has only been seen in non-avian hosts. Additionally, the increased abundance of Clostridium at high elevations suggests a potential microbial response to lower oxygen levels. This study emphasizes the importance of incorporating multiple statistical models and abiotic factors such as elevation in empirical microbiome research, and is the first to describe an avian gut microbiome from the island of Sulawesi.

Phylogenetic relationships of southern Wallacean ranid frogs (Anura: Ranidae: Hylarana).

Frogs in the family Ranidae are diverse in Asia and are thought to have dispersed to the Sahul Shelf approximately 10 million years ago, where they radiated into more than a dozen species. Ranid species in the intervening oceanic islands of Wallacea, such as Hylarana florensis and H. elberti from the Lesser Sundas and H. moluccana from eastern Wallacea, are assumed to belong to the subgenus Papurana, yet this has not been confirmed with molecular data. We analyzed mitochondrial DNA of Hylarana species from five islands spanning the reported ranges of H. florensis and H. elberti and compared them to confirmed Papurana species and closely related subgenera within Hylarana. We find that the Lesser Sunda H. florensis and H. elberti form a clade that is sister to the rest of the Australo-Papuan Papurana assemblage. Species delimitation analyses and divergence time estimates suggest that populations of H. florensis on Lombok may be distinct from those on Flores at the species level. Likewise, populations of H. elberti on Sumba and Timor may be distinct from each other and from those on Wetar, tshe type locality of H. elberti. Samples from Babar Island thought to be members of H. elberti in fact belong to the wide-ranging H. daemeli, which occurs in northern Australia, across New Guinea, and on the neighboring island of Tanimbar. These results suggest that the Lesser Sundas may have served as a stepping-stone for colonization of the Sahul Shelf and that species diversity of Papurana frogs is underestimated in the Lesser Sundas.

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.

Diverge and Conquer: Phylogenomics of southern Wallacean forest skinks (Genus: Sphenomorphus) and their colonization of the Lesser Sunda Archipelago.

The archipelagos of Wallacea extend between the Sunda and Sahul Shelves, serving as a semipermeable two-way filter influencing faunal exchange between Asia and Australo-Papua. Forest skinks (Genus Sphenomorphus) are widespread throughout southern Wallacea and exhibit complex clinal, ontogenetic, sexual, and seasonal morphological variation, rendering species delimitation difficult. We screened a mitochondrial marker for 245 Sphenomorphus specimens from this area to inform the selection of 104 samples from which we used targeted sequence capture to generate a dataset of 1154 nuclear genes (∼1.8 Mb) plus complete mitochondrial genomes. Phylogenomic analyses recovered many deeply divergent lineages, three pairs of which are now sympatric, that began to diversify in the late Miocene shortly after the oldest islands are thought to have become emergent. We infer a complex and nonstepping-stone pattern of island colonization, with the group having originated in the Sunda Arc islands before using Sumba as a springboard for colonization of the Banda Arcs. Estimates of population structure and gene flow across the region suggest total isolation except between two Pleistocene Aggregate Island Complexes that become episodically land-bridged during glacial maxima. These historical processes have resulted in at least 11 Sphenomorphus species in the region, nine of which require formal description. This fine-scale geographic partitioning of undescribed species highlights the importance of utilizing comprehensive genomic studies for defining biodiversity hotspots to be considered for conservation protection.

Whos your daddy? On the identity and distribution of the paternal hybrid ancestor of the parthenogenetic gecko Lepidodactylus lugubris (Reptilia: Squamata: Gekkonidae).

The widespread parthenogenetic gecko Lepidodactylus lugubris is comprised of several clonal lineages, at least one of which has been known for some time to have originated from hybridization between its maternal ancestor, Lepidodactylus moestus, and a putatively undescribed paternal ancestor previously known only from remote islands in the Central Pacific. By integrating new genetic sequences from multiple studies on Lepidodactylus and incorporating new genetic sequences from previously sampled populations, we recovered a phylogenetic tree that shows a close genetic similarity between the generally hypothesized paternal hybrid ancestor and a recently described species from Maluku (Indonesia), Lepidodactylus pantai. Our results suggest that the paternal hybrid ancestor of at least one parthenogenetic clone of L. lugubris is conspecific with L. pantai and that the range of this species extends to Palau, the Caroline Islands, the Kei Islands, Wagabu, and potentially other small islands near New Guinea. Deeper genetic structure in the western (Palau, Maluku) versus eastern (eastern Melanesia, Micronesia, Polynesia) part of this species range suggests that the western populations likely dispersed via natural colonization, whereas the eastern populations may be the result of human-mediated dispersal. The potential taxonomic affinities and biogeographic history should be confirmed with further morphological and genetic analyses, including research on L. woodfordi from its type locality, which would have nomenclatural priority if found to be conspecific with L. pantai. We recommend referring to the wide-ranging sexual species as Lepidodactylus pantai until such a comparison can be made.

Phylogenomic Analysis Reveals Dispersal-Driven Speciation and Divergence with Gene Flow in Lesser Sunda Flying Lizards (Genus Draco).

The Lesser Sunda Archipelago offers exceptional potential as a model system for studying the dynamics of dispersal-driven diversification. The geographic proximity of the islands suggests the possibility for successful dispersal, but this is countered by the permanence of the marine barriers and extreme intervening currents that are expected to hinder gene flow. Phylogenetic and species delimitation analyses of flying lizards (genus Draco) using single mitochondrial genes, complete mitochondrial genomes, and exome-capture data sets identified 9-11 deeply divergent lineages including single-island endemics, lineages that span multiple islands, and parapatrically distributed nonsister lineages on the larger islands. Population clustering and PCA confirmed these genetic boundaries with isolation-by-distance playing a role in some islands or island sets. While gdi estimates place most candidate species comparisons in the ambiguous zone, migration estimates suggest 9 or 10 species exist with nuclear introgression detected across some intra-island contact zones. Initial entry of Draco into the archipelago occurred at 5.5-7.5 Ma, with most inter-island colonization events having occurred between 1-3 Ma. Biogeographical model testing favors scenarios integrating geographic distance and historical island connectivity, including an initial stepping-stone dispersal process from the Greater Sunda Shelf through the Sunda Arc as far eastward as Lembata Island. However, rather than reaching the adjacent island of Pantar by dispersing over the 15-km wide Alor Strait, Draco ultimately reached Pantar (and much of the rest of the archipelago) by way of a circuitous route involving at least five overwater dispersal events. These findings suggest that historical geological and oceanographic conditions heavily influenced dispersal pathways and gene flow, which in turn drove species formation and shaped species boundaries. [Biogeography; genomics, Indonesia; lizards; phylogeography; reptiles].

Molecular phylogeny of Asian pipesnakes, genus Cylindrophis Wagler, 1828 (Squamata: Cylindrophiidae), with the description of a new species from Myanmar.

Cylindrophis is a genus of secretive, semi-fossorial, non-venomous snakes comprising 14 species, characterized by a generally cylindrical body, uniform scales (with barely enlarged ventrals), and vestiges of pelvic and limb bones, the latter terminating in a claw lateral to the vent. We reconstructed a concatenated molecular phylogeny of seven taxa of Cylindrophis taxa based on one nuclear (R35) and two mitochondrial (16S, ND2) genes. Analyses recovered the Sri Lankan endemic C. maculatus as sister to all other sampled Cylindrophis. The mainland Southeast Asian species C. burmanus and C. jodiae form successive sister lineages to a monophyletic Wallacean island group containing C. boulengeri, C. isolepis, and C. yamdena. We also describe a new species of Cylindrophis, morphologically similar to C. burmanus, from Kachin State in northern Myanmar. Cylindrophis slowinskii sp. nov. is distinguished from all congeners by the following combination of characters: 19 dorsal scale rows at midbody, 216-220 ventrals, eight subcaudals, a dark venter with > 60 very narrow diffuse pale blotches, and a pale bar running along the posterior border of the prefrontals. In our phylogeny, the new species is strongly supported as the sister species of C. burmanus. It is the 15th currently recognised species in the genus, and the fourth from mainland Southeast Asia.

Rivers of Indochina as potential drivers of lineage diversification in the spotted flying lizard (Draco maculatus) species complex.

Southeast Asia hosts a rich concentration of biodiversity within multiple biodiversity hotspots. Indochina, a region with remarkably high levels of in situ diversification, possesses five major rivers (Ayeyarwady, Chiang Mai, Mekong, Red, and Salween), several of which coincide with phylogenetic breaks of terrestrial taxa. Draco maculatus possesses a range that stretches across Indochina, which widespread geographic distribution along with potential discrete variation within subspecies alludes to the possibility of this taxon constituting multiple divergent lineages. Using sequence data from three mitochondrial (12S, 16S, and ND2) and three nuclear (BDNF, CMOS, and PNN) genes, we provide the first estimated phylogeny of this hypothesized species complex and examine its phylogeographic architecture with maximum likelihood and Bayes factor delimitation (BFD) approaches. Our results support multiple divergent lineages with phylogenetic breaks coincident with rivers, indicating that river barriers may be contributing to the elevated levels of in situ diversification of Indochina.