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.

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.

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.].

Sexual dimorphism and reproductive biology of the Asian bockadam snake (Cerberus schneiderii) in West Java.

Although they are among the most abundant snakes on Earth, and are heavily exploited for their skins and meat, Asian bockadams (or "dog-faced water snakes", Cerberus schneiderii) have attracted relatively little study across their wide geographic range. Based on dissection of 3,382 snakes brought to processing facilities in and around the city of Cirebon in West Java, Indonesia, we document facets of the biology of these mangrove-dwelling aquatic homalopsids. Females attain larger body sizes than do males, and are heavier-bodied (due in part to greater fat reserves) but have shorter tails relative to snout-vent length. Males showed testicular enlargement late in the year (August-November) but both reproductive and non-reproductive females were found year-round. Litters were large (3 to 45 offspring), especially in larger females. The commercial harvest falls mainly on adult snakes of both sexes, with seasonal variation in sex ratios. Life-history traits such as early maturation and frequent production of large litters render this species resilient to commercial harvesting. Future research should explore reasons for strong variation among facilities in the sex ratios of snakes, potentially identifying ways to focus the harvest on the sex (males) whose numbers are less critical for population viability.

Melanesia holds the world's most diverse and intact insular amphibian fauna.

Identifying hotspots of biological diversity is a key step in conservation prioritisation. Melanesia-centred on the vast island of New Guinea-is increasingly recognised for its exceptionally species-rich and endemic biota. Here we show that Melanesia has the world's most diverse insular amphibian fauna, with over 7% of recognised global frog species in less than 0.7% of the world's land area, and over 97% of species endemic. We further estimate that nearly 200 additional candidate species have been discovered but remain unnamed, pointing to a total fauna in excess of 700 species. Nearly 60% of the Melanesian frog fauna is in a lineage of direct-developing microhylids characterised by smaller distributions than co-occurring frog families, suggesting lineage-specific high beta diversity is a key driver of Melanesian anuran megadiversity. A comprehensive conservation status assessment further highlights geographic concentrations of recently described range-restricted threatened taxa that warrant urgent conservation actions. Nonetheless, by world standards, the Melanesian frog fauna is relatively intact, with 6% of assessed species listed as threatened and no documented extinctions; and thus it provides an unparalleled opportunity to understand and conserve a megadiverse and relatively intact insular biota.

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.

Population structure, genomic diversity and demographic history of Komodo dragons inferred from whole-genome sequencing.

Population and conservation genetics studies have greatly benefited from the development of new techniques and bioinformatic tools associated with next-generation sequencing. Analysis of extensive data sets from whole-genome sequencing of even a few individuals allows the detection of patterns of fine-scale population structure and detailed reconstruction of demographic dynamics through time. In this study, we investigated the population structure, genomic diversity and demographic history of the Komodo dragon (Varanus komodoensis), the world's largest lizard, by sequencing the whole genomes of 24 individuals from the five main Indonesian islands comprising the entire range of the species. Three main genomic groups were observed. The populations of the Island of Komodo and the northern coast of Flores, in particular, were identified as two distinct conservation units. Degrees of genomic divergence among island populations were interpreted as a result of changes in sea level affecting connectivity across islands. Demographic inference suggested that Komodo dragons probably experienced a relatively steep population decline over the last million years, reaching a relatively stable Ne during the Saalian glacial cycle (400-150 thousand years ago) followed by a rapid Ne decrease. Genomic diversity of Komodo dragons was similar to that found in endangered or already extinct reptile species. Overall, this study provides an example of how whole-genome analysis of a few individuals per population can help define population structure and intraspecific demographic dynamics. This is particularly important when applying population genomics data to conservation of rare or elusive endangered species.

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].

Exploring the vertebrate fauna of the Bird's Head Peninsula (Indonesia, West Papua) through DNA barcodes.

Biodiversity knowledge is widely heterogeneous across the Earth's biomes. Some areas, due to their remoteness and difficult access, present large taxonomic knowledge gaps. Mostly located in the tropics, these areas have frequently experienced a fast development of anthropogenic activities during the last decades and are therefore of high conservation concerns. The biodiversity hotspots of Southeast Asia exemplify the stakes faced by tropical countries. While the hotspots of Sundaland (Java, Sumatra, Borneo) and Wallacea (Sulawesi, Moluccas) have long attracted the attention of biologists and conservationists alike, extensive parts of the Sahul area, in particular the island of New Guinea, have been much less explored biologically. Here, we describe the results of a DNA-based inventory of aquatic and terrestrial vertebrate communities, which was the objective of a multidisciplinary expedition to the Bird's Head Peninsula (West Papua, Indonesia) conducted between 17 October and 20 November 2014. This expedition resulted in the assembly of 1005 vertebrate DNA barcodes. Based on the use of multiple species-delimitation methods (GMYC, PTP, RESL, ABGD), 264 molecular operational taxonomic units (MOTUs) were delineated, among which 75 were unidentified and an additional 48 were considered cryptic. This study suggests that the diversity of vertebrates of the Bird's Head is severely underestimated and considerations on the evolutionary origin and taxonomic knowledge of these biotas are discussed.

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.

High expressions of the cytoglobin and PGC-1α genes during the tissue regeneration of house gecko (Hemidactylus platyurus) tails.

BACKGROUND: The tissue regeneration process requires high oxygen and energy levels. Cytoglobin (Cygb) is a member of the globin family, which has the ability to bind oxygen, plays a role in dealing with oxidative stress, and carries oxygen into the mitochondria. Energy production for tissue regeneration is associated with mitochondria-especially mitochondrial biogenesis. The peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha protein helps to regulate mitochondrial biogenesis. House geckos (Hemidactylus platyurus) are reptiles that have the ability to regenerate the tissue in their tails. House geckos were selected as the animal models for this study in order to analyze the association of Cygb with oxygen supply and the association of PGC-1α with energy production for tissue regeneration. RESULTS: The growth of house gecko tails showed a slow growth at the wound healing phase, then followed by a fast growth after wound healing phase of the regeneration process. While Cygb mRNA expression reached its peak at the wound healing phase and slowly decreased until the end of the observation. PGC-1α mRNA was expressed and reached its peak earlier than Cygb. CONCLUSIONS: The expressions of both the Cygb and PGC-1α genes were relatively high compared to the control group. We therefore suggest that Cygb and PGC-1α play an important role during the tissue regeneration process.

Expression and role of HIF-1α and HIF-2α in tissue regeneration: a study of hypoxia in house gecko tail regeneration.

The house gecko (Hemidactylus platyurus) has evolved the ability to autotomize its tail when threatened. The lost part is then regrown via epimorphic regeneration in a process that requires high energy and oxygen levels. Oxygen demand is therefore likely to outstrip supply and this can result in relative hypoxia in the tissues of the regenerating tail. The hypoxic state is stabilized by the Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α proteins. We induced tail autotomy in 30 mal H. platyurus adults using a standard procedure and then collected samples of the regenerated tail tissue on days 1, 3, 5, 8, 10, 13, 17, 21, 25, and 30 post autotomy. For each sample, mRNA expression was analyzed by qPCR, proteins were analyzed using Western Blot tests and immunohistochemistry, and the histological structure was analyzed using Hematoxylin and Eosin staining. On day 1, HIF-1α mRNA expression increased and the tissue was dominated by leucocyte and erythrocyte cells. HIF-1α mRNA expression peaked on day 3, at which time some cells were actively proliferating, migrating, and differentiating. At the same time as HIF-1α expression decreased, HIF-2α mRNA expression increased, as did overall cellular activity. HIF-2α expression increased more gradually but was present over a longer period of time than HIF-1α. We hypothesize that HIF-1α helps to initially stimulate the tissue regeneration process while HIF-2α functionally takes over the role of HIF-1α after HIF-1α succumbs to the oxygen conditions, but we suspect that both HIF-1α and HIF-2α play a role in overcoming the tissue's hypoxic state.

Habitat preference modulates trans-oceanic dispersal in a terrestrial vertebrate.

The importance of long-distance dispersal (LDD) in shaping geographical distributions has been debated since the nineteenth century. In terrestrial vertebrates, LDD events across large water bodies are considered highly improbable, but organismal traits affecting dispersal capacity are generally not taken into account. Here, we focus on a recent lizard radiation and combine a summary-coalescent species tree based on 1225 exons with a probabilistic model that links dispersal capacity to an evolving trait, to investigate whether ecological specialization has influenced the probability of trans-oceanic dispersal. Cryptoblepharus species that occur in coastal habitats have on average dispersed 13 to 14 times more frequently than non-coastal species and coastal specialization has, therefore, led to an extraordinarily widespread distribution that includes multiple continents and distant island archipelagoes. Furthermore, their presence across the Pacific substantially predates the age of human colonization and we can explicitly reject the possibility that these patterns are solely shaped by human-mediated dispersal. Overall, by combining new analytical methods with a comprehensive phylogenomic dataset, we use a quantitative framework to show how coastal specialization can influence dispersal capacity and eventually shape geographical distributions at a macroevolutionary scale.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

Cyrtodactylus tahuna sp. nov., a new bent-toed gecko (Reptilia: Squamata: Gekkonidae) from Sangihe Island, North Sulawesi, Indonesia.

Cyrtodactylus tahuna sp. nov. is a new bent-toed gecko we describe herein based on three specimens from Sangihe, North Sulawesi, Indonesia, an island situated in the northern corner of the Wallacea biodiversity hotspot. The new species is a medium sized Cyrtodactylus with a SVL of up to 78.5 mm in adult males and 79.2 mm in females. It is easily distinguished from all but four species (Cyrtodactylus fumosus, C. halmahericus, C. papuensis, and C. tambora) occurring on Sulawesi as well as in the Moluccas and the Lesser Sunda Islands, by possessing precloacal and femoral pores, enlarged precloacal and femoral scales, and lacking transversely enlarged median subcaudal scales. It differs from C. fumosus by the presence of tubercles on the ventrolateral fold, more unkeeled dorsal tubercles (19 versus 4-7) and a pit-like precloacal depression in males (versus groove in males); from C. halmahericus by presence of smaller scales between the enlarged precloacal and femoral scales (versus a continuous series of enlarged precloacofemoral scales) and a continuous series of precloacofemoral pores, as well as the presence of a pit-like precloacal depression in male (versus groove in males), from C. papuensis by possessing a pit-like precloacal depression in males (versus a groove ); and from C. tambora by the presence of tubercles on the dorsal surface of the brachium and the possession of femoral pores. Our contribution, along with several other recent descriptions of bent-toad geckos from Southeast Asia, clearly indicating that the diversity of the genus Cyrtodactylus in Indonesia is still underestimated.

Squeezing water from a stone: high-throughput sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards.

We used Massively Parallel High-Throughput Sequencing to obtain genetic data from a 145-year old holotype specimen of the flying lizard, Draco cristatellus. Obtaining genetic data from this holotype was necessary to resolve an otherwise intractable taxonomic problem involving the status of this species relative to closely related sympatric Draco species that cannot otherwise be distinguished from one another on the basis of museum specimens. Initial analyses suggested that the DNA present in the holotype sample was so degraded as to be unusable for sequencing. However, we used a specialized extraction procedure developed for highly degraded ancient DNA samples and MiSeq shotgun sequencing to obtain just enough low-coverage mitochondrial DNA (721 base pairs) to conclusively resolve the species status of the holotype as well as a second known specimen of this species. The holotype was prepared before the advent of formalin-fixation and therefore was most likely originally fixed with ethanol and never exposed to formalin. Whereas conventional wisdom suggests that formalin-fixed samples should be the most challenging for DNA sequencing, we propose that evaporation during long-term alcohol storage and consequent water-exposure may subject older ethanol-fixed museum specimens to hydrolytic damage. If so, this may pose an even greater challenge for sequencing efforts involving historical samples.