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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.

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.].

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.

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.

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.

An integrative taxonomic analysis of Sundaic populations of Gekko smithii from the Thai-Malaya Peninsula, Sumatra, and Borneo recovered four deeply divergent mitochondrial lineages that are separated by major geographic barriers (mountains and seaways). Furthermore, they bear a number of concordant statistically significant differences in meristic and morphometric features, morphospatial separation in multivariate space, and discrete differences in color pattern. Gekko smithii sensu stricto is restricted to southern Thailand south of the Isthmus of Kra and Peninsular Malaysia west of the Banjaran (mountain range) Titiwangsa, being that the type locality is on Penang Island, Penang. Gekko hulk sp. nov. is a new species from extreme southern Thailand and Peninsular Malaysia east of the Banjaran Titiwangsa and five east coast islands—the type locality being Pulau (island) Tioman, Pahang. Gekko cf. albofasciolatus is tentatively used to include Bornean populations west of the Iran Mountains in Sabah and Sarawak which, in the absence of molecular data, cannot unequivocally be separated morphologically from G. albofasciolatus from the type locality at Banjarmasin, Kalimantan, Indonesia east of the Iran Mountains. In the absence of molecular data, G. albomaculatus is resurrected to include mainland Sumatran, Nias Island, and Banyak Islands populations which, based on their morphology, cannot be separated from descriptions of G. albomaculatus from the type locality of Bangka Island, 15 km off the southeast coast of mainland Sumatra. Further integrative analyses of all Sumatran and Bornean populations are currently underway as well as the enigmatic Wallacean populations from Sulawesi. Data are presented that strongly suggest all references to G. smithii from Java stem from a 151 year-old misidentification of a specimen of G. gecko of unknown provenance. Additionally, there are no vouchered records of G. smithii from Myanmar. The phylogeographic patterns of Sundaic populations of the G. smithii complex are concordant with those of a plethora of other Sundaic lineages.

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].

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 (547 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.

Abstract: The Lesser Sunda Archipelago consists of hundreds of oceanic islands located in southern Wallacea. The Sunda ratsnake, Coelognathus subradiatus, is endemic to the Lesser Sundas and is found on most of the major islands. Mitochondrial DNA was sequenced from snakes representing five of the major islands revealing that levels of sequence divergence between islands range from 2-7%. Phylogenetic analyses recover what can be interpreted as a three-lineage polytomy consisting of lineages from 1) Alor, 2) Sumbawa + Flores, and 3) Timor + Wetar. The archipelago was colonized from the Sunda Shelf ∼7 Myr with subsequent population divergence occurring ∼4.5 Myr, likely resulting in insular species formation.

Wallace's and Lydekker's Lines both describe important biogeographic barriers in the Indo-Australian Archipelago, with Wallace's Line demarcating the boundary of the Greater Sunda Shelf and Lydekker's Line indicating the edge of the Sahul continental shelf. Despite their similarities, Wallace's Line has been much more heavily studied than has Lydekker's Line, yet provides an interesting system for testing the source of fauna into eastern Wallacea. New collections of Northern Water Dragons, Tropicagama temporalis, from several islands in Maluku, eastern Indonesia now allow for an assessment of the phylogeography of the species and the ability to test if New Guinean or Australian populations served as the source for over-water dispersal across Lydekker's Line into Maluku. We collected specimens from remote islands in eastern Indonesia, sequenced the mitochondrial ND2 gene, and aligned the data to previously sequenced specimens on GenBank. We conducted several phylogenetic and divergence time analyses to investigate the source population and timing of dispersal. We found low genetic diversity among the islands in Maluku, and these samples showed little genetic divergence from New Guinea samples. The New Guinea and Maluku populations diverged less than 1 million years ago (Ma) and together diverged from the Australian population between 2.3 and 4.7 Ma. These results, along with patterns in other taxa, illustrated that, despite Australia's close geographic proximity to many of the islands in southeastern Indonesia, New Guinea has been the more frequent source of Wallacean fauna from Sahul.

The Lesser Sundas Archipelago is comprised of two parallel chains of islands that extend between the Asian continental shelf (Sundaland) and Australo-Papuan continental shelf (Sahul). These islands have served as stepping stones for taxa dispersing between the Asian and Australo-Papuan biogeographical realms. While the oceanic barriers have prevented many species from colonizing the archipelago, a number of terrestrial vertebrate species have colonized the islands either by rafting/swimming or by human introduction. Here, we examine phylogeographic structure within the Lesser Sundas for three snake, two lizard and two frog species that each has a Sunda Shelf origin. These species are suspected to have recently colonized the archipelago, though all have inhabited the Lesser Sundas for over 100 years. We sequenced mtDNA from 231 samples to test whether there is sufficiently deep genetic structure within any of these taxa to reject human-mediated introduction. Additionally, we tested for genetic signatures of population expansion consistent with recent introduction and estimated the ages of Lesser Sundas clades, if any exist. Our results show little to no genetic structure between populations on different islands in five species and moderate structure in two species. Nucleotide diversity is low for all species, and the ages of the most recent common ancestor for species with monophyletic Lesser Sundas lineages date to the Holocene or late Pleistocene. These results support the hypothesis that these species entered the archipelago relatively recently and either naturally colonized or were introduced by humans to most of the larger islands in the archipelago within a short time span.

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.

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 (

Aim: The Lesser Sunda Islands are situated between the Sunda and Sahul Shelves, with a linear arrangement that has functioned as a two-way filter for taxa dispersing between the Asian and Australo-Papuan biogeographical realms. Distributional patterns of many terrestrial vertebrates suggest a stepping-stone model of island colonization. Here we investigate the timing and sequence of island colonization in Asian-origin fanged frogs from the volcanic Sunda Arc islands with the goal of testing the stepping-stone model of island colonization. Location: The Indonesian islands of Java, Lombok, Sumbawa, Flores and Lembata. Taxon: Limnonectes dammermani and L. kadarsani (Family: Dicroglossidae). Methods: Mitochondrial DNA was sequenced from 153 frogs to identify major lineages and to select samples for an exon-capture experiment. We designed probes to capture sequence data from 974 exonic loci (1,235,981 bp) from 48 frogs including the outgroup species, L. microdiscus. The resulting data were analysed using phylogenetic, population genetic and biogeographical model testing methods. Results: The mtDNA phylogeny finds L. kadarsani paraphyletic with respect to L. dammermani, with a pectinate topology consistent with the stepping-stone model. Phylogenomic analyses of 974 exons recovered the two species as monophyletic sister taxa that diverged ~7.6 Ma with no detectable contemporary gene flow, suggesting introgression of the L. dammermani mitochondrion into L. kadarsani on Lombok resulting from an isolated ancient hybridization event ~4 Ma. Within L. kadarsani, the Lombok lineage diverged first while the Sumbawa and Lembata lineages are nested within a Flores assemblage composed of two parapatrically distributed lineages meeting in central Flores. Biogeographical model comparison found strict stepping-stone dispersal to be less likely than models involving leap-frog dispersal events. Main conclusions: These results suggest that the currently accepted stepping-stone model of island colonization might not best explain the current patterns of diversity in the archipelago. The high degree of genetic structure, large divergence times, and absent or low levels of migration between lineages suggests that L. kadarsani represents five distinct species.

Biogeographical reconstructions of the Indo-Australian Archipelago (IAA) have suggested a recent spread across the Sunda and Sahul shelves of lineages with diverse origins, which appears to be congruent with a geological history of recent tectonic uplift in the region. However, this scenario is challenged by new geological evidence suggesting that the Sunda shelf was never submerged prior to the Pliocene, casting doubt on the interpretation of recent uplift and the correspondence of evidence from biogeography and geology. A mismatch between geological and biogeographical data may occur if analyses ignore the dynamics of extinct lineages, because this may add uncertainty to the timing and origin of clades in biogeographical reconstructions. We revisit the historical biogeography of multiple IAA taxa and explicitly allow for the possibility of lineage extinction. In contrast to models assuming zero extinction, we find that all of these clades, including plants, invertebrates and vertebrates, have a common and widespread geographic origin, and each has spread and colonized the region much earlier than previously thought. The results for the eight clades re-examined in this article suggest that they diversified and spread during the early Eocene, which helps to unify the geological and biological histories of IAA. [ABSTRACT FROM AUTHOR]

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.

(a) A map of Wallacea showing islands invaded by Duttaphrynus melanostictus in red, islands inhabited by Varanus komodoensis in blue, and localities of genetic samples in yellow points. (b) A D. melanostictus from Lombok Island. (c) Environmental niche model for the Sunda Islands clade of D. melanostictus projected into Wallacea. Green color indicates very high suitability, yellow color indicates high suitability, and orange color indicates moderate suitability.

Lepidodactylus pantai is a new species of gecko from the Kei Islands, Maluku, Indonesia that is closely associated with intertidal habitats. This species does not fit cleanly into any of the three species groups described for the genus because it possesses the unique combination of both divided terminal scansors on all toes and a nearly completely cylindrical tail without fringes or evidence of dorsoventral compression. A phylogenetic analysis including this species demonstrates that it is the sister taxon of a population from Palau, and that this clade is sister to the clade containing Group III species for which we have molecular data.

The Asian common toad (Duttaphrynus melanostictus) is a human commensal species that occupies a wide variety of habitats across tropical Southeast Asia. We test the hypothesis that genetic variation in D. melanostictus is weakly associated with geography owing to natural and human-mediated dispersal facilitated by its commensal nature. Phylogenetic and population genetic analyses of mitochondrial and nuclear DNA sequence variation, and predictive species distribution modelling, unexpectedly recovered three distinct evolutionary lineages that differ genetically and ecologically, corresponding to the Asian mainland, coastal Myanmar and the Sundaic islands. The persistence of these three divergent lineages, despite ample opportunities for recent human-mediated and geological dispersal, suggests that D. melanostictus actually consists of multiple species, each having narrower geographical ranges and ecological niches, and higher conservation value, than is currently recognized. These findings also have implications for the invasion potential of this human commensal elsewhere, such as in its recently introduced ranges on the islands of Borneo, Sulawesi, Seram and Madagascar.

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. [ABSTRACT FROM AUTHOR]