(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.

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.

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.

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.

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.

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 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].

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 Wallaces 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.

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.

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.