Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona).

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale.

Molecular phylogenetics of sub-Saharan African natricine snakes, and the biogeographic origins of the Seychelles endemic Lycognathophis seychellensis.

Phylogenetic relationships of sub-Saharan African natricine snakes are understudied and poorly understood, which in turn has precluded analyses of the historical biogeography of the Seychelles endemic Lycognathophis seychellensis. We inferred the phylogenetic relationships of Seychelles and mainland sub-Saharan natricines by analysing a multilocus DNA sequence dataset for three mitochondrial (mt) and four nuclear (nu) genes. The mainland sub-Saharan natricines and L. seychellensis comprise a well-supported clade. Two maximally supported sets of relationships within this clade are (Limnophis,Natriciteres) and (Afronatrix,(Hydraethiops,Helophis)). The relationships of L. seychellensis with respect to these two lineages are not clearly resolved by analysing concatenated mt and nu data. Analysed separately, nu data best support a sister relationship of L. seychellensis with (Afronatrix,(Hydraethiops,Helophis)) and mt data best support a sister relationship with all mainland sub-Saharan natricines. Methods designed to cope with incomplete lineage sorting strongly favour the former hypothesis. Genetic variation among up to 33 L. seychellensis from five Seychelles islands is low. Fossil calibrated divergence time estimates support an overseas dispersal of the L. seychellensis lineage to the Seychelles from mainland Africa ca. 43-25 million years before present (Ma), rather than this taxon being a Gondwanan relic.

A new Microhyla species (Anura: Microhylidae) from riparian evergreen forest in the eastern Himalayan state of Arunachal Pradesh, India.

A new frog species of the genus Microhyla (Anura, Microhylidae) is described from riparian mid-elevation (860 m asl) evergreen forest in Namdapha National Park, located in the eastern Himalayan state of Arunachal Pradesh, India. The new species can be morphologically distinguished from other congeners by a suite of characters such as adult size, dorsal and lateral colouration and markings, snout shape, foot webbing, and digit tip morphology. Phylogenetically, the new species is more closely related to some of the smallest known members of the genus. It forms a deeply divergent sister lineage to the clade containing members of the Microhyla zeylanica species group that are restricted to Peninsular India and Sri Lanka, and shows sequential relationship with Southeast Asian species M. superciliaris, followed by clade containing M. aurantiventris + M. butleri. The discovery indicates that novel taxa representing distinct evolutionary lineages still remain to be formally described in the genus Microhyla, especially from less explored regions such as the eastern Himalayan forests in Northeast India.

Cryptic diversity within the Megophrys major species group (Amphibia: Megophryidae) of the Asian Horned Frogs: Phylogenetic perspectives and a taxonomic revision of South Asian taxa, with descriptions of four new species.

The Megophrys major species group (MMSG) is composed of typically medium to large sized frogs. Within the genus, it is the most geographically widespread clade ranging from the western Himalayas to southern Indochina. In this study, we examined in detail the extent of cryptic diversity within the MMSG-Indian populations based on molecular data (up to ten genes) using multigene concatenation and coalescent-based phylogenetic techniques, species delimitation analyses and extensive morphological data.Molecular evidence suggests a high level of hidden cryptic diversity within the MMSG, particularly within the M. major species complex (MMC), highlighting overlapping distributions, a case of potential mitochondrial transfer between two species, and tree topology discordance between phylogenetic methods and mitochondrial and nuclear data sets. Most analyses indicated distinct eastern and western clades in the MMC, and that the western clade may further divide into a northern and a southern subclade.A detailed taxonomic review of Indian members of the Megophrys major species group is provided. Previously undocumented complex nomenclatural issues involving known species are highlighted and resolved. Megophrys monticola is taxonomically redefined for the first time as a valid species since its synonymy with M. parva in 1893. The taxonomic status of two recently described species, Xenophrys katabhako and X. sanu are discussed in light of increased molecular and morphological sampling, and are synonymised with M. monticola. Megophrys monticola and M. robusta are redescribed based on their original type specimens and recently collected material. Megophrys major is neotypified and M. robusta lectotypified to remove prevailing nomenclatural instability. Four new large sized Indian MMC species are formally described from the Northeast Indian states of Arunachal Pradesh, Meghalaya and Nagaland, and Myanmar. All South Asian MMSG species are morphologically diagnosed from known congeners in the group. The geographic distributions of all taxa discussed are significantly redefined based on the revised taxonomy and extensive literature review. Morphological and molecular evidence suggests that Megophrys major sensu stricto might be endemic to Northeast India; M. glandulosa is formally removed from the Indian and Bhutan species checklists. Numerous misidentifications in literature are highlighted and corrected. In order to reduce future misidentifications of species reported from surrounding regions, high definition images of the holotypes of three Chinese species, M. glandulosa, M. medogensis and M. zhangi are provided for the first time, and a detailed description of Myanmar specimens of M. glandulosa is also given. This study provides the principle foundation for further research into the taxonomic status of the remaining, currently undescribed MMC taxa from Southeast Asia.

Delineating Microhyla ornata (Anura, Microhylidae): mitochondrial DNA barcodes resolve century-old taxonomic misidentification.

Microhyla ornata, a species originally described from the southwest coast of India in 1841, was long reported to be wide-ranging throughout South, Southeast, and East Asia. Although the name M. ornata is restricted to populations from South Asia, the species is still considered to occur widely in India and its neighboring regions. To clarify the identity and geographical distribution of M. 'ornata', we performed DNA barcoding using a fragment of the mitochondrial 16S rRNA gene from 62 newly obtained samples. Our results show that this taxon is restricted to Peninsular India and Sri Lanka, whereas, populations from the other parts represent three different species - M. mukhlesuri, M. mymensinghensis, and M. nilphamariensis, creating new country records for India. Our work reemphasizes the benefits of DNA barcoding for rapidly identifying populations of widespread species and provides insights into the patterns of genetic differentiation in the M. 'ornata' species complex of South Asia.

On the taxonomic status of Ichthyophis husaini Pillai & Ravichandran, 1999 (Amphibia: Gymnophiona: Ichthyophiidae).

Extensive surveys at and around the type locality of Ichthyophis husaini Pillai & Ravichandran, 1999 (an 'unstriped' Ichthyophis) in North East India between 2008 and 2014 yielded tens of striped but no unstriped specimens of Ichthyophis. Contrary to the original description, we observed a lateral stripe on the holotype of I. husaini. The holotype of I. husaini is very similar to the holotype of the striped species I. garoensis Pillai & Ravichandran, 1999, which was collected from a locality only 20 km away from the type locality of I. husaini. The holotypes of these two species are also very similar to 12 newly collected specimens of striped Ichthyophis from this area. We conclude that the holotypes of I. husaini and I. garoensis represent but a single species, and we designate Ichthyophis husaini Pillai & Ravichandran, 1999 as a junior subjective synonym of Ichthyophis garoensis Pillai & Ravichandran, 1999. This taxonomic action and newly reported material extends the known geographic range of I. garoensis. It also reduces the number of Ichthyophis species of North East India to eight, and restricts unstriped members of the genus within this region to a single species (I. sikkimensis Taylor, 1960) that is known only from the far west of the region.

Frankixalus, a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles.

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic 'Polypedates' jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.

Taxonomic review of the tree frog genus Rhacophorus from the Western Ghats, India (Anura: Rhacophoridae), with description of ontogenetic colour changes and reproductive behavior.

A taxonomic revision of the Western Ghats species from the genus Rhacophorus is presented. Based on museum studies and new collections from localities spanning the known range of Western Ghats Rhacophorus, we review the four known species of this genus, their type specimens, current taxonomic status and their geographic distribution on the basis of morphological and molecular data. The holotypes of Rhacophorus calcadensis, R. lateralis and R. nialabaricus are redescribed. The previously unidentified holotype of Rhacophorus inalabaricus is herein fixed. Descriptions of ontogenetic colour change (OCC) in the Western Ghats Rhacophorus are provided and we conjecture the taxonomic utility of OCC. Additionally we provide observations on nesting behaviour of each species, and report multiple male participation during amplexus, oviposition and foam nest construction in R. lateralis and R. malabaricus.

Systematics of the caecilian family Chikilidae (Amphibia: Gymnophiona) with the description of three new species of Chikila from northeast India.

A taxonomic review of the monogeneric northeast Indian caecilian family Chikilidae is presented based on 64 specimens. Chikila fuilleri (Alcock, 1904), known previously only from a single specimen collected more than 100 years ago, is rediagnosed and characterised based on recent collections. We describe three additional species new to science, Chikila alcocki sp. nov., Chikila darlong sp. nov., and Chikila gaiduwani sp. nov. This species-level taxonomy is consistent with mitochondrial DNA sequence data. A key to the species of Chikila is presented.

Discovery of a new family of amphibians from northeast India with ancient links to Africa.

The limbless, primarily soil-dwelling and tropical caecilian amphibians (Gymnophiona) comprise the least known order of tetrapods. On the basis of unprecedented extensive fieldwork, we report the discovery of a previously overlooked, ancient lineage and radiation of caecilians from threatened habitats in the underexplored states of northeast India. Molecular phylogenetic analyses of mitogenomic and nuclear DNA sequences, and comparative cranial anatomy indicate an unexpected sister-group relationship with the exclusively African family Herpelidae. Relaxed molecular clock analyses indicate that these lineages diverged in the Early Cretaceous, about 140 Ma. The discovery adds a major branch to the amphibian tree of life and sheds light on both the evolution and biogeography of caecilians and the biotic history of northeast India-an area generally interpreted as a gateway between biodiversity hotspots rather than a distinct biogeographic unit with its own ancient endemics. Because of its distinctive morphology, inferred age and phylogenetic relationships, we recognize the newly discovered caecilian radiation as a new family of modern amphibians.