Evolution of Sri Lanka's Giant Danios (Teleostei: Cyprinidae: Devario): Teasing apart species in a recent diversification.

The small, colourful freshwater fishes of the cyprinid genus Devario are among the many vertebrate groups that appear to have diversified on Sri Lanka, a continental Indian Ocean island, which is part of the Western Ghats-Sri Lanka Biodiversity Hotspot. Despite Sri Lanka having been connected with India via a wide isthmus intermittently until the Plio-Pleistocene and almost continuously since then, during sea-level low-stands, the number of species of Devario on Sri Lanka is comparable with that on the Indian Peninsula, some 25 times its size. Here, from a sampling of 27 Devario populations across Sri Lanka's major river basins and climatic zones, we present and analyze a phylogeny based on mitochondrial and nuclear DNA data. We show that five species of Devario occur on the island, all but one of which are endemic: Devario malabaricus is widespread throughout the lowlands and parts of central hills of Sri Lanka and southern India. A new narrow-range endemic, here described as D. memorialis sp. nov., was discovered in a remnant rainforest habitat at Aranayake (Ma Oya basin) in this study. It is immediately distinguished from Sri Lankan congeners by having only 8 (vs 9-12) branched dorsal-fin rays and by uncorrected pairwise genetic distances of more than 4.0% and 7.8% for cytochrome c oxidase subunit 1 and cytochrome b, respectively. Our results provide strong support for the monophyly of the entire Sri Lankan diversification of Devario. Divergence-timing analysis suggests the common ancestor dispersed to the island in the late Miocene, with the insular diversification in the island's south-western wet zone taking place during the Plio-pleistocene. There are signs of gene flow between the Indian and Sri Lankan populations of D. malabaricus until the late Pleistocene. Phylogenetic and haplotype-network analyses suggest basin-centric phylogeographic structure within the endemic species; D. malabaricus, however, shows little such structure in the island. Molecular and morphological analyses failed to identify D. annnataliae and D. udenii confidently as distinct species: they are considered synonyms of D. micronema. The morphological variation observed within D. micronema is likely attributable to polymorphism. The discordance between the mitochondrial and nuclear phylogenies for some samples of Devario in the present study suggest signs of mitochondrial introgression.

Diversification of shrub frogs (Rhacophoridae, Pseudophilautus) in Sri Lanka - Timing and geographic context.

Pseudophilautus comprises an endemic diversification predominantly associated with the wet tropical regions of Sri Lanka that provides an opportunity to examine the effects of geography and historical climate change on diversification. Using a time-calibrated multi-gene phylogeny, we analyze the tempo of diversification in the context of past climate and geography to identify historical drivers of current patterns of diversity and distribution. Molecular dating suggests that the diversification was seeded by migration across a land-bridge connection from India during a period of climatic cooling and drying, the Oi-1 glacial maximum around the Eocene-Oligocene boundary. Lineage-through-time plots suggest a gradual and constant rate of diversification, beginning in the Oligocene and extending through the late Miocene and early Pliocene with a slight burst in the Pleistocene. There is no indication of an early-burst phase of diversification characteristic of many adaptive radiations, nor were there bursts of diversification associated with favorable climate shifts such as the intensification of monsoons. However, a late Miocene (8.8 MYA) back-migration to India occurred following the establishment of the monsoon. The back migration did not trigger a diversification in India similar to that manifest in Sri Lanka, likely due to occupation of available habitat, and consequent lack of ecological opportunity, by the earlier radiation of a sister lineage of frogs (Raorchestes) with similar ecology. Phylogenetic area reconstructions show a pattern of sister species distributed across adjacent mountain ranges or from different parts of large montane regions, highlighting the importance of isolation and allopatric speciation. Hence, local species communities are composed of species from disparate clades that, in most cases, have been assembled through migration rather than in situ speciation. Lowland lineages are derived from montane lineages. Thus, the hills of Sri Lanka acted as species pumps as well as refuges throughout the 31 million years of evolution, highlighting the importance of tropical montane regions for both the generation and maintenance of biodiversity.

Correction: Molecular species delimitation of shrub frogs of the genus Pseudophilautus (Anura, Rhacophoridae).

[This corrects the article DOI: 10.1371/journal.pone.0258594.].

Biogeography and evolutionary history of Puntius sensu lato (Teleostei: Cyprinidae) in Sri Lanka.

Sri Lanka's biota is derived largely from Southeast Asian lineages which immigrated via India following its early-Eocene contact with Laurasia. The island is now separated from southeastern India by the 30 km wide Palk Strait which, during sea-level low-stands, was bridged by the 140 km-wide Palk Isthmus. Consequently, biotic ingress and egress were mediated largely by the climate of the isthmus. Because of their dependence on perennial aquatic habitats, freshwater fish are useful models for biogeographic studies. Here we investigate the timing and dynamics of the colonization of-and diversification on-Sri Lanka by a group of four closely-related genera of cyprinid fishes (Puntius sensu lato). We construct a molecular phylogeny based on two mitochondrial and two nuclear gene markers, conduct divergence timing analyses and ancestral-range estimations to infer historical biogeography, and use haplotype networks to discern phylogeographic patterns. The origin of Puntius s.l. is dated to ~ 20 Ma. The source of diversification of Puntius s.l. is Sri Lanka-Peninsular India. Species confined to perhumid rainforests show strong phylogeographic structure, while habitat generalists show little or no such structure. Ancestral range estimations for Plesiopuntius bimaculatus and Puntius dorsalis support an 'Out of Sri Lanka' scenario. Sri Lankan Puntius s.l. derive from multiple migrations across the Palk Isthmus between the early Miocene and the late Pleistocene. Species dependent on an aseasonal climate survived aridification in rainforest refugia in the island's perhumid southwest and went on to recolonize the island and even southern India when pluvial conditions resumed. Our results support an historical extinction of Sri Lanka's montane aquatic fauna, followed by a recent partial recolonization of the highlands, showing also that headwater stream capture facilitated dispersal across basin boundaries.

The role of climate and islands in species diversification and reproductive-mode evolution of Old World tree frogs.

Large diversifications of species are known to occur unevenly across space and evolutionary lineages, but the relative importance of their driving mechanisms, such as climate, ecological opportunity and key evolutionary innovations (KEI), remains poorly understood. Here, we explore the remarkable diversification of rhacophorid frogs, which represent six percent of global amphibian diversity, utilize four distinct reproductive modes, and span a climatically variable area across mainland Asia, associated continental islands, and Africa. Using a complete species-level phylogeny, we find near-constant diversification rates but a highly uneven distribution of species richness. Montane regions on islands and some mainland regions have higher phylogenetic diversity and unique assemblages of taxa; we identify these as cool-wet refugia. Starting from a centre of origin, rhacophorids reached these distant refugia by adapting to new climatic conditions ('niche evolution'-dominant), especially following the origin of KEIs such as terrestrial reproduction (in the Late Eocene) or by dispersal during periods of favourable climate ('niche conservatism'-dominant).

Molecular phylogeny and phylogeography of ricefishes (Teleostei: Adrianichthyidae: Oryzias) in Sri Lanka.

Ricefishes of the genus Oryzias occur commonly in the fresh and brackish waters in coastal lowlands ranging from India across Southeast Asia and on to Japan. Among the three species of Oryzias recorded from peninsular India, two widespread species, O. carnaticus and O. dancena, have previously been reported from Sri Lanka based on museum specimens derived from a few scattered localities. However, members of the genus are widespread in the coastal lowlands of Sri Lanka, a continental island separated from India by the shallow Palk Strait. Although recent molecular phylogenies of Adrianichthyidae represent near-complete taxon representation, they lack samples from Sri Lanka. Here, based on sampling at 13 locations representative of the entire geographic and climatic regions of the island's coastal lowlands, we investigate for the first time the molecular phylogenetic relationships and phylogeography of Sri Lankan Oryzias based on one nuclear and two mitochondrial markers. Sri Lankan Oryzias comprise two distinct non-sister lineages within the javanicus species group. One of these is represented by samples exclusively from the northern parts of the island; it is recognized as O. dancena. This lineage is recovered as the sister group to the remaining species in the javanicus group. The second lineage represents a species that is widespread across the island's coastal lowlands. It is recovered as the sister group of O. javanicus and is identified as O. cf. carnaticus. Ancestral-range estimates suggest two independent colonizations of Indian subcontinent and Sri Lanka by widespread ancestral species of Oryzias during two discrete temporal windows: late Miocene and Plio-Pleistocene. No phylogeographic structure is apparent in Sri Lankan Oryzias, suggesting that there are no strong barriers to gene flow and dispersal along the coastal floodplains, as is the case also for other generalist freshwater fishes in the island.

Molecular species delimitation of shrub frogs of the genus Pseudophilautus (Anura, Rhacophoridae).

Sri Lanka is an amphibian hotspot of global significance. Its anuran fauna is dominated by the shrub frogs of the genus Pseudophilautus. Except for one small clade of four species in Peninsular India, these cool-wet adapted frogs, numbering some 59 extant species, are distributed mainly across the montane and lowland rain forests of the island. With species described primarily by morphological means, the diversification has never yet been subjected to a molecular species delimitation analysis, a procedure now routinely applied in taxonomy. Here we test the species boundaries of Pseudophilautus in the context of the phylogenetic species concept (PSC). We use all the putative species for which credible molecular data are available (nDNA-Rag-1; mt-DNA- 12S rRNA, 16S rRNA) to build a well resolved phylogeny, which is subjected to species delimitation analyses. The ABGD, bPTP, mPTP and bGMYC species delimitation methods applied to the 16S rRNA frog barcoding gene (for all species), 12S rRNA and Rag-1 nDNA grouped P. procax and P. abundus; P. hallidayi and P. fergusonianus; P. reticulatus and P. pappilosus; P. pleurotaenia and P. hoipolloi; P. hoffmani and P. asankai; P. silvaticus and P. limbus; P. dilmah and P. hankeni; P. fulvus and P. silus.. Surprisingly, all analyses recovered 14 unidentified potential new species as well. The geophylogeny affirms a distribution across the island's aseasonal 'wet zone' and its three principal hill ranges, suggestive of allopatric speciation playing a dominant role, especially between mountain masses. Among the species that are merged by the delimitation analyses, a pattern leading towards a model of parapatric speciation emerges-ongoing speciation in the presence of gene flow. This delimitation analysis reinforces the species hypotheses, paving the way to a reasonable understanding of Sri Lankan Pseudophilautus, enabling both deeper analyses and conservation efforts of this remarkable diversification. http://zoobank.org/urn:lsid:zoobank.org:pub:DA869B6B-870A-4ED3-BF5D-5AA3F69DDD27.

Molecular phylogeny and phylogeography of the freshwater-fish genus Pethia (Teleostei: Cyprinidae) in Sri Lanka.

BACKGROUND: Sri Lanka is a continental island separated from India by the Palk Strait, a shallow-shelf sea, which was emergent during periods of lowered sea level. Its biodiversity is concentrated in its perhumid south-western 'wet zone'. The island's freshwater fishes are dominated by the Cyprinidae, characterized by small diversifications of species derived from dispersals from India. These include five diminutive, endemic species of Pethia (P. bandula, P. cumingii, P. melanomaculata, P. nigrofasciata, P. reval), whose evolutionary history remains poorly understood. Here, based on comprehensive geographic sampling, we explore the phylogeny, phylogeography and morphological diversity of the genus in Sri Lanka. RESULTS: The phylogenetic analyses, based on mitochondrial and nuclear loci, recover Sri Lankan Pethia as polyphyletic. The reciprocal monophyly of P. bandula and P. nigrofasciata, and P. cumingii and P. reval, is not supported. Pethia nigrofasciata, P. cumingii, and P. reval show strong phylogeographic structure in the wet zone, compared with P. melanomaculata, which ranges across the dry and intermediate zones. Translocated populations of P. nigrofasciata and P. reval in the Central Hills likely originate from multiple sources. Morphological analyses reveal populations of P. nigrofasciata proximal to P. bandula, a narrow-range endemic, to have a mix of characters between the two species. Similarly, populations of P. cumingii in the Kalu basin possess orange fins, a state between the red-finned P. reval from Kelani to Deduru and yellow-finned P. cumingii from Bentara to Gin basins. CONCLUSIONS: Polyphyly in Sri Lankan Pethia suggests two or three colonizations from mainland India. Strong phylogeographic structure in P. nigrofasciata, P. cumingii and P. reval, compared with P. melanomaculata, supports a model wherein the topographically complex wet zone harbors greater genetic diversity than the topographically uniform dry-zone. Mixed morphological characters between P. bandula and P. nigrofasciata, and P. cumingii and P. reval, and their unresolved phylogenies, may suggest recent speciation scenarios with incomplete lineage sorting, or hybridization.

Phylogenetic diversity of Sri Lankan freshwater crabs and its implications for conservation.

As part of a Global Biodiversity Hotspot, the conservation of Sri Lanka's endemic biodiversity warrants special attention. With 51 species (50 of them endemic) occurring in the island, the biodiversity of freshwater crabs is unusually high for such a small area (65,600 km(2)). Freshwater crabs have successfully colonized most moist habitats and all climatic and elevational zones in Sri Lanka. We assessed the biodiversity of these crabs in relation to the different elevational zones (lowland, upland and highland) based on both species richness and phylogenetic diversity. Three different lineages appear to have radiated simultaneously, each within a specific elevational zone, with little interchange thereafter. The lowland and upland zones show a higher species richness than the highland zone while--unexpectedly--phylogenetic diversity is highest in the lowland zone, illustrating the importance of considering both these measures in conservation planning. The diversity indices for the species in the various IUCN Red List categories in each of the three zones suggest that risk of extinction may be related to elevational zone. Our results also show that overall more than 50% of Sri Lanka's freshwater crab species (including several as yet undescribed ones), or approximately 72 million years of evolutionary history, are threatened with extinction.

A commentary on the taxonomic review of Sri Lankan Devario by Batuwita et al. 2017 (Teleostei: Danionidae).

We address several problems arising from 'A review of the genus Devario in Sri Lanka (Teleostei: Cyprinidae), with description of two new species', a paper authored by S. Batuwita, M. de Silva and S. Udugampola and published in 2017 in the journal FishTaxa (2(3): 156-179). The neotypes they designate for Perilampus malabaricus Jerdon and Perilampus mysoricus Jerdon are inconsistent with article 75.3 of the International Code of Zoological Nomenclature ('the Code') and are hence invalid. Devario udenii, which they describe as a new species, is shown to be indistinguishable from D. micronema sensu Batuwita et al. The characters by which they distinguish another new species, D. annnataliae, are shown to be self-contradictory, making it impossible to distinguish from its congeners; it is treated as a species inquirendum. The diagnoses provided for D. malabaricus, D. micronema and D. monticola are ambiguous and self-contradictory, rendering them unusable. Much of the material examined, stated to be in the collection of the National Museum of Sri Lanka, is not deposited in that institution: such material as is deposited is inconsistent with the specimen data published by Batuwita et al.

The identity of the Sri Lankan Amblypharyngodon (Teleostei, Cyprinidae).

Morphological and molecular analyses of specimens representative of the geographic range of the cyprinid genus Amblypharyngodon in Sri Lanka suggest the presence of only a single species in the island, for which the name Amblypharyngodongrandisquamis Jordan & Starks, 1917, is available. Amblypharyngodongrandisquamis is a species endemic to Sri Lanka, distributed across the lowlands of both of the island's main climatic zones. It is distinguished from all other species of Amblypharyngodon, including the three species recorded from peninsular India (A.mola, A.microlepis, and A.melettinus), by a suite of characters that includes a body depth of 26.9-31.2% of the standard length (SL), 42-56 scales in the lateral series (of which usually 8-16 are pored), 20-24 circumpeduncular scales, 14-17 scale rows between the origins of the dorsal and pelvic fins, a dorsal-fin height of 21.1-27.6% SL, 18-19 caudal vertebrae and an eye diameter of 22.7-30.5% of the head length. Amblypharyngodongrandisquamis differs from A.melettinus and A.mola by uncorrected pairwise genetic distances of more than 9% and 6%, respectively, for the mitochondrial cytochrome oxidase subunit 1 (COI) gene.

Undocumented translocations spawn taxonomic inflation in Sri Lankan fire rasboras (Actinopterygii, Cyprinidae).

A recent (2013) taxonomic review of the freshwater-fish genus Rasboroides, which is endemic to Sri Lanka, showed it to comprise four species: R. vaterifloris, R. nigromarginatus, R. pallidus and R. rohani. Here, using an integrative-taxonomic analysis of morphometry, meristics and mitochondrial DNA sequences of cytochrome b (cytb) and cytochrome oxidase subunit 1 (coi), we show that R. nigromarginatus is a synonym of R. vaterifloris, and that R. rohani is a synonym of R. pallidus. The creation and recognition of unnecessary taxa ('taxonomic inflation') was in this case a result of selective sampling confounded by a disregard of allometry. The population referred to R. rohani in the Walawe river basin represents an undocumented trans-basin translocation of R. pallidus, and a translocation into the Mahaweli river of R. vaterifloris, documented to have occurred ca 1980, in fact involves R. pallidus. A shared haplotype suggests the latter introduction was likely made from the Bentara river basin and not from the Kelani, as claimed. To stabilize the taxonomy of these fishes, the two valid species, R. vaterifloris and R. pallidus, are diagnosed and redescribed, and their distributions delineated. We draw attention to the wasteful diversion of conservation resources to populations resulting from undocumented translocations and to taxa resulting from taxonomic inflation. We argue against translocations except where mandated by a conservation emergency, and even then, only when supported by accurate documentation.

Barcoding snakeheads (Teleostei, Channidae) revisited: Discovering greater species diversity and resolving perpetuated taxonomic confusions.

Snakehead fishes of the family Channidae are predatory freshwater teleosts from Africa and Asia comprising 38 valid species. Snakeheads are important food fishes (aquaculture, live food trade) and have been introduced widely with several species becoming highly invasive. A channid barcode library was recently assembled by Serrao and co-workers to better detect and identify potential and established invasive snakehead species outside their native range. Comparing our own recent phylogenetic results of this taxonomically confusing group with those previously reported revealed several inconsistencies that prompted us to expand and improve on previous studies. By generating 343 novel snakehead coxI sequences and combining them with an additional 434 coxI sequences from GenBank we highlight several problems with previous efforts towards the assembly of a snakehead reference barcode library. We found that 16.3% of the channid coxI sequences deposited in GenBank are based on misidentifications. With the inclusion of our own data we were, however, able to solve these cases of perpetuated taxonomic confusion. Different species delimitation approaches we employed (BIN, GMYC, and PTP) were congruent in suggesting a potentially much higher species diversity within snakeheads than currently recognized. In total, 90 BINs were recovered and within a total of 15 currently recognized species multiple BINs were identified. This higher species diversity is mostly due to either the incorporation of undescribed, narrow range, endemics from the Eastern Himalaya biodiversity hotspot or the incorporation of several widespread species characterized by deep genetic splits between geographically well-defined lineages. In the latter case, over-lumping in the past has deflated the actual species numbers. Further integrative approaches are clearly needed for providing a better taxonomic understanding of snakehead diversity, new species descriptions and taxonomic revisions of the group.

The Sri Lankan torrent toads (Bufonidae: Adenominae: Adenomus): species boundaries assessed using multiple criteria.

The bufonid genus Adenomus, an endemic of the montane and lowland rainforests of central and south-western Sri Lanka, has been considered to comprise of three species, viz. A. kelaartii, A. dasi and A. kandianus, the last of which has been recently highlighted as "the world's rarest toad". We conducted a survey across the known range of Adenomus and used multiple criteria to delineate species boundaries within the genus. These include: a molecular phylogeny based on a 16S ribosomal RNA gene fragment; an examination of the external morphology of adults and larvae, and the skeletal morphology of adults; a bioacoustic analysis; and ecological niche modelling. We show that Adenomus is monophyletic and that it comprises only two species: A. kelaartii and A. kandianus, with A. dasi being a junior synonym of the latter. For the two valid species of Adenomus, we provide detailed osteological descriptions; clarify the distribution patterns; and provide genetic data to facilitate their scientific conservation management. 

Validation of the South Asian cichlid genus Pseudetroplus Bleeker (Pisces: Cichlidae).

The South Asian Cichlidae are composed of two clades that together represent the sister group of the Madagascan genus Paretroplus Bleeker. Chaetodon suratensis Bloch and Etroplus canarensis Day are retained in Etroplus Cuvier, while Chaetodon maculatus Bloch is allocated to Pseudetroplus Bleeker. Pseudetroplus is distinguished from Etroplus in having, among other characters, 11 (vs. 12-13) pleural ribs; 26-27 (vs. 28-29) vertebrae; the anterior half of the median suture between the lower pharyngeal jaw serrated (vs. smooth); the first 6 anal-fin pterygiophores arranged anterior to the first 3 (vs. 2) haemal spines; the supraoccipital-exoccipital prong extending ventrally about half-way across the foramen magnum (vs. not extending into the foramen magnum); and the anterior jaw teeth tricuspid, acuminate (vs. unicuspid, spatulate). Microgaster Swainson is a synonym of Pseudetroplus and a junior homonym of Microgaster Latreille in Hymenoptera. 

Rostral horn evolution among agamid lizards of the genus Ceratophora endemic to Sri Lanka.

The first phylogenetic hypothesis for the Sri Lankan agamid lizard genus Ceratophora is presented based on 1670 aligned base positions (472 parsimony informative) of mitochondrial DNA sequences, representing coding regions for eight tRNAs, ND2, and portions of ND1 and COI. Phylogenetic analysis reveals multiple origins and possibly losses of rostral horns in the evolutionary history of Ceratophora. Our data suggest a middle Miocene origin of Ceratophora with the most recent branching of recognized species occurring at the Pliocene/Pleistocene boundary. Haplotype divergence suggests that an outgroup species, Lyriocephalus scutatus, dates at least to the Pliocene. These phylogenetic results provide a framework for comparative studies of the behavioral ecological importance of horn evolution in this group.

Local endemism within the Western Ghats-sri Lanka biodiversity hotspot.

The apparent biotic affinities between the mainland and the island in the Western Ghats-Sri Lanka biodiversity hotspot have been interpreted as the result of frequent migrations during recent periods of low sea level. We show, using molecular phylogenies of two invertebrate and four vertebrate groups, that biotic interchange between these areas has been much more limited than hitherto assumed. Despite several extended periods of land connection during the past 500,000 years, Sri Lanka has maintained a fauna that is largely distinct from that of the Indian mainland. Future conservation programs for the subcontinent should take into account such patterns of local endemism at the finest scale at which they may occur.