Hidden hotspots of amphibian biodiversity in China.

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

Diversification of rhacophorid frogs provides evidence for accelerated faunal exchange between India and Eurasia during the Oligocene.

The accretion of the Indian subcontinent to Eurasia triggered a massive faunal and floral exchange, with Gondwanan taxa entering into Asia and vice versa. The traditional view on the Indian-Asian collision assumes contact of the continental plates during the Early Eocene. Many biogeographic studies rely on this assumption. However, the exact mode and timing of this geological event is still under debate. Here we address, based on an extensive phylogenetic analysis of rhacophorid tree frogs, if there was already a Paleogene biogeographic link between Southeast Asia and India; in which direction faunal exchange occurred between India and Eurasia within the Rhacophoridae; and if the timing of the faunal exchange correlates with one of the recently suggested geological models. Rhacophorid tree frogs showed an early dispersal from India to Asia between 46 and 57 Ma, as reconstructed from the fossil record. During the Middle Eocene, however, faunal exchange ceased, followed by increase of rhacophorid dispersal events between Asia and the Indian subcontinent during the Oligocene that continued until the Middle Miocene. This corroborates recent geological models that argue for a much later final collision between the continental plates. We predict that the Oligocene faunal exchange between the Indian subcontinent and Asia, as shown here for rhacophorid frogs, also applies for other nonvolant organisms with an Indian-Asian distribution, and suggest that previous studies that deal with this faunal interchange should be carefully reinvestigated.

Amphibians crossing the Bering Land Bridge: evidence from holarctic treefrogs (Hyla, Hylidae, Anura).

Based on an updated, time-calibrated phylogeny and applying biogeographical model testing and diversification analysis, we re-examined systematics and biogeography of the Holarctic treefrog genus Hyla with a focus on the East Asian species. We analyzed four mitochondrial genes (12S and 16S rRNA, tRNA(Leu), ND1) and one nuclear gene (POMC) for 192 samples representing 30 species of Hyla. Based on our results we suggest that H. ussuriensis is a synonym of H. japonica. Specimens from Sakhalin and Kunashir Islands might represent a cryptic species within H. japonica. We confirm earlier hypotheses that the genus Hyla originated during the Eocene to Early Oligocene and that Eurasian species originated from two independent dispersal events from North America via the Bering Land Bridge. Middle Eocene to Oligocene dispersal gave rise to the most recent common ancestor of the West Palearctic H. arborea-group and the East Palearctic, newly defined, H. chinensis-group. The Northeast Asian H. japonica-group resulted from a second wave of colonization from the Nearctic. A trans-Atlantic dispersal route could be excluded. Dispersal of the H. arborea-group to the western Palearctic coincides with the closure of the Turgai Strait at the end of the Oligocene. Diversification of Hyla decreased at the end of the Middle Miocene, possibly coinciding with the end of the Mid Miocene Climatic Optimum and the advent of cooler and drier climates in the Northern Hemisphere.

DNA barcoding of Vietnamese bent-toed geckos (Squamata: Gekkonidae: Cyrtodactylus) and the description of a new species.

Species of bent-toed gecko (Cyrtodactylus) in Vietnam have been described at a rate of nearly four species per year since 2007 mostly based on morphological data. A tool that guides species delimitation will accelerate the rate of documentation, and at a time when the recognition of species greatly benefits conservation. We use DNA barcoding using COI (550 bp) to re-examine the levels of genetic divergence and taxonomic status of 21 described species of Vietnamese bent-toed geckos. Tree-based analyses resolve all sampled species and identify potential undescribed taxa. Kimura 2-parameter genetic distances between the described species average 21.0±4.2% and range from 4.3% to 28.7%. Further, our analyses discover two potentially new species from Vietnam, two from Laos and one from China. Herein we describe the new species Cyrtodactylus puhuensis sp. nov. from Vietnam on the basis of both genetics and morphology. Genetically, it differs from the remaining species by an average K2P distance of 24.0±1.8%. Morphologically, the new species is diagnosed by its medium-size (snout-vent length 79.24 mm and tail length 82.59 mm, for the single known individual), in having a series of moderately enlarged transverse subcaudals and a series of moderately enlarged femoral scales that extend from precloacal scales, in possessing femoral scales without pores, with males having five precloacal pores, and in exhibiting 8 supralabials, 10 infralabials, 23 narrow subdigital lamellae on its fourth toe, and 36 transverse ventrals.

Genealogy and Demographic History of a Widespread Amphibian throughout Indochina.

Relatively little is known about spatial patterns of cryptic diversity in tropical species and the processes that generate them. Few studies examine the geographic distribution of genetic lineages in Southeast Asia, an area hypothesized to harbor substantial cryptic diversity. We investigated the evolutionary history of Asian tree frogs of the Polypedates leucomystax complex (n = 172) based on 1800 bp of the mtDNA genes ND1 and cytochrome b and tested hypotheses pertaining to climate, geology, and dispersal patterns. Analyses revealed substantial genetic diversity and lineage divergence throughout the region with evidence for widespread sympatric lineages and a general north versus south clustering. Relaxed molecular clock analysis and tests for demographic expansion identified an initial cladogenesis during the Miocene with subsequent Plio-Pleistocene diversification, with the former corresponding to periods of increased aridity and the onset of monsoonal weather systems. Rates of diversification were relatively constant until the Early Pleistocene when rates increased exponentially. We found equivocal evidence for isolation-by-distance and a potential role of some landscape features as partial barriers to dispersal. Finally, our analyses showed that divergence between insular and mainland populations occurred before Homo sapiens colonized Southeast Asia, suggesting that historical human-mediated dispersal did not drive insular diversification. Our results suggested that demographic expansion in the Late Pleistocene resulted in widespread sympatric lineages in the P. leucomystax complex throughout southern China and Indochina and further clarified the evolutionary history of lineages within P. leucomystax.

Description of a New Cobra (Naja Laurenti, 1768; Squamata, Elapidae) from China with Designation of a Neotype for Naja atra.

Taxonomic frameworks for medically important species such as cobras (genus Naja Laurenti, 1768; Squamata, Elapidae) are essential for the medical treatment of snake bites and accurate antivenin development. In this paper, we described the former N. kaouthia populations recorded from China as a new species and designated a neotype for N. atra-based morphological and mitochondrial phylogenetic analysis. The new species N. fuxisp. nov. was morphologically diagnosed from N. kaouthia by (1) regular single narrow crossband present on the middle and posterior parts of the dorsum (3-15, 7.9 ± 2.7, n = 32) and the dorsal surface of the tail (1-6, 4.2 ± 1.1, n = 32) of both adults and juveniles, buff-colored with dark fringes on both edges, vs. South Asian populations (n = 39) and Southeast Asian populations (n = 35) without cross bands, with irregular cross bands or multiple light-colored crossbands pairs, or densely woven lines; (2) small scales between the posterior chin shields, usually three (40%) or two (37%), rarely four (13%), or one (10%) (n = 30) vs. mostly one (81%) and rarely two (19%) (n = 28); (3) ventrals 179-205 (195.4 ± 6.7, n = 33) vs. South Asian populations 179-199 (188.7 ± 5.9, n = 12); Southeast Asian populations 168-186 (177.8 ± 4.9, n = 18). Phylogenetically, the new species forms an independent sister clade to the clade including N. atra, N. kaouthia, N. oxiana and N. sagittifera. Furthermore, the subspecies N. naja polyocellata should be resurrected and recognized as a full species, N. polyocellatacomb. nov., and the subspecies N. sumatrana miolepis should be resurrected.

From mainland to islands: colonization history in the tree frog Kurixalus (Anura: Rhacophoridae).

The origin and colonization history of Kurixalus, a genus of small arboreal tree frogs breeding exclusively in shallow swamps, is under disputed. On the basis of comprehensive sampling program, the evolutionary history of Kurixalus is investigated based on 3 mitochondrial genes. Our results indicate that the genus Kurixalus originated in the Asian mainland and subsequently arrived at its current distribution in Borneo, Taiwan, Ryukyu, and Hainan islands by a series of dispersal events. Moreover, the colonization of Taiwan from mainland Asia has occurred 2 times. The initial colonization of Taiwan occurred at 3.46-8.68 Mya (95% highest posterior density), which rejects the hypothesis that Kurixalus probably originated from Taiwan during the early Oligocene and favors the model of Neogene-origin rather than the model of Quaternary-origin for Taiwanese Kurixalus. Kurixalus eiffingeri has dispersed from Taiwan to the Ryukyus once or 2 times pending more data. Both transoceanic dispersal and landbridge dispersal have played a role in the colonization process; the former resulted in the colonization of Taiwan and the Ryukyus and the latter led to the colonization of Borneo and Hainan.

Shared response to changes in drainage basin: Phylogeography of the Yunnan small narrow-mouthed frog, Glyphoglossus yunnanensis (Anura: Microhylidae).

AIM: With the late Cenozoic uplift of the Qinghai-Tibetan Plateau (QTP), drainage of the southeastern edge of the QTP changed significantly. However, the impact of this dramatic change on the geographical distribution and genetic diversity of endemic organisms is still poorly understood. Here, we examined the geographical patterns of genetic variation in the Yunnan small narrow-mouthed frog, Glyphoglossus yunnanensis (Microhylidae), and two alternative hypotheses were tested: That is, the geographical distribution of genetic variation was determined by either the contemporary drainage basin or historical drainage basins. LOCATION: The Mountains of southwest China. MATERIALS AND METHODS: Analyses were based on 417 specimens collected from across the distribution of the species. We reconstructed the genealogy (Bayesian and maximum parsimony methods) and assessed demographic history based on DNA sequencing data from mitochondrial and nuclear markers. We also mapped the genetic diversity and estimated the divergence times by a relaxed clock model. RESULTS: The species has maintained a relatively stable population size without recent population expansion. Four major maternal lineages were identified with good support, one representing a possible cryptic species and the other three showing further subdivision. The distribution of these deeply differentiated lineages/sublineages corresponded well to geographical regions. The secondary contact zones and phylogeographic breaks in distinct lineages of G. yunnanensis were almost concordant with those of Nanorana yunnanensis. MAIN CONCLUSIONS: Lineage division conformed to the hypothesis of drainage system evolution, that is, the phylogeographic pattern of G. yunnanensis was shaped by historical drainage patterns. Concordance in phylogeographic patterns may suggest a shared response to common hydrogeological history and also might indicate that there was more contribution of the drainage history than ecological or life-history traits in structuring genetic variation between these two disparate codistributed taxa G. yunnanensis and N. yunnanensis.

Review of the genus Brachytarsophrys (Anura: Megophryidae), with revalidation of Brachytarsophrys platyparietus and description of a new species from China.

The genus-level recognition of monophyletic short-legged toads ( Brachytarsophrys) has been recently implicated in the taxonomic debate of Megophrys sensu lato. In the present study, Brachytarsophrys is reasonably regarded as a distinct genus based on significant morphological differentiations and recent molecular analyses. Furthermore, a comprehensive review of this genus is performed, with two species groups proposed based on morphological differences and phylogenetic relationships. Particularly, Brachytarsophrys platyparietus is removed as a synonym of Brachytarsophrys carinense and considered a valid species due to significant genetic divergence and distinct morphological differences. In addition, a new species, Brachytarsophrys orientalis sp. nov., is described based on a series of specimens collected from southeastern China. This work takes the member species of the genus Brachytarsophrys to seven, suggesting that the diversity of Brachytarsophrys is underestimated. In addition, the genus levels of other monophyletic groups within the subfamily Megophryinae are discussed.

Author Correction: Review of the genus Brachytarsophrys (Anura: Megophryidae), with revalidation of Brachytarsophrys platyparietus and description of a new species from China.

Following the publication of our paper (Li et al., 2020), it has come to our attention that, due to our oversight, the contributions of some colleagues were not reflected in the "ACKNOWLEDGEMENTS" section. Hereby, we wish to add the following to the "ACKNOWLEDGEMENTS" section as a correction.

New insights to the molecular phylogenetics and generic assessment in the Rhacophoridae (Amphibia: Anura) based on five nuclear and three mitochondrial genes, with comments on the evolution of reproduction.

The phylogenetic relationships among 12 genera of treefrogs (Family, Rhacophoridae), were investigated based on a large sequence data set, including five nuclear (brain-derived neurotrophic factor, proopiomelanocortin, recombination activating gene 1, tyrosinase, rhodopsin) and three mitochondrial (partial 12S and 16S ribosomal RNA and the complete valine t-RNA) genes. Phylogenetic analysis of the nuclear gene sequences resolved three major clades. The first group included Philautus, Pseudophilautus, Kurixalus, Gracixalus, and Theloderma moloch; Pseudophilautus and Kurixalus were sister taxa. The second group consisted of Nyctixalus and Theloderma. The third group contained Feihyla, Polypedates, Rhacophorus, and Chiromantis vittatus; Polypedates and Feihyla were sister taxa. Analyses of the nuclear and mitochondrial genes supported the following results: (1) Genus Liuixalus formed the sister group of all other rhacophorines. (2) Philautus, Theloderma, and Chiromantis were not resolved as monophyletic genera. Four groups, including Philautus ocellatus and P. hainanus, P. longchuanensis and P. gryllus, P. banaensis, and P. quyeti nested well within the genera Liuixalus, Pseudophilautus, Kurixalus, and Gracixalus, respectively. (3) Theloderma moloch and Chiromantis vittatus did not cluster with other species of Theloderma and Chiromantis, respectively. Foam nesting evolved only once, as did laying eggs in a jelly-like matrix containing some bubbles. Terrestrial direct development evolved twice in the Rhacophoridae.

Phylogeny of the Asian spiny frog tribe Paini (Family Dicroglossidae) sensu Dubois.

The anuran tribe Paini, family Dicroglossidae, is known in this group only from Asia. The phylogenetic relationships and often the taxonomic recognition of species are controversial. In order to stabilize the classification, we used approximately 2100 bp of nuclear (rhodopsin, tyrosinase) and mitochondrial (12S, 16S rRNA) DNA sequence data to infer the phylogenetic relationships of these frogs. Phylogenetic trees reconstructed using Bayesian inference and maximum parsimony methods supported a monophyletic tribe Paini. Two distinct groups (I,II) were recovered with the mtDNA alone and the total concatenated data (mtDNA+nuDNA). The recognition of two genera, Quasipaa and Nanorana, was supported. Group I, Quasipaa, is widespread east of the Hengduan Mountain Ranges and consists of taxa from relatively low elevations in southern China, Vietnam and Laos. Group II, Nanorana, contains a mix of species occurring from high to low elevation predominantly in the Qinghai-Tibetan Plateau and Hengduan Mountain Ranges. The occurrence of frogs at high elevations appears to be a derived ecological condition. The composition of some major species groups based on morphological characteristics strongly conflicts with the molecular analysis. Some possible cryptic species are indicated by the molecular analyses. The incorporation of genetic data from type localities helped to resolve some of the taxonomic problems, although further combined analyses of morphological data from type specimens are required. The two nuDNA gene segments proved to be very informative for resolving higher phylogenetic relationships and more nuclear data should be explored to be more confident in the relationships.

A new species of the genus Leptolalax (Anura: Megophryidae) from Mt. Gaoligongshan of western Yunnan Province, China.

A new species of the genus Leptolalax is described from the Tengchong Section of Gaoligongshan National Nature Reserve, Tengchong County, Yunnan Province, China. The new species, Leptolalax tengchongensis sp. nov., can be distinguished from its congeners by a combination of the following characters: (1) small size (SVL 23.9-26.0 mm in males, 28.8-28.9 mm in females); (2) dorsal skin shagreened and scattered with fine, round reddish tubercles; (3) toes with rudimentary webbing and narrow lateral fringes; (4) tympanum distinctly discernible, almost entirely black; (5) ventrolateral glands indistinct; (6) flanks with several distinct and large dark blotches; (7) ventral surfaces white, scattered with distinct irregular dark speckling; (8) iris not bicolored, uniformly dark brown and scattered with minute, coppery reticulations throughout. To date, the new species has only been found at its type locality in evergreen broadleaf forests at elevations between 2000-2100 m.

A new species of the genus Liuixalus (Anura: Rhacophoridae) from southern China.

A new tree frog species of the genus Liuixalus was described from Heishiding Nature Reserve, Guangdong Province, China based on a combination of morphological characters and molecular phylogenetic analyses. Liuixalus feii sp. nov. is distinguished from its congeners by a combination of following characters: small size (SVL 16.2-17.6 mm in adult males and 18.0-18.7 mm in adult females); snout obtusely pointed; tympanum distinct, about half size of eye diameter; nostril closer to eye than to the tip of snout; fingers free of webbing; toe III longer than toe V; toes weakly webbed; tibiotarsal articulation reaching the naris or loreal; dorsal skin smooth and scattered with fine granulars; a subtle longitudinal median ridge present on dorsum; weak skin folds present on dorsal surface of body and thighs; supratympanic fold distinct and curved; ventral surface dull white with more or less irregular dark spots; iris bicolored. The new species appears to be forest-dependent and to date has only been found on the forest floor in primary forests at elevations between 350-800 m. Based on our molecular analyses, we consider Liuixalus catbaensis as a junior synonym of L. calcarius. Thus, with the description of the new species, the genus Liuixalus hitherto contains five recognized species, four of which are endemic to China.

[A new record of a Gekkonid (Cyrtodactylus wayakonei) from Yunnan, China].

The bent-toed gecko (Cyrtodactylus wayakonei Nguyen, Kingsada, Rosler, Auer and Ziegler, 2010) has been recorded for the first time in China. Here, we provide descriptions of specimens from Yunnan, China. This species was distinguished from all congeners by the following characteristics: 7-8 supralabials; 9-10 infralabials; dorsal tubercles smooth to slightly keeled; 17-19 rows of dorsal tubercles; 6-8 precloacal pores in both sexes; femoral scales not enlarged; no femoral pores; subcaudals somewhat enlarged and broadened; and tail tubercles flat and smooth.

Is chytridiomycosis an emerging infectious disease in Asia?

The disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused dramatic amphibian population declines and extinctions in Australia, Central and North America, and Europe. Bd is associated with >200 species extinctions of amphibians, but not all species that become infected are susceptible to the disease. Specifically, Bd has rapidly emerged in some areas of the world, such as in Australia, USA, and throughout Central and South America, causing population and species collapse. The mechanism behind the rapid global emergence of the disease is poorly understood, in part due to an incomplete picture of the global distribution of Bd. At present, there is a considerable amount of geographic bias in survey effort for Bd, with Asia being the most neglected continent. To date, Bd surveys have been published for few Asian countries, and infected amphibians have been reported only from Indonesia, South Korea, China and Japan. Thus far, there have been no substantiated reports of enigmatic or suspected disease-caused population declines of the kind that has been attributed to Bd in other areas. In order to gain a more detailed picture of the distribution of Bd in Asia, we undertook a widespread, opportunistic survey of over 3,000 amphibians for Bd throughout Asia and adjoining Papua New Guinea. Survey sites spanned 15 countries, approximately 36° latitude, 111° longitude, and over 2000 m in elevation. Bd prevalence was very low throughout our survey area (2.35% overall) and infected animals were not clumped as would be expected in epizootic events. This suggests that Bd is either newly emerging in Asia, endemic at low prevalence, or that some other ecological factor is preventing Bd from fully invading Asian amphibians. The current observed pattern in Asia differs from that in many other parts of the world.

Validity and systematic position of Rana altaica (Rana: ranidae): results of a phylogenetic analysis.

In order to evaluate the phylogenetic position and validity of Rana altaica, we investigated the phylogeny of brown frogs in Eurasia by Bayesian Inference and Maximum Parsimony analyses of a fragment from the mitochondrial DNA gene Cytochrome b. Both analyses resolved R. altaica as nesting deeply within R. arvalis. Most samples of the nominal R. altaica from the Altai region and specimens from Central Siberia shared a haplotype with R. arvalis based on the network analysis. The matrilineal relationships suggested that R. altaica should be considered as a junior synonym of R. arvalis. Furthermore, our study suggested that the species group division of Chinese brown frogs should be re-evaluated within a phylogenetic context.

Phylogenetic relationships of the mustache toads inferred from mtDNA sequences.

Species of the genus Vibrissaphora are unique among all anurans in that males exhibit external cornified spines in the maxillary region during the breeding season. They were separated from species of the genus Leptobrachium based on this unique character. We construct a phylogeny using the 16S, ND4, and cytochrome b mitochondrial genes of 42 individuals from eight species of Vibrissaphora and five species of Leptobrachium from mainland China, Southeast Asia, and Hainan Island. Species of both Oreolalax and Scutiger were used as outgroups. The results indicate that: L. huashen and L. chapaense form a clade that is nested within Vibrissaphora, and L. hainanense is the sister taxon to the clade comprising all Vibrissaphora plus L. chapaense and L. huashen; V. boringiae is grouped with a clade consisting of V. leishanensis, V. liui, and V. yaoshanensis; and V. yaoshanensis is a species separate from V. liui. We propose taxonomic changes that reflect these findings. Also based on the resulting phylogenetic trees, we propose that the mustache toads originated in the trans-Himalayan region of southwest China, and that the evolution of maxillary spines, large body size, and reverse sexual size dimorphism in these frogs was influenced by intrasexual selection due to adopting a resource-defense polygyny mating system.