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.

Cryptic diversity and phylogeography of the Rhabdophis nuchalis group (Squamata: Colubridae).

Previous studies, have found that the rapid uplift of the Tibetan plateau accelerated the diversification of species. However, there are few relevant biogeographic data for the Colubridae in this region. We conducted a comprehensive study of the Rhabdophis nuchalis Group, which presently contains four nominal species, R. nuchalis, R. pentasupralabialis, R. leonardi, and R. chiwen. Building upon previous studies with specimens we have recently examined, greater interspecific and intraspecific diversity has been revealed. Here we address three questions: (1) Do the intraspecific differences represent only geographic variation within lineages, or are there cryptic species? (2) What are the interspecific relationships among members of the R. nuchalis Group? (3) What has been the biogeographic history of this species group? To resolve these questions we used four mitochondrial gene sequences and one nuclear sequence to investigate the molecular phylogenetic and geographic relationships among populations. Our molecular analysis reveals cryptic species diversity within the R. nuchalis Group, and seven clades were identified in the analysis. Ancestral area estimation suggests that the R. nuchalis Group originated in the Hengduan Mountains approximately 6.24 Mya and expanded its range northward to the Qinling-Daba Mountains. The Sichuan Basin appears to have been a barrier to migration. Species divergence seems to have been related to the rapid uplift of the Qinghai-Tibet Plateau.

Multiple independent structural dynamic events in the evolution of snake mitochondrial genomes.

BACKGROUND: Mitochondrial DNA sequences have long been used in phylogenetic studies. However, little attention has been paid to the changes in gene arrangement patterns in the snake's mitogenome. Here, we analyzed the complete mitogenome sequences and structures of 65 snake species from 14 families and examined their structural patterns, organization and evolution. Our purpose was to further investigate the evolutionary implications and possible rearrangement mechanisms of the mitogenome within snakes. RESULTS: In total, eleven types of mitochondrial gene arrangement patterns were detected (Type I, II, III, III-A, III-B, III-B1, III-C, III-D, III-E, III-F, III-G), with mitochondrial genome rearrangements being a major trend in snakes, especially in Alethinophidia. In snake mitogenomes, the rearrangements mainly involved three processes, gene loss, translocation and duplication. Within Scolecophidia, the OL was lost several times in Typhlopidae and Leptotyphlopidae, but persisted as a plesiomorphy in the Alethinophidia. Duplication of the control region and translocation of the tRNALeu gene are two visible features in Alethinophidian mitochondrial genomes. Independently and stochastically, the duplication of pseudo-Pro (P*) emerged in seven different lineages of unequal size in three families, indicating that the presence of P* was a polytopic event in the mitogenome. CONCLUSIONS: The WANCY tRNA gene cluster and the control regions and their adjacent segments were hotspots for mitogenome rearrangement. Maintenance of duplicate control regions may be the source for snake mitogenome structural diversity.

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.

Assessing species boundaries and the phylogenetic position of the rare Szechwan ratsnake, Euprepiophis perlaceus (Serpentes: Colubridae), using coalescent-based methods.

Delimiting species and clarifying phylogenetic relationships are the main goals of systematics. For species with questionable taxonomic status, species delimitation approaches using multi-species coalescent models with multiple loci are recommended if morphological data are unavailable or unhelpful. Moreover, these methods will also reduce subjectivity based on genetic distance or requirement of monophyletic genetic lineages. We determine the validity and phylogenetic position of a rare and long controversial species of Chinese reptile, the Szechwan ratsnake (Euprepiophis perlaceus), using multi-locus data from multiple individuals and coalescent-based approaches. Species were first delimited using Bayesian Phylogenetics & Phylogeography (BP&P), Brownie and Bayes Factor model comparison approaches, while relationships among species were estimated using species tree inference in (*)BEAST. Results indicate that Euprepiophis perlaceus is a distinct species sister to Euprepiophis mandarinus. Despite gene tree discrepancy, the coalescent model-based approaches used here demonstrate the taxonomic validity and the phylogenetic position of Euprepiophis perlaceus. These approaches objectively test the validity of questionable species diagnoses based on morphological characters and determine their phylogenetic position.

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.

A new species of Hemiphyllodactylus (Squamata, Gekkonidae) from southwest Yunnan, China.

A new species of gekkonid, Hemiphyllodactylusgengmaensissp. nov., is described based on six specimens from Gengma Dai and Wa Autonomous County, Yunnan, China. The new species can be distinguished from its congeners by a significant genetic divergence of greater than 9.7% in the mitochondrial ND2 gene and a combination of the following characters: a maximum SVL of 43.24mm; 8 or 9 chin scales; six circumnasal scales; 2 or 3 internasal scales; 9-11 supralabial scales; 8 or 9 infralabial scales; 11-18 dorsal scales; 8-10 ventral scales; a manual lamellar formula of 5-5-5-4 or 5-6-5-4 and a pedal lamellar formula of 5-5-6-5; 20-25 precloacal and femoral pore-bearing scales contiguous in males; dark postorbital stripes or striping on body; dark dorsal transverse blotches present; and a brown postsacral mark bearing anteriorly projecting arms. The discovery of this new species brings the number of Hemiphyllodactylus species in China to 15.

Description of a New Species of the Pareas hamptoni Complex from Yunnan, China, with Confirmation of P. hamptoni Sensu Stricto in China (Squamata, Pareidae).

We describe a new species of the genus Pareas, based on three specimens collected from Guanyinshan Provincial Nature Reserve in Yuanyang County, Honghe Prefecture, Yunnan Province, China. The new species is distinguished from its congeners by one preocular, one postocular or postocular fused with subocular, loreal not bordering the orbit, one row enlarged vertebral scales, five rows keeled mid-dorsal scales at the middle of the body, 189-192 ventral scales and 72-89 subcaudal scales. The dorsal surfaces of the head and body are yellowish red or yellowish brown, and the belly and ventral surfaces of the head and tail are pinkish yellow or yellow with more or less small black spots. Phylogenetic analyses of mitochondrial DNA recovered the new species being the sister taxon to P. hamptoni sensu stricto. The genetic divergences between the new species and P. hamptoni sensu stricto were 4.2% in the Cyt b sequences and 5.0% in the ND4 sequences. In addition, based on specimens collected from Honghe and Wenshan prefectures, we confirmed that P. hamptoni sensu stricto is distributed in China.

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.

Four new species of the genus Diploderma Hallowell, 1861 (Squamata, Agamidae) from China.

Four new species of Diploderma are described from Sichuan and Yunnan provinces, southwestern China, based on an integrative taxonomic approach, combining morphological and genetic data. The first new species from Danba County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.flaviceps, but it can be diagnosed from the latter by having a relatively much shorter tail and by a genetic distance of 4.4% in the ND2 gene; the second new species from Muli County, Sichuan Province, is phylogenetically closely related to D.daochengense, D.yongshengense, and D.yulongense, but it can be diagnosed from the latter three species by having a pale yellow gular spot and by genetic distances of 5.6-6.7% in the ND2 gene; the third new species from Jiulong County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.angustelinea, but it can be diagnosed from the latter by having a relatively much longer tail and by a genetic distance of 2.8% in the ND2 gene; and the last new species from Weixi County, Yunnan Province, is phylogenetically closely related to D.aorun, but it can be diagnosed from the latter by having a pale yellow gular spot and by a genetic distance of 2.9% in the ND2 gene. Our work brings the number of species within the genus Diploderma to 46.

A new species of Cyrtodactylus Gray, 1827 (Squamata, Gekkonidae) from southwestern Yunnan, China.

A new species of the Cyrtodactyluschauquangensis species group is described based on four specimens collected from the karst formations of Menglian County, Puer City, Yunnan Province, China. The new species can be separated from all other congeners by having a unique combination of morphological characters: a medium-sized body; ventrolateral folds present with interspersed small tubercles; seven precloacal pores in a continuous series in males, absent in females; enlarged femoral scales and femoral pores absent; two postcloacal tubercles on each side; and one or two rows of enlarged subcaudals. Genetically, the new species most closely related to C.wayakonei and the uncorrected sequence divergences of the ND2 gene and its flanking tRNAs between the new species and investigated congeners range from 7.2% to 18.4%.

Three new species of Diploderma Hallowell, 1861 (Squamata, Agamidae) from the Hengduan Mountain Region, south-western China.

Three new species of Diploderma are described from the Hengduan Mountain Region in south-western China, based on morphological and genetic data. The first new species from Yulong County, Yunnan Province is morphologically most similar and phylogenetically closely related to D.brevicauda, but it can be diagnosed from the latter by having a relatively longer tail; the second new species from Xiangcheng County, Sichuan Province is phylogenetically closely related to D.bowoense, but it can be diagnosed from the latter by the absence of a distinct gular spot; and the third new species from Yongsheng County, Yunnan Province is phylogenetically closely related to D.yulongense, but it can be diagnosed from the latter by having different colourations of the ventral and ventrolateral surfaces of the body. Taxonomy and diversity survey are the basis of species conservation, our discoveries contributing to better conservation of the species of this genus.

Confirmation of Tylototritonziegleri Nishikawa, Matsui & Nguyen, 2013 in China, with discussion on the relationship between T.verrucosus Anderson, 1871 and T.panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 (Caudata, Salamandridae).

Background: The distribution of the Ziegler's Crocodile Newt Tylototritonziegleri Nishikawa, Matsui & Nguyen, 2013 in China has been controversial. This species was originally recorded uncertainly from Guangxi Autonomous Region, China. Subsequently, this species was recorded from Yunnan and Guangdong provinces, China. Thereafter, the record from Guangdong was denied and the record from Yunnan was questioned. New information: Two specimens of Tylototriton Anderson, 1871 were collected from Wenshan Prefecture, Yunnan Province, China, in 2020. Phylogenetically, the sequences of these two specimens clustered with the sequences of T.ziegleri (including the holotype) from its type locality with strong support, and morphologically agree well with the original description of T.ziegleri. We confirm the record of T.ziegleri in China and present detailed collection site and morphological description of the specimens from China. In addition, we found that T.panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 may be the synonym of T.verrucosus Anderson, 1871. We discussed the relationship between T.verrucosus and T.panwaensis.

First record of Calotesvindumbarbatus Wagner, Ihlow, Hartmann, Flecks, Schmitz & Böhme, 2021 (Squamata: Agamidae) from China, with revised diagnosis of this species.

BACKGROUND: Three new species were recently described from the Calotesmystaceus Duméril & Bibron, 1837 complex. Of the three new species, C.vindumbarbatus Wagner, Ihlow, Hartmann, Flecks, Schmitz & Böhme, 2021 was known only from northern Myanmar. NEW INFORMATION: Seven specimens of lizard were collected from Tongbiguan Nature Reserve, western Yunnan, China. Phylogenetically, these specimens clustered with the type specimens of Calotesvindumbarbatus from Myanmar with strong support and showed inappreciable genetic divergence from the type specimens of C.vindumbarbatus. We report the first country record of C.vindumbarbatus from China. In addition, a supplementary description, based on the newly-collected specimens and revised diagnosis of this species, was provided.

Genomic insights into the evolution of the critically endangered soft-shelled turtle Rafetus swinhoei.

The 2019 death of the female Swinhoes soft-shelled turtle (Rafetus swinhoei) individual in China put this species in a dire conservation state. To avoid the loss of genetic information from the female individual and facilitate the protection and preservation of R. swinhoei, we sequenced and assembled its draft chromosome-level genome by combining BGISEQ short reads, Nanopore long reads, and Hi-C sequencing reads. We characterized its potential ZZ/ZW sex determination system. Phylogeny and divergence time analyses indicated that R. swinhoei has a close relationship with Pelodiscus sinensis, and they shared a common ancestor ~54.4 Ma. Population history analyses indicated that R. swinhoei has the lowest heterozygosity among all turtles examined, and its population size has declined dramatically over the past 10 million years. Many core genes involved in autophagy and DNA damage response, such as Rab5a and Parp1, were under positive selection and/or evolving rapidly, and these genes may make important contributions to the long lifespan and evolutionary adaptation of R. swinhoei. The loss of several tooth-related genes in turtle genomes explains the genetic basis of their toothless phenotype. This study not only provides a genomic resource for scientists to study the species using the strategy of comparative genomics, but also provides a data basis for us to find more potential R. swinhoei individuals in the wild.

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.

Complete Mitogenome of Oreolalax Omeimontis Reveals Phylogenetic Status and Novel Gene Arrangement of Archaeobatrachia.

Species of the genus Oreolalax displayed crucial morphological characteristics of vertebrates transitioning from aquatic to terrestrial habitats; thus, they can be regarded as a representative vertebrate genus for this landing phenomenon. But the present phylogenetic status of Oreolalax omeimontis has been controversial with morphological and molecular approaches, and specific gene rearrangements were discovered in all six published Oreolalax mitogenomes, which are rarely observed in Archaeobatrachia. Therefore, this study determined the complete mitogenome of O. omeimontis with the aim of identifying its precise phylogenetic position and novel gene arrangement in Archaeobatrachia. Phylogenetic analysis with Bayesian inference and maximum likelihood indicates O. omeimontis is a sister group to O. lichuanensis, which is consistent with previous phylogenetic analysis based on morphological characteristics, but contrasts with other studies using multiple gene fragments. Moreover, although the duplication of trnM occurred in all seven Oreolalax species, the translocation of trnQ and trnM occurred differently in O. omeimontis to the other six, and this unique rearrangement would happen after the speciation of O. omeimontis. In general, this study sheds new light on the phylogenetic relationships and gene rearrangements of Archaeobatrachia.

A new species of the genus Pareas (Squamata, Pareidae) from Yunnan, China.

A new species of Pareas is described from Yunnan Province, China, based on morphological comparisons and molecular data. Genetically, the new species is most closely related to the recently-described Pareas geminatus, for which we present new topotypic findings. The genetic divergence (uncorrected p-distance) of the cytb gene between the new species and congeners ranged from 6.14% to 24.68%. Morphologically, the new species can be distinguished from P. geminatus and all other congeners. Our work brings the total number of species within the genus Pareas to 22.

A new species of Cyrtodactylus Gray, 1827 (Squamata, Gekkonidae) from Yunnan, China.

A new species of Cyrtodactylus is described on the basis of five specimens collected from the karst formations of Zhenkang County, Yunnan Province, China. Cyrtodactylus zhenkangensis sp. nov. is recognized by having a unique combination of morphological characters, the most diagnostic being: 12-15 enlarged femoral scales on each thigh; 2-5 femoral pores on each thigh in males, 0-3 pitted scales on each thigh in females; eight or nine precloacal pores in a continuous row or separated by one poreless scale in males, 7-9 pitted scales in females; subcaudals enlarged, arranged alternately as single and double on anterior and mostly single at middle and posterior; dorsal surface of head with obvious reticulations. Phylogenetic analyses show that the new species is a member of the C. wayakonei species group and a sister taxon to a clade consisting of C. wayakonei and C. martini based on Maximum Likelihood analyses and Bayesian Inference and differs from its congeners by at least 12.0% genetic divergence in a fragment of the COI gene.