A large-scale systematic framework of Chinese snakes based on a unified multilocus marker system.

Snakes are one of the most diverse groups of terrestrial vertebrates, with approximately 3500 extant species. A robust phylogeny and taxonomy of snakes is crucial for us to know, study and protect them. For a large group such as snakes, broad-scale phylogenetic reconstructions largely rely on data integration. Increasing the compatibility of the data from different researches is thus important, which can be facilitated by standardization of the loci used in systematic analyses. In this study, we proposed a unified multilocus marker system for snake systematics by conflating 5 mitochondrial markers, 19 vertebrate-universal nuclear protein coding (NPC) markers and 72 snake-specific noncoding intron markers. This marker system is an addition to the large squamate conserved locus set (SqCL) for studies preferring a medium-scale data set. We applied this marker system to over 440 snake samples and constructed the currently most comprehensive systematic framework of the snakes in China. Robust snake phylogenetic relationships were recovered at both deep and shallow evolutionary depths, demonstrating the usefulness of this multilocus marker system. Discordance was revealed by a parallel comparison between the snake tree based on the multilocus marker system and that based on only the mitochondrial loci, highlighting the necessity of using multiple types of markers to better understand the snake evolutionary histories. The divergence times of different snake groups were estimated with the nuclear data set. Our comprehensive snake tree not only confirms many important nodes inferred in previous studies but also contributes new insights into many snake phylogenetic relationships. Suggestions are made for the current Chinese snake taxonomy.

Large-scale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae).

Southeast Asia and southern China (SEA-SC) harbor a highly diverse and endemic flora and fauna that is under increasing threat. An understanding of the biogeographical history and drivers of this diversity is lacking, especially in some of the most diverse and threatened groups. The Asian leaf-litter frog genus Leptolalax Dubois 1980 is a forest-dependent genus distributed throughout SEA-SC, making it an ideal study group to examine specific biogeographic hypotheses. In addition, the diversity of this genus remains poorly understood, and the phylogenetic relationships among species of Leptolalax and closely related Leptobrachella Smith 1928 remain unclear. Herein, we evaluate species-level diversity based on 48 of the 53 described species from throughout the distribution of Leptolalax. Molecular analyses reveal many undescribed species, mostly in southern China and Indochina. Our well-resolved phylogeny based on multiple nuclear DNA markers shows that Leptolalax is not monophyletic with respect to Leptobrachella and, thus, we assign the former to being a junior synonym of the latter. Similarly, analyses reject monophyly of the two subgenera of Leptolalax. The diversification pattern of the group is complex, involving a high degree of sympatry and prevalence of microendemic species. Northern Sundaland (Borneo) and eastern Indochina (Vietnam) appear to have played pivotal roles as geographical centers of diversification, and paleoclimatic changes and tectonic movements seem to have driven the major divergence of clades. Analyses fail to reject an "upstream" colonization hypothesis, and, thus, the genus appears to have originated in Sundaland and then colonized mainland Asia. Our results reveal that both vicariance and dispersal are responsible for current distribution patterns in the genus.

A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae).

The horned toad assemblage, genus Megophrys sensu lato, currently includes three groups previously recognized as the genera Atympanophrys, Xenophrys and Megophrys sensu stricto. The taxonomic status and species composition of the three groups remain controversial due to conflicting phenotypic analyses and insufficient phylogenetic reconstruction; likewise, the position of the monotypic Borneophrys remains uncertain with respect to the horned toads. Further, the diversity of the horned toads remains poorly understood, especially for widespread species. Herein, we evaluate species-level diversity based on 45 of the 57 described species from throughout southern China, Southeast Asia and the Himalayas using Bayesian inference trees and the Generalized Mixed Yule Coalescent (GMYC) approach. We estimate the phylogeny using both mitochondrial and nuclear DNA data. Analyses reveal statistically significant mito-nuclear discordance. All analyses resolve paraphyly for horned toads involving multiple strongly supported clades. These clades correspond with geography. We resurrect the genera Atympanophrys and Xenophrys from the synonymy of Megophrys to eliminate paraphyly of Megophrys s.l. and to account for the morphological, molecular and biogeographic differences among these groups, but we also provide an alternative option. Our study suggests that Borneophrys is junior synonym of Megophrys sensu stricto. We provide an estimation of timeframe for the horned toads. The mitochondrial and nuclear trees indicate the presence of many putative undescribed species. Widespread species, such as Xenophrys major and X. minor, likely have dramatically underestimated diversity. The integration of morphological and molecular evidence can validate this discovery. Montane forest dynamics appear to play a significant role in driving diversification of horned toads.

A new species of the genus Rhabdophis Fitzinger, 1843 (Squamata: Colubridae) from Guangdong Province, southern China.

A new species, Rhabdophis guangdongensis sp. nov., is described from the Guangdong Province, China. It can be easily distinguished from other known congeners by cyt b and c-mos sequences, and by the following combination of morphological characters: body size small; head distinct from the neck; 20 maxillary teeth, the three most posterior teeth strongly enlarged, and not separated by diastemata from other teeth; six supralabials, the third and fourth touching the eye; seven infralabials, the first four in contact with anterior chin shields; dorsal scales in 15 rows throughout the body, weakly keeled, the outer row smooth; 126 ventrals; 39 paired subcaudals; anal scale divided; 44 pairs of narrow dorsolateral black cross-bars on body and 15 pairs on tail; body and tail with two dorsolateral longitudinal brownish-red lines, respectively with a series of white spots in cross-bars. The description of this new species brings the total number of described species of this genus to 21 and represents the tenth known Rhabdophis species in China.

Description of two new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from Heishiding Nature Reserve, Fengkai, Guangdong, China, based on molecular and morphological data.

Two new species, Megophrys acuta sp. nov. and Megophrys obesa sp. nov., are described based on a series of specimens collected from Heishiding Nature Reserve, Fengkai County, Guangdong Province, China. They can be distinguished from other known congeners occurred in southern and eastern China by morphological characters and molecular divergence in the mitochondrial 16S rRNA gene. M. acuta is characterized by small and slender body with adult females measuring 28.1-33.6 mm and adult males measuring 27.1-33.0 mm in snout-vent length; snout pointed, strongly protruding well beyond margin of lower jaw; canthus rostralis well developed and sharp; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the pupil of eye. M. obesa is characterized by stout and slightly small body with adult females measuring 37.5-41.2 mm, adult male measuring 35.6 mm in snout-vent length; snout round in dorsal view; canthus rostralis developed; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the posterior margin of eye. The discovery of these two new species further confirms that the diversity of this genus has been significantly underestimated. At present the genus Megophrys contains 56 species of which 35 species are distributed in China.

Contrasting nidification behaviors facilitate diversification and colonization of the Music frogs under a changing paleoclimate.

In order to cope with the complexity and variability of the terrestrial environment, amphibians have developed a wide range of reproductive and parental behaviors. Nest building occurs in some anuran species as parental care. Species of the Music frog genus Nidirana are known for their unique courtship behavior and mud nesting in several congeners. However, the evolution of these frogs and their nidification behavior has yet to be studied. With phylogenomic and phylogeographic analyses based on a wide sampling of the genus, we find that Nidirana originated from central-southwestern China and the nidification behavior initially evolved at ca 19.3 Ma but subsequently lost in several descendants. Further population genomic analyses suggest that the nidification species have an older diversification and colonization history, while N. adenopleura complex congeners that do not exhibit nidification behavior have experienced a recent rapid radiation. The presence and loss of the nidification behavior in the Music frogs may be associated with paleoclimatic factors such as temperature and precipitation. This study highlights the nidification behavior as a key evolutionary innovation that has contributed to the diversification of an amphibian group under past climate changes.

A new species of the Gekko japonicus group (Squamata: Sauria: Gekkonidae) from the border region between China and Vietnam.

We describe a new species of the genus Gekko on the basis of 25 specimens from southern China and northern Vietnam. Gekko adleri sp. nov. is distinguished from the remaining congeners by a combination of the following characters: size moderate (SVL < 80 mm); nares in contact with rostral; internasal single, smaller than supranasal; postmentals enlarged; interorbital scales between anterior corners of the eyes 27-36; dorsal tubercle rows 7-11; ventral scales between mental and cloacal slit 168-190; midbody scale rows 123-144; ventral scale rows 35-44; subdigital lamellae on first toe 11-14, on fourth toe 11-15; finger and toe webbing present at base; tubercles absent on upper surface of fore limbs; tubercles on tibia 0-8; precloacal pores 17-21 in males; postcloacal tubercle single; tubercles present on dorsal surface of tail base; subcaudals enlarged; dorsal surface of body with four or five narrow light bands between shoulder and sacrum.

Discovery of a new cryptic Achalinus Peters, 1869 (Serpentes, Xenodermidae) species from Hunan Province, China.

A new species, Achalinusshenisp. nov., from central Hunan Province is described, based on the results of molecular systematics and morphological characters according to five specimens. Our molecular phylogeny inferred from the mitochondrial CO1 gene fragment revealed that this new species is most closely related to A.yunkaiensis, but a considerable amount of genetic divergence exists between them (p-distance ranging from 5.8% to 6.4%) and much distinct genetic divergence exists compared with other known Achalinus species (p-distance ranging from 10.4% to 15.8%), supporting its validity. Morphologically, it can be distinguished from its congeners by: (1) dorsal scales strongly keeled, 23 rows throughout the body, the outmost row smooth and significantly enlarged; (2) tail relatively short, TaL/TL 0.183 ~ 0.224; (3) the suture between internasals subequal to the suture between prefrontals; (4) loreal one, subrectangular, LorH/LorL 0.53 ~ 0.57; (5) ventrals 161-170, anal entire, subcaudals 55-61, not paired; (6) the length of supraocular equal to or longer than the length of upper anterior temporal; and (7) vertebral line inconspicuous and subcaudal streak absent. Currently, 27 species of Achalinus are known in the world, amongst which 20 species are distributed in China. Moreover, a key to species of the genus Achalinus is provided in this study.

Generic classification of Asian horned toads (Anura: Megophryidae: Megophryinae) and monograph of Chinese species.

The subfamily Megophryinae, as a representative batrachian group of the Oriental Realm and one of the most diverse groups of amphibians, has attracted considerable attention due to continued conjecture regarding its generic classification and failure to reach a satisfactory consensus. China boasts the richest diversity of Asian horned toads, containing some two thirds of the total species cataloged. However, most species have a complicated taxonomic history, resulting in multiple misidentifications. As such, an overall clarification of historical records and regional checklists is required. In the current investigation, we established the phylogeny of the Asian horned toads and performed detailed examinations with redefinitions of several important morphological traits. Based on the phylogenetic relationships and morphological differences, we propose a new ten-genus classification for the Asian horned toad subfamily Megophryinae: i.e., Brachytarsophrys, Atympanophrys, Grillitschia, Sarawakiphrys gen. nov., Jingophrys gen. nov., Xenophrys, Megophrys, Pelobatrachus, Ophryophryne, and Boulenophrys. Revisions on the diagnosability, distribution, and content of each genus are provided. Furthermore, we present a careful review of the taxonomic history of Asian horned toad species from China and provide a monograph of congeners, including six species of Brachytarsophrys, four species of Atympanophrys, five species of Jingophrys gen. nov., 10 species of Xenophrys, two species of Ophryophryne, and 60 species of Boulenophrys. Finally, we discuss the importance of traditional morphological traits based on multiple populations in taxonomic work as well as taxonomic inflation caused by the genetic species delimitation.

Description of three new Boulenophrys species from eastern Guangdong, China, emphasizing the urgency of ecological conservation in this region (Anura, Megophryidae).

The hilly region in eastern Guangdong, China lacks comprehensive scientific investigations for decades, especially in terms of herpetofauna. In recent years, several highly threatened amphibians have been gradually discovered from this region. In this work, three new species of the genus Boulenophrys are described, which are endemic from only one or two known localities in eastern Guangdong. These discoveries enrich the diversity of Boulenophrys in eastern Guangdong. With the large number of threatened urodeles and anuran species occurring in this densely populated area, the unique herpetological diversity in eastern Guangdong is facing the impacts of habitat degradation and fragmentation, and conservation actions are urgently required.

A new species of Torrent frog (Anura, Ranidae, Amolops) from the Coastal Hills of Southeastern China.

The Amolops populations in the coastal hills in eastern Guangdong and southern Fujian, China, were controversially recorded as A. hongkongensis or A. daiyunensis before. In this study, based on the morphological examination and phylogenetic analysis of the specimens from these areas, a new species, Amolops teochew sp. nov., is described. Amolops teochew sp. nov. can be distinguished reliably from A. hongkongensis and A. daiyunensis by a combination of characteristics morphologically and distinct divergences genetically. The description of the new species highlights the Amolops diversity in the limited hilly region of southeastern China, which is remarkably higher than that in the more extensive inland region of southeastern China.

Four new species of Panophrys (Anura, Megophryidae) from eastern China, with discussion on the recognition of Panophrys as a distinct genus.

The diversity of Panophrys horned toads is considered highly underestimated with a large number of undescribed cryptic species. In this work, we describe four Panophrys species from eastern China which were proposed as cryptic species by molecular data in previous study, additionally provide new information on the biogeography of these four species. Panophrys daiyunensis sp. nov. from southern Fujian, Panophrys daoji sp. nov. from eastern Zhejiang, Panophrys sanmingensis sp. nov. from the hilly area among Fujian, Jiangxi and Guangdong, and Panophrys tongboensis sp. nov. from northeastern Jiangxi, can be distinguished from all recognized congeners by a combination of morphological characteristics. The descriptions of these four new species take the recognized species of Panophrys to 51, which is the largest genus within the Asian horned toads subfamily Megophryinae. Considered as an appropriate arrangement for the Asian horned toads currently and applied in this study to describe the new species, the generic recognition of Panophrys is also discussed.

A new species of Music frog (Anura, Ranidae, Nidirana) from Mt Daming, Guangxi, China.

Nidiranaguangxiensis sp. nov., a new music frog species, is proposed, based on a series of specimens collected from Mt Daming, Guangxi, southern China. The new species is close to N.yeae, N.daunchina, N.yaoica, and N.chapaensis from southwestern and south-central China and northern Indochina, while the relationships among these species remain unresolved. Nidiranaguangxiensis sp. nov. can be distinguished from all known congeners by the genetic divergences in the mitochondrial 16S and COI genes, the behavior of nest construction, the advertisement call containing 6-11 rapidly repeated regular notes, and a combination of morphological characteristics. Furthermore, the Nidirana populations recorded in Guangxi are clarified in this work, providing valuable new information on the knowledge of the genus Nidirana.

Three new species of the genus Boulenophrys (Anura, Megophryidae) from southern China.

The diversity of Asian horned toads is considered highly underestimated and to contain a large number of undescribed cryptic species. In this work, we describe three new species of Boulenophrys from south China, namely, Boulenophrys yaoshanensis sp. nov. from central Guangxi, Boulenophrys yingdeensis sp. nov. from northern Guangdong, and Boulenophrys yunkaiensis sp. nov. from western Guangdong. These three new species can be distinguished from all recognized congeners by a combination of morphological characteristics and significant genetic divergences. These descriptions increase the number of recognized species of Boulenophrys to 61. In addition, an updated checklist of the Asian horned toads of the subfamily Megophryinae is provided in this study.

A sheep in wolf's clothing: Elaphe xiphodonta sp. nov. (Squamata, Colubridae) and its possible mimicry to Protobothrops jerdonii.

Based on combined morphological and osteological characters and molecular phylogenetics, we describe a new species of the genus Elaphe that was discovered from the south slope of the Qinling Mountains, Shaanxi, China, namely Elaphe xiphodonta sp. nov. It is distinguished from the other congeners by a combination of the following characters: dorsal scales in 21-21-17 rows, the medial 11 rows keeled; 202-204 ventral scales, 67-68 subcaudals; two preoculars (including one subpreocular); two postoculars; two anterior temporals, three posterior temporals; reduced numbers of maxillary teeth (9+2) and dentary teeth (12); sharp cutting edges on the posterior or posterolateral surface of the rear maxillary teeth and dentary teeth; dorsal head yellow, three distinct markings on the head and neck; a distinct black labial spot present in supralabials; dorsum yellow, 46-49 complete (or incomplete) large black-edged reddish brown blotches on the body and 12-19 on the tail, two rows of smaller blotches on each ventrolateral side; ventral scales yellow with mottled irregular black blotches, a few irregular small red spots dispersed on the middle of the ventral. Based on molecular phylogenetic analyses, the new species forms the sister taxon to E. zoigeensis. The discovery of this new species increases the number of the recognized species in the genus Elaphe to 17.

A new species of the genus Lycodon (Serpentes, Colubridae) from Guangxi, China.

A new species of colubrid snake, Lycodon cathaya sp. nov., is described based on two adult male specimens collected from Huaping Nature Reserve, Guangxi, southern China. In a phylogenetic analyses, the new species is shown to be a sister taxon to the clade composed of L. futsingensis and L. namdongensis with low statistical support, and can be distinguished from all known congeners by the significant genetic divergence in the mitochondrial cytochrome b gene fragment (p-distance ≥ 7.9%), and morphologically by the following combination of characters: (1) dorsal scales in 17-17-15 rows, smooth throughout; (2) supralabials eight, third to fifth in contact with eye, infralabials nine; (3) ventral scales 199-200 (plus two preventral scales), subcaudals 78; (4) loreal single, elongated, in contact with eye or not, not in contact with internasals; (5) a single preocular not in contact with frontal, supraocular in contact with prefrontal, two postoculars; (6) maxillary teeth 10 (4+2+2+2); (7) two anterior temporals, three posterior temporals; (8) precloacal plate entire; (9) ground color from head to tail brownish black, with 31-35 dusty rose bands on body trunk, 13-16 on tail; (10) bands in 1-2 vertebral scales broad in minimum width; (11) bands separate ground color into brownish black ellipse patches arranged in a row along the top of body and tail; (12) elliptical patches in 3-6 scales of the vertebral row in maximum width; (13) ventral surface of body with wide brownish black strip, margined with a pair of continuous narrow greyish white ventrolateral lines. With the description of the new species, 64 congeners are currently known in the genus Lycodon, with 16 species occurring in China.

A new species of the Rana japonica group (Anura, Ranidae, Rana) from China, with a taxonomic proposal for the R. johnsi group.

Rana jiulingensis sp. nov., a new species from Hunan and Jiangxi, southeastern China, is described. The new species is assigned to the R. japonica group. The clade comprising R. jiulingensis sp. nov. and R. dabieshanensis from Anhui is the sister taxon of R. omeimontis from Sichuan. Rana jiulingensis sp. nov. can be distinguished by the significant divergences in the 16S and COI genes, and the combination of following morphological characters: body size medium, SVL 48.3-57.8 mm in adult males and 48.2-57.5 mm in adult females; dorsolateral fold straight; digits without circummarginal grooves; dorsal skin smooth; tibio-tarsal articulation reaching forward beyond the tip of snout; heels overlapping; webbing formula of toes: I 1⅓ - 2 II 1⅓ - 2⅓ III 1½ - 2⅔ IV 3 - 1⅓ V; absence of vocal sacs in males; and presence of creamy white nuptial pad with tiny hoar spines on the finger I and reddish tubercles on loreal and temporal regions in breeding males. Furthermore, based on our results and the previous literature, R. zhengi is synonymized with R. sangzhiensis, and a new species group, the Rana johnsi group, is proposed for the R. johnsi and R. sangzhiensis. Currently, the Rana contains 41 recognized species, and the phylogenetic placements of several species remain unresolved.

Four new species of Asian horned toads (Anura, Megophryidae, Megophrys) from southern China.

Recent phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtDNA genealogy has revealed a series of cryptic species of the subgenus Panophrys within genus Megophrys from southern and eastern China. This study demonstrates that the Panophrys specimens from the hilly areas among Guangdong, Guangxi and Hunan can be morphologically distinguished from all recognized congeners, thereby providing additional supports for the recognitions of four new species of Panophrys, namely Megophrys (Panophrys) mirabilis Lyu, Wang & Zhao, sp. nov. from northeastern Guangxi, Megophrys (Panophrys) shimentaina Lyu, Liu & Wang, sp. nov. from northern Guangdong, and Megophrys (Panophrys) xiangnanensis Lyu, Zeng & Wang, sp. nov. and Megophrys (Panophrys) yangmingensis Lyu, Zeng & Wang, sp. nov. from southern Hunan. The descriptions of these species take the number of Megophrys species to 101, 46 of which belong to the subgenus Panophrys.

Two new Leptobrachella species (Anura, Megophryidae) from the Yunnan-Guizhou Plateau, southwestern China.

Two new toad species of the genus Leptobrachella are described from the Yunnan-Guizhou Plateau of China, based on the combination of molecular and morphological data. The description of Leptobrachella aspera Wang, Lyu, Qi & Wang, sp. nov. from Huanglianshan Nature Reserve represents the thirteenth Leptobrachella species known from Yunnan Province, and the description of Leptobrachella dorsospina Wang, Lyu, Qi & Wang, sp. nov. from Yushe Forest Park represents the sixth Leptobrachella species known from Guizhou Province. These new discoveries further emphasize the extremely high diversity of the Leptobrachella toads in these regions.