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.

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.

Discovery of a new limestone karst-restricted odorous frog from northern Guangdong, China (Anura, Ranidae, Odorrana).

Karstic landscapes play an important role in biodiversity formation and often contain high levels of endemism. However, site-endemic taxa in karstic landscapes are being threatened by exploitation and weak legal protection. In this study, we describe Odorranaconcelata Wang, Zeng, & Lin, sp. nov., a limestone karst-restricted odorous frog from northern Guangdong, China. This new species shows distinctive genetic divergence and morphological differences from its congeners. Phylogenetic results suggest that the new species represents an independent lineage that is grouped with O.lipuensis and O.liboensis based on the mitochondrial 16S and 12S ribosomal RNA genes. We recommend the new species be listed as Vulnerable (VU) in the IUCN categorization as it is only known from the type locality with limited microhabitats and is threatened by habitat degradation.

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.

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.

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.

A new species of Nidirana from the N. pleuraden group (Anura, Ranidae) from western Yunnan, China.

The Music Frog genus Nidirana was recently resurrected as a distinct genus and contains 14 species distributed in subtropical eastern and southeastern Asia. The species diversity of Nidirana is dramatically underestimated, and half of its species was described in the last five years. In this study, Nidirana occidentalis sp. nov., a new species of Music Frog from western Yunnan, China, is proposed based on morphological and molecular evidences. The new species was previously misidentified as N. pleuraden, but can be distinguished from the true N. pleuraden from eastern Yunnan, and all other congeners, by a combination of morphological characteristics, and significant divergence in the mitochondrial genes (≥ 5.1% in 16S and ≥ 8.9% in CO1). Nidirana occidentalis sp. nov. is assigned to the N. pleuraden group on the basis of morphological characters, but its phylogenetic placement remains unresolved due to weak branch support. Geographically, these two species are isolated by the Red River in Yunnan, supporting the hypothesis that the Red River is an important geographical barrier that drives speciation in flora and fauna. Nidirana occidentalis sp. nov. represents the second species of N. pleuraden group and the 15th species of the genus.

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.

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.

Re-examination of the Chinese record of Opisthotropis maculosa (Squamata, Natricidae), resulting in the first national record of O. haihaensis and description of a new species.

The taxonomic status of the previous record of Opisthotropis maculosa Stuart & Chuaynkern, 2007 from Guangdong and Guangxi, southern China, is revised based on the comparison of morphological and molecular data collected from the Chinese specimens and the holotype of O. maculosa from Thailand and O. haihaensis Ziegler, Pham, Nguyen, Nguyen, Wang, Wang, Stuart & Le, 2019 from Vietnam. Results reveal that the population from Shiwandashan Nature Reserve in southern Guangxi, China belongs to O. haihaensis, and represents the first national record for China; the populations from western Guangdong and southeastern Guangxi are described as a new species, Opisthotropis hungtai sp. nov. We suggest that O. maculosa should be removed from the Chinese herpetofauna checklist. The new national record of O. haihaensis and the description of the new species bring the total number of Opisthotropis to 13 in China.

Phylogenetic relationships of the Chinese torrent frogs (Ranidae: Amolops) revealed by phylogenomic analyses of AFLP-Capture data.

The torrent frog genus Amolops contains nearly sixty species distributed in swift mountain streams throughout southeast Asia. The taxonomy of this genus has proven complicated due to unstable morphological diagnostic characters. The relationships of Amolops species and species groups were not readily resolved with a small number of molecular markers. Here, we applied the novel AFLP-Capture approach and acquired two large datasets (242 anonymous nuclear sequences and the mitochondrial genome) from 70 Chinese Amolops samples to study their relationships. The phylogenies inferred from the nuclear data and the mitochondrial data were both robust and revealed a primary phylogenetic split between eastern and western Chinese Amolops species. The relationships of the six species groups were clarified. While the three species groups in east China (the A. ricketti, A. daiyunensis and A. hainanensis groups) were monophyletic, the three species groups in the west (the A. mantzorum, A. monticola and A. marmoratus groups) were not monophyletic, suggesting a need for further investigation and revision. The robust phylogenies also provided new insights into species relationships, especially for the A. mantzorum group, which has been difficult to resolve due to multiple speciation events occurring approximately 7-8 million years ago. The divergence times estimated with the nuclear data indicated that the ancestor of the Chinese Amolops appeared in the late Eocene or early Oligocene, and that speciation events in the Chinese Amolops were often related to geological events (e.g. the uprising of mountains and the formation of islands). By including the mitochondrial sequences from GenBank, a more comprehensive Amolops phylogeny was constructed that reflected the origin of the Chinese Amolops. Based on all these results, a dispersal scenario of the torrent frogs was hypothesized. Our research serves as the first example of using AFLP-Capture to obtain a large amount of data for shallow-scale phylogenetic and taxonomic studies, which should be useful for other nonmodel organism groups.

A new species of the genus Achalinus from southwestern Guangdong Province, China (Squamata: Xenodermatidae).

A new species of xenodermatid snake, Achalinus yunkaiensis J. Wang, Y. Li Y.Y. Wang, sp. nov. was described based on a series of specimens collected from Dawuling Forestry Station located in the Yunkai Mountains of western Guangdong Province. It can be distinguished from known congeners by a significant genetic divergence at the mitochondrial CO1 gene fragment examined (p-distance ≥ 12.0%) and the following combination of characters: (1) dorsal scales strongly keeled, in 23 rows throughout the body, the most outer row on each side smooth and significantly enlarged; (2) tail relatively shorter, TaL/TL ratio 18.5-20.0%; (3) maxillary teeth 20-22; (4) length of suture between internasals subequal to that between the prefrontals; (5) nasal divided into two sections by nasal cleft, posterior one half as long as anterior; (6) loreal elongated, nearly twice as wide as high; (7) supralabials six; (8) infralabials six; (9) temporals 2+2+3 (rarely 2+2+4), the two anterior temporals in contact with eye; (10) ventrals 151-162, subcaudals 49-56 arranged in single row, not paired; (11) cloacal entire; (12) uniform brown (in adults) or black (in juveniles) above, tinged weakly iridescent, with a longitudinal dark-colored vertebral line; (13) light brown (in adults) or greyish white (in juveniles) beneath; and (14) dorsum with a longitudinal dark brown vertebral stripe from posterior margin of parietals to tail tip. Currently, 13 species are known in the genus Achalinus, with seven from mainland China.

A new species of Amolops (Anura: Ranidae) from China, with taxonomic comments on A. liangshanensis and Chinese populations of A. marmoratus.

Amolops shuichengicus sp. nov., a new species of the A. mantzorum group is described from Guizhou, southwest China, on the basis of significant molecular divergences in 16S + CO1 genes and the combination of morphological characteristics: small body size, SVL 34.6-39.6 mm in adult males and 48.5-55.5 mm in adult females; dorsal skin relatively smooth; presence of vomerine teeth; presence of cream maxillary gland from lower edge of eye to the anterior of supratympanic fold; presence of supratympanic folds and glandular dorsolateral folds; tympanum indistinct; absence of a circummarginal groove on the disk of the first finger; presence of supernumerary tubercles below the base of fingers III and IV; absence of outer metatarsal tubercle and tarsal glands; males without vocal sacs. In addition, evidenced by the phylogenetic analyses in this study and literature data, we suggest that A. liangshanensis should be synonymized with A. loloensis and the records of A. marmoratus in Yunnan, China should be referred to A. afghanus. Following our proposal, the genus Amolops contains 57 species, with 32 recorded from China.

Description of six new species of the subgenus Panophrys within the genus Megophrys (Anura, Megophryidae) from southeastern China based on molecular and morphological data.

The diversity of the subgenus Panophrys within the genus Megophrys has been revealed to be extremely underestimated from southeastern China. Herpetological surveys coupled with extensive sampling in a longitudinal mountain belt located in southeastern China resulted in the discoveries of six new species of the subgenus Panophrys. Furthermore, the new discoveries support the findings of "micro-endemism", "sympatric phenomenon" and "sympatric but distant phylogenetically" which appear to be common among Panophrys species, and also indicates that the Asian horned toads would be good candidates for studies on speciation and biogeography, and additionally emphasizes the conservation difficulties of these toads.

Sequence capture using AFLP-generated baits: A cost-effective method for high-throughput phylogenetic and phylogeographic analysis.

Target sequence capture is an efficient technique to enrich specific genomic regions for high-throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP-based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome-scale data (hundreds or thousands of loci).

Description of a new species of Gracixalus (Amphibia: Anura: Rhacophoridae) from Guangdong Province, southeastern China.

A new species of tree frog, G. guangdongensis sp nov., is described based on a series of specimens collected from Dawuling Forest Station, Mount Nankun and Nanling Nature Reserve of Guangdong Province, southeastern China. The new species is distinguished from all known congeners by a significant genetic divergence at the mitochondrial 16S rRNA gene fragment examined (p-distance ≥ 4.6%) and the following combination of morphological characters: relatively small body size (SVL 26.1-34.7 mm in adult males, 34.9-35.4 mm in adult females); upper eyelid and dorsum lacking spines; supratympanic fold and tympanum distinct; dorsal and lateral surface rough, sparsely scattered with tubercles; ventral skin granular; tibiotarsal projection absent; toe-webbing moderately developed, finger webbing rudimentary; heels slightly overlapping when flexed hindlimbs are held at right angles to the body axis; brown to beige above, with an inverse Y-shaped dark brown marking extendeing from the interorbital region to the centre of the dorsum; males with a single subgular vocal sac and protruding nuptial pads with minute granules on the dorsal surface of the base of first finger. The discovery and description of Gracixalus guangdongensis sp. nov. represents the 14th species known in this genus.

A new species of Amolops (Anura: Ranidae) from southwestern Guangdong, China.

A new species, Amolops yunkaiensis sp. nov. is described based on a series of specimens from Ehuangzhang Nature Reserve and Yunkaishan Nature Reserve, southwestern Guangdong Province, China. The new species can be distinguished from all known congeners by molecular divergence in the mitochondrial 16S rRNA, 12S rRNA and CO1 genes, and a combination of the following characters: relatively small body size, SVL 31.8-34.1 mm in adult males, 35.2-39.0 mm in adult females; numerous raised large warts on dorsum and flanks; dorsal body olive-brown or light brown with dark brown blotches; absence of vomerine teeth; absence of tarsal glands; presence of a pair of subgular vocal sacs, nuptial spines on the first finger, and sparse translucent tubercles on the lower jaw, forechest, posterior belly and ventral thigh in male. Hence, the genus Amolops contains 52 species, 29 of which occur in China.