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.

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs.

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.

First national record of Microhylahmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler and Nguyen, 2022 (Anura, Microhylidae, Microhyla) in China.

Background: To date, 10 species of the genus Microhyla have been recorded in China, of which six were distributed in Yunnan Province. Microhylahmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler, and Nguyen, 2022 was also speculated to be distributed in Xishuangbana, Yunnan Province, China. However, there is no evidence of documentation of M.hmongorum. New information: We report the first country record of Microhylahmongorum, based on specimens collected from Yunnan border region. Morphologically, the specimen was consistent with the original descriptions of M.hmongorum. Phylogenetically, the sequences of the specimens from China clustered with the sequence of type specimens of M.hmongorum from Vietnam, with uncorrected pairwise distances of 0.9% at the 16S gene fragment analysed. Therefore, we report M.hmongorum as a new record species in China.

Confirmation of Leptobrachellaventripunctata (Fei, Ye, and Li, 1990), based on molecular and morphological evidence in Thailand.

BACKGROUND: Thailand is considered a global biodiversity hotspot that is known to harbour a striking diversity of endemic species. However, several research studies have determined that the level of amphibian diversity in the country has been significantly underestimated. The megophryid genus Leptobrachella Smith, 1925 is currently known to include 89 species that are primarily distributed throughout southern China and Southeast Asia; however, only seven species have been found in Thailand. NEW INFORMATION: Based on an integrative approach encompassing genetic and morphological analyses, we have concluded that the population identified from Chiang Rai Province of Thailand is conspecific with Leptobrachellaventripunctata (Fei, Ye, and Li, 1990). Importantly, this is the first confirmation record of this species, based on molecular and morphological evidence in Thailand. The discovery of this species reaffirms that the diversity within the genus has been underestimated with many species yet to be discovered. In addition, the findings of our study further highlight the lack of existing knowledge on amphibian taxonomy and an underestimation of the biodiversity that exists along these national border areas.

Integrative taxonomy reveals a new species of Leptobrachella (Anura: Megophryidae) from the mountains of northern Thailand.

The genus Leptobrachella is one of the most speciose and taxonomically troubling groups of Asian anurans. Herein, we describe a new species of Leptobrachella from Doi Inthanon, Chiang Mai Province, Thailand based on the integration of morphological, acoustic and molecular data. The new species, Leptobrachella murphyi sp. nov., is morphologically distinguished from its congeners on the basis of body size, dorsal skin texture and coloration, ventral coloration and pattern, degree of webbing and fringing on the fingers and toes, iris coloration in life, pattern of markings on flanks and pattern of body macroglands. It also differs from its phylogenetically close congeners by an uncorrected p-distance of >9.6% for a fragment of 16S rRNA. The advertisement call of the new species consists of 4.54.7 kHz (at 15 C) and without a distinct introductory note. Leptobrachella murphyi sp. nov. likely occurs across the Thanon Thong Chai Range and analyses provide evidence of unknown biodiversity and species composition on Doi (mountain) Inthanon. In addition, the congeneric species L. minima was also confirmed in Doi Inthanon. The coexistence pattern of Leptobrachella in Doi Inthanon deserves further study. As Thailands highest mountain and biodiversity reservoir, the need for further biological exploration is urgent given ongoing habitat loss and degradation.

Molecular phylogeny and morphological comparisons of the genus Hebius Thompson, 1913 (Reptilia: Squamata: Colubridae) uncover a new taxon from Yunnan Province, China, and support revalidation of Hebius septemlineatus (Schmidt, 1925).

We describe a new species of the genus Hebius and provide evidence for the validity of H. septemlineatuscomb. nov. Morphological and molecular analyses of Hebius specimens collected in Yunnan Province, China, revealed three distinct lineages, namely the newly described Hebius weixiensissp. nov., as well as H. octolineatus (Boulenger, 1904), and H. septemlineatuscomb. nov. (Schmidt 1925), which is removed from synonymy with H. octolineatus. Based on mitochondrial genealogy, Hebius weixiensissp. nov. is sister to H. septemlineatuscomb. nov., while H. octolineatus is sister to H. bitaeniatus. The new species and H. septemlineatuscomb. nov. showed considerable genetic divergence from their recognized congeners (uncorrected P-distance ≥3.9%). Furthermore, the new species and H. septemlineatuscomb. nov. can be diagnosed from closely related congeners by a combination of pholidosis characters.

A new snake species of the genus Gonyosoma Wagler, 1828 (Serpentes: Colubridae) from Hainan Island, China.

A new species of the genus Gonyosoma Wagler, 1828 is described herein based on six specimens from the Diaoluoshan Mountains, Hainan Island, Hainan Province, China. The new species, Gonyosoma hainanensesp. nov., is most similar to its continental sister species, Gonyosoma boulengeri (Mocquard, 1897). Both taxa have a scaled protrusion on the anterior portion of the rostrum, distinct from other congeners. However, Gonyosoma hainanensesp. nov. can be distinguished from G. boulengeri by two significant morphological characters: (1) black orbital stripe absent in adults (vs. present in G. boulengeri); and (2) two loreals (vs. one loreal in G. boulengeri). The new species is also genetically divergent and forms a unique clade from its sister species and all other congeners based on sequences of the mitochondrial gene cytochrome b (cyt b).

Multi-locus genetic analyses of Quasipaa from throughout its distribution.

Montane frogs of the genus Quasipaa Dubois, 1992 occur from southern China to Southeast Asia (Frost 2021). Analyses of mtDNA (Cytb) and nuDNA data (Rag1, Rag2, Rhod, Tyr) for samples from 93 localities throughout its distribution yield a phylogeny. Clades A and B occur in Southeast Asia, clade C in northern Yangtze River, China, clade D in southwestern China, and clades E and F in southeastern China. Results place Q. yei within monophyletic Quasipaa and identify two new species. Based on nuDNA data, the basal split of clade A and B indicates an Indochinese origin of Quasipaa. The west-east diversification of five species across South China (Q. spinosa, Q. exilispinosa, Q. jiulongensis, Q. shini, Q. boulengeri) corresponds to topographic terrains II and III of China. Divergence of species from southeastern China (Q. shini, Q. jiulongensis, Q. spinosa, Q. exilispinosa) and southwestern China (Q. boulengeri) dates to 15.30-16.56 Ma (million years ago). A principal component analysis (PCA) and t-test involving 19 bioclimatic variables identifies significantly different environmental conditions between the two regions. Species' distribution models (SDM) for Q. spinosa and Q. boulengeri identify the best areas to be eastern and western South China, respectively. Thus, environmental variation appears to have influenced the genetic divergence and distributions of Quasipaa in South China. Mito-nuclear discordance indicates that some individuals of Q. exilispinosa and Q. spinosa hybridized historically.

Two new species and a new country record of the genus emAchalinus/em (Reptilia: Squamata: Xenodermidae) from China.

Combining the results from morphological and molecular analyses, we explore the taxonomy of the genus Achalinus from Southwest China. As a result, we describe two new species, A. panzhihuaensis sp. nov. and A. yangdatongi sp. nov. from southern Sichuan and southern Yunnan provinces, respectively, and we record a new country record, A. emilyae, from Guangxi Zhuang A. R.. The mitochondrial genealogy suggests that A. panzhihuaensis sp. nov. is sister to A. meiguensis, while A. yangdatongi sp. nov. clusters with the sister species A. juliani and A. ater. Both new species show considerable genetic divergence from their recognized congeners (uncorrected p-distance 6.2 % in COI gene). Furthermore, both new species can be diagnosed from closely related congeners by a combination of pholidosis characters. With our discovery, we provide a revised key to the 13 species from China and discuss some of the remaining issues regarding the taxonomy of the genus in China.

New country records for three species of frog from Myanmar including two genera (Nasutixalus and Oreolalax).

Myanmar, a biodiversity hotspot, harbors a striking diversity and endemism of species. Despite this, its herpetofauna remains one of the least explored in continental Asia due to restrictions of crossing political boundaries and infrastructure in remote regions. Many species in adjacent China and India are hypothesized to occur in Myanmar but records are wanting. Recent fieldwork found the frogs Polypedates braueri, Nasutixalus jerdonii and Oreolalax jingdongensis there, and the latter two species represent new generic records for Myanmar. All major morphological characters of these populations match the original descriptions. In addition, our matrilineal genealogy based on DNA barcoding confirms their identities. Overall, these findings confirm that the amphibian diversity is underestimated and this has important implications for conservation. Analyses indicate that northern Myanmar is a biogeographic corridor for the Himalayas, southern China, and northeastern India.

How little is known about "the little brown frogs": description of three new species of the genus Leptobrachella (Anura: Megophryidae) from Yunnan Province, China.

Asian leaf-litter toads of the genus Leptobrachella represent a great anuran diversification in Asia. Previous studies have suggested that the diversity of this genus is still underestimated. During herpetological surveys from 2013 to 2018, a series of Leptobrachella specimens were collected from the international border areas in the southern and western parts of Yunnan Province, China. Subsequent analyses based on morphological and molecular data revealed three distinct and previously unknown lineages, which we formally describe as three new species herein. Among them, we describe a new species that occurs at the highest known elevation for Leptobrachella in China. Four species of Leptobrachella, including two new species, are found in the same reserve. Furthermore, our results suggest that the population from Longchuan County, Yunnan, may represent an additional new species of Leptobrachella, although we tentatively assigned it to Leptobrachella cf. yingjiangensis due to the small sample size examined. Lastly, we provide the first description of females of L. yingjiangensis. Our results further highlight that both micro-endemism and sympatric distributions of species are common patterns in Leptobrachella, that contribute to taxonomic and conservation challenges in these frogs. We provide an identification key for Leptobrachella known to occur in Yunnan. Given the lack of knowledge on species diversity of Leptobrachella along international border areas, we recommend that future studies include trans-boundary collaborative surveys.

An integrative phylogenomic approach illuminates the evolutionary history of Old World tree frogs (Anura: Rhacophoridae).

Rhacophoridae are one of the most speciose and ecologically diverse families of amphibians. Resolution of their evolutionary relationships is key to understanding the accumulation of biodiversity, yet previous hypotheses based on Sanger sequencing exhibit much discordance amongst generic relationships. This conflict precludes the making of sound macroevolutionary conclusions. Herein, we conduct the first phylogenomic study using broad-scale sampling and sequences of 352 nuclear DNA loci obtained using anchored hybrid enrichment targeted sequencing. The robust time-calibrated phylogenetic hypothesis clarifies several long-disputed relationships and facilitates the testing of evolutionary hypotheses on spatiotemporal diversification and reproductive modes. The major extant lineages of Rhacophoridae appear to have radiated in mainland Asia, and the spatiotemporal process corresponds with several common accumulations of biodiversity in Asia. Analyses do not detect any case of "Out of Himalaya" in Rhacophoridae. All transitions of reproductive modes appear to have evolved in an ordered, gradual sequence associated with gaining independence of standing water for larval development. The different reproductive modes are phylogenetically conserved and the completion of their transitions appear to have occurred over a period of ~30 Ma, which does not fit a pattern of a rapid burst of diversification. Innovations in reproductive modes associate statistically with the uneven distribution of species-richness between clades, where higher diversification is linked to increased terrestrial modes of reproduction. These results strengthen the hypothesis that breeding innovations drive diversification by providing new opportunities for ecological release and dispersion.

A new species of the genus Raorchestes (Anura: Rhacophoridae) from Yunnan Province, China.

A new bush frog species, Raorchestes cangyuanensis sp. nov., from Cangyuan, Yunnan Province, China, is described based on morphological and molecular analyses. It differs from all known congeners by a combination of the following characters: body size small, adult snout-vent length (SVL) 16.1-20.0 mm in males (n=3); tympanum indistinct; tips of all fingers and toes expanded into discs with circummarginal grooves; rudimentary webbing between toes; fingers and toes with lateral dermal fringes; inner and outer metacarpal tubercles present; heels meeting when limbs held at right angles to body; crotch with a distinct black patch; discs of fingers and toes orange; male with external single subgular vocal sac and reddish nuptial pad at the base of first finger.

A new species of Mountain Dragon (Reptilia: Agamidae: Diploderma) from the D. dymondi complex in southern Sichuan Province, China.

Mountain Dragons of the genus Diploderma Hallowell, 1861 were recently resurrected from the paraphyletic genus Japalura sensu lato (Wang et al., 2019a). Despite the recent split, Diploderma still represents one of the most diverse groups of agamid lizard from Asia, including 25 species recognized currently, with most species found in China ((Wang et al., 2019a, 2019b). Although increasing attention has been paid to cryptic diversity within the genus in Southwest China during the past decade, most studies have focused on a single species complex, D. flaviceps, only (Manthey et al., 2012; Wang et al., 2015, 2016, 2017, 2019a), with few studies on other congeners that also have widespread distributions. One such example is D. dymondi (Boulenger, 1906).

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.

Efficacy of noninvasive evaluations in monitoring inflammatory bowel disease activity: A prospective study in China.

AIM: To optimize the efficacy of noninvasive evaluations in monitoring the endoscopic activity of inflammatory bowel disease (IBD). METHODS: Fecal calprotectin (FC), clinical activity index (CDAI or CAI), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and procalcitonin (PCT) were measured for 136 IBD patients. Also, FC was measured in 25 irritable bowel syndrome (IBS) patients that served as controls. Then, endoscopic activity was determined by other two endoscopists for colonic or ileo-colonic Crohn's disease (CICD) with the "simple endoscopic score for Crohn's disease" (SES-CD), CD-related surgery patients with the Rutgeerts score, and ulcerative colitis (UC) with the Mayo score. The efficacies of these evaluations to predict the endoscopic disease activity were assessed by Mann-Whitney test, χ2 test, Spearman's correlation, and multiple linear regression analysis. RESULTS: The median FC levels in CD, UC, and IBS patients were 449.6 (IQR, 137.9-1344.8), 497.9 (IQR, 131.7-118.0), and 9.9 (IQR, 049.7) µg/g, respectively (P < 0.001). For FC, CDAI or CAI, CRP, and ESR differed significantly between endoscopic active and remission in CICD and UC patients, but not in CD-related surgery patients. The SES-CD correlated closely with levels of FC (r = 0.802), followed by CDAI (r = 0.734), CRP (r = 0.658), and ESR (r = 0.557). The Mayo score also correlated significantly with FC (r = 0.837), CAI (r = 0.776), ESR (r = 0.644), and CRP (r = 0.634). For FC, a cut-off value of 250 µg/g indicated endoscopic active inflammation with accuracies of 87.5%, 60%, and 91.1%, respectively, for CICD, CD-related surgery, and UC patients. Moreover, clinical FC activity (CFA) calculated as 0.8 × FC + 4.6 × CDAI showed higher area under the curve (AUC) of 0.962 for CICD and CFA calculated as 0.2 × FC + 50 × CAI showed higher AUC (0.980) for UC patients than the FC. Also, the diagnostic accuracy of FC in identifying patients with mucosal inflammation in clinical remission was reflected by an AUC of 0.91 for CICD and 0.96 for UC patients. CONCLUSION: FC is the most promising noninvasive evaluation for monitoring the endoscopic activity of CICD and UC. CFA might be more accurate for IBD activity evaluation.

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.

Taxonomic revision of the Chinese Limnonectes (Anura, Dicroglossidae) with the description of a new species from China and Myanmar.

Phylogenetic reconstructions derived from DNA sequence data play a central role in documenting the number of species in a complex. Such analyses are pointing to the existence of many cryptic species, especially in poorly understood groups such as the genus Limnonectes, and the L. kuhlii species complex in particular. To understand the Limnonectes frogs of China, we reconstruct the major matrilineal genealogy of Limnonectes from China and Southeast Asia based on 12S rRNA, tRNAVal and 16S rRNA gene sequences. Based on new data we recognize five species of Limnonectes in China including L. bannaensis, L. fujianensis, L. fragilis, L. taylori (new record), and a new species from southern China and Myanmar. Phylogenetically, the new species is more closely related to the clade comprising L. taylori, L. megastomias, L. isanensis, L. nguyenorum, and L. jarujini from Thailand than to other Chinese species. This study supports previous findings of sympatric members of a species complex that are not each other's closest relatives.