Additive partitioning of multispecies distributional aggregation of local assemblages.

The distribution of species is not random in space. At the finest-resolution spatial scale, that is, field sampling locations, distributional aggregation level of different species would be determined by various factors, for example spatial autocorrelation or environmental filtering. However, few studies have quantitatively measured the importance of these factors. In this study, inspired by the statistical properties of a Markov transition model, we propose a novel additive framework to partition local multispecies distributional aggregation levels for sequential sampling-derived field biodiversity data. The framework partitions the spatial distributional aggregation of different species into two independent components: regional abundance variability and the local spatial inertia effect. Empirical studies from field amphibian surveys through line-transect sampling in southwestern China (Minya Konka) and central-southern Vietnam showed that local spatial inertia was always the dominant mechanism structuring the local occurrence and distributional aggregation of amphibians in the two regions with a latitudinal gradient from 1200 to nearly 4000 m. However, regional abundance variability is still nonnegligible in highly diverse tropical regions (i.e. Vietnam) where the altitude is not higher than 2000 m. In summary, we propose a novel framework that shows that the multispecies distributional aggregation level can be structured by two additive components. The two partitioned components could be theoretically independent. These findings are expected to deepen our understanding of the local community structure from the perspective of both spatial distribution and regional diversity patterns. The partitioning framework might have potential applications in field ecology and macroecology research.

Impact of Climate Change on the Distribution of Three Rare Salamanders (Liua shihi, Pseudohynobius jinfo, and Tylototriton wenxianensis) in Chongqing, China, and Their Conservation Implications.

The Wushan Salamander (Liua shihi), Jinfo Salamander (Pseudohynobius jinfo), and Wenxian Knobby Salamander (Tylototriton wenxianensis) are rare national Class II protected wild animals in China. We performed MaxEnt modeling to predict and analyze the potential distribution and trends of these species in Chongqing under current and future climate conditions. Species distribution data were primarily obtained from field surveys, supplemented by museum collections and the existing literature. These efforts yielded 636 records, including 43 for P. jinfo, 23 for T. wenxianensis, and 570 for L. shihi. Duplicate records within the same 100 m × 100 m grid cell were removed using ENMTools, resulting in 10, 12, and 58 valid distribution points for P. jinfo, T. wenxianensis, and L. shihi, respectively. The optimization of feature class parameters (FC) and the regularization multiplier (RM) were applied using R package "ENMeval 2.0" to establish the optimal model with MaxEnt. The refined models were applied to simulate the suitable distribution areas for the three species. The results indicate that the current suitable habitat area for L. shihi accounted for 9.72% of the whole region of the Chongqing municipality. It is projected that, by 2050, the proportion of suitable habitat will increase to 12.54% but will decrease to 11.98% by 2070 and further decline to 8.80% by 2090. The current suitable habitat area for P. jinfo accounted for 1.08% of the whole region of the Chongqing municipality, which is expected to decrease to 0.31%% by 2050, 0.20% by 2070, and 0.07% by 2090. The current suitable habitat area for T. wenxianensis accounted for 0.81% of the whole region of the Chongqing municipality, which is anticipated to decrease to 0.37% by 2050, 0.21% by 2070, and 0.06% by 2090. Human disturbance, climate variables, and habitat characteristics are the primary factors influencing the distribution of three salamander species in Chongqing. The proximity to roads significantly impacts L. shihi, while climate conditions mainly affect P. jinfo, and the distance to water sources is crucial for T. wenxianensis. The following suggestions were made based on key variables identified for each species: (1) For L. shihi, it is imperative to minimize human disturbances and preserve areas without roads and the existing vegetation within nature reserves to ensure their continued existence. (2) For P. jinfo, the conservation of high-altitude habitats is of utmost importance, along with the reduction in disturbances caused by roads to maintain the species' ecological niche. (3) For T. wenxianensis, the protection of aquatic habitats is crucial. Additionally, efforts to mitigate the impacts of road construction and enhance public awareness are essential for the preservation of this species and the connectivity of its habitats.

Gut microbiota reflect adaptation of cave-dwelling tadpoles to resource scarcity.

Gut microbiota are significant to the host's nutrition and provide a flexible way for the host to adapt to extreme environments. However, whether gut microbiota help the host to colonize caves, a resource-limited environment, remains unknown. The nonobligate cave frog Oreolalax rhodostigmatus completes its metamorphosis within caves for 3-5 years before foraging outside. Their tadpoles are occasionally removed from the caves by floods and utilize outside resources, providing a contrast to the cave-dwelling population. For both cave and outside tadpoles, the development-related reduction in their growth rate and gut length during prometamorphosis coincided with a shift in their gut microbiota, which was characterized by decreased Lactobacillus and Cellulosilyticum and Proteocatella in the cave and outside individuals, respectively. The proportion of these three genera was significantly higher in the gut microbiota of cave-dwelling individuals compared with those outside. The cave-dwellers' gut microbiota harbored more abundant fibrolytic, glycolytic, and fermentative enzymes and yielded more short-chain fatty acids, potentially benefitting the host's nutrition. Experimentally depriving the animals of food resulted in gut atrophy for the individuals collected outside the cave, but not for those from inside the cave. Imitating food scarcity reproduced some major microbial features (e.g. abundant Proteocatella and fermentative genes) of the field-collected cave individuals, indicating an association between the cave-associated gut microbiota and resource scarcity. Overall, the gut microbiota may reflect the adaptation of O. rhodostigmatus tadpoles to resource-limited environments. This extends our understanding of the role of gut microbiota in the adaptation of animals to extreme environments.

C15:0 and C17:0 partially mediate the association of milk and dairy products with bladder cancer risk.

The relationship between saturated fatty acids (SFA) and bladder cancer (BC) risk has been conflicting. Our aim was to investigate the relationship between erythrocyte membrane SFA and BC risk. A total of 404 participants were enrolled in the study (including 112 cases and 292 controls). A validated food frequency questionnaire was used to assess the food intake. The constitutive composition of fatty acids in the erythrocyte membrane was measured by gas chromatography. After adjustment for BC risk factors, SFA had no significant association with BC risk. However, C18:0 was positively linked with BC risk with an odds ratio (OR; 95% CI) of 2.99 (1.37-6.53). In contrast, very-long-chain saturated fatty acids (VLCSFA), especially C24:0, were negatively related to BC risk with an OR (95% CI) of 0.28 (0.12-0.65) for VLCSFA and 0.33 (0.15-0.75) for C24:0. Higher total odd-chain SFA (C15:0 and C17:0) were associated with a lower risk of BC with OR (95% CI) of 0.18 (0.076-0.44), 0.18 (0.068-0.47), 0.34 (0.14-0.81), respectively. After subgroup analysis, the protective effects C15:0 and C17:0 were still remained. Receiver operating characteristic analysis displayed that the combination of C15:0 and C17:0 indexes increased the accurate predictive rate of BC risk. Further mediation effect analysis showed that C15:0 and C17:0 could be used as partial mediation effectors for milk and dairy products and bladder carcinogenesis. Overall, the combination of odd-chain SFA (C15:0 and C17:0) in the erythrocyte membrane could serve as a reliable mediator and predictor, indicating a relationship between a high intake of milk and dairy products and a lower risk of BC.

A new Asian leaf litter toad of the genus Leptobrachella (Amphibia, Anura, Megophryidae) from central south China.

A new species of the Asian leaf litter toad genus Leptobrachella from central south China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and nuclear RAG1 gene sequences indicated the new species as an independent clade in the genus. The new species could be distinguished from its congeners by a combination of the following characters: body of medium size (SVL 29.2-34.2 mm in 15 adult males and 34.4-43.1 mm in seven adult females); distinct black spots present on flanks; toes rudimentary webbed, with wide lateral fringes; ventral belly white with distinct nebulous brown speckling on ventrolateral flanks; skin on dorsum shagreened with fine tiny granules or short ridges; iris copper above, silver below; heels overlapped when thighs are positioned at right angles to the body; tibia-tarsal articulation reaches the middle eye; dorsal surface of tadpole semi-transparent light brown, spots on tail absent, keratodont row formula I: 3+3/2+2: I; call series basically consist of repeated long calls, at dominant frequency (5093 ± 412 Hz).

A wide hybrid zone mediated by precipitation contributed to confused geographical structure of Scutiger boulengeri.

Confused geographical structure of a population and mitonuclear discordance are shaped by a combination of rapid changes in population demographics and shifts in ecology. In this study, we generated a time-calibrated phylogeny of Scutiger boulengeri, an endemic Xizang alpine toad occurring in mountain streams on the Qinghai-Xizang (Tibet) Plateau (QTP). Based on three mitochondrial DNA (mtDNA) genes, eight clades were assigned to three deeply divergent lineages. Analysis of nuclear DNA (nuDNA) genes revealed three distinct clusters without geographic structure, indicating significantly high rates of gene flow. Coalescent theory framework analysis (approximate Bayesian computation model DIYABC and Migrate-N) suggested that divergence of the main intraspecific clusters was the result of hybridization after secondary contact in the Holocene around 0.59 million years ago (Ma). The ratio of mtDNA F ST (fixation index) to nuDNA F ST was 2.3, thus failing to show male-biased dispersal. Geographic cline analysis showed that a wide hybrid zone was initially established in southwestern China, without significant reproductive isolation but with strong introgression in S. boulengeri, suggesting high hybrid fitness. Furthermore, mtDNA genes exhibited isolation by distance (IBD) while nuDNA genes exhibited significant isolation by environment (IBE). Results suggested that mitonuclear discordance may have initially been caused by geographic isolation, followed by precipitation-mediated hybridization, producing a wide hybrid zone and geographic structure confusion of nuDNA genes in S. boulengeri. This study indicated that complicated historical processes may have led to specific genetic patterns, with a specific climate factor facilitating gene flow in the system.

A new Asian lazy toad of the genus Scutiger Theobald, 1868 (Anura, Megophryidae) from southern Tibet, China.

In this study, a new species named Scutigerluozhaensissp. nov. is described from Luozha, southern Tibet, China. Genetic analysis based on two mitochondrial genes 16S rRNA and COI and the nuclear gene RAG1 revealed that the new species belongs to an independent phylogenetic clade close to S.gongshanensis and S.nyingchiensis and shares no RAG1 haplotype with other species. Morphological comparisons based on examined specimens and literatures indicated that it can be diagnosed from congeners by the following combination of characters: (1) body moderate, male body length 47.0-67.2 mm (n = 13), female body length 49.8-66.2 mm (n = 8); (2) maxillary teeth and budding absent; (3) numerous tiny dense nuptial spines present on dorsal surface of fingers I, II and inner surface of finger III of males in breeding condition with similar size; (4) spine patches on belly of males in breeding condition absent; (5) spines on inner surface of forearm and upper arm of males in breeding condition absent; (6) small patches of black spines present near armpit of males in breeding condition absent; (7) adult males without vocal sac; (8) some large warts and tubercles on dorsum gathered into short skin ridges with several spines present on top; (9) space between upper eyelids wider than upper eyelids; (10) spots or irregular cross bands on limbs absent; (11) webbing between toes rudimentary; (12) coloration of dorsal body olive brown to bronze.

A new species of Achalinus Peters, 1869 (Squamata, Xenodermidae) from Hunan Province, China.

A new species, Achalinushunanensissp. nov., is described from middle and western Hunan Province based on the results of molecular systematics and morphological characters. It diverges from known congeners by a significant genetic divergence (p-distance 3.2%-16.9% based on CO1 mitochondrial gene), and it can be distinguished from all known congeners by the following morphological characters: (1) all dorsal scales strongly keeled, 23 rows throughout the body, the outmost one strongly keeled and enlarged; (2) tail relatively short, TaL/TL 0.221 ~ 0.225; (3) maxillary teeth 23; (4) the suture between internasals 2 × as long as that between prefrontals; (5) loreal one, subrectangular, LorH/LorL 0.62 ~ 0.70; (6) supralabials 6, the 4th and 5th touch the eye; (7) the two anterior temporals in contact with eye; (8) ventrals 163-165, subcaudals 69-72, not paired. This raises the number of known species of Achalinus to 24.

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.

One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy.

Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N 3-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results.

Description of a new species of the newt genus Tylototriton sensu lato (Amphibia: Urodela: Salamandridae) from southwestern China.

A new species of the genus Tylototriton sensu lato from Tongzi County, Guizhou Province, China was described. Molecular phylogenetic analyses based on mitochondrial 16S and ND2 gene sequences indicated the new species as the most closely related species of T. dabienicus in Henan. The new species could be identified from its congeners by a combination of the following morphological characters: (1) body size medium (TOL 120.5135.1 mm and SVL 61.165.9 mm in males, and TOL 123.5127.6 mm and SVL 66.769.2 mm in females); (2) gular fold present; (3) the tail length shorter than the snout-vent length; (4) the distal ends and ventral surfaces of digits, peripheral area of cloaca, and the lower margin of tail orange; (5) the distal tips of the limbs greatly overlapping when the fore and hind limbs being pressed along the trunk; (6) fingertips reaching to the level beyond the snout when the forelimbs being stretched forward; (7) nodule-like warts on body sides continuous and no obvious. The new species is known only from the montane forests of Huanglian Nature Reserve, Tongzi County, Guizhou Province, China. We recommend the new species to be listed as Critically Endangered.

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy.

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene. It plays an essential role in many biological systems as a vital driving force for protein folding, stability, and receptor-ligand interaction/recognition. To date, the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional (3D) protein structures and corresponding computational calculations. However, their experimental verification and quantification remain sparse at the molecular level, mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins. Here, we use atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of protein neutrophil gelatinase-associated lipocalin (also known as NGAL, siderocalin, lipocalin 2) that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols. Based on a site-specific cysteine engineering and anchoring method, we first characterized the stability and unfolding pathways of apo-NGAL. Then, the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized. AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites, F122 and F133. Here, NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect. Thus, our work demonstrates high-precision detection of the weak cation-π interaction in NGAL. Electronic Supplementary Material: Supplementary material (additional SDS-PAGE, UV-vis, protein sequences, and more experimental methods) is available in the online version of this article at 10.1007/s12274-021-4065-9.

Direct Measurements of the Cobalt-Thiolate Bonds Strength in Rubredoxin by Single-Molecule Force Spectroscopy.

Cobalt is a trace transition metal. Although it is not abundant on earth, tens of cobalt-containing proteins exist in life. Moreover, the characteristic spectrum of Co(II) ion makes it a powerful probe for the characterization of metal-binding proteins through the formation of cobalt-ligand bonds. Since most of these natural and artificial cobalt-containing proteins are stable, we believe that these cobalt-ligand bonds in the protein system are also mechanically stable. To prove this, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to directly measure the rupture force of Co(II)-thiolate bond in Co-substituted rubredoxin (CoRD). By combining the chemical denature/renature method for building metalloprotein and cysteine coupling-based polyprotein construction strategy, we successfully prepared the polyprotein sample (CoRD)n suitable for single-molecule studies. Thus, we quantified the strength of Co(II)-thiolate bonds in rubredoxin with a rupture force of ∼140 pN, revealing that it is a mechanostable chemical bond. In addition, the Co-S bond is more labile than the Zn-S bond in proteins, similar to the result from the metal-competing titration experiment.

Exploring cryptic biodiversity in a world heritage site: a new pitviper (Squamata, Viperidae, Crotalinae) from Jiuzhaigou, Aba, Sichuan, China.

This study presents a comprehensive morphological comparison along with molecular phylogeny of the genus Gloydius based on five mitochondrial genes (12S, 16S, COI, cytb, and ND4). The specimens collected from Jiuzhaigou National Nature Reserve are shown to be a new species, Gloydiuslateralis sp. nov. Zhang, Shi, Jiang & Shi based on a combination of morphological and molecular accounts. G.lateralis sp. nov. differs from other congeneric species by a series of diagnostic morphological characteristics and forms a strongly supported monophyletic group. The new species is phylogenetically closely related to G.swild, another recently described species from Heishui, Aba, Sichuan.

Enzymatic Protein-Protein Conjugation through Internal Site Verified at the Single-Molecule Level.

Enzymes are widely used for protein ligation because of their efficient and site-specific connections under mild conditions. However, many enzymatic ligations are restricted to connections between protein termini while protein-protein conjugation at a specific internal site is limited. Previous work has found that Sortase A (SrtA) conjugates small molecules/peptides to a pilin protein at an internal lysine site via an isopeptide bond. Herein, we apply this noncanonical ligation property of SrtA for protein-protein conjugation at a designed YPKH site. Both a small protein domain, I27, and a large protein, GFP, were ligated at the designed internal site. Moreover, besides characterization by classic methods at the ensemble level, the specific ligation site at the interior YPKH motif is unambiguously verified by atomic force microscopy-based single-molecule force spectroscopy, showing the characteristic unfolding signature at the single-molecule level. Finally, steered molecular dynamics simulations also agreed with the results.

A new species of the genus Rana sensu lato Linnaeus, 1758 (Anura, Ranidae) from Wuyi Mountain, Fujian Province, China.

A new species of the frog genus Rana sensu lato from Wuyi Mountain, Fujian Province, China is described. Molecular phylogenetic analyses clustered the new species into the R.johnsi group and indicated that it was genetically divergent from its closely related species. The new species could be distinguished from its congeners by a combination of the following characters: body size medium, SVL 41.4-45.6 mm (42.9 ± 1.9 mm, n = 4) in adult males and 47.6-50.3 mm (n = 2) in adult females; adult male with a pair of internal subgular vocal sacs; lateroventral grooves present on tip of toes; webbing on fourth toes reaching the tip of toe; transverse skin ridges distinctly present on the dorsal surface of thigh and tibia, the number large (mean 26.5 ± 2.7, range 22-29, n = 6); breeding males possess creamy white nuptial pad with tiny velvety spines on the dorsal surface of the first finger, divided into three parts.

Neotype designation and redescription of Tropidophorus guangxiensis Wen, 1992 (Squamata: Sauria: Scincidae), with description of a new subspecies from central South China.

The genus Tropidophorus consists of small semi-aquatic lizards that dwell in lowland forest steams (Barbour, 1921; Bauer & Jackman, 2008). Here, we designate the neotype and re-describe T. guangxiensis Wen, 1992 based on newly collected topotypic specimens. We also describe a new subspecies from Xuefeng Mountain, Hongjiang County, Hunan Province, central South China. Based on two mitochondrial genes (12S rRNA and 16S rRNA), the phylogenetic position of T. guangxiensis is allocated for the first time. Additionally, our data strongly support that the new subspecies is phylogenetically closely related to T. g. guangxiensis. We also present a morphological identification key for known species and subspecies of Tropidophorus in China.

N501Y mutation of spike protein in SARS-CoV-2 strengthens its binding to receptor ACE2.

SARS-CoV-2 has been spreading around the world for the past year. Recently, several variants such as B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), which share a key mutation N501Y on the receptor-binding domain (RBD), appear to be more infectious to humans. To understand the underlying mechanism, we used a cell surface-binding assay, a kinetics study, a single-molecule technique, and a computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction, with a faster association rate and a slower dissociation rate. Atomic force microscopy (AFM)-based single-molecule force microscopy (SMFS) consistently quantified the interaction strength of RBD with the mutation as having increased binding probability and requiring increased unbinding force. Molecular dynamics simulations of RBD-ACE2 complexes indicated that the N501Y mutation introduced additional π-π and π-cation interactions that could explain the changes observed by force microscopy. Taken together, these results suggest that the reinforced RBD-ACE2 interaction that results from the N501Y mutation in the RBD should play an essential role in the higher rate of transmission of SARS-CoV-2 variants, and that future mutations in the RBD of the virus should be under surveillance.

Multiple lines of evidence reveal a new species of Krait (Squamata, Elapidae, Bungarus) from Southwestern China and Northern Myanmar.

Kraits of the genus Bungarus Daudin 1803 are widely known venomous snakes distributed from Iran to China and Indonesia. Here, we use a combination of mitochondrial DNA sequence data and morphological data to describe a new species from Yingjiang County, Yunnan Province, China: Bungarus suzhenae sp. nov. Phylogenetically, this species forms a monophyletic lineage sister to the Bungarus candidus/multicinctus/wanghaotingi complex based on cyt b and ND4 genes but forms a sister species pair with the species B. magnimaculatus Wall & Evans, 1901 based on COI gene fragments. Morphologically, B. suzhenae sp. nov. is similar to the B. candidus/multicinctus/wanghaotingi complex but differs from these taxa by a combination of dental morphology, squamation, coloration pattern, as well as hemipenial morphology. A detailed description of the cranial osteology of the new species is given based on micro-CT tomography images. We revised the morphological characters of B. candidus/multicinctus/wanghaotingi complex and verified the validity of three species in this complex. The distribution of these species was revised; the records of B. candidus in China should be attributed to B. wanghaotingi. We also provide an updated key to species of Bungarus.

Description of a new toad of Megophrys Kuhl amp; Van Hasselt, 1822 (Amphibia: Anura: Megophryidae) from western Yunnan Province, China.

A new species of genus Megophrys from Gaoligong Mountains, Yunnan Province, China is described. Phylogenetic analyses based on mitochondrial DNA and nuclear DNA all clustered the new species as an independent clade nested into the subgenus Panophrys. The smallest genetic distance based on 16S rRNA gene between the new species and its congeners was 3.0%. The new species could be identified from its congeners by a combination of following characters: moderate body size (SVL 31.0-34.8 mm in males); vomerine ridge weak, vomerine teeth absent; dorsal skin relatively smooth; tongue slightly notched behind; tympanum rounded and relatively large, 0.54 times of eye length; a horn-like tubercle on edge of each upper eyelid small; tibio-tarsal articulation reaches middle eye when leg stretched forward; finger tips rounded, not expanded to small pad; toes with narrow fringes and rudimentary webbing; ventral hindlimbs semitransparent purplish with greyish white pigments; ventral body scattered with distinct dark patches in the middle.