Genetic diversity and haplotype distribution patterns analysis of cytb and RAG2 sequences in Rana hanluica from southern China.

Rana hanluica: an endemic amphibian of China, is found in the hills and mountains south of the Yangtze River. In this comprehensive study, we collected 162 samples from 14 different localities to delve into the genetic diversity of Rana hanluica using mitochondrial Cytb and nuclear RAG2 as genetic markers. Our findings reveal that the Nanling Mountains, specifically regions like Jiuyi Shan, Jinggang Shan, Mang Shan, and Qiyun Shan, are genetic hotspots harboring remarkable diversity. The research results also indicate that there is gene flow among the various populations of the species, and no distinct population structure has formed, which may be due to migration. Moreover, populations in some regions, as well as the overall population, show signs of a possible genetic bottleneck, which we speculate may have been caused by climate change. However, given the exploratory nature of our study, further investigations are warranted to confirm these observations. Through phylogenetic analyses, we uncovered indications that R. hanluica might have originated within the Nanling region, dispersing along the east-west mountain ranges, with a significant contribution originating from Jiuyi Shan. The genetic distributions uncovered through our research reflect historical migratory patterns, evident in the distinct haplotypes of the RAG2 gene between the western and eastern parts of the studied area. Moreover, Heng Shan and Yangming Shan exhibited unique genetic signatures, possibly influenced by geographic isolation, which has shaped their distinct genotypes. The insights gained from this study hold profound implications for conservation efforts. By identifying regions rich in genetic diversity and crucial gene flow corridors, we can develop more effective conservation strategies. Preserving these genetically diverse areas, especially within the Nanling Mountains, is vital for maintaining the evolutionary potential of R. hanluica. In conclusion, our research has laid a solid foundation for understanding the genetic landscape of R. hanluica, shedding light on its origins, population structures, and evolutionary trajectories. This knowledge will undoubtedly guide future research endeavors and inform conservation strategies for this endemic amphibian.

Tadpoles of four sympatric megophryinid frogs (Anura, Megophryidae, Megophryinae) from Mangshan in southern China.

Sympatric distribution and potentially long larval development time make the assignment of tadpoles confusing in Asian-horned frogs of the subfamily Megophryinae. In this study, we used molecular data to identify four syntopic megophryinid tadpoles from Mangshan on the border between Hunan and Guangdong provinces in southern China: Brachytarsophryspopei, Boulenophrysshimentaina, Bo.cf.ombrophila, and Bo.nanlingensis. A detailed re-description of the Br.popei tadpoles is provided as well as the first descriptions of three Boulenophrys tadpoles based on external morphology and coloration. An effort is attempted to distinguish these tadpoles by coloration patterns: the dorsal pattern, ventral pattern, and pattern on tail are useful for field identification of these tadpoles. However, the variation of color pattern could sometimes make species delineation difficult. Researchers are encouraged to document coloration in life with photographs and the collection of tadpoles of different development stages and sizes advocated in order to better understand how color may change during larval development.

[Potential geographical distribution of Rana hanluica in China under climate change]. / 气候变化下寒露林蛙在中国的潜在地理分布.

Global warming in the last few decades had strong impacts on biodiversity and geographi-cal distribution of different animal species worldwide, especially amphibians. Rana hanluica, a frog species endemic in China, is still classified as Least Concerned in the Red List of Threatened Species because few studies have been conducted on this species. To understand the survival of Rana hanluica population, we used maximum entropy models (MaxEnt) to analyze its distribution across regions under current climatic conditions based on 47 distribution records and 20 environmental factors. We investigated the changes in distribution of this species under different climate scenarios in China (2050s and 2070s). Finally, current and future suitable habitats for R. hanluica were mode-led, and the impacts of environmental factors in shaping its distribution were evaluated. The results showed that the prediction accuracy of the MaxEnt model was high, and AUC value of the receiver operating curve was 0.993. The total suitable habitat area for R. hanluica was 36.36×104 km2, mainly located in Hunan and Guizhou provinces in China. The major environmental factors influencing the geographic distribution of R. hanluica were precipitation of dryest month and altitude. Under the future climate scenario (2050 and 2070) with two representative concentration pathways (RCPs, SSP1-2.5, SSP5-8.5), the suitable habitat of R. hanluica was reduced in different degrees, resulting in a decreasing trend of the total suitable habitat area. The center of gravity in highly suitable habitat of R. hanluica shifted to high-latitude regions, with the core distribution area in Hunan Province.

A new species of Odorrana (Anura, Ranidae) from Hunan Province, China.

A new species, Odorrana sangzhiensis sp. nov., is described, based on five specimens from Sangzhi County, Zhangjiajie City, Hunan Province, China. Molecular phylogenetic analyses, based on mitochondrial 12S rRNA and 16S rRNA gene sequences, strongly support the new species as a monophyletic group nested into the O. schmackeri species complex. The new species can be distinguished from its congeners by a combination of the following characters: (1) body size medium (SVL: 42.1-45.1 mm in males, 83.3-92.7 mm in females); (2) dorsolateral folds absent; (3) tympanum diameter 1.53 times as long as the width of the disc of finger III in females; 2.3 times in males; (4) dorsal skin green with dense granules and sparse irregular brown spots; males with several large warts on dorsum; (5) two metacarpal tubercles; (6) relative finger lengths: I ≤ II < IV < III; (7) tibiotarsal articulation beyond the tip of the snout; (8) ventral surface smooth in females; throat and chest having pale spinules in adult males; (9) dorsal limbs green or yellow green with brown transverse bands; and (10) paired external vocal sacs located at corners of the throat, finger I with light yellow nuptial pad in males. This discovery increases the number of Odorrana species to 59 and those known from China to 37.

A new species of Leptobrachella (Anura: Megophryidae) Smith, 1925 from Wuling Mountains in Hunan Province, China.

A new species of Leptobrachella, Leptobrachella wulingensis sp. nov. is described based on specimens collected from the Wuling mountains in Hunan Province, China. The new species can be distinguished from its congeners by a combination of having a SVL body size range of 24.5-32.8 mm in four adult males and 29.9-38.5 mm in three adult females; dorsal surface brown to reddish brown with indistinct markings; ventral surface creamy white, often with pale brown speckling on chest and margins; flanks with small to moderate black spots; skin on dorsum shagreened with sparse large warts, sometimes with short longitudinal ridges; toes with rudimentary webbing and narrow lateral fringes; dermal ridges under toes interrupted at the articulations; and iris bicolored with a bright orange or golden upper half, fading to silver in the lower half. Uncorrected sequence divergence between L. wulingensis sp. nov. and homologous 16S rRNA sequences available for all known species in the genus are ≥ 2.3%-2.9%.

Habitat association in the critically endangered Mangshan pit viper (Protobothrops mangshanensis), a species endemic to China.

Habitat directly affects the population size and geographical distribution of wildlife species, including the Mangshan pit viper (Protobothrops mangshanensis), a critically endangered snake species endemic to China. We searched for Mangshan pit viper using randomly arranged transects in their area of distribution and assessed their habitat association using plots, with the goals of gaining a better understanding of the habitat features associated with P. mangshanensis detection and determining if the association with these features varies across season. We conducted transect surveys, found 48 individual snakes, and measured 11 habitat variables seasonally in used and random plots in Hunan Mangshan National Nature Reserve over a period of 5 years (2012-2016). The important habitat variables for predicting Mangshan pit viper detection were fallen log density, shrub density, leaf litter cover, herb cover and distance to water. In spring, summer and autumn, Mangshan pit viper detection was always positively associated with fallen log density. In summer, Mangshan pit viper detection was related to such habitats with high canopy cover, high shrub density and high herb cover. In autumn, snakes generally occurred in habitats near water in areas with high fallen log density and tall shrubs height. Our study is the first to demonstrate the relationship between Mangshan pit viper detection and specific habitat components. Mangshan pit viper detection was associated with habitat features such as with a relatively high fallen log density and shrub density, moderately high leaf litter cover, sites near stream, and with lower herb cover. The pattern of the relationship between snakes and habitats was not consistent across the seasons. Identifying the habitat features associated with Mangshan pit viper detection can better inform the forestry department on managing natural reserves to meet the habitat requirements for this critically endangered snake species.

Comparative analysis and characterization of the gut microbiota of four farmed snakes from southern China.

BACKGROUND: The gut microbiota plays an important role in host immunity and metabolic homeostasis. Although analyses of gut microbiotas have been used to assess host health and foster disease prevention and treatment, no comparative comprehensive study, assessing gut microbiotas among several species of farmed snake, is yet available. In this study, we characterized and compared the gut microbiotas of four species of farmed snakes (Naja atra, Ptyas mucosa, Elaphe carinata, and Deinagkistrodon acutus) using high-throughput sequencing of the 16S rDNA gene in southern China and tested whether there was a relationship between gut microbiotal composition and host species. RESULTS: A total of 629 operational taxonomic units across 22 samples were detected. The five most abundant phyla were Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, and Actinobacteria, while the five most abundant genera were Bacteroides, Cetobacterium, Clostridium, Plesiomonas, and Paeniclostridium. This was the first report of the dominance of Fusobacteria and Cetobacterium in the snake gut. Our phylogenetic analysis recovered a relatively close relationship between Fusobacteria and Bacteroidetes. Alpha diversity analysis indicated that species richness and diversity were highest in the gut microbiota of D. acutus and lowest in that of E. carinata. Significant differences in alpha diversity were detected among the four farmed snake species. The gut microbiotas of conspecifics were more similar to each other than to those of heterospecifics. CONCLUSION: This study provides the first comparative study of gut microbiotas among several species of farmed snakes, and provides valuable data for the management of farmed snakes. In farmed snakes, host species affected the species composition and diversity of the gut microbiota.

Phylogenetic surveys on the newt genus Tylototriton sensu lato (Salamandridae, Caudata) reveal cryptic diversity and novel diversification promoted by historical climatic shifts.

Global climatic transitions and Tibetan Plateau uplifts are hypothesized to have profoundly impacted biodiversity in southeastern Asia. To further test the hypotheses related to the impacts of these incidents, we investigated the diversification patterns of the newt genus Tylototriton sensu lato, distributed across the mountain ranges of southeastern Asia. Gene-tree and species-tree analyses of two mitochondrial genes and two nuclear genes revealed five major clades in the genus, and suggested several cryptic species. Dating estimates suggested that the genus originated in the early-to-middle Miocene. Under different species delimitating scenarios, diversification analyses with birth-death likelihood tests indicated that the genus held a higher diversification rate in the late Miocene-to-Pliocene era than that in the Pleistocene. Ancestral area reconstructions indicated that the genus originated from the northern Indochina Peninsula. Accordingly, we hypothesized that the Miocene Climatic Transition triggered the diversification of the genus, and the reinforcement of East Asian monsoons associated with the stepwise uplifts of the Tibetan Plateau promoted the radiation of the genus in southeastern Asia during the Miocene-to-Pliocene period. Quaternary glacial cycles likely had limited effects on speciation events in the genus, but mainly had contributions on their intraspecific differentiations.

Stepping-stones and dispersal flow: establishment of a meta-population of Milu (Elaphurus davidianus) through natural re-wilding.

The Milu (Père David's deer, Elaphurus davidianus) became extinct in China in the early 20(th) century but was reintroduced to the country. The reintroduced Milu escaped from a nature reserve and dispersed to the south of the Yangtze River. We monitored these accidentally escaped Milu from 1995 to 2012. The escaped Milu searched for vacant habitat patches as "stepping stones" and established refuge populations. We recorded 122 dispersal events of the escaped Milu. Most dispersal events occurred in 1998, 2003, 2006 and 2010. Milu normally disperse in March, July and November. Average dispersal distance was 14.08 ± 9.03 km, with 91.41% shorter than 25 km. After 5 generations, by the end of 2012, 300 wild Milu were scattered in refuge populations in the eastern and southern edges of the Dongting Lake. We suggest that population density is the ultimate cause for Milu dispersal, whereas floods and human disturbance are proximate causes. The case of the Milu shows that accidentally escaped animals can establish viable populations; however, the dispersed animals were subject to chance in finding "stepping stones". The re-wilded Milu persist as a meta-population with sub-populations linked by dispersals through marginal habitats in an anthropogenic landscape.

[Construction of an indicator system for evaluating the protection efficacy of national nature reserves in China: A case study on terrestrial vertebrates (excluding migratory birds)].

The protection efficacy of nature reserves is a key element in achieving targets of biodiversity conservation. It is therefore very important to develop a scientific, systematic, and accurate index system for evaluating the protection efficacy of national nature reserves in China. Using methods of frequency statistics, expert consultation, analytic hierarchy process, and demonstration survey, we present a novel index system for evaluating the protection efficacy of Chinese national nature reserves for terrestrial vertebrates (excluding migratory birds) over a 10-year period. The indicator system included one target layer, two system layers, nine factor layers, and forty index layers. The system layer included ecological effectiveness evaluation (with a score of 60%) and management effectiveness evaluation (score of 40%). The ecological effectiveness evaluation was a comprehensive, dynamic evaluation of the target species, population, habitat, and ecological system. The management effectiveness evaluation was focused on the effectiveness of patrol and monitoring. The additional part aimed to analyze the impact of humans on the target species, population and nature resources of the nature reserve. This study combined the ecological effectiveness evaluation and the management effectiveness evaluation for the first time, highlighted the importance of time and space changes, distinguished the influence of natural factors from human factors, and integrated them into the evaluation results. By emphasizing quantifiable indicators, this evaluation index system could vastly assist the protection of nature reserves by improving management effectiveness, biodiversity conservation, and macroscopic decision-making.

[Effects of Germanium Concentrations on Germanium Accumulation and Biotransformation of Polysaccarified Germanium in Cordyceps militaris].

OBJECTIVE: To study the effects of Germanium (Ge) concentration on Ge accumulation and biotransformation of polysaccarified Ge (PG) in Cordyceps militaris. METHODS: Solid and liquid culture were used in this study. RESULTS: In the solid culture conditions, when the Ge concentration of medium was 200 mg/L, the sporophore biomass of Cordyceps militaris was the maximum; and when Ge concentration was 300 mg/L,the amount of biotransformation of PG in sporophore was the highest; and when the Ge concentration is 250 mg/L, conversion rate of organic germanium (OG) in sporophore reached the highest value. In the liquid culture conditions, when the Ge concentration was 250 mg/L, the mycelium biomass of Cordyceps militaris was the maximum; and when Ge concentration was 150 mg/L, the amount of organic conversion of PG in mycelium was the most; and conversion rate of OG in mycelium was the highest in media with the Ge concentration of 200 mg/L. This study showed the germanium concentrations in 150 - 300 mg/L was more suitable for Ge accumulation and biotransformation of PG in Cordyceps militaris. In general, the biotransformation capacity to germanium of sporophore was stronger than that of mycelium of Cordyceps militaris. CONCLUSION: Germanium can significantly affect Ge accumulation and biotransformation of PG in Cordyceps militaris (P < 0.05) at different concentration. This result has practical value for Ge enriched cultivation of fruiting body in Cordyceps militaris.

Mitochondrial genome of Protobothrops mangshanensis (Squamata: Viperidae: Crotalinae).

Protobothrops mangshanensis is a venomous pit viper species endemic to Hunan province in China. In this study, the complete mitochondrial genome of P. mangshanensis had been determined. The circle genome with the 17,230 bp total length contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 control regions. Overall base composition of the complete mtDNA was 32.27% A, 24.16% T, 30.34% C and 13.23% G. All the genes in P. mangshanensis were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand.

[Study on monosaccharide composition of intracellular polysacchride and contents of cordycepin and cordyceps polysacchride produced by Cordyceps militaris induced by blue light].

OBJECTIVE: To study effects of blue light irradiation on monosaccharide composition of intracellular polysacchride and contents of cordycepin and cordyceps polysacchride of mycelium and sporocarp in Cordyceps militaris. METHODS: The monosaccharide composition of intracellular polysacchride of mycelium and sporocarp in Cordyceps militaris as materials were determined by gas chromatography after 144 h blue light irradiation. The contents of cordycepin and cordyceps polysacchride of mycelium and sporocarp in Cordyceps militaris were detected at different blue light irradiation periods. At the same time, the growth of mycelium and sporocarp in Cordyceps militaris were observed during blue light irradiation. RESULTS: Mycelium polysaccharide in Cordyceps militaris was a kind of heteropolysaccharide containing four kinds of monosaccharide and fruiting body polysaccharide was a kind of heteropolysaccharide containing five kinds of monosaccharide. Whether blue light irradiation or dark culture, the content changes of cordyceps polysacchride in two groups showed similar patterns in the test of mycelium polysaccharides. The content changes of cordyceps polysacchride in two groups were basically the same in the detection of sporocarp polysacchride. Cordycepin content in the two set of experiments of blue light irradiation all showed a clear upward trend in the detection of mycelium and sporocarp in Cordyceps militaris. CONCLUSION: The blue light irradiation has certain effect on the species and quantity of monosaccharide in intracellular polysaccharide. The content increase of cordycepin and cordyceps polysacchride in Cordyceps militaris are promoted by blue light irradiation. Blue light can help the morphogenesis and promote the differentiation and growth of sporocarp in Cordyceps militaris. This study is the first report about the effect of blue light on the type and quantity of the monosaccharide composition in polysaccharide of Cordyceps militaris, which will lay the foundation for further study on the metabolism of active substance in Cordyceps militaris by blue light irradiation.

Changes in attitudes toward wildlife and wildlife meats in Hunan Province, central China, before and after the severe acute respiratory syndrome outbreak.

Two surveys of opinions about wildlife conservation were carried out in Hunan Province, China, before and after the severe acute respiratory syndrome (SARS) epidemic. Hunan is the northern neighbor of Guangdong Province, where the first SARS case was reported. The custom of consuming wild meat was not common in Hunan 30 years ago. However, in recent years, consumption of wildlife such as snakes has spread to northern China. We handed out 1300 questionnaires between 22 February and 10 May 2002, before the SARS epidemic. Survey sites included the provincial capital, major cities, towns, and villages in Hunan. Another 1300 questionnaires were distributed between 10 March and 10 May 2004, after the SARS epidemic. The 2004 survey covered the same sites as the 2002 survey. Questionnaire recovery rates for the 2002 and 2004 surveys were 81.2% and 84.6%, respectively. The valid return rate was 73.0%. Frog, snake, hare, and pheasant were the wild meats most frequently eaten by local people. At the time of the first survey, more than 80% of interviewees claimed to have eaten frogs. That number had reduced to 60% by the time of the second survey. Monkey, Chinese pangolin, and bear paw were the wild meats least frequently eaten. Although palm civet was suspected to be the carrier of SARS, it was interesting to note that, after the SARS epidemic, the proportion of people surveyed who ate palm civets had declined only slightly. The results of the surveys indicate a low level of conservation consciousness; however, after the SARS and bird flu epidemics, there were obvious changes in the conservation consciousness of people in Hunan Province. We recommend some measures that could be taken to change the habits of people who consume wildlife.