Abstract:[Objectives] Pastor roseus is the important natural enemy of grassland grasshoppers in Xinjiang, China. This study aims to clarify the breeding ecological characteristics of P. roseus. [Methods] The study was conducted at the third company at the horse ranch and Songshutang community in Yizhou District, Hami City, Xinjiang. During the breeding season of P. roseus from 2022 to 2023, we combined the grasshopper occurrence data provided by the “monitoring and reporting workstation to prevention and control of grasshopper and mouse in Hami District” with the results of our field investigation, to analyze the factors influencing the breeding site selection and migration time of P. roseus. Ten nests of P. roseus were selected each year to monitor the reproductive behavior and nestling development using infrared cameras. The total recording duration was 38 d, with a valid data duration of 2 337 h (n = 20 nests). The behavioral characteristics of nesting, egg-laying, hatching and brooding were analyzed using single factor analysis of variance and Friedman test in SPSS 26 software. At the same time, growth and development indicators of weight and tarsometatarsal length of nestlings were fitted based on the logistic growth curve model, and growth curves were plotted. [Results] 1) The population densities of grasshoppers in the study areas were higher than that in other areas during 2022﹣2023 (Table 1). No P. roseus was seen in other areas with low grasshopper densities. Both immigration and emigration times of P. roseus coincided with the grasshopper occurrence time during the two years. 2) After mating, females laid white or cyan eggs (Fig. 2). The clutch size was 3﹣7, and the hatching rate was 88.89% (n = 54 eggs). 3) Eggs were incubated alternately by the male and female, and nestlings were fed by both parents. The daily feeding peak for nestlings was from 10:00 to 12:00, and feeding times during this period were significantly higher than those during other periods (P < 0.05) (Table 3). 4) Growth of body weight and tarsometatarsus of nestlings were consistent with the logistic models (Fig. 5). [Conclusion] The selection of breeding sites and migration time of P. roseus was mainly related to population density of grasshoppers. Both males and females raise offspring. The nestlings are altricial, and the growth and development trend is S-shaped. The results provided basic parameters for the protection and attraction of P. roseus.

Abstract:[Objectives] Establishing nature reserves is not only an effective measure for biodiversity conservation but also plays a crucial role in maintaining and protecting ecosystems, natural resources and landscapes. Over the past two decades, significant changes have occurred in China’s network of nature reserves and land use. Reeves’s Pheasant Syrmaticus reevesii is an endangered species that is endemic to China. Assessing changes in protected areas and land use types within the important distribution area of the Reeves’s Pheasant in the Dabie Mountains is vital for the survival of this species. [Methods] This study combines field surveys with Geographic Information Systems (GIS), species distribution models, and gap analysis methods to evaluate the effectiveness of national nature reserves in protecting the habitat of Reeves’s Pheasant in the Dabie Mountains over the past two decades. [Results] The lengths of roads and areas of building area within and around the national nature reserves in the Dabie Mountains showed an increasing trend from 2001—2002 to 2018—2019 (Figs. 2, 3). In 2001—2002, the primary influencing factors on suitable habitats for Reeves’s Pheasant were distances to farmland, railways, and national roads, whereas in 2018—2019, elevation and distances to wind turbines became more prominent factors. Moreover, during 2018—2019, there was a declining trend in the proportion of suitable habitat for Reeves’s Pheasant within the national nature reserves in the Dabie Mountains (Fig. 5, Table 4). [Conclusion] The findings indicate that the national nature reserves in the Dabie Mountains are being affected by the expansion of the human footprint, highlighting the challenge of balancing conservation with economic and social development in reserve management. Future planning and management of nature reserves should prioritize a deep understanding of the ecological needs of endangered species, rational zoning of reserve functions, and enhancement of conservation effectiveness to ensure sustainable coexistence between nature and human activities.

Abstract:[Objectives] Asian OpenbillAnastomus oscitans is a newly recorded species in China, with the first sighting occurring in 2006. Since then, it has rapidly expanded its distribution to encompass over ten provinces and regions, including Yunnan, Guizhou, Sichuan and Gansu, etc., and has already become a regionally common species in some areas. Nevertheless, fundamental data regarding the population sources and movement patterns were still limited. [Methods] From November 2015 to July 2018, 11 storks were captured using the leg-harness method and equipped with satellite trackers in the Changqiao Hai Wetland of Mengzi, Yunnan Province, and the Caohai Wetland of Weining, Guizhou Province. The home ranges of onestorkwere calculated using the dynamic Brownian bridge model and its spatial movement patterns and habitat utilization were analyzed. [Results] Eight of the tracked storks died accidentally or did not leave the capture site, and only 3 exhibited longer-distance movements, of which one individual (ID no. 2647) successfully returned to its traditional breeding ground (Fig. 1). The stork (ID no. 2647) departed from Mengzi, Yunnan, China on August 29, 2018, migrating southwestward through Vietnam and Laos, and arrived in Chiang Rai, central Thailand on September 16, 2018, covering a distance of 775.06 km over 18 d. After a 95-day stay in Chiang Rai, it left on December 19, 2018, and moved eastward along the Mekong River bordering Laos and Thailand. It lost contact in Nghe An province, Vietnam, on March 6, 2019, after traveling 620.16 km over 79 d (Fig. 1). During its migration, the storkhad a total of 6 night roosting sites and 2 important stopover sites. Except for the last roosting site in farmland, all night roosting sites were in forests. It flew during the day and rested in forests at night. (Table 1). While in Chiang Rai, its total home range was 304.32 km2, with a core area of 0.97 km2, accounting for 3.2% of the total area (Fig. 2). The main habitat types used by the storks were cropland (79.72%) and forest (16.83%). [Conclusion] This study reveals the spatial connections between Asian Openbill populations in China and those in Thailand, Laos, and Vietnam, providing foundational data for further exploration of the distribution and dispersal patterns of this species.

Abstract:[Objectives] Approximately 10 billion birds of 1 800 species migrate globally. From 1970 to 2020, ecological conservation efforts in North America and Europe have resulted in a significant increase in the population of large migratory waterbirds and raptors, gradually becoming the primary cause of bird strike accidents and symptoms. Data from the US civil aviation sector indicate a significant correlation between the average weight of birds and the frequency of accidents, with bird weight serving as a predictive indicator of bird strike risk. [Methods] We conducted a comparative analysis of large waterbird population estimates across China and East Asia from 2008 to 2020, and examined the population trends in China and the underlying drivers of these ecological changes. Moreover, we compared the number and characteristics of birdstrike accidents in civil aviation between China and the United States, using one-way ANOVA. [Results] From 2008 to 2020, the number of large overwintering waterfowls in six groups in East Asia increased from 650 000 to 1130 000, of which China experiencing an increase from 420 000 to 530 000 (Appendix 1). The factors contributing to this increase include increased utilization of farmland by birds, strengthened bird conservation efforts and the expansion of protected areas, as well as the prohibition of certain chemical substances and the expansion of aquaculture. Over the past 30 years, the number of civil aviation takeoffs and landings in the United States has increased by 1.1 times, leading to a 7.3-fold increase in bird strike incidents. In China, there has been a 4.6-fold increase in civil aviation takeoffs and landings over the past 20 years, resulting in a 14.3-fold increase in bird strike incidents and a 9.3-fold increase in accidents and incidents (Figs. 1, 2). Although the rate of bird strike incidents per ten thousand flights in China is lower than that in the United States, the number of accidents and incidents is higher, attributed to measures such as bird strike warnings and airport habitat management adopted in the United States (Figs. 1, 2, Table 1). [Conclusion] China serves as crucial stopover and wintering grounds for large wintering waterbirds, with millions of migratory birds pass through Chinese airspace annually. International trend analysis suggests that large birds, such as waterbirds, may pose significant flight safety hazards during their migration and breeding seasons. Therefore, China needs to conduct long-term bird monitoring, identify hotspots of bird activity and their spatiotemporal variations, and continuously strengthen proactive prevention efforts. This will help to reduce the reliance on traditional passive interventions, and minimizing the risk of bird strikes on aircraft.

Abstract:[Objectives] To support fish resource protection and ecological restoration, this study investigated the age, growth and reproduction of Pseudogyrinocheilus procheilus based on monthly samples collected in the Chishui River between March 2023 and February 2024. [Methods] Scales were used as material for age determination, and von Bertalanffy growth functions were fitted based on the ages determined from scales and the measured body length data. The Chi-square test is used for further validation of the goodness-of-fit of the equation. [Results] The results indicated that the age of P. procheilus ranged from 1 to 5 years, with a predominance of 2 and 3 years of age (Fig. 1), and a sex ratio of 1︰0.53 (female︰male). The relationship between body length and weight was described by the equations W_♀ = 3.948 × 10^﹣6 L^3.317 for females and W_♂ = 2.137 × 10^﹣5 L^2.989 for males (Fig. 3). The t-test of the allometric growth index (b) was further analyzed, uncovering the b value of females is significantly greater than 3 (P < 0.01), indicating a positive allometric growth pattern. Differently, males exhibited a b value of 2.989, suggesting an isometric growth pattern. The von Bertalanffy functions were L_t♀ = 324.89[1﹣e^{﹣0.139(t+ 2.216)}] for females and L_t♂ = 236.25[1﹣e^{﹣0.239(t+ 1.828)}] for males (Fig. 4). The growth rate and acceleration of body length show no inflection point, with growth rate decreasing with age and the acceleration remaining negative. In contrast, the growth rate and acceleration of body weight exhibited inflection points, with inflection ages (t_i) of 6.41 years for females and 3.40 years for males (Fig. 5). The analysis indicated that the first sexual maturation and age occurred at a length of 170.35 mm and an age of 3.12 years for females, and 155.28 mm and 2.65 years for males (Fig. 6), respectively. Analysis of the gonadal somatic index and ovarian development period indicated that spawning occurred from May to September, peaking in May and June (Fig. 7). Studies on egg diameter frequency distributions (Fig. 9) and histological observations of ovarian development (Fig. 8) revealed that P. procheilus employs a single-batch spawning strategy. The mean absolute fecundity and relative fecundity were 4 496 eggs per female and 31 eggs per gram, respectively, with absolute fecundity positively correlated with body length and binomially correlated with body weight (Fig. 11). [Conclusion] Based on the above conclusions, we believe that fishing has a great impact on fish like P. procheilus, which have low individual fecundity and high growth inflection age. Maintaining a long-term fishing ban is the basic measure for fish protection in the upper reaches of the Chishui River.

Abstract:[Objectives] The Siberian Flying Squirrel Pteromys volans is an arboreal, nocturnal, and gliding rodent. It plays an important role in the dispersal of forest seeds and the maintenance of ecosystem balance. In northeast China, the population and distribution of the Siberian Flying Squirrels are decreasing due to deforestation and habitat loss. As an important indicator species for sustainable forest management, it has been listed in the Catalog of Terrestrial wildlife Species with Significant Ecological, Scientific, and Social Value in China. It has also been listed as “Vulnerable (VU)” in the Red List of China’s Vertebrate. There is an urgent need to further evaluate genetic changes of the population, especially genetic diversity and gene flow, to enhance the pertinence of conservation and management. [Methods] From 2018 to 2023, we collected hair samples from 20, 26, 15 and 27 Siberian Flying Squirrel individuals from four geographical populations (Fangzheng, Bin County, Weihe and Muling) in southern Heilongjiang Province, northeast China, respectively (Fig. 1 and Table 1), and evaluated their genetic diversity and structure based on the complete sequences of mtDNA control region (1 070 bp). [Results] The results showed that 155 variation sites and 67 haplotypes were detected in the control region sequence of these 88 individuals. In the populations, the haplotype diversity (H_d) and nucleotide diversity (P_i) were 0.992 (0.945﹣0.986) and 1.772% (1.221%﹣1.974%), respectively (Table 2). Only one haplotype (H_20) was shared by two populations (Bin County and Weihe), and there were 52 haplotypes with only one individual, and the proportion of rare haplotypes is 77.16% (52/67) (Figs. 2 and 3). The genetic differentiation coefficient (F_ST) and gene flow (N_m) between populations were 0.051 3﹣0.135 7 (P < 0.05) and 3.19﹣9.24, respectively (Tables 3﹣5). The Mantel test indicated that genetic and geographic distances were not significantly positively correlated (r = 0.413, P > 0.05). [Conclusion] Thegenetic diversity of the populations was at a high level, among which the Weihe and Muling populations were the highest, followed by the Bin County population, and the Fangzheng population was the lowest. This may suggest that the Changbai Mountains were the refuge for Siberian Flying Squirrels during the last glacial period. However, the high proportion of rare haplotypes indicated a risk of declining genetic diversity of the Siberian Flying Squirrel population in the future. There were significant moderate levels of genetic differentiation among the four populations, and the distance isolation model could not effectively explain the current population genetic pattern. The phylogenetic tree constructed by maximum likelihood, Bayesian inference method and haplotype network indicated that there was no obvious phylogeographic separation among the four geographic populations (Figs. 2 and 3). This result implies that the geographical barriers between the populations may be recent. It is suggested that conservation efforts for the Siberian Flying Squirrels should be improved, and that habitat restoration and ecological corridor construction should be carried out in the field, to improve gene exchange between individuals and accelerate population restoration.

Abstract:[Objectives] The genus Paedocypris comprises the smallest known fish species and one of the smallest vertebrates, with adult sizes ranging between 8﹣10 mm. Previous studies have indicated that the loss of Hox gene family members and the reduction of intron length and repetitive sequences might be potential causes for the miniaturization of the Paedocypris species and the simplification of their genomes, as demonstrated through draft genome assembly and comparative genomic analysis of P. carbunculus and P. micromegethes. The objectives of this study include characterizing the full-length transcriptome of the two species, P. carbunculus and P. micromegethes, identifying potential regulatory changes that could contribute to their miniaturized stature, with a particularly focus on Hox family and developmental gene expression patterns and gene structure. [Methods] All samples were purchased from an aquarium market in Guangzhou, comprising seven P. carbunculus and eleven P. micromegethes live specimens (Fig. 1). RNA was extracted from whole fish, and the purity, concentration, and integrity were tested. RNA from the seven P. carbunculus and the eleven P. micromegethes was mixed respectively and used for the construction of a full-length transcriptome library. The sequencing was performed on the PacBio Sequel platform. Quality control of full-length transcriptome sequencing data from the two Paedocypris species were performed. Then, we described the data quality, mapping statistics, annotation statistics and transcript classification of the full-length transcriptome data. By employing different software and pipelines, we annotated the gene structure, transcription factors, alternative splicing sites, long non-coding RNAs (LncRNAs), and gene fusions. To characterize the expression situation of Hox family genes in Paedocypris, we downloaded 81 zebrafish Hox family gene coding sequences belonging to 48 different Hox genes and align to the full-length transcriptome sequences of Paedocypris and other four species using Blastn with a threshold e-value < 10^﹣20. To explore the expression of genes in important developmental pathways in the Paedocypris genus, we selected seven key GO developmental pathways encompassing 1 581 non-redundant zebrafish genes and align with the full-length transcriptome data of Paedocypris and the other four species for analysis. To explore whether there are significant structural differences between the developmental-related genes mapped in the Paedocypris genus and other genes on the Paedocypris genome, based on the GO annotation information of the Paedocypris reference genome, we conducted significance tests (t-test) on the developmental-related genes mapped in the Paedocypris genus against all annotated genes on the Paedocypris reference genome for alternative splicing, polyadenylation, exon count, and fusion genes. [Results] In this study, we conducted full-length transcriptome sequencing on whole-body samples of P. carbunculus and P. micromegethes (Tables 1﹣4), obtaining the first full-length transcriptome library for Paedocypris. After correction and deduplication, 38 651 and 23 165 corrected consensus sequences were obtained, with average lengths of 2 413 bp and 2 460 bp, respectively. Among these sequences, 35 883 (92.84%) and 20 381 (87.98%) were mapped to the Paedocypris reference genome (Table 2). The classification and characterization analyses of the full-length transcriptome data for these two species resulted in 19 352 and 11 139 full-length transcripts, respectively (Tables 4 and 5). In the full-length transcriptome data of P. carbunculus and P. micromegethes, 9 404 and 6 068 alternative splicing events were identified, predominantly of the A5 type (Table 6). We also observed that the majority of genes (74.55% and 84.75%, respectively) had only one alternative polyadenylation site. Annotation and classification of transcription factors revealed that the most abundant transcription factor family in Paedocypris was zf-C2H2, followed by ZBTB. Additionally, we predicted 1 382 and 1 649 LncRNAs in the two Paedocypris species, respectively (Fig. 2). Comparative analysis of the full-length transcriptome data between Paedocypris and carp, silver carp, salmon, and rainbow trout revealed that the number of expressed genes in the Hox gene family in Paedocypris was significantly lower than that in the other four fish species (Fig. 3). We also identified that Paedocypris has the most expressed developmental genes belonging to the seven GO terms than the four fish species (Fig. 3). We found that those developmental-related genes have fewer gene fusion events than other genes in the full-length transcriptome of Paedocyrpis (Fig. 4). [Conclusion] We provide a full-length transcriptome landscape of the genus Paedocypris by performing full-length transcriptome sequencing. Data quality, mapping statistics, annotation statistics, and transcript classification of the full-length transcriptome data of Paedocypris are described. The analysis highlights that the loss of Hox genes at the expression level may be one of the functional reasons for the miniaturization of Paedocypris. This study provides new insights into the molecular mechanisms underlying the miniaturization of Paedocypris.

Abstract:[Objectives] This study aims to study the regulatory role of exosomes derived from Bactrian Camel Camelus bactrianus plasma in the invasion and metastasis of hepatocellular carcinoma cells. [Methods] In this study, the plasma exosomes of Bactrian Camels were separated by ultracentrifugation, the morphology and ultrastructure of the exosomes were observed by transmission electron microscopy, and the particle size and distribution of the exosomes were determined by nanoparticle tracking analyzer, the exosomes were co-cultured with hepatoma cells (MHCC-97H and Huh7 cells) after labeling with fluorescent dye PKH67, and the uptake of plasma-derived exosomes by hepatoma cells was observed by scanning confocal microscopy (LSCM). [Results] The obtained plasma exosomes of Bactrian Camels were all cup-shaped or round-shaped, with a diameter of about 100 nm (Fig. 1):the main peaks of particle size were about 124.6 nm and 117.9 nm, respectively, and the overall size distribution ranged from 50 nm to 200 nm (Fig. 2). Exosomes carrying PKH67 markers were observed to enter MHCC-97H and Huh7 cells under LSCM, and mainly distributed in the cytoplasm of cell membranes and perinucleus (Fig. 3). [Conclusion] In this study, exosomes derived from Bactrian Camel plasma were successfully extracted, and the uptake of exosomes by hepatoma cells was observed, suggesting that hepatocellular carcinoma cells may be one of the target cells of Bactrian exosomes.

Abstract:[Objectives] The Luopanshan Pig Sus scrofa domestica, an indigenous local breed of China, is found mainly in the Tongnan region of Chongqing. The population of purebred Luopanshan Pigs has significantly declined due to the widespread impact of African swine fever and intense competition from other breeds, putting them at risk of endangerment. To conserve this invaluable genetic resource, the establishment of a somatic cell genetic resource bank is essential. [Methods] In this study, we successfully established a fibroblast cell line from the ear margin of Luopanshan Pigs using the tissue block adhesion method. Fluorescent staining was utilized to detect mycoplasma contamination and to identify the fibroblast marker vimentin protein. Chromosomal karyotype analysis and G-banding were conducted to assess genetic stability. In addition, flow cytometry was employed to evaluate key cell characteristics, including apoptosis rate, cell cycle distribution, reactive oxygen species levels, and cellular senescence. Statistical analyses were performed using GraphPad Prism software (version 9.5), with one-way ANOVA, two-way ANOVA and Student’s t-test being used to determine significant differences. [Results] The study showed that primary fibroblasts migrated from tissue blocks within 2 to 5 d, exhibited an elongated, spindle-shaped morphology (Fig. 1). After 8 to 12 d of continued cultivation, the cell density reached 90%, allowing successful passaging. The first five generations exhibited robust growth, forming a dense monolayer within 2 to 3 d post-passage. However, from the sixth generation onwards, cell volume gradually increased, leading to an irregular morphology characterized by enhanced lamellipodia and filopodia formation, alongside a significant decrease in growth rate. Cell fusion was observed after approximately 4 to 5 d. (Fig. 2). To assess cell purity, fluorescent staining for vimentin (a fibroblast marker) and nuclear DNA was performed, revealing a fibroblast purity exceeding 98% in the third passages (Fig. 3). Hoechst staining confirmed the absence of mycoplasma DNA, indicating no contamination (Fig. 4). The Luopanshan Pig fibroblasts have a chromosome number of 2n = 38, comprising 18 pairs of autosomes. Specifically, there are five pairs of submetacentric chromosomes (chromosomes 1﹣5), two pairs of subtelocentric chromosomes (chromosomes 6 and 7), five pairs of metacentric chromosomes (chromosomes 8﹣12), and six pairs of telocentric chromosomes (chromosomes 13﹣18). In additional, there is one pair of sex chromosomes, which are classified as metacentric chromosomes (Fig. 5). The relative lengths of each chromosome are detailed in Table 2. The viability of the third-generation cells before cryopreservation and after recovery exceeded 85%, with no significant differences observed. The ninth generation showed a viability surpassing 80% before cryopreservation, the viability remained above 70% after recovery. Before cryopreservation and after recovery, the proliferation rate of third generation cells showed a rapid increase, following a standard “S” shaped growth curve. In contrast, the growth curve of the senescent cells of the ninth-generation cells showed a gradual increase in an “S” shape (Fig. 6). The results of the flow cytometry analysis showed a significant increase in the proportion of cells in the gap 1 phase (G1) phase after the third generation, compared to the conditions before cryopreservation, there was a slight decrease in the proportion of cells in the gap 2 phase (G2) and synthesis phase (S), but this difference was not statistically significant. Although there was a slight increase in the rate of apoptosis after recovery, this did not reach statistical significance. After the recovery of the ninth generation, there was a significant increase in the proportion of cells in G1 phases and a significant decrease in the percentage of cells in S phase. There was also a slight increase in the proportion of cells in G2 phase, but this difference did not reach statistical significance. Furthermore, a significant increase in the apoptosis rate was also observed (Fig. 7). Finally, compared to third generation cells, significantly increased levels of reactive oxygen species (ROS) and senescence were observed in ninth generation cells, with marked increases after recovery (Figs. 8, 9). [Conclusion] In this study, we successfully established a cell line of marginal fibroblasts from the ear tissue of Lopangshan pigs. By comparing the growth characteristics of third- and ninth-generation fibroblasts before cryopreservation and after recovery, we found that low-generation fibroblasts exhibited greater stability in terms of growth after recovery. Therefore, low-generation fibroblasts are more suitable for long-term ultra-low temperature preservation and provide stable and excellent materials for subsequent genetic studies compared to high generation fibroblasts.

Abstract:With more field work and investigations being conducted, many cryptic species have been described in the genus Leptobrachella, indicating that the species diversity of Leptobrachella was underestimated. L. sungi is known from Northern Vietnam and southern Guangxi of China. In June 2020, a specimen of the genusLeptobrachella resembling L. sungi wascollectedfrom Shuitouhou Mountain, Hekou Yao Autonomous County, Yunnan Province, China (22°37′43″ N，103°52′39″ E). In this study, we examined the specimen to provide additional data on taxonomy and distribution of genus Leptobrachella. The newly collected Leptobrachella specimen from Hekou, Yunnan was identified based on morphological characters and molecular phylogenetic analyses. Morphologically, this specimen was measured and compared with type specimens of L. sungi. Measurements included:snout-vent length (SVL), head length (HL), head width (HW), snout length (SL), internarial distance (IND), interorbital distance (IOD), upper eyelid width (UEW), eye diameter (ED), nostril-eye distance (DNE), tympanum diameter (TD), forearm and hand length (FHL), tibia length (TL), foot length (FL), and length of foot and tarsus (TFL). Fragment encoding 16S rRNA was amplified and sequenced. The Kimura 2-parameter model was used to calculate the genetic distances between species of the genus Leptobrachella in MEGA 7 based on 16S rRNA gene. Subsequently, Bayesian phylogenetic analysis was conducted based on the sequences of mitochondrial 16S rRNA gene in MrBayes v.3.2.6. Morphologically, the specimen is basically consistent with type specimens of L. sungi (Figs. 2, 3, Table 2). Molecularly, the Bayesian phylogenetic tree showed that the specimen was clustered together with topotypes of L. sungi with a posterior probability of 1.00, and the genetic distance between them was 0.0% (Appendix 1, Fig. 4). The above results indicate that the specimen collected from Shuitouhou Mountain, Hekou County, Yunnan Province is L. sungi, which is a new record in Yunnan Province. This study extended the distribution area of L. sungi from north of Vietnam northward to Yunnan Province, China and provided new information for biogeography and biodiversity research.

Abstract:This study was carried out in Cheng County, Longnan City, Gansu Province in April 2023, with the aim of understanding the diversity and distribution patterns of Chiroptera species in the region and providing fundamental data for biodiversity conservation. During the survey, four bat specimens (3 ♂, 1 ♀) were collected by hand netting and the morphology and skull characteristics of these specimens were measured using electronic digital calipers. The Cyt b and COI gene sequences of three of the bat specimens (specimen numbers:GS2023023, GS2023029, and GS2023038) were sequenced and uploaded to the NCBI database (Table 1). A phylogenetic tree was constructed using the maximum likelihood method in IQ-TREE, and intraspecific and interspecific and intergeneric genetic distances for the Fanjingshan Tube-nosed Bat Murina fanjingshanensis Cyt b and COI gene were calculated based on the Kimura 2-parameter model. The following morphological measurements were taken:head and body length 47.99﹣49.51 mm, forearm length 40.15﹣42.60 mm, tibia length 16.97﹣19.30 mm, hind-foot length 9.95﹣10.53 mm (Table 2). The greatest length of the skull is 18.17﹣19.30 mm; the braincase height is 7.51﹣8.97 mm; braincase slightly swollen. Sagittal crest and lambdoid crests are not evident (Fig. 1 and Table 2). The dorsum consists of underfur and guard hairs. The base of the guard hairs is dark gray to black, the shaft is light brown, and the tips are dark brown with a reddish brown (Fig. 1). The above characteristics are consistent with the appearance and skull characteristics of M. fanjingshanensis. Phylogenetic analysis based on Cyt b and COI gene sequences showed that it was clustered with M. fanjingshanensis specimens from Guizhou (type specimen) and Hunan (Fig. 2). The genetic distance analysis showed that the intra-species genetic distance of M. fanjingshanensis was remarkably low, with a value less than 1%; the inter-species genetic distance of the same genus ranged from 2% to 22%; and the inter-generic genetic distance was 16%﹣23% (Appendices 1 and 2). The integration of external morphology, skull characteristics, and molecular phylogenetics supports the identification of M. fanjingshanensis, marking a new record of Chiroptera distribution in Gansu Province.

Abstract:In May 2023 and June 2024, six Amolops specimens were collected in Huagaoxi National Nature Reserve, Xuyong County, Sichuan Province. Due to the large number of Amolops species and the existence of highly similar relatives, molecular identification was conducted.Morphometric measurements were taken on the collected specimens followed by extraction, sequencing, and analysis targeting mitochondrial COI gene segments derived from muscle tissue for phylogenetic inference at intra-genus level.The maximum likelihood tree of COI gene fragments from four different populations of A. sinensis showed that the Xuyong specimen from Sichuan and type locality Shimentai sequence from Guangdong were clustered together, and the genetic distance between them was 0﹣0.7%, which is significantly smaller than the average genetic distance within the Amolops species (3.1%), and the average genetic distance between them and the outgroup (closely related species Nidirana pleuraden, Odorrana schmackeri, Hylarana guentheri) of 8.5%. Meanwhile, the distribution area of A. sinensis was predicted using MaxEnt, with an average test AUC of 0.986 and a standard deviation of 0.007 after repeated runs.The species previously identified as A. ricketti, which was distributed in Sichuan Province, should be revised as A. sinensis, representing a new species record of amphibians in Sichuan Province. This study further expanded the altitude range of A. sinensis, extending the low altitude to 400 m in comparison with the previous record (500﹣1 300 m). The discovery will play an important role in understanding the potential range and phylogeography of the species.

Abstract:The number of leukocytes in the circulating blood of amphibians is related to their health status, and the ratio of neutrophils to lymphocytes (N/L ratio) can be considered as a surrogate indicator of their stress level. In this paper, we reviewed the common blood sampling positions, methods and precautions for blood sampling in amphibians, summarized the effects of multiple factors including species, developmental stage, urbanization, environmental pollutants, temperature and disease on the percentages of neutrophils and lymphocytes and the N/L ratio, and the relationship between the N/L ratio and stress response capacity in amphibians. Combined with amphibian’s life history characteristics, at least three further areas of work should be urgently carried out:1) Optimalization and standardization of blood sampling technique for the tadpoles, juvenile and adult amphibians, and making it suitable for amphibian research in the field; 2) It is optimal to clearly define the relationship between a single factor and the percentage of neutrophils, the percentage of lymphocytes, and their ratio in the blood of tadpoles, juvenile and adult amphibians; Meanwhile, it is also important to clarify the relationship between the health status of amphibian and these above-mentioned indicators in the short term or for a longer period under the combination of multiple factors; 3) From the immunological point of view, to clarify whether there is a trade-off relationship between different components of the immune system and between immune function and other energy-consuming physiological activities for some key life history stages from aquatic to terrestrial phase transitions, and to provide a novel idea for estimating the future fate of amphibian population. The percentage of neutrophils and lymphocytes, as well as their ratio in amphibian blood is affected by many factors, and current studies focus on intraspecific comparisons under the condition of a single factor. It is necessary to cooperate with other physiological indicators, and to interpret their ecological significances carefully if these indicators will be applied under the conditions of multiple factors or for interspecific comparative studies.

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