Abstract:[Objectives] Understanding the activity patterns and suitable habitat distribution of Dholes Cuon alpines is crucial for comprehending its survival strategies in response to environmental changes and formulating effective conservation measures. [Methods] From January 2020 to July 2024, we used both infrared camera trapping and transect methods to conduct comprehensive surveys in the Altun-Qimantag Mountains. Through kernel density estimation, we analyzed the daily activity patterns of Dholes during rainy (May-August) and dry seasons (September-April), and evaluated their suitable habitat distribution along with protection gaps using species distribution models. The analysis was performed in R (version 4.4.2), primarily relying on the activity and biomod2 packages. [Results] (1) The 134 camera traps deployed across the study area accumulated 67 864 effective camera trap days, capturing 118 independent valid records (89 independent records in the dry season and 29 in the rainy season) of Dholes at 31 locations (Table 1), with September showing the highest record frequency. (2) Dholes exhibited typical small group-living behavior with average group size of 1.6 ± 1.4 ind. (maximum observed group size: 8 ind.), displaying crepuscular bimodal activity patterns (Fig. 2). (3) Among 10 models in biomod2 package in R 4.4.2, the Generalized Boosting Model (GBM) performed the best and the MAXENT model performed the worst. The ensemble model demonstrated excellent predictive accuracy, with the TSS and AUC values of combined model being 0.987 and 0.997, respectively (Fig. 3). (4) Variable contribution analysis revealed that annual temperature range (Bio7, 72.87%), snow water equivalent (Swe, 37.75%), and mean temperature of driest quarter (Bio9, 32.16%) were the dominant factors influencing habitat suitability (Figs. 3, 4). (5) The total suitable habitat area covered 18 484.28 kmsup>2 (moderately suitable: 13 497.47 kmsup>2; highly suitable: 4 986.81 kmsup>2), with only 45.44% being within protected areas. Core distribution areas were concentrated in Lop Nur and An’nanba Nature Reserve, along with the eastern slopes of Qimantag Mountains (Fig. 5). [Conclusion] This study provides the systematic documentation of ecological adaptation characteristics of Dholes in the Altun-Qimantag Mountains, offering scientific foundations for regional conservation planning and ecosystem management of the proposed Kunlun Mountains National Park.

Abstract:[Objectives] The Amur Tiger and Amur Leopard are endangered protected animals, and using efficient means to identify and monitor them is of great significance for the conservation of species diversity.In order to solve the problems of tree occlusion, background interference, and difficulty in nighttime identification encountered in infrared camera monitoring in the wild, this study builds a deep learning model incorporating attention mechanism as a basic framework for target recognition, providing an efficient and accurate method for wildlife identification. [Methods] This study captured video images of wild animals by deploying automatic infrared cameras in the Northeast China Tiger and Leopard National Park. Eight hundred videos were selected for keyframe extraction. After noise removal, image enhancement, and image calibration, a dataset composed of 11 020 images was constructed for five species: Amur Tiger, Amur Leopard, Wild Boar, Sika Deer, and Roe Deer. This study proposed a convolutional neural network model integrating an attention mechanism module and realizing local cross-channel communication to reduce the impact of complex background environments on target recognition. This model achieved precise identification of animals in different scenarios, including day and night, different angles, and different scenes. The recognition performance of the model was evaluated via metrics such as average precision, recall, accuracy, and F1 score. [Results] The mean average precision value of the YOLO_v5m algorithm was 86.67%. After introduction of transfer learning, the mean average precision value was increased to 91.16%, and the time consumption was shortened by 106 min. Among the four types of attention mechanisms: CA, CBAM, SE, and ECA, the CA attention mechanism exhibited the best performance, achieving the average accuracy of 93.72%, which was 1.85%, 1.78%, and 1.05% higher than the other attention mechanisms, respectively (Fig. 5). [Conclusion] The deep learning model proposed in this study, which integrates transfer learning and attention mechanism, has the advantages of high accuracy and strong robustness, balancing training speed and recognition accuracy. By deploying infrared cameras to capture images of wild animals, this study can better test the potential of the model under the real living conditions of wild animals. The improved model in this study is more suitable for the identification of Amur Tigers and Amur Leopards in complex backgrounds.

Abstract:[Objectives] The safe operation of airports is an important guarantee for air transportation and social traffic. Bird activity is one of the main factors threatening airport safety, and bird strikes can lead to serious aircraft damage and even casualties. Therefore, clarifying the ecological habits and predation relationships of birds in the airport area is of great significance for formulating effective bird strike prevention measures. Hence, this study constructs and analyzes the bird-insect predation network at Hefei Xinqiao International Airport. [Methods] We collected the struck bird samples from the perimeter and internal protective facilities of Hefei Xinqiao International Airport between October 2020 and September 2022. After dissecting and identifying these samples, we selected and recorded insects from the stomach contents. Species identification was conducted with reference to relevant books and reference. Insects were classified and counted at the family level. We used complete insects as individual records and, to avoid repetition, pieced together insect fragments and counted insects based on single body parts. Data were statistically analyzed in Excel. Network-level and species-level indicators were calculated, and the network robustness under different disturbances was analyzed by the “bipartite” package in R 4.4.1, and the key species of the network were identified. [Results] A total of 21 bird species samples were collected, and insects belonging to 39 families of 9 orders were detected. We recorded 120 types of 1 630 bird-insect predation interactions, generating a bipartite network (Fig. 2). Network-level analysis indicated that compared to the null model (Table 1), the network showed decreased connectance (0.147), weighted nestedness (15.287), and niche overlap (0.205), which suggested fewer connections between birds and insects and more independent interspecies interactions in the network. However, the robustness (0.621), species specialization (0.745), and modularity (0.601) were high, indicating a higher level of specialization in species interactions and a more stable network. The network comprised four modules, within which the interactions were intensive (Fig. 3). Node-level analysis revealed that key bird species in the network were Egretta garzetta, Ardea alba, Vanellus vanellus, Anas zonorhyncha, and Pica serica, which showed the centrality indices higher than other birds (Appendix 1). Insects significantly affecting the network stability included species of Carabidae, Gryllotalpidae, Cricotidae, and Pieridae (Appendix 2). On the basis of species degree, we obtained bird species extinction curves by removing insects from both directions. Compared with the random network, the interaction network showed decreased robustness after removal of insects with high species degree first. [Conclusion] In the man-made ecosystem of an airport, the bird-insect predation network has high modularity and specialization, being stable. Different bird species may target specific insect groups for predation. Key bird species in the network were identified, and changes in important insect species in the network can cause drastic changes in bird populations. It is recommended that future airport bird prevention work focus on these species, control insect populations from the perspective of species interaction networks, and reduce the distribution of birds near airports.

Abstract:[Objectives] Hippocampus abdominalis is the largest of all known seahorses. To characterize the early embryonic development of H. abdominalis and provide a biological basis for captive breeding and rearing, we observed the embryonic development of this species. [Methods] We selected males with mating intervals within 0.5 h and conducted the experiment at the water temperature of 18.5 ± 0.5 ℃. Morphological observation was carried out on fertilized eggs and litters collected in the brood pouch every 30 min after the start of the experiment. The embryonic developmental status at each stage was recorded. [Results] The findings revealed that the peak mating period of H. abdominalis primarily occurred between 11:00 and 13:00, with significantly fewer or no mating behaviors observed at other time points. The eggs are oblong, measuring 2.63 to 3.37 mm in length and 1.28 to 1.75 mm in width. Upon fertilization, the yolk contracts into a spherical shape, creating a perivitelline space, with the yolk diameter shrinking to half of the egg’s long axis, while the overall size of the fertilized egg remains unchanged. The development process was categorized based on the external morphology and characteristic features of the embryos into fifteen distinct stages: cleavage stage, blastula stage, gastrula stage, neurula stage, optic vesicle and muscle ganglion formation stage, tail bud formation stage, heart formation stage, brain formation stage, ocular melanin appearance stage, dorsal fin and pectoral fin formation stage, ventricle and atrium formation stage, yolk sac circulation stage, hatching stage, pre-larval stage, and larval stage (Figs. 1, 2). Calculations indicate that after approximately 16 d and 12 h in the brood pouch, the embryos sequentially emerge. They then undergo approximately 4 days of further development within the pouch before being expelled when the yolk sac is nearly depleted, transitioning into the larval stage. The initial larvae measure between 10.58 and 13.02 mm in total length upon expulsion (Table 1). [Conclusion] This detailed developmental timeline of H. abdominalis offers valuable insights into the unique reproductive strategies and early life stages of this species, providing a foundational reference for further studies on seahorse biology, reproductive ecology, and conservation efforts. The comprehensive characterization of the development from fertilization to the larval stage underscores the complexity and specificity of embryonic development of seahorses.

Abstract:[Objectives] We observed the testicular microstructure of Feirana taihangnicus throughout the annual reproductive cycle to investigate its reproductive pattern. [Methods] We used histological techniques (including hematoxylin-eosin method and micro-measurement technique) to observe the testicular microstructures of F. taihangnicus in different months. The diameter of seminiferous tubules and the area of testicular interstitial regions were measured and recorded. The relevant data were analyzed by ANOVA and multiple comparisons. All statistics were performed in SPSS 22.0. [Results] The diameter of seminiferous tubules, the structure of seminiferous tubules, and the area of testicular interstitial regions in F. taihangnicus exhibited distinct annual cyclical changes. The diameter of seminiferous tubules was at its maximum when F. taihangnicus emerged from hibernation in March. During the breeding season in April, the diameter of seminiferous tubules showed no significant change compared with that at the time of emergence from hibernation (Tables 2, 3). In May, when the breeding season ended, the diameter of seminiferous tubules reached the minimum value in the annual cycle. From July to August, the peak period of spermatogenesis, the diameter of seminiferous tubules increased significantly (Tables 2, 3). In October, as F. taihangnicus was about to enter hibernation, the diameter of seminiferous tubules was already close to the maximum value (Tables 2, 3). The spermatogenetic cycle of F. taihangnicus showed a discontinuous type. It took one year from spermatogonial proliferation to spermiation. The spermatogenetic cycle of F. taihangnicus comprised five stages with significant features. Spermatogonial proliferation lasted from June to July, when spermatogonia proliferated extensively and formed numerous spermatogenic cysts within the seminiferous tubules, and a new round of spermatogenesis started. The meiotic division period of spermatocytes was from July to August. The primary spermatogonias within spermatogenic cysts underwent two successive meiotic divisions to form a large number of spermatids. Sperm formation was from August to September, when the spermatids within spermatogenic cysts underwent a process of maturation known as spermiogenesis to form bundles of spermatozoa. Sperm storage was from October to March of the following year. The spermatozoa were distributed in bundles within seminiferous tubules and matured after hibernation. As the maturity of spermatozoa increased, the motility gradually strengthened, and thus the arrangement in seminiferous tubules became more disordered. Sperm expulsion was from the end of April to May of the following year, when most of the spermatozoa in the seminiferous tubules were expelled from the body. Meanwhile, the diameter of seminiferous tubules was significantly reduced, and a small amount of spermatozoa and shed sertoli cells remained in the lumen. While only a single layer of spermatogonia and a few sertoli cells remained on the tubule wall (Fig. 3). During the breeding period, the interstitial regions of the testis were clearly visible. There were many interstitial cells around the seminiferous tubules, which were large in volume. Shortly after the end of the breeding period, the area of testicular interstitial regions reached its peak, and then gradually decreased, reaching its minimum before hibernation. During hibernation, there was no significant change in the interstitial area. After emerging from hibernation, the interstitial area showed almost unchanged compared with that before hibernation (Tables 5, 6). Interstitial cells showed marked morphological changes before and after breeding, playing a key role in regulating sex hormone synthesis and secretion. This might explain the appearance and disappearance of secondary sexual characteristics in male F. taihangnicus. [Conclusion] Significant seasonal variations were observed in the testicular microstructure of F. taihangnicus. The spermatogenesis in F. taihangnicus started in June and ended in May next year. The spermatogenetic cycle of F. taihangnicus is similar to that of other reported anuran amphibians, both consists of five stages: spermatogonial proliferation, meiosis of spermatocytes, sperm formation, sperm storage, and sperm release. However, there are some differences in the start and end time of each stage between different species (Table 7). The results enrich the basic information of amphibian reproductive biology and lay a foundation for the breeding and conservation of F. taihangnicus.

Abstract:[Objectives] Studying the changes of cell and tissue morphology in the tail regeneration process of Gekko reevesii during winter can help understand the mechanism of tail regeneration in G. reevesii. [Methods] The squeezing method was employed to induce tail self-amputation of G. reevesii, and the tail regeneration trends and influencing factors of G. reevesii at different time points (0 d, 0.5 d, 1 d, 3 d,7 d, 10 d, 15 d, 20 d, 30 d, 45 d, 60 d, and 90 d) after tail self-amputation were analyzed. Then, paraffin and frozen sections were prepared to analyze the characteristics of cells and tissues of the original tail and the regenerating tail in G. reevesii, respectively. The generalized linear mix model was adopted to analyze the effects of temperature on weight changes before and after tail regeneration. [Results] When G. reevesii amputated its tail, the skin on one side of the self-amputation site first cracked, and then the muscles were pulled out. Finally, the tail vertebrae were broken. During days 0﹣3 after tail amputation (Fig. 4), the main characteristics were small blood loss from the tail artery, no tissue contraction, exposure of vertebrae and fat, formation of blood clots, and collapse of the outer skin around the wound site to reduce the wound size. During days 3﹣30 after tail amputation (Fig. 5), the main characteristics were the contraction of exposed soft tissue (e.g. adipose tissue and axial muscle tissue), collapse of the tunica, and greater prominence of the intervertebral remnants due to the lack of muscle tissue. During days 30﹣45 after tail amputation (Fig. 6), the main features were the disappearance of exudate clots and exposure of newly formed wound epithelium, relatively flat self-section, no longer existence of the previously protruding vertebral residue, and not obvious growth. During days 45﹣90 after tail amputation (Fig. 7), although the wound healed, no tail regeneration was observed in most individuals. However, on days 60 and 90, it was found that two individuals formed regenerated tail blastema, with an average regeneration rate of 0.01 mm/d (Fig. 2). Temperature did not affect the weight changes before and after tail amputation (B = 0.001，95% CI ﹣0.006 to 0.004，Wald x2 = 0.130，P = 0.719). [Conclusion] The above results indicate that after tail amputation of G. reevesii in winter, most individuals do not regenerate the tail and only keep wound healing at a low rate.

Abstract:[Objectives] Bufo gargarizans, a nationally protected amphibian species with significant ecological and medicinal value in China, faces critical conservation challenges due to declining wild populations. Its artificial breeding is essential for sustainable resource utilization, particularly for producing Bufonis Venenum—a key component in Chinese patent medicines such as Liushen Pills and Shexiang Baoxin Pills. However, outbreaks of mass mortality in captive breeding facilities threaten conservation and pharmaceutical efforts. This study investigated the etiology of explosive mortality events occurring in June-July 2024 at a B. gargarizans farm in Shandong Province, China, where environmental conditions (26﹣35 ℃) coincided with seasonal rainfall. [Methods] Eighty-four moribund individuals of B. gargarizans were subjected to necropsy, pathological examination, and molecular diagnostics. Gross examination revealed systemic abnormalities: pulmonary edema, hepatomegaly with pallor, hydrops of the gallbladder, and intestinal edema. Histopathological analysis identified severe pulmonary lesions, including multifocal calcification, extensive tissue vacuolization, and nuclear vacuolization (Fig. 9), indicating profound structural damage. Nematodes were exclusively localized in the lungs, with no detection in the liver, kidneys or gastrointestinal tract. Morphological characterization integrated light microscopy (LM; Fig. 3, 4) and scanning electron microscopy (SEM; Fig. 5). The parasites exhibited fusiform bodies (2.866﹣8.817 mm length × 0.177﹣0.320 mm width), transverse cuticular annulations, funnel-shaped buccal capsules, cylindrical esophagi, and conical tails with terminal papillae, which were consistent with those of Rhabdias spp. Comparative morphometrics against other Rhabdias species (Table 2) further supported taxonomic assignment. [Results] Molecular identification confirmed the pathogen as R. nipponica. Genomic DNA was extracted from nematodes, and ITS (730 bp) and COI (436 bp) genes were amplified with primers NC5/NC2 and COI-F/COI-R, respectively (Table 1). BLAST alignment of the obtained sequences (GenBank accession number: PQ669203 for ITS; GenBank accession number: PQ669209 for COI) showed 99.65% similarity to R. nipponica ITS (GenBank accession number: AB818377) and 98.66% similarity to R. nipponica COI (GenBank accession number: LC671279). Phylogenetic analysis (neighbor-joining method, 1 000 bootstrap replicates) placed the isolates within the R. nipponica clade with strong support (95% for ITS, 63% for COI; Figs. 7, 8). Crucially, PCR screening excluded viral co-infections (LMBV, MRV, RGV, and FV3; Fig. 2). [Conclusion] Epidemiological analysis revealed high parasitism: 65 of 84 (77.38%) B. gargarizans individuals were infected, harboring 283 nematodes (mean intensity: 4.4 nematodes/toad). The extensive pulmonary damage observed—attributed to mechanical obstruction, inflammatory responses, and impaired gas exchange—directly correlated with clinical symptoms (lethargy, anorexia, and dyspnea) and mortality. This study provides the first conclusive evidence of R. nipponica as a primary pathogen causing epizootic mortality in farmed B. gargarizans. The integration of histopathology, morphometrics, and molecular phylogenetics establishes a robust diagnostic framework for lung nematodiasis. Our findings underscore the urgent need for targeted anthelmintic protocols (e.g., ivermectin or fenbendazole, validated in related studies) and environmental disinfection strategies to mitigate transmission. This work lays a foundation for biosecurity measures essential for sustainable farming of B. gargarizans, aligning with national policies promoting non-food wildlife utilization for medicinal conservation.

Abstract:[Objectives] To analyze the structure and characteristics of the mitochondrial genome of the genus Micromys and elucidate the phylogenetic relationships of this genus through mitochondrial genomic analysis, we conducted mitochondrial genome sequencing for M. minutus, M. erythrotis, and M. pygmaeus. [Methods] The mitochondrial genome structure, base composition, and codon usage were analyzed by SnapGene 6.0.2 and PhyloSuite 1.2.2. The secondary structure of tRNA was analyzed via the online tool tRNAscan-SE. On the basis of the concatenated sequences of 13 protein coding genes, the maximum likelihood and Bayesian inference phylogenetic trees were constructed in MEGA 11.0 and MrModeltest2 v2.4, respectively. [Results] The complete mitochondrial genome sequences of M. minutus, M. erythrotis, and M. pygmaeus obtained in this study were 16 239 bp, 16 240 bp, and 16 239 bp, respectively. Their structures all included 22 tRNA-encoding genes, 13 protein-coding genes, 2 rRNA-encoding genes, one origin of light strand replication (OL), and one control region D-loop. The codon usage patterns of M. minutus and M. pygmaeus were similar, while M. erythrotis showed great differences in codon usage from the other two species (Fig. 1). The 20 types of tRNA in the three species could form a cloverleaf secondary structure, while trnK and trnS1 could not fold into a cloverleaf structure due to the absence of D-loop and dihydrouridine arm. All mismatches in the tRNA secondary structures were U-G mismatches. The genetic distances across different parts of the mitochondrial genome indicated that M. minutus and M. pygmaeus had a smaller genetic distance (Table 1), and the phylogenetic tree also showed that the two species were in the same branch, indicating a closer phylogenetic relationship (Fig. 2). [Conclusion] The mitochondrial genome structure and characteristics of the three species of Micromys in this study are consistent with those of vertebrates. The analysis of the mitochondrial genome revealed that M. minutus and M. pygmaeus have a closer kinship, while M. erythrotis is distant from the other two species.

Abstract:Coral reef ecosystems harbors high biodiversity, which makes them among the most biologically complex and valuable ecosystems on the planet. Coral reef fish represents a crucial functional group within these ecosystems. However, the species diversity of coral reef fish in the South China Sea remains largely underestimated. During an investigation of coral reef fish samples from the South China Sea, we identified a new record of coral reef fish in China through morphological comparison and molecular identification. We employed a comparative analysis of 18 morphological characteristics alongside molecular identification with COI as the molecular maker. MitoZ was used to assemble the complete mitochondrial genome of the record after whole genome resequencing. After extraction of the COI gene, a maximum likelihood phylogenetic tree was constructed in RAxML, and a Bayesian inference tree was constructed in Beast2. Genetic distances were estimated via the Kimura 2-parameter (K2P) model implemented in MEGA 11. Thirteen coding genes were extracted and concatenated to construct the maximum likelihood phylogenetic tree in PhyloSuite. We identified the species as a new record of coral reef fish in China, which was Pomacentrus cheraphilus (collected from Sanya City, Hainan Province). The morphological characteristics of the newly collected specimens were described and measured (Table 1 and Fig. 1), and their phylogenetic relationships were analyzed (Figs. 2﹣4 and Appendices 1 and 2). The discovery of the new record of coral reef fish in China enriches the diversity of China’s marine fish fauna and indicates that further enhancement of surveys and conservation efforts is required for the biodiversity of coral reef ecosystems in the South China Sea.

Abstract:This study aimed to document and analyze a newly discovered bat specimen of the genus Barbastella in Hebei Province, China and elucidate its taxonomic status through integrated morphological and molecular methodologies. In September 2024, a male Barbastella bat (specimen number: HEB-2024-058) was captured via a mist net in Hezhang Township, Shexian County, Handan City, Hebei Province. Comprehensive external morphological and cranial measurements were recorded, and high-resolution photographs were taken to facilitate detailed morphological comparisons. Muscle tissue was collected for DNA extraction, and the mitochondrial ND1 gene was amplified and sequenced. Phylogenetic analysis was conducted based on the published sequences of B. darjelingensis, B. beijingensis, and B. barbastellus, with Plecotus auritus and P. austriacus serving as outgroups. A maximum likelihood tree was constructed in Mega 11.0 to evaluate phylogenetic relationships. The specimen exhibits a forearm length of 39.9 mm and ears measuring 13.5 mm in length (Appendix 1). The ears are characterized by a nearly square morphology, with their inner edges converging across the forehead (Fig. 1). Notably, the margins of the ears lack lobes. Both the dorsal and ventral surfaces are covered in hairs that display a dark brownish-black coloration with slightly lighter tips. The hind-foot length, at 4.8 mm, is notably short, measuring less than one-third of the tibia length, which is 18.6 mm. Cranially, the specimen presents a greatest skull length of 15.0 mm, with a condylobasal length of 14.7 mm, which surpasses that of B. darjelingensis and B. beijingensis (Fig. 2). However, its upper canine width (3.4 mm) and condylocanine length (12.6 mm) are comparatively smaller. These morphological characteristics closely correspond with those of B. darjelingensis. Phylogenetic analysis of the ND1 gene further corroborated this classification, as the Hebei specimen formed a well-supported clade with B. darjelingensis, distinct from B. beijingensis (Fig. 3). Integrative morphological and molecular analyses have confirmed the specimen as B. darjelingensis, marking the first documented occurrence of this species in Hebei Province. This discovery expands the known geographical distribution of B. darjelingensis within China and highlights the ecological significance of the Taihang Mountains as a viable habitat for this species. Further investigation is necessary to evaluate its population dynamics and conservation needs.

Abstract:A total of 25 Barbastella bats (KT13742, KT13743, KT13753, PY13770, and BX13777﹣BX13797) were captured by our research group in June and July 2013 in Pingliang City of Gansu Province, Guyuan City of Ningxia Hui Autonomous Region, and Xianyang City of Shaanxi Province. They are medium-sized bats with forearm length ranging from 42.00 to 45.58 mm. The hind-foot length is less than half of the tibia length, and the length of the 3rd metacarpal bone to the 5th metacarpal bone decreases in descending order (Table 1). These bats have nearly a square outline of ears with transverse ridges (Fig. 1a). Their ears join across the forehead, with a small lobe on the middle outer edge of each pinna (Fig. 1b). The length of skulls ranges from 14.81 to 15.74 mm (Table 1), while both sagittal and lambdoidal crests are weak (Fig. 1d). The dental formula is 2.1.2.3/3.1.2.3 = 34 (Fig. 1c). With 1 000 bootstrap replicates, phylogenetic trees were reconstructed based on cytochrome b (Cyt b) and NADH dehydrogenase subunit 1 (ND1) gene sequences. The specimens in this study clustered with B. beijingensis, rather than B. darjelingensis in both cladograms of Cyt b (Fig. 2) and ND1 (Fig. 3) genes. By comparing external morphology, skull characteristics, and phylogenetic data with B. beijingensis and B. darjelingensis, these bats are identified as B. beijingensis, which are new records of Chiroptera for Gansu Province, Ningxia Hui Autonomous Region, and Shaanxi Province. The above specimens are preserved in the Institute of Zoology, Guangdong Academy of Sciences.

Abstract:Rana sangzhiensis was initially described by Shen Yuhui in 1986 from Hunan, China. On August 27, 2024, a female subadult specimen (GXNU090506) was collected from Rongshui Miao Autonomous County, Liuzhou, Guangxi. Subsequently, on October 15, 2024, a male adult specimen (GXNU102338) was collected from the same location. Morphological comparison revealed similarities to R. sangzhiensis (Figs. 1, 2). In addition, the 16S ribosomal RNA (16S rRNA) gene sequences were analyzed, with sequencing peak maps evaluated and manually corrected. Additionally, 60 sequences from 18 Rana species were retrieved from GenBank for comparison (Table S1). Phylogenetic analyses were conducted using Bayesian inference (BI) and Maximum likelihood (ML) methods via PhyloSuite. Species boundaries were delineated via the Bayesian Poisson Tree Processes (bPTP). The phylogenetic analysis based on the 16S rRNA gene indicated that the collected specimens clustered with R. sangzhiensis specimens (Figs. 3, 4), exhibiting uncorrected pairwise distances (p-distance) of 0.2% (Table 2). This p-distance was notably lower than the interspecific p-distances observed within the genus Rana (0.8%﹣8.4%) and significantly lower than the p-distance to the outgroup (11.6%). Integrating morphological characteristics with molecular phylogenetic evidence, we conclude that the two specimens collected from Guangxi belong to R. sangzhiensis. This finding represents a new record for amphibian distribution in Guangxi and extends the known distribution range of the R. johnsi group.

Abstract:During a field survey of amphibians in Meihua Mountain National Nature Reserve, Longyan City, Fujian Province in October 2024, four specimens (1 male and three females) of Rana were collected. The study aims to accurately identify the species of these specimens through morphological and phylogenetic comparisons, enriching the diversity of amphibians in Fujian Province. Morphological identification was performed by comparing the diagnostic features described in the database of Amphibia China (http://www.amphibiachina.org/), as well as by comparing the measurements of 23 morphological indicators. Meanwhile, molecular phylogenetic identification was performed based on mitochondrial 16S ribosomal RNA (16S rRNA) gene fragments. The 16S rRNA gene fragments were amplified with primer pairs of L3975: 5′-CGC CTG TTT ACC AAA AAC AT-3′ and H4551: 5′-CCG GTC TGA ACT CAG ATC ACG T-3′. Corresponding sequences of 37 species of Rana were downloaded from GenBank (Table 1) and R. luteiventris and R. muscosa were selected as outgroups. DNA sequences were aligned and trimmed by Clustal W and the nucleotide substitution model GTR + I + G was selected based on the Bayesian information criterion (BIC) in MEGA 11. RaxmlGUI 2.0 and MrBayes 3.2.4 were used to build maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees, respectively. The uncorrected genetic distances within Rana were calculated via MEGA 11. The measurements of 23 morphological indicators were shown in Table 2, which indicated that snout-vent length of the collected specimens ranged from 59.74 to 63.04 mm; the head length was longer than the head width, and the snout was blunt pointed, obviously extending beyond the lower lip; the lengths of lower arm and hand were less than half of the body length; the hindlimbs were long, and the tibial tarsal joint extended anteriorly beyond the snout. The characteristics of skin and coloration: dorsal skin smooth, with scattered brown maculations in females; ventral skin smooth, light yellow, throat with scattered red punctate speckles and the posterior femur and its ventral surface with flat tubercles; dorsal and lateral surface olive-yellow in breeding males and reddish brown in breeding females; the dorsal skin on both hindlimbs with 9 or 10 neat and narrow black-brown horizontal stripes, without male lines (Figs. 1, 2). The morphological characteristics are consistent with the diagnosis characteristics of R. hanluica. Additionally, our specimens are larger than the topotype and those collected from other localities (Table 3). The new sequences of our specimens were deposited in GenBank with the accession number PQ608565﹣PQ608568. The BI and ML phylogenetic trees based on 16S rRNA gene fragments had identical topologies, which showed that our specimens and R. hanluica from Guangxi, Chongqing, and Hunan formed a monophyletic clade with the posterior probability of 0.99 and bootstrap support value of 100% (Fig. 3). The uncorrected p-distances among them were zero, which were much smaller than the minimum genetic distance (2.20%) between these samples and other Rana species. Based on the comprehensive comparisons of morphology and phylogenetics, the collected specimens were identified as R. hanluica, a newly record species of amphibian in Fujian Province. This discovery further enriches the geographical distribution information of R. hanluica and the diversity of amphibians in Fujian Province.

Abstract:Wild birds play a significant role in the maintenance and transmission of various zoonotic pathogens. Among them, the Oriental Magpie Pica serica is considered a representative terrestrial wild bird species. Elucidating its role in the transmission of zoonotic pathogens is of particular importance. In this study, deceased Oriental Magpies were collected from the campus of Capital Normal University, and comprehensive morphological and molecular analyses were performed on their intestinal parasitic nematodes. First, necropsies of deceased Oriental Magpies revealed a significant number of cylindrical parasites, measuring 4 to 15 cm in length, in their intestines. A stereoscopic zoom microscope (SZM) and a scanning electron microscope (SEM) were subsequently used to examine the morphological characteristics. Genomic DNA was then extracted from Ascaridia galli, and the cytochrome oxidase subunit I (COX1) gene was amplified via polymerase chain reaction (PCR). Positive clones were sequenced to identify target DNA inserts. The resulting sequences were assembled manually via BioEdit (v7.0.9) and aligned with A. galli reference sequences from GenBank by BLAST. A phylogenetic tree was constructed via the Neighbor-Joining (NJ) method with 1 000 bootstrap replicates in MEGA 9, along with a barcoding gap assessment. The parasite exhibited a three-lipped anterior oral aperture, a visible anus at the tail end, and a vulva anterior to the anus, which suggested that this specimen was female A. galli (Fig. 1). Subsequent phylogenetic analysis indicated that the parasite sequence obtained clustered with reference sequences of A. galli from chicken hosts (Fig. 2), with sequence similarity greater than 90%, conclusively identifying the parasite as A. galli. This study identified A. galli in Oriental Magpies inhabiting urban natural environments, thereby expanding current knowledge of the parasite’s host range and highlighting the important role of wild urban-dwelling birds in public health surveillance.

Abstract:[Objectives] The species diversity of terrestrial birds serves as a crucial indicator for assessing terrestrial ecosystem health and holds significant implications for formulating biodiversity conservation strategies. However, due to the high vigilance of terrestrial birds and the complexity of their habitats, conventional species diversity survey methods often have low accuracy because individuals are difficult to be observed directly in field surveys. In contrast, the survey methods based on molecular scatology do not require direct sighting and allow accurate species identification through fecal DNA analysis. However, the lack of standardized and quality-controlled procedure limits its application in species diversity research. This study aims to establish a species diversity survey protocol for terrestrial birds based on molecular scatology, covering the entire process from fecal sample collection to data analysis. [Methods] This study focused on the birds of Galliformes in the Xiaozhaizigou National Nature Reserve, Sichuan Province. Field surveys were conducted from 2021 to 2023 via the line transect method, covering 27.7% of the total area of the reserve. The surveyed areas were predominantly evergreen coniferous and broad-leaved forests, with elevations ranging from 1 438 to 3 810 m (Fig. 1). During the survey period, a total of 116 avian fecal samples were collected. Species were identified based on the molecular markers COI and Cyt b. The species identification protocol was optimized by integrating several key steps to enhance the success rate of species identification. These steps included selecting the freshness of samples, extracting avian DNA, and determining the minimum DNA concentration threshold required for identification. The Cochran-Armitage trend test was employed to examine the correlation between fecal sample freshness and species identification success rate. [Results] 1) The success rate of species identification in fresh and semi-fresh avian fecal samples was high (fresh: 100%; semi-fresh: 84.4%), whereas that in the old samples significantly decreased to 45.8%. The Cochran-Armitage trend test revealed a positive correlation between fecal sample freshness and species identification success rate (x² = 21.227, df = 1, P < 0.001). 2) The minimum DNA concentration threshold for species identification using avian feces was 3 mg/L. DNA re-extraction is recommended in the case of the DNA concentration below this value. 3) The intraspecies genetic distances based on the COI gene sequence ranged from 0.001 7 for Tragopan temminckii to 0.012 7 for Lophophorus lhuysii, with a mean of 0.007 7. In contrast, the interspecies genetic distances varied from 0.125 (between Tetraophasis obscurus and Pucrasia macrolopha) to 0.250 (between Chrysolophus pictus and Tragopan temminckii), with an average of 0.177. The interspecies genetic distance was 23 times greater than the intraspecies genetic distance, meeting the “10 × rule”, which states that an interspecies genetic distance greater than 10 times the intraspecies genetic distance can be used to differentiate species (Table 2). Furthermore, the phylogenetic analysis revealed that all species formed monophyletic groups, demonstrating that the established protocol can effectively distinguish bird species (Fig. 3). Ultimately, this study successfully identified 87 samples (75% success rate), encompassing 10 bird species, including 6 species of Galliformes (Table 1), and established a species diversity survey protocol based on avian molecular scatology (Fig. 4). [Conclusion] This study analyzes the relationship between the avian fecal freshness and species identification success rate, and optimizes the species identification protocol through a combination of multiple steps to improve identification success rate. The standardized survey protocol based on molecular scatology provides technical support for bird species diversity monitoring in nature reserves, and can be applied to biodiversity assessment and conservation management.

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