Isolation and characterisation of 17 microsatellite DNA loci from RAD reduced-representation genomes for Asian warty newts, genus Paramesotriton (Caudata: Salamandridae).

Asian warty newts, genus Paramesotriton, are endemic to southern China and northern Vietnam. Despite the achievements in biodiversity, molecular systematics and biogeography of species in this genus, population genetic diversity studies are lacking due to the lack of economical and available genetic markers. In this study, we developed 17 highly polymorphic microsatellite loci from RAD simplified genomic data for the Asian warty newts, genus Paramesotriton and successfully completed cross-species amplification tests on 20 samples of four species of Paramesotriton. These microsatellite markers can be used as important tools to study population genetic structure, levels of gene flow, population differentiation, mating systems and landscape genetics within the genus Paramesotriton and, thus, to make scientific conservation decisions and actions for the conservation of these rare and endangered amphibians.

A preliminary study on home range and daily travel distance of François' langur in a high-altitude area.

François' langur (Trachypithecus francoisi) is an indicator species living in Karst rocky habitats. To understand the François' langurs ecological adaptation to high-altitude habitat, we used the field tracking method to collect home-range data and daily travel distance of François' langur at high-altitude (800-1400 m) areas of Kuankuoshui National Nature Reserve between April 2008 and March 2009. The results revealed the following: (1) according to the grid cell method, the home range of François' langurs was estimated to be 50.7 ha. The area in the winter-spring season was larger than in the summer-autumn season (37.7 vs. 27.7 ha). According to the minimum convex polygon method by GIS, the home range of François' langurs in the winter-spring season and the summer-autumn season was 123.5 and 68.8 ha, respectively. The whole-year home range of François' was clearly larger than the grid cell method (140.4 vs. 50.7 ha). (2) The home range of François' langurs had clear superposition. Langur's core areas were concentrated in three small areas, which only occupied 18.2% of the home range. (3) The langur had a short daily travel distance (230-1115 m) and significant seasonal differences. The summer-autumn season moving distances being obviously shorter than the winter-spring season (517 vs. 785 m). (4) With the decrease in the availability of food resource in winter-spring season, the home range and daily travel distance of François' langurs significantly increased. (5) Living in the high altitudes, François' langurs tended to form a larger group (≥ 8 individuals), which is larger than other groups at lower altitude, had the larger home range, and had shorter daily travel distance. Our results indicate that colobines' survival strategies tend to form a larger home range and shorter day-traveling distance to adapt to high-altitude and low-temperature habitat.

Mitochondrial genome of a typical cavefish of Sinocyclocheilus anatirostris.

Sinocyclocheilus anatirostris Lin and Luo, 1986 is a member of the endemic Chinese genus Sinocyclocheilus Fang, 1936, living in dark caves with absence of eyes and scales. Muscle tissue was collected from cavefish samples from Guangxi, China, and complete mitogenome was sequenced. This is the first report of the mitogenome of S. anatirostris. This mitogenome consists of 13 protein-coding genes (PCGs), two rRNA genes (12S rRNA and 16S rRNA), 22 tRNA genes, a control region (CR), and comprises 31.2% A, 24.4% T, 16.7% G, and 27.7% C bases. Phylogenetically, S. anatirostris is closely related to the Sinocyclocheilus furcodorsalis, and originated in the late Miocene, ∼6.07 Ma.

Oreonectesdamingshanensis (Cypriniformes, Nemacheilidae), a new species of stream fish from Guangxi, Southwest China.

In this work, a new species of the genus Oreonectes is described, named Oreonectesdamingshanensis Yu, Luo, Lan, Xiao & Zhou, sp. nov., collected from the Damingshan Mountains of the Guangxi Zhuang Autonomous Region, China. Phylogenetic trees constructed based on the mitochondrial Cyt b showed that the new species represents an independent evolutionary lineage, with uncorrected genetic distances (p-distance) from congeners ranging from 6.1% to 8.9%. Morphologically, the new species can be distinguished from five other species of the genus by a combination of characters. The discovery of this new species raises the number of known species of Oreonectes from five to six. Our study suggests that O.platycephalus may be a complex containing multiple species and that previously recorded areas need to be further delimited and reevaluated.

Integrated phylogenetic analyses reveal the evolutionary, biogeographic, and diversification history of Asian warty treefrog genus Theloderma (Anura, Rhacophoridae).

Asian warty treefrogs, genus Theloderma, are morphologically variable arboreal frogs endemic to Southeast Asia and Southern China. However, integrated systematic studies are lacking, and knowledge of the genus in terms of diversity, origin, and historical diversification remains limited. To address these knowledge gaps, we used three mitochondrial and five nuclear gene fragments to reconstruct the Theloderma phylogeny, estimate divergence times, and examine the biogeography of the genus. Phylogenetic and species delimitation analyses suggest that the genus Theloderma comprises three major clades corresponding to two subgenera and seven species groups, and mPTP identified at least 12 putative cryptic species, suggesting that species diversity has been underestimated. Biogeographic analyses indicated that most recent common ancestor of Theloderma originated in the Indochina Peninsula during the Middle Oligocene (ca. 27.77 Ma) and the splitting of Clade A to C occurred in the Late Oligocene (ca. 23.55-25.57 Ma). Current biogeographic patterns result from two distinct processes: in situ diversification in the Indochina Peninsula and dispersal in multiple areas, namely southward dispersal to the Malay Peninsula and Borneo, northeastward dispersal to Southern China, northward dispersal to the Himalayas, and dispersal from Southern China to the Indochina Peninsula. Ancestral character reconstruction suggests that the ancestor of Theloderma may have possessed a small body size, rough dorsal skin, and absence of vomerine teeth and hand webbing, and that these four characters have undergone multiple evolutions. Principal component analysis based on eight bioclimatic variables did not clearly distinguish the three major clades of Theloderma, suggesting that species in these clades may occupy similar climatic ecological niches. Our research highlights the importance of orogeny and paleoclimatic changes, in shaping amphibian biodiversity in mountain ecosystems.

Phylogenetic analysis of combined mitochondrial genome and 32 nuclear genes provides key insights into molecular systematics and historical biogeography of Asian warty newts of the genus Paramesotriton (Caudata: Salamandridae).

The Paramesotriton Chang, 1935 genus of Asian warty newts is the second most diverse genus in the family Salamandridae, currently containing 14 recognized species from northern Vietnam to southwest-central and southern China. Although species of this genus have been included in previous phylogenetic studies, the origin and interspecific relationships of the genus are still not fully resolved, especially at key nodes in the phylogeny. In this study, we sequenced mitochondrial genomes and 32 nuclear genes from 27 samples belonging to 14 species to reconstruct the interspecific phylogenetic relationships within Paramesotriton and explore its historical biogeography in southern China. Both Bayesian inference and maximum-likelihood analyses highly supported the monophyly of Paramesotriton and its two recognized species groups ( P. caudopunctatus and P. chinensis groups) and further identified five hypothetical phylogenetic cryptic species. Biogeographic analyses indicated that Paramesotriton originated in southwestern China (Yunnan-Guizhou Plateau/South China) during the late Oligocene. The time of origin of Paramesotriton corresponded to the second uplift of the Himalayan/Qinghai-Xizang (Tibetan) Plateau (QTP), rapid lateral extrusion of Indochina, and formation of karst landscapes in southwestern China. Principal component analysis (PCA), independent sample t-tests, and niche differentiation using bioclimatic variables based on locations of occurrence suggested that Paramesotriton habitat conditions in the three current regions (West, South, and East) differ significantly, with different levels of climatic niche differentiation. Species distribution model (SDM) predictions indicated that the most suitable distribution areas for the P. caudopunctatus and P. chinensis species groups are western and southern/eastern areas of southern China. This study increases our knowledge of the taxonomy, biodiversity, origin, and suitable distribution areas of the genus Paramesotriton based on phylogenetic, biogeographic, and species distribution models.

A species of the genus Panophrys (Anura, Megophryidae) from southeastern Guizhou Province, China.

Herein, we describe Panophrys congjiangensis sp. nov. obtained from the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China. Phylogenetic analyses based on the mitochondrial genes 16S rRNA and COI indicated that this new species represented an independent lineage, closely related to P. leishanensis. The uncorrected genetic distances between the new species and its closest congener, P. leishanensis, were 3.0% for 16S rRNA and 8.4% for COI. The new species is distinguished from its congeners by a combination of the following morphological characteristics (1) medium body size (SVL 28.6-33.4 mm in males and 38.4-40.2 mm in females); (2) a small horn-like tubercle at the edge of each upper eyelid; (3) the tympanum distinctly visible (TD/ED ratio 0.47-0.66); (4) vomerine teeth absent; (5) the tongue not notched behind; (6) a narrow and unobvious lateral fringe on toes; (7) relative finger lengths II < I < V < III; (8) rudimentary webs on toes; (9) hindlimbs slender, heels overlapping when thighs are positioned at right angles to the body; (10) two metacarpal tubercles on the palm, with the inner metatarsal tubercle long and oval-shaped; (11) the tibiotarsal articulation reaching the nostril when the leg is adpressed and stretched forward; (12) dorsal skin rough with numerous orange-red granules, ventral surface smooth; (13) a single internal subgular vocal sac present in males; and (14) in breeding males, weak gray-black nuptial pads with black nuptial spines present on the dorsal surface of the bases of the first and second fingers. To date, the new species is only known from the type locality.

Reassignment of 'Triplophysa' jiarongensis (Cypriniformes: Nemacheilidae) to Oreonectes Günther, 1868.

Specimens identified as Oreonectes jiarongensis (Cypriniformes: Nemacheilidae) were collected from a karst cave in Jiarong Town and Banzhai Township, Libo County, Guizhou, China. Several lines of morphological and molecular evidence suggested that this species was similar to species of Oreonectes Günther 1868 and not closely related to species of Triplophysa Rendal, 1933. The anterior and posterior nostrils of 'Triplophysa' jiarongensis were separated by a short distance, and there was no secondary sexual dimorphism in male specimens. The Bayesian phylogenetic analysis based on cytochrome b recovered 'T.' jiarongensis in a well-supported clade with Oreonectes daqikongensis, O. shuilongensis, O. furcocaudalis and O. platycephalus, sister to the Triplophysa clade. In addition, the genetic distances between 'T.' jiarongensis and species of Oreonectes were low (O. daqikongensis: 0.114; O. shuilongensis: 0.106; O. platycephalus: 0.180), while distances to species of Triplophysa were higher (T. dorsalis: 0.233; T. yarkandensis: 0.282). Therefore, we reassign 'T.' jiarongensis to Oreonectes. Based on this result, it is now clear that species of both Oreonectes and Triplophysa inhabit the same underground river system in Guizhou. However, as species of Oreonectes are only known from the southern part of Guizhou, it may be that O. jiarongensis inhabits the most northerly part of the range of this genus.

"Juggling" Behavior in Wild Hainan Gibbons, a New Finding in Nonhuman Primates.

Many species of primates use tools and manipulate objects. Environmental objects, such as sticks and branches, are used in locomotion, display, conflict, nesting, and foraging. This study presents observations regarding endangered male Hainan gibbons (Nomascus hainanus) selecting sticks and then throwing and catching them repeatedly. This act of Hainan gibbons was termed as "juggling" behavior. This study is the first record of branch use of this kind in free-living gibbons. While it is impossible to experiment on this only remaining population of Hainan gibbons, the deliberate acquisition and then throwing and catching of a stick raises myriad questions regarding their function. The study determined that the juggling behavior of Hainan gibbons, in the process of their brachiation, helps them accurately judge the distance and support strength of an object. It was also found that an adult individual's proficiency in juggling behavior was much higher than that of a youth. Of all gibbon species, the juggling behavior of Hainan gibbon has a high degree of behavior refinement. Gibbons have the longest forearm than any other primates, which helps them in such performances-a unique mechanism that allows them to perform such unique activities, including juggling.

Non-specific alarm calls trigger mobbing behavior in Hainan gibbons (Nomascus hainanus).

Alarm calls are important defensive behaviors. Here, we report the acoustic spectrum characteristics of alarm calls produced by Hainan gibbons (Nomascus hainanus) inhabiting Bawangling National Nature Reserve in Hainan, China. Analysis of call data collected from 2002-2014 shows that alarm calls are emitted by all family group members, except infants. Alarm behavior included simple short alarming calls (7-10 min) followed by longer variable-frequency mobbing calls lasting 5-12 min. The duration of individual alarming and mobbing calls was 0.078 ± 0.014 s and 0.154 ± 0.041 s at frequency ranges of 520-1000 Hz and 690-3920 Hz, respectively. Alarming call duration was positively associated with group size. The alarm calls can trigger mobbing behavior in Hainan gibbons; this is a defense way of social animals, and first report among the primates' species. The system of vocal alarm behavior described in this critically endangered species is simple and effective.

A new blind species of the cave genus Oreonectes from Guizhou, China (Nemacheilinae).

This study aimed to describe a new specimen of cavefish collected from a karst cave in the Daqikong area of Libo County, Guizhou. Twenty-six cavefish specimens were collected and identified as a new species of Balitoridae: Nemacheilinae, and named Oreonectes daqikongensissp. n. A genetic analysis was performed and showed that its genetic distances from Oreonectes shuilongensis and Oreonectes platycephalus are higher than intraspecific distances. Discovery of this species will be helpful to understand the distribution of Oreonectes.

A new species of the genus Oreonectes (Cypriniformes: Nemacheilidae) from Guizhou, China.

This study examined the morphological and phylogenetic characteristics of Oreonectes shuilongensis sp. nov. The genus Oreonectes is distributed only in southwestern China and northern Vietnam.  Sixteen specimens of cavefish were captured in the Shuilong Township in Guizhou Province of China and identified as a new species of Oreonectes (Cypriniformes: Nemacheilidae).  Maximum likelihood was used to construct phylogenetic trees based on data for Nemacheilidae species accessible on GenBank. Molecular phylogenetic analyses showed that O. shuilongensis is closely related to O. platycephalus from which it is distinguished by having 6 vs. 5 branched anal rays, ratio of body length to body height 7.0-7.9 vs. 5.6-7.0, ratio of head length to distance between eyes 3.5-4.9 vs. 2.0-2.4, naked vs. scaly body, densely distributed fine black stains except on abdomen, and semitransparent body. Oreonectes shuilongensis is the only species of the genus not distributed in Guangxi Province. This study has contributed to the overall knowledge about the distribution of Oreonectes.

The analysis for mitochondrial genome sequencing of Triplophysa lewanggensis.

Triplophysa lewanggensis revealed that the complete length of its mitochondrial genome was 16,568 bp, composed of A (31.7%), T (27.1%), G (15.9%), C (25.4%), and A + T (58.8%). Its genetic constitution and arrangement were consistent with other Triplophysa, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control area (D-loop). All genes were encoded by the H-strand, except for one protein-coding gene (ND6) and eight tRNA genes (tRNAGln, tRNAAla, tRNACys, tRNAAsn, tRNATyr, tRNASer(UCN), tRNAGlu, tRNAPro) are encoded by the L chain, and the remaining genes are encoded by the H chain. The phylogenetic tree was divided into two main clades: one just including Triplophysa and the other including Oreonectes, Homatula, Schistura, Nemacheilus, Traccatichthys. T. lewanggensis is distributed in Southwest Guizhou Province attached to Yunnan-Guizhou Plateau, T. xiangxiensis is distributed in Hunan Province, they are near relatives, but their kinship with Triplophysa distributed in Tibetan Plateau is farther.

Complete sequence determination and analysis of mitochondrial genome in Oreonectes daqikongensis.

Oreonectes daqikongensi revealed that the complete length of its mitochondrial genome was 16,578 bp, composed of A (30.7%), T (25.6%), G (16.2%), C (27.5%) and A + T (56.3%). Its genetic constitution and arrangement were consistent with those of other Osteichthyes, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control area (D-loop). All genes were encoded by the H-strand, except for 1 protein-coding gene (ND6) and 8 tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Cys, tRNA-Asn, tRNA-Tyr, tRNA-Ser, tRNA-Glu and tRNA-Pro) are encoded by the L-strand. The mitochondrial genes were arranged very closely. There were four gene overlapping regions, with an overall length of 21 bp and a base covering number range of 1-14 bp, and 23 intergenic regions, with an overall length of 147 bp and an intergenic length range of 1-19 bp. There were 10 gene pairs that were neither overlapping nor intergenic.