Molecular Phylogeography and Population Genetic Structure of an Endangered Species Pachyhynobius shangchengensis (hynobiid Salamander) in a Fragmented Habitat of Southeastern China

The salamander Pachyhynobius shangchengensis (Hynobiidae) is a vulnerable species restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes in the Mount Dabieshan region in southeastern China. However, molecular phylogeography and population genetic structure of P. shangchengensis remain poorly investigated. In this study, we explored the genetic structure and phylogeography of P. shangchengensis based on partial sequences of the mitochondrial DNA (mtDNA) cytochrome b and cytochrome c oxidase subunit I genes. Fifty-one haplotypes and four clades were found among 93 samples. Phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographic regions separated by complicated topography and long distances. The distinct geographic distributions of all lineages and the estimated divergence time suggest spatial and temporal separation coinciding with climatic changes during the Pleistocene. Analysis of molecular variance indicated that most of the observed genetic variation occurred among the four groups, implying long-term interruption of gene flow, and the possible separation of P. shangchengensis into four management units for conservation.     

The Impact of Stream Landscape on Genetic Structure and Dispersal Patterns in Stream Salamander(Pachyhynobius shangchengensis)

Compared with other terrestrial environments,the stream environment generally presents a linear spatial structure and relatively simple environment. In a stream landscape, the dispersal direction of stream-type organisms usually presents a linear structure along the stream,which results in the limited dispersal and the genetic differentiation of stream-type organisms across different stream sections. The Shangcheng Stout Salamander(Pachyhynobius shangchengensis) is a narrowly distributed stream salamander in Dabie Mountains of East China.In the present study, we tested for the impact of stream landscape(i.e. waterfalls and underground river) on genetic structure and dispersal pattern in P. shangchengensis based on 12 nuclear microsatellite loci from 195 individuals in 3 populations(A, B and C) from three closely connected sections within one stream. Genetic diversity results suggested that Population B contains relatively high genetic diversity for P. shangchengensis when compared to the other populations(A and C). Detectable genetic differentiation was found(FST = 0.008, P = 0.007) among three populations, which was also supported by the Structure, FCA analysis and relatedness estimates of each pair of individuals among populations. The assignment test suggested that P. shangchengensis has philopatric males and female-biased dispersal(mean female Alc =.0.031, SE= 0.225; mean male Alc = 0.026, SE = 0.198). Female-biased dispersal was also supported by analyses for each sex (i.e. Spatial autocorrelation, Genetic distance, Relatedness analysis). Our study indicated that small and isolated populations(A and C) had relatively low genetic diversity due to the limited population size. For stream salamanders,landscape features(i.e. waterfalls and underground river)can influence the ability of an individual to disperse through the landscape, and consequently influence the formation of strong genetic differentiation of P. shangchengensis.     

The Breeding Ecology of a Critically Endangered Salamander,Hynobius amjiensis(Caudata:Hynobiidae),Endemic to Eastern China

Hynobius amjiensis is a critically endangered salamander species(IUCN Red List) endemic to eastern China. It currently has three known populations: one in Longwangshan, Zhejiang Province(type locality), and two in Qingliangfeng between Anhui and Zhejiang Provinces. We examined the relatively unstudied breeding ecology of this species in the field and at laboratory from March 2007 to May 2014. Adult males and females were year-round terrestrial, except for the February–April breeding season. During this period, we captured only a total of 16 breeding adults(11 males and 5 females). As few as 100 breeding females were estimated based on the number of egg sacs observed since 2007. This number was significantly reduced from the estimated number between 1992 and 1998. Males(mean total length = 16.21 cm, mean body mass = 18.8 g) were slightly smaller than females(16.51 cm, 19.2 g). Size of breeding pools ranged from 0.2 m~2 to 1.2 m~2(0.1–1.2 m depths). Each female deposits a pair of egg sacs by attaching the adhesive tips of the sacs to aquatic plants or dead twigs. Fifteen pairs of egg sacs had an average length of 28.6 cm and a diameter of 3.3 cm. On average, each egg sac contained 75 eggs with a diameter of 0.3 cm. Our field survey revealed that H. amjiensis used oviposition sites in small, cool, and weakly acidic pools at high elevations(1 300–1 600 m) where peat moss was abundant. Reduction in wetland size and disappearance of suitable breeding pools suggest that this salamander species is under threat of extinction, particularly at Longwangshan, where 5 of the 9 breeding pools have either dried up or disappeared. Combined size of the remaining 4 pools is less than 2 m~2. We urge immediate implementation of more effective conservation measures and suggest that preservation priority should be given to habitat that contains suitable breeding pools.     

Seasonal Dynamics of Male and Female Reproductive Systems in the Siberian Salamander,Salamandrella keyserlingii (Caudata,Hynobiidae)

It is not well known how low temperatures, like a subarctic steppe–tundra climate, influence reproductive traits of ectothermic vertebrates. To begin answering this question, we studied male and female reproductive systems of Salamandrella keyserlingii inhabiting a Tomsk population(southeast of Western Siberia), Russia, in ecological and physiological terms. In males, before spermiation, the testicular size and weight in late April–early May were greatest of all. Spermiation occurred during breeding immigration in spring when mean air temperature was above 10°С, and at the same time rain fell. After spermiation, the testicular size and weight decreased sharply, and the diameter of the vasa deferentia increased. "Spawning"(i.e., simultaneous extrusion of sperm and oviposition) occurred from late April to late May, and this duration fluctuated in temperature and humidity. The testicular size and weight increased in summer. Sperm mass was detected in the testes by the smear method in April–September, except in June when single fragmented unrealized sperm was detected and in July when spermatids were detected. In females, ovarian weight was greatest in spring before ovulation. From late June, vitellogenesis began in ovarian follicles, in which mint green yolks accumulated. Melanin deposited in the surface of the ovary from July when oviducts were hypertrophying. In contrast, some large-sized females did not show any sexual maturity shortly before hibernation(although these females may be subadults). These results suggest that low temperatures in Siberia induce early timing of gamete maturation in females, but the females' reproductive cycle might also be biennial. A reproductive cycle in males was annual with the completion of the gamete maturation process in August.     

拟小鲵属(有尾目:小鲵科)一新种--水城拟小鲵

在比较和研究拟小鲵属(Pseudohynobius)已知二物种后,确定贵州省水城县石龙乡产拟小鲵为一新种,即水城拟小鲵(PseudohynobiusshuichengensisTian,LietGusp.nov)。新种与黄斑拟小鲵(P.flavomaculatus)的犁骨齿列形状相似;但新种体尾及四肢背面紫褐色,无黄色斑块;四肢长,前后肢贴体相向时,掌、部前半重叠;而黄斑拟小鲵体尾及四肢背面紫褐色,有土黄色斑块;四肢甚短,前后肢贴体相向时,指、趾仅相遇或略重叠。秦巴拟小鲵(P.tsinpaensis)背面棕褐色,体尾及四肢背面有不规则云状斑;四肢较短,前后肢贴体相向时,指、趾末端仅相遇。本文统计并记述了新种的外形、头骨特征、生物学资料。     

Age and body size of Salamandrella keyserlingii (Caudata: Hynobiidae): a difference in altitudes, latitudes, and temperatures

The debate surrounding Bergmann??s rule, in which the body size of animals is predicted to be larger in cooler environments, is still open concerning ectotherms. Our goal was to test this rule in the broadest ranging amphibian species Salamandrella keyserlingii. We determined age and body size in a cooler region (Darhadyn, Mongolia: mean yearly air temperature?=??C8.31 °C) using skeletochronology, and compared their differences in altitude, latitude, and temperature with those of a warmer area (Kushiro, Japan: 7.98 °C). In Darhadyn, both sexes reached sexual maturity at 5?C6?years of age (growth coefficient: male?=?0.585, female?=?0.266), 2?C3?years later than those in Kushiro (male?=?1.341, female?=?1.129). Mean body size was smaller in Darhadyn (53.08?mm) than in Kushiro (57.63?mm) for males despite their constant metamorphic size around 30?mm. We also analyzed data available from published studies for 27 populations within the geographic range of this species from 43 to 69°N across a 2,900-km long latitudinal gradient. The analysis indicated an intraspecific tendency to decrease body size with increased latitude from 43 to 57°N, to increase size from 57 to 69°N, and to decrease body size with decreased temperature from 8 to ?C7?°C and increase size from ?C7 to ?C15?°C. This pattern does not follow the intraspecific extension of Bergmann??s rule and may follow the converse of Terentjev??s optimum rule??a rule formulated to be an inverted-U shaped curve between increased latitude (or decreased temperature) and increased body size.     

Aggregation and site tenacity under downed logs in Salamandrella keyserlingii (Caudata: Hynobiidae)

Darhadyn Wetland, Mongolia, is located in a subarctic steppe–tundra climate, where salamanders are exposed to both extremely hot and cold environments. We hypothesized that log refugia provide an adaptive advantage to salamanders in these environments because the habitat availability hypothesis predicts that restricted habitat availability can cause aggregation that plays a role in the initial stages of the evolution of complex sociality. To test this hypothesis, we assessed whether a rare salamander species aggregated under a limited shelter resource. Many small vertebrates use terrestrial burrow refuges. While many salamander species also use burrows, Salamandrella keyserlingii at Darhadyn used only downed log refuges (i.e., restricted habitat availability). Some individuals displayed site tenacity to remain under a specific log, and one or more of the same individuals were located continually or frequently under the same logs. The majority of recapture events (96.8 %, 121/125) did not show any movements of salamanders between logs. Of the 300 capture events, 66 % were aggregating. The maximum number of individuals sharing a log refuge on the same day (i.e., sharing group size) ranged from 2 to 9. Individuals were resident nonrandomly in downed log refuges, i.e., an aggregation pattern was nonrandom. Based on these findings, we provided conservation measures such that total number of individuals captured per log over the course of the study (maximum number = 32) had a positive relationship to 1 of 7 explanatory variables (i.e., log decaying class in ascending order). Thus, the retention of decaying downed logs is important for the conservation of this species.     

Two New Species of Fire-Bellied Newts (Caudata,Salamandridae, Cynops) from Southeastern China

The fire-bellied newt genus Cynops contains ten known species distributed in China and Japan in East Asia.In this work,two new Cynops species are described,namely Cynops jiaoren sp.nov.distributed in northern Guangdong and Cynops maguae sp.nov.distributed in eastern Jiangxi.The two new species can be distinctly distinguished from their congeners by the independent phylogenetic placements and a combination of morphological characteristics.The discovery of these two new species in the Southeast Ch...     

The complete mitochondrial genome sequence of the Kori salamander Hynobius yangi (Caudata: Hynobiidae)

The complete mitochondrial genome (JN415127) was sequenced and annotated newly from an individual of Hynobius yangi populations which were collected in Ulju-gun, Korea. The total length of the H. yangi genome is 16,403 bp, with a total base composition of 33.5% A, 32.3% T, 20.5% C, and 13.6% G.     

Sexual Dimorphism of the Jilin Clawed Salamander,Onychodactylus zhangyapingi,(Urodela:Hynobiidae:Onychodactylinae) from Jilin Province,China

Sexual dimorphism in size and shape is common in many organisms, and is a key evolutionary feature. In this study, we analyzed morphometric data of the Jilin clawed salamander Onychodactylus zhangyapingi, an endemic Chinese salamander, to examine sexual size and shape dimorphism. The morphometric data included 14 characteristics of 13 females and 11 males and was analyzed using univariate and multivariate methods. Our results showed that sexual dimorphism occurs not only in body size, but also in body shape. Males have a longer snout-vent length than females, a rarely reported pattern of male-biased sexual size dimorphism. Females have a larger space between the axilla and groin than males, while males have longer and larger tails compared to females. The sexual dimorphism in body size and shape can be explained by existing theories, but there is little data for the mating system, behavior, reproduction, or ecology of O. zhangyapingi, so further studies are required.     

爪鲵消化系统的解剖学和组织学初步研究

本文报道了爪鲵消化系统的形态学和组织学结构特点。爪鲵口腔底部具有肌肉质的舌,食管很短,胃是呈纺锤形的长囊,胃壁较厚,粘膜厚,胃腺发达。消化管肌层皆为平滑肌,环肌明显多于纵肌。肝脏较大,分左、中、右三叶;有胆囊;胰腺长带状,胰管与胆管汇合后与小肠最前部的十二指肠相连。     

Phylogenetic relationships of two Salamandrella species as revealed by mitochondrial DNA and allozyme variation (Amphibia: Caudata: Hynobiidae)

We conducted molecular phylogenetic analyses to confirm taxonomic relationships and to delimit distributional ranges of Siberian salamanders, Salamandrella keyserlingii and Salamandrella schrenckii, and to elucidate the origin of the isolated population of this species complex on Hokkaido, Japan. Phylogenetic trees constructed by MP, NJ, ML, and Bayesian methods, using complete sequences of mitochondrial cytochrome b genes, all indicated monophyly of Salamandrella and of each of the two species. Identical relationships were found on UPGMA, NJ, and CONTML trees derived from electrophoretic analysis of variation in 18 inferred allozyme loci. Populations from Hokkaido and northeastern China proved to be S. keyserlingii, while populations from Khabarovsk and Lazovsky are S. schrenckii. Genetic differentiations of S. keyserlingii within Sakhalin, and between Sakhalin and Hokkaido, are substantial. The Hokkaido population is hypothesized to have been isolated on the island since early Pleistocene, much earlier than isolation of sympatric anuran populations from their Sakhalin relatives. In contrast, the continental populations of S. keyserlingii are only slightly differentiated from some Sakhalin populations, and are thought to have expanded their ranges in the late Pleistocene.     

Episodes of adaptive evolution of mitochondrial genome in asiatic salamanders (Amphibia,Caudata, Hynobiidae)

To elucidate the effect of natural selection on the evolution of mitochondrial DNA (mtDNA) in Asiatic salamanders of the family Hynobiidae, nucleotide sequences of 12 protein-coding genes were analyzed. Using a mixed effects model of evolution, it was found that, in spite of the pronounced effect of negative selection on the mtDNA evolution in Hynobiidae (which is typical for the animals in general), two phylogenetic clusters, the West Asian one, represented by the genera Ranodon and Paradactylodon, and North Eurasian one, represented by the genus Salamandrella, were formed under the influence of episodic positive selection. Analysis of protein sequences encoded by the mitochondrial genome also supported the influence of positive selection on the evolution of Hynobiidae at some stages of their cladogenesis. It is suggested that the signatures of adaptive evolution detected in the mtDNA of Hynobiidae were determined by the complex and long-lasting history of their formation, accompanied by adaptation to the changing environment.     

COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae)

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.     

Ranodon cf.Sibiriens (Amphibia,Caudata) from the upper pliocene of Southern Kazakhstan: The first fossil record of the family Hynobiidae

PalZ - Remains of a hynobiid salamanderRanodon cf.Sibiriens are reported from the Upper Pliocene (Early Villafranchian, mammal zone MN 16) of the Zaili Alatau Range, southern Kazakhstan. Vertebrae...