Hidden hotspots of amphibian biodiversity in China.

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

Large X-effects are absent in torrent frogs with nascent sex chromosomes.

Sex chromosomes are popularized as a special role in driving speciation. However, the empirical evidence from natural population processes has been limited to organisms with degenerated sex chromosomes, where hemizygosity is mainly considered to act as the driver of reproductive isolation. Here, we examined several hybrid zones of torrent frog Amolops mantzorum species complex, using an approach by mapping species-diagnostic loci onto the reference genome to compare sex-linked versus autosomal patterns of introgression. We find little support in sex-linked incompatibilities for large X-effects for these populations in hybrid zones with homomorphic sex chromosomes, due to the absence of the hemizygous effects. As expected, the large X-effects were not found in those with heteromorphic but newly evolved sex chromosomes, owing to the absence of strong genetic differences between X and Y chromosomes. The available data so far on amphibians suggest little role for sex-linked genes in speciation. The large X-effects in those with nascent sex chromosomes may not be as ubiquitous as presumed across the animal kingdom.

Heterogeneous Evolution of Sex Chromosomes in the Torrent Frog Genus Amolops.

In sharp contrast to birds and mammals, in numerous cold-blooded vertebrates, sex chromosomes have been described as homomorphic. This sex chromosome homomorphy has been suggested to result from the high turnovers often observed across deeply diverged clades. However, little is known about the tempo and mode of sex chromosome evolution among the most closely related species. Here, we examined the evolution of sex chromosome among nine species of the torrent frog genus Amolops. We analyzed male and female GBS and RAD-seq from 182 individuals and performed PCR verification for 176 individuals. We identified signatures of sex chromosomes involving two pairs of chromosomes. We found that sex-chromosome homomorphy results from both turnover and X-Y recombination in the Amolops species, which simultaneously exhibits heterogeneous evolution on homologous and non-homologous sex chromosomes. A low turnover rate of non-homologous sex chromosomes exists in these torrent frogs. The ongoing X-Y recombination in homologous sex chromosomes will act as an indispensable force in preventing sex chromosomes from differentiating.

UCE Phylogenomics, detection of a putative hybrid population, and one older mitogenomic node age of Batrachuperus salamanders.

The prevalence of incomplete lineage sorting complicates the examination of hybridization and species-level paraphyly with gene trees of a small number of loci. In Asian mountain salamanders of the genus Batrachuperus, possible hybridization and species paraphyly had been identified by utilizing mitochondrial genealogy and fixed allozyme differences. Here we sampled 2909 UCEs in 44 local populations from all six Batrachuperus species, inferred gene and species trees, compared them with mitochondrial and allozyme results, and examined the potential hybridization and species paraphyly. The clustering pattern of single-locus trees, increased proportion of heterozygous SNPs, allele frequency-based migration edge estimation, and intrapopulation long branches (as expected from an increase of genetic lineage and nucleotide diversity) support that an eastern B. karlschmidti population has experienced admixture with B. tibetanus. On the 2909-UCE concatenated and species trees, lower nodal supports were observed when similar proportions of loci agreed with alternative topologies, i.e., a reciprocal monophyly between a Pengxian lineage and the remainder of B. pinchonii (0.379) or a paraphyly of the latter with respect to Pengxian (0.362). The UCE phylogenomics agreed with the relatively recent groupings in the allozyme dendrogram. Despite incomplete lineage sorting, the mitochondrial trees were similar to the UCE trees for deeper relationships of the genus. However, one significant branch-length level discordance was identified. The branch between the common ancestor of B. daochengensis and B. yenyuanensis and common ancestor of the genus was approximately three times shorter on the mitochondrial tree than on the UCE tree, suggesting that the split of the mitochondrial lineages was likely a few million years earlier than the split of species. This finding supports considering possible ancestral polymorphism when interpreting different divergence dates estimated from mitochondrial and genome-wide data.

Dynamics behind disjunct distribution, hotspot-edge refugia, and discordant RADseq/mtDNA variability: insights from the Emei mustache toad.

BACKGROUND: The distribution of genetic diversity and the underlying processes are important for conservation planning but are unknown for most species and have not been well studied in many regions. In East Asia, the Sichuan Basin and surrounding mountains constitute an understudied region that exhibits a "ring" of high species richness overlapping the eastern edge of the global biodiversity hotspot Mountains of Southwest China. We examine the distributional history and genetic diversification of the Emei mustache toad Leptobrachium boringii, a typical "ring" element characterized by disjunct ranges in the mountains, by integrating time-calibrated gene tree, genetic variability, individual-level clustering, inference of population splitting and mixing from allele frequencies, and paleoclimatic suitability modeling. RESULTS: The results reveal extensive range dynamics, including secondary contact after long-term isolation via westward dispersal accompanied by variability loss. They allow the proposal of a model that combines recurrent contractions caused by Quaternary climatic changes and some failed expansions under suitable conditions for explaining the shared disjunct distribution pattern. Providing exceptional low-elevation habitats in the hotspot area, the eastern edge harbors both long-term refugial and young immigrant populations. This finding and a synthesis of evidence from other taxa demonstrate that a certain contributor to biodiversity, one that preserves and receives low-elevation elements of the east in this case, can be significant for only a particular part of a hotspot. By clarifying the low variability of these refugial populations, we show that discordant mitochondrial estimates of diversity can be obtained for populations that experienced admixture, which would have unlikely left proportional immigrant alleles for each locus. CONCLUSIONS: Dispersal after long-term isolation can explain much of the spatial distribution of genetic diversity in this species, while secondary contact and long-term persistence do not guarantee a large variation. The model for the formation of disjunct ranges may apply to many other taxa isolated in the mountains surrounding the Sichuan Basin. Furthermore, this study provides insights into the heterogeneous nature of hotspots and discordant variability obtained from genome-wide and mitochondrial data.

Chromosomal Evolution in the Amolops mantzorum Species Group (Ranidae; Anura) Narrated by Repetitive DNAs.

In an attempt to analyze the organization of repetitive DNAs in the amphibian genome, 7 microsatellite motifs and a 5S rDNA sequence were synthesized and mapped in the karyotypes of 5 Amolops species. The results revealed nonrandom distribution of the microsatellite repeats, usually in the heterochromatic regions, as found in other organisms. These microsatellite repeats showed rapid changes among Amolops species, documenting the recent evolutionary history within this lineage. In contrast, 5S rDNA was localized in chromosomes 5 of all species, suggesting that these chromosomes are homologous within the monophyletic clade. Furthermore, the heteromorphic X and Y sex chromosomes (chromosomes 5) of A.mantzorum, had identical patterns of 5S rDNA, indicating that the subtelocentric Y resulted from a pericentric inversion. Several microsatellite repeats were found in the heteromorphic sex chromosomes, verifying the association of repetitive DNAs with sex chromosome differentiation in A. mantzorum.

Diversification and historical demography of the rapid racerunner (Eremias velox) in relation to geological history and Pleistocene climatic oscillations in arid Central Asia.

Late Cenozoic stepwise aridification has transformed Central Asia into an arid environment, and the Pleistocene climatic oscillations exerted further ecological impact. Therefore, both aridification and glaciation would have considerably influenced the evolution of many midlatitude species in arid Central Asia (ACA). However, strong biotic evidence supporting this role is still lacking. Here, we test this perspective using a phylogeographic study of Eremias velox, which is an arid-adapted lizard, across ACA using sequences from mitochondrial cytochrome b and 12S rRNA genes. Phylogenetic analyses of the concatenated data, including 595 specimens from 107 localities, revealed ten geographically correlated lineages that diverged by 1.1-15.4% for the cytochrome b gene and 1.0-10.3% for the 12S rRNA gene, which were estimated to have coalesced ∼6.23 million years ago. Ancestral area estimations suggested that E. velox originated from the Iranian Plateau and then dispersed into Central Asia. The intensification of aridification across Central Asia during the Late Pliocene may have facilitated the rapid radiation of this arid-adapted lizard throughout this vast territory. Subsequently, the geological events (e.g., uplift of the Kopet-Dagh, Tianshan and Greater Caucasus Mountains) and glacial oscillations during the Pleistocene triggered the progressive diversification of E. velox. The most recent common ancestor of the Caucasus-Central Asia clade was dated to approximately 2.05 Ma. Specifically, the diversification between the Caucasus clade (VI, VII) and the Central Asia clade (VIII, IX, X), and within the Central Asia clade may have been established and partially maintained by repeated transgressions of the Caspian Sea during the Pleistocene and Holocene. In contrast to demographic and/or range contractions in response to climatic changes during the Last Glacial Maximum (LGM) of the populations (Clades VI and X) from the Caucasus-Central Asia clade, mitochondrial evidence and ecological niche modeling support the signature of demographic and range expansions during the LGM for the Clade V populations (E. v. roborowskii, being endemic to the Turpan Depression). The effect of Pleistocene climatic changes on the historical demography of this arid-adapted species may be lineage-specific, depending predominantly on animal physiology and geography. Finally, we discuss the taxonomic implications, such as the appearance of the Turkmenistan populations as a distinct species, and E. v. roborowskii deserving a full species status.

Cloning, Expression and Effects of P. americana Thymosin on Wound Healing.

The American cockroach (Periplaneta americana) is a medicinal insect. Its extract is used clinically to promote wound healing and tissue regeneration, but the effective medicinal components and mechanisms are not yet clear. It has been reported that human thymosin beta 4 (Tß4) may accelerate skin wound healing, however, the role of P. americana thymosin (Pa-THYs) is still poorly understood. In the present study, we identify and analyze the DNA sequences of Pa-THYs by bioinformatics analysis. Then we clone, express, and purify the Pa-THYs proteins and evaluate the activity of recombinant Pa-THYs proteins by cell migration and proliferation assays in NIH/3T3 cells. To elucidate the role of Pa-THYs in wound healing, a mouse model is established, and we evaluate wound contraction, histopathological parameters, and the expressions of several key growth factors after Pa-THYs treatment. Our results showed that three THY variants were formed by skipping splicing of exons. Pa-THYs could promote fibroblast migration, but have no effect on fibroblast proliferation. In wound repair, Pa-THYs proteins could effectively promote wound healing through stimulating dermal tissue regeneration, angiogenesis, and collagen deposition. On the molecular mechanism, Pa-THYs also stimulated the expression of several key growth factors to promote wound healing. The data suggest that Pa-THYs could be a potential drug for promoting wound repair.

Microsatellite development from genome skimming and transcriptome sequencing: comparison of strategies and lessons from frog species.

BACKGROUND: Even though microsatellite loci frequently have been isolated using recently developed next-generation sequencing (NGS) techniques, this task is still difficult because of the subsequent polymorphism screening requires a substantial amount of time. Selecting appropriate polymorphic microsatellites is a critical issue for ecological and evolutionary studies. However, the extent to which assembly strategy, read length, sequencing depth, and library layout produce a measurable effect on microsatellite marker development remains unclear. Here, we use six frog species for genome skimming and two frog species for transcriptome sequencing to develop microsatellite markers, and investigate the effect of different isolation strategies on the yield of microsatellites. RESULTS: The results revealed that the number of isolated microsatellites increases with increased data quantity and read length. Assembly strategy could influence the yield and the polymorphism of microsatellite development. Larger k-mer sizes produced fewer total number of microsatellite loci, but these loci had a longer repeat length, suggesting greater polymorphism. However, the proportion of each type of nucleotide repeats was not affected; dinucleotide repeats were always the dominant type. Finally, the transcriptomic microsatellites displayed lower levels of polymorphisms and were less abundant than genomic microsatellites, but more likely to be functionally linked loci. CONCLUSIONS: These observations provide deep insight into the evolution and distribution of microsatellites and how different isolation strategies affect microsatellite development using NGS.

Sex chromosomal dimorphisms narrated by X-chromosome translocation in a spiny frog (Quasipaa boulengeri).

BACKGROUND: In the general model of sex chromosome evolution for diploid dioecious organisms, the Y (or W) chromosome is derived, while the homogametic sex presumably represents the ancestral condition. However, in the frog species Quasipaa boulengeri, heteromorphisms caused by a translocation between chromosomes 1 and 6 are not related to sex, because the same heteromorphic chromosomes are found both in males and females at the cytological level. To confirm whether those heteromorphisms are unrelated to sex, a sex-linked locus was mapped at the chromosomal level and sequenced to identify any haplotype difference between sexes. RESULTS: Chromosome 1 was assigned to the sex chromosome pair by mapping the sex-linked locus. X-chromosome translocation was demonstrated and confirmed by the karyotypes of the progeny. Translocation heteromorphisms were involved in normal and translocated X chromosomes in the rearranged populations. Based on phylogenetic inference using both male and female sex-linked haplotypes, recombination was suppressed not only between the Y and normal X chromosomes, respectively the Y and translocated X chromosomes, but also between the normal and translocated X chromosomes. Both males and females shared not only the same translocation heteromorphisms but also the X chromosomal dimorphisms in this frog. CONCLUSIONS: The reverse of the typical situation, in which the X is derived and the Y has remained unchanged, is known to be very rare. In the present study, X-chromosome translocation has been known to cause sex chromosomal dimorphisms. The X chromosome has gone processes of genetic differentiation and/or structural changes by chance, which may facilitate sex chromosome differentiation. These sex chromosomal dimorphisms presenting in both sexes may represent the early stages of sex chromosome differentiation and aid in understanding sex chromosome evolution.

A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae).

In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA.

Microsatellites mapping for non-model species with chromosomal rearrangement: a case study in the frog Quasipaa boulengeri (Anura: Dicroglossidae).

Gene mapping is an important resource for understanding the evolution of genes and cytogenetics. Model species with a known genetic map or genome sequence allow for the selection of genetic markers on a desired chromosome, while it is hard to locate these markers on chromosomes of non-model species without such references. A frog species, Quasipaa boulengeri, shows chromosomal rearrangement polymorphisms, making itself a fascinating model for chromosomal speciation mediated by suppressed recombination. However, no markers have been located on its rearranged chromosomes. We present a complete protocol to map microsatellites based on mechanical microdissection and chromosome amplification techniques. Following this protocol, we mapped 71 microsatellites of Q. boulengeri at the chromosome level. In total, eight loci were assigned to rearranged chromosomes, and the other 63 loci might attach to other chromosomes. These microsatellites could be used to compare the gene flow and verify the chromosomal suppressed recombination hypothesis in Q. boulengeri. This integrated protocol could be effectively used to map genes to chromosomes for non-model species.

Intraspecific rearrangement of mitochondrial genome suggests the prevalence of the tandem duplication-random loss (TDLR) mechanism in Quasipaa boulengeri.

BACKGROUND: Tandem duplication followed by random loss (TDRL) is the most frequently invoked model to explain the diversity of gene rearrangements in metazoan mitogenomes. The initial stages of gene rearrangement are difficult to observe in nature, which limits our understanding of incipient duplication events and the subsequent process of random loss. Intraspecific gene reorganizations may represent intermediate states, and if so they potentially shed light on the evolutionary dynamics of TDRL. RESULTS: Nucleotide sequences in a hotspot of gene-rearrangement in 28 populations of a single species of frog, Quasipaa boulengeri, provide such predicted intermediate states. Gene order and phylogenetic analyses support a single tandem duplication event and a step-by-step process of random loss. Intraspecific gene rearrangements are not commonly found through comparison of all mitochondrial DNA records of amphibians and squamate reptiles in GenBank. CONCLUSIONS: The intraspecific variation in Q. boulengeri provides insights into the rate of partial duplications and deletions within a mitogenome, and reveals that fixation and gene-distribution in mitogenomic reorganization is likely non-adaptive.

A reinvestigation of phylogeny and divergence times of Hynobiidae (Amphibia, Caudata) based on 29 nuclear genes.

Although several recent studies have investigated the major phylogenetic relationships within Hynobiidae, their evolutionary history remains partially resolved and the phylogenetic positions of some genera, particularly Pachyhynobius and Salamandrella are still disputed. Notably, previous studies relied primarily on mitochondrial DNA data and concatenated analyses; thus, a new investigation based on multiple nuclear genes and species-tree inference is needed. Here, we provide an in-depth phylogenetic analysis, based on 29 nuclear genes comprising 29,232bp of data from a comprehensive taxonomic sampling (24 hynobiids and 7 outgroups), using both concatenated and species-tree methods. Our results robustly resolved most genus-level relationships within Hynobiidae, including the placement of Salamandrella as the sister group to a clade containing Batrachuperus, Liua and Pseudohynobius, and the placement of Pachyhynobius as the sister group to a clade containing all hynobiids excluding Onychodactylus, Paradactylodon and Ranodon. Time estimates based on our data suggest that the major group of living hynobiids (excluding Onychodactylus) originated approximately 40Ma, ∼50% younger than estimates from mtDNA data (62.5Ma) but 10% older than estimates from three nuclear genes (36Ma). Our results highlight the benefits of using a large number of nuclear loci to infer both phylogeny and time for relatively old lineages.

The evolution of mitochondrial genomes in modern frogs (Neobatrachia): nonadaptive evolution of mitochondrial genome reorganization.

BACKGROUND: Although mitochondrial (mt) gene order is highly conserved among vertebrates, widespread gene rearrangements occur in anurans, especially in neobatrachians. Protein coding genes in the mitogenome experience adaptive or purifying selection, yet the role that selection plays on genomic reorganization remains unclear. We sequence the mitogenomes of three species of Glandirana and hot spots of gene rearrangements of 20 frog species to investigate the diversity of mitogenomic reorganization in the Neobatrachia. By combing these data with other mitogenomes in GenBank, we evaluate if selective pressures or functional constraints act on mitogenomic reorganization in the Neobatrachia. We also look for correlations between tRNA positions and codon usage. RESULTS: Gene organization in Glandirana was typical of neobatrachian mitogenomes except for the presence of pseudogene trnS (AGY). Surveyed ranids largely exhibited gene arrangements typical of neobatrachian mtDNA although some gene rearrangements occurred. The correlation between codon usage and tRNA positions in neobatrachians was weak, and did not increase after identifying recurrent rearrangements as revealed by basal neobatrachians. Codon usage and tRNA positions were not significantly correlated when considering tRNA gene duplications or losses. Change in number of tRNA gene copies, which was driven by genomic reorganization, did not influence codon usage bias. Nucleotide substitution rates and dN/dS ratios were higher in neobatrachian mitogenomes than in archaeobatrachians, but the rates of mitogenomic reorganization and mt nucleotide diversity were not significantly correlated. CONCLUSIONS: No evidence suggests that adaptive selection drove the reorganization of neobatrachian mitogenomes. In contrast, protein-coding genes that function in metabolism showed evidence for purifying selection, and some functional constraints appear to act on the organization of rRNA and tRNA genes. As important nonadaptive forces, genetic drift and mutation pressure may drive the fixation and evolution of mitogenomic reorganizations.

Genetic Structure and Population History of the Zaisan Toad-Headed Agama (Phrynocephalus melanurus) Inferred from Mitochondrial DNA.

The agamid lizard Phrynocephalus melanurus is restricted to Northwest China (Dzungar Basin) and the adjacent Eastern Kazakhstan (Zaisan and Alakol basins). To elucidate the phylogeography of P. melanurus, we obtained the mitochondrial DNA COI segments of 175 sampled lizards from 44 localities across the whole distribution. Phylogenetic analyses revealed two main Clades comprising five geographically structured lineages (I, IIa, IIb1, IIb2, and IIb3) that fit an isolation-by-distance (IBD) model. The divergence from the most recent common ancestor was dated to ~1.87 million years ago (Ma). Demographic analyses demonstrated lineage-specific response to past climate change: stable population for Clade I, Subclade IIb1; past population expansion for IIb3 since 0.18 Ma, respectively. Bayesian phylogeographic diffusion analyses detected initial spreading at the Saur Mount vicinity, approximately 1.8 Ma. Historical species distribution model (SDM) projected expansion of the suitable habitat in the last interglacial and shift and contraction in the last glacial maximum and Holocene epochs. The SDM predicted a drastic reduction in suitable area throughout the range as a response to future climate change. Our findings suggest that the evolution of P. melanurus followed a parapatric divergence with subsequent dispersal and adaptation to cold and dry environments during the Quaternary. Overall, this work improves our understanding of the lineage diversification and population dynamics of P. melanurus, providing further insights into the evolutionary processes that occurred in Northwest China and adjacent Eastern Kazakhstan.

Exploring patterns and extent of bias in estimating divergence time from mitochondrial DNA sequence data in a particular lineage: a case study of salamanders (order Caudata).

In the practice of molecular dating, substitution saturation will bias the results if not properly modeled. Date estimates based on commonly used mitochondrial DNA sequences likely suffer from this problem because of their high substitution rate. Nevertheless, the patterns and extent of such expected bias remain unknown for many major evolutionary lineages, which often differ in ages, available calibrations, and substitution rates of their mitochondrial genome. In this case study of salamanders, we used estimates based on multiple nuclear exons to assess the effects of saturation on dating divergences using mitochondrial genome sequences on a timescale of ~200-300 My. The results indicated that, due to saturation for older divergences and in the absence of younger effective calibration points, dates derived from the mitochondrial data were considerably overestimated and systematically biased toward the calibration point for the ingroup root. The overestimate might be as great as 3-10 times (about 20 My) older than actual divergence dates for recent splitting events and 40 My older for events that are more ancient. For deep divergences, dates estimated were strongly compressed together. Furthermore, excluding the third codon positions of protein-coding genes or only using the RNA genes or second codon positions did not considerably improve the performance. In the order Caudata, slowly evolving markers such as nuclear exons are preferred for dating a phylogeny covering a relatively wide time span. Dates estimated from these markers can be used as secondary calibrations for dating recent events based on rapidly evolving markers for which mitochondrial DNA sequences are attractive candidates due to their short coalescent time. In other groups, similar evaluation should be performed to facilitate the choice of markers for molecular dating and making inferences from the results.

Coalescence patterns of endemic Tibetan species of stream salamanders (Hynobiidae: Batrachuperus).

Orogenesis of topographically diverse montane regions often drives complex evolutionary histories of species. The extensive biodiversity of the eastern edge of the Tibetan Plateau, which gradually decreases eastwardly, facilitates a comparison of historical patterns. We use coalescence methods to compare species of stream salamanders (Batrachuperus) that occur at high and low elevations. Coalescent simulations reveal that closely related species are likely to have been influenced by different drivers of diversification. Species living in the western high-elevation region with its northsouth extending mountains appear to have experienced colonization via dispersal followed by isolation and divergence. In contrast, species on the eastern low-elevation region, which has many discontinuous mountain ranges, appear to have experienced fragmentation, sometimes staged, of wide-ranging ancestral populations. The two groups of species appear to have been affected differently by glaciation. High-elevation species, which are more resistant to cooler temperatures, appear to have experienced population declines as recently as the last glaciation (0.016-0.032Ma). In contrast, salamanders dwelling in the warmer and wetter habitats at low-elevation environs appear to have been affected less by the relatively recent, milder glaciation, and more so by harsher, extensive glaciations (0.5-0.175 Ma). Thus, elevation, topography and cold tolerance appear to drive evolutionary patterns of diversification and demography even among closely related taxa. The comparison of multiple species in genealogical analyses can lead to an understanding of the evolutionary drivers.

Using Sex-Linked Markers via Genotyping-by-Sequencing to Identify XX/XY Sex Chromosomes in the Spiny Frog (Quasipaa boulengeri).

We used genotyping-by-sequencing (GBS) to identify sex-linked markers in 43 wild-collected spiny frog (Quasipaa boulengeri) adults from a single site. We identified a total of 1049 putatively sex-linked GBS-tags, 98% of which indicated an XX/XY system, and finally confirmed 574 XY-type sex-linked loci. The sex specificity of five markers was further validated by PCR amplification using a large number of additional individuals from 26 populations of this species. A total of 27 sex linkage markers matched with the Dmrt1 gene, showing a conserved role in sex determination and differentiation in different organisms from flies and nematodes to mammals. Chromosome 1, which harbors Dmrt1, was considered as the most likely candidate sex chromosome in anurans. Five sex-linked SNP makers indicated sex reversals, which are sparsely present in wild amphibian populations, in three out of the one-hundred and thirty-three explored individuals. The variety of sex-linked markers identified could be used in population genetics analyses requiring information on individual sex or in investigations aimed at drawing inferences about sex determination and sex chromosome evolution.

The complete mitogenome of the Amolops jinjiangensis (Anura: Ranidae).

We describe the mitochondrial genome sequence of a torrent frog, Amolops jinjiangensis. The mitogenome was extracted and assembled for the first time by restriction site-associated DNA sequencing (RAD-seq). The total length is 17,780 bp, containing 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and one control region. The gene rearrangement was detected as the W-OL-ANCY gene cluster which consisted with several published Amolops mitogenomes. The phylogenetic tree was constructed based on 13 protein-coding genes of A. jinjiangensis and 11 closely related species by Bayesian analyses.