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.

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.

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.

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.

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.

The complete mitochondrial genome of Batrachuperus sp. 2 (Caudata: Hynobiidae).

We describe the mitochondrial genome sequence of a mountain salamander, Batrachuperus sp.2 (Hynobiidae). The mitogenome was extracted and assembled from a large genome by next-generation sequencing. The mitochondrial genome sequences were 16,394 bp in size with 37 genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding control region (D-loop). The overall base composition of Batrachuperus sp.2 is 34.1% for A, 19.3% for C, 13.5% for G, 33.1% for T, and 32.8% for GC content. The phylogenetic tree was constructed based on 13 protein-coding genes of Batrachuperus sp.2 and 6 closely related species by RAxML.

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.

A new species of the genus Batrachuperus (Urodela: Hynobiidae) from Southwestern China.

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

Assigning the Sex-Specific Markers via Genotyping-by-Sequencing onto the Y Chromosome for a Torrent Frog Amolops mantzorum.

We used a genotyping-by-sequencing (GBS) approach to identify sex-linked markers in a torrent frog (Amolops mantzorum), using 21 male and 19 female wild-caught individuals from the same population. A total of 141 putatively sex-linked markers were screened from 1,015,964 GBS-tags via three approaches, respectively based on sex differences in allele frequencies, sex differences in heterozygosity, and sex-limited occurrence. With validations, 69 sex-linked markers were confirmed, all of which point to male heterogamety. The male specificity of eight sex markers was further verified by PCR amplifications, with a large number of additional individuals covering the whole geographic distribution of the species. Y chromosome (No. 5) was microdissected under a light microscope and amplified by whole-genome amplification, and a draft Y genome was assembled. Of the 69 sex-linked markers, 55 could be mapped to the Y chromosome assembly (i.e., 79.7%). Thus, chromosome 5 could be added as a candidate to the chromosomes that are particularly favored for recruitment in sex-determination in frogs. Three sex-linked markers that mapped onto the Y chromosome were aligned to three different promoter regions of the Rana rugosaCYP19A1 gene, which might be considered as a candidate gene for triggering sex-determination in A. mantzorum.

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.

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.

The mitochondrial genome of broad-folded frog (Hylarana latouthii).

The broad-folded frog, Hylarana latouthii, is an endemic freshwater frog in southern China. In the present study, the mitochondrial DNA sequence of the H. latouchii was first determined. The genome was 17,291 bp in length, which contains 37 genes (13 protein-coding genes, 2 ribosomal tRNAs, 22 transfer RNAs) and a putative CR (D-loop). The phylogenetic tree was constructed based on the 13 protein-coding genes of H. latouchii and 11 closely species.

The Origin and Evolution of Chromosomal Reciprocal Translocation in Quasipaa boulengeri (Anura, Dicroglossidae).

Chromosomal rearrangements have long fascinated evolutionary biologists for being widely implicated in causing genetic differentiation. Suppressed recombination has been demonstrated in various species with inversion; however, there is controversy over whether such recombination suppression would facilitate divergence in reciprocal translocation with reduced fitness. In this study, we used the spiny frog, Quasipaa boulengeri, whose western Sichuan Basin populations exhibit translocation polymorphisms, to test whether the genetic markers on translocated (rearranged) or normal chromosomes have driven this genetic differentiation. We also investigated its overall genetic structure and the possibility of chromosomal fixation. Whole-chromosome painting and genetic structure clustering suggested a single origin of the translocation polymorphisms, and high-throughput sequencing of rearranged chromosomes isolated many markers with known localizations on chromosomes. Using these markers, distinct patterns of gene flow were found between rearranged and normal chromosomes. Genetic differentiation was only found in the translocated chromosomes, not in normal chromosomes or the mitochondrial genome. Hybrid unfitness cannot explain the genetic differentiation, as then the differentiation would be observed throughout the whole genome. Our results suggest that suppressed recombination drives genetic differentiation into a balanced chromosomal polymorphism. Mapping to a reference genome, we found that the region of genetic differentiation covered a wide range of translocated chromosomes, not only in the vicinity of chromosomal breakpoints. Our results imply that the suppressed recombination region could be extended by accumulation of repetitive sequences or capture of alleles that are adapted to the local environment, following the spread and/or fixation of chromosomal rearrangement.

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.

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.

Examining the interglacial high-elevation refugia scenario in East Asian subtropical mountain systems with the frog species Leptobrachium liui.

The effects of Quaternary climatic oscillations on the distributions of organisms in different parts of the world are not equally well understood, limiting the ability to understand the determinants of biodiversity. Compared with the mountain regions in southern Europe and southwestern North America, such effects on high-elevation species in the East Asian subtropical mountain systems located in southern and southeastern China have seldom been addressed. In this study, using Leptobrachium liui (Megophryidae), we made one of the earliest attempts to examine the interglacial high-elevation refugia scenario in these Asian mountains. Based on our current understanding of the study system, we formulated a hypothesis that these frogs of western origin were distributed more widely and continuously during glacial phases, allowing eastward dispersal, and that they are currently isolated in interglacial refugia at higher elevations. Microsatellite data and mitochondrial and nuclear sequence data were obtained with extensive sampling followed by the synthesis of phylogeographic and population genetic analyses and modeling of the species distribution. The analyses revealed a sequential eastward divergence of microsatellite clusters and gene lineages accompanied by a decline in genetic diversity. Molecular dating estimates revealed divergence events in the Pleistocene, and a reduction in local populations was inferred to have occurred at a time comparable to the end of the last glacial. Strong genetic isolation by distance reflecting a more continuous historical distribution was detected. Furthermore, environmental niche models inferred a wide planar distribution during the last glacial maximum, providing further support for the hypothesis.

Suppressed Recombination of Sex Chromosomes Is Not Caused by Chromosomal Reciprocal Translocation in Spiny Frog (Quasipaa boulengeri).

Chromosome rearrangements (CRs) are perceived to be related to sex chromosome evolution, but it is a matter of controversy whether CRs are the initial causative mechanism of suppressed recombination for sex differentiation. The early stages of sex chromosome evolution in amphibians may represent intermediate states of differentiation, and if so, they potentially shed light on the ultimate cause of suppressed recombination and the role of CRs in sex chromosome differentiation. In this paper, we showed that sex determination differs among 16 populations of spiny frog (Quasipaa boulengeri), in which individuals have normal and rearranged chromosomes caused by reciprocal translocation. In eastern areas, without translocation, genetic differentiation between sexes was relatively low, suggesting unrestricted recombination. In comparison, in western populations that have both normal and translocated chromosomes, a male-heterogametic system and lack of X-Y recombination were identified by male-specific alleles and heterozygote excess. However, such genetic differentiation between sexes in western populations was not directly related to karyotypes, as it was found in individuals with both normal and translocated karyotypes. In the western Sichuan Basin, male-specific and translocation-specific allelic frequency distributions suggested that recombination of sex-differentiation ceased in all populations, but recombination suppression caused by translocation did not exist in some populations. Combined with phylogenetic inference, this indicated that the establishment of sex-linkage had taken place independently of reciprocal translocation, and translocation was not the ultimate cause of sex chromosome differentiation. Furthermore, comparison of the genetic diversity of alleles on Y chromosomes, X chromosomes, and autosomes in western populations showed a reduction of effective population size on sex chromosomes, which may be caused by reciprocal translocation. It indicates that, although it is not the ultimate cause of recombination suppression, reciprocal translocation may enhance sex chromosome differentiation.