In sharp contrast with birds and mammals, sex chromosomes have been described as homomorphic in cold-blooded vertebrates. This sex-chromosome homomorphy has been suggested to result from high turnovers when they are often observed across deeply diverged clades. However, little is known about the tempo and mode of sex chromosome evolution among most closely related species. Here, we examine patterns of sex chromosome evolution among nine species of the torrent frog genus Amolops. Through the analysis of male and female GBS and RAD-seq from 182 individuals and of PCR verification for 176 individuals, we identify signatures of sex chromosomes involving two pairs of chromosomes. We find that the sex-chromosome homomorphy results from both turnover and X–Y recombination in the Amolops species, simultaneously exhibiting heterogeneous evolution on homologous and non-homologous sex chromosomes. The lower turnover rate of non-homologous sex chromosomes exists in these torrent frogs, whereas the ongoing X–Y recombination in homologous sex chromosomes would act as an indispensable force to prevent the sex chromosomes from differentiations. 

Genomic DNA of the 182 samples from nine species of Amolops was subjected to GBS and RAD-seq by the Novogene. Clean Illumina reads were demultiplexed, trimmed, and filtered by the process_radtags function in STACKS [v 2.4].  We used RStudio 4.1.0 to generate candidate sex-linked markers for each individual. 

catalog.fa.zip and catalog.tags.tsv.zip

Stacks2.41 denovo_map.pl pipeline was used to construct a catalog of loci from GBS and RAD-seq reads of samples, by changing the parameters M (M controls the number of mismatches allowed between the two alleles of a heterozygote sample), n (n controls the number of mismatches allowed between any two alleles of the population) and m (m controls the number of identical reads required to initiate a new putative allele). M and n allow for assembly of polymorphic loci within individuals and fixed differences among individuals at a locus, respectively. We used optimal parameters and the [M2n1m2] parameter to construct catalogs of loci from nine species of Amolops' GBS data.  At the same time, we used the [M2n1m2] parameter to construct catalogs of loci from two species of Amolops' RAD-seq data. In addition, to test whether there were shared sex-linked markers among the species of Amolops, data from nine species of Amolops were obtained, and a unified component analysis of denovo_map.pl pipeline was performed (parameters: -M = 2, -n = 1, and -m = 2).

matches.zip

Samples' stacks are matched to the catalog to create a matches.tsv file for each sample. We counted the matches to the catalog to find the depth of each locus in each individual sample. 

populations.sumstats.zip

Stacks2.41 populations program provides summary statistics for each locus containing SNPs. To identify sex markers, we split samples into male and female: males were assigned to Pop M, females were assigned to Pop F. 

Putative sex-linked marker.zip

Our goal was to isolate single nucleotide polymorphisms (SNP) and sex-limited markers. The popations.sumstats.tsv file was used to identify the putative sex-linked SNP loci. Similarly, the match between each individual and catalog was calculated in the matches.tsv file to determine the loci depth of each individual; then, the loci that existed in all individuals of one sex and not in the other were screened out, which are called putative sex-limited GBS/RAD-seq loci. Sex-linked markers were screened with three approaches followed by Brelsford et al. (2017), respectively based on (i) sex differences in allele frequencies, (ii) sex differences in heterozygosity, and (iii) sex-limited occurrence. We used RStudio 4.1.0 to generate candidate alleles for each individual.

Sequence.zip

After amplification in additional samples (10 male,10 female) respectively from nine species of Amolops, the PCR products were sequenced at Sangon Sequencing Center (Shanghai, China). All male individuals and female individuals showed heterozygosity and homozygosity at the polymorphic sites, respectively.

Unified component analysis.zip

With the SNP-based analysis of the nine species unified dataset, one shared sex-linked locus was found between only a species pair of A. xinduqiao and A. sp. All male genotypes for this sex-linked locus were T/A heterozygous while all female genotypes were T/T homozygous, which confirmed that the marker was gender-diagnostic in both species (Clocus746278).