Heterogeneous evolution of sex chromosomes in the torrent frog genus Amolops

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).