DOI: 10.5061/dryad.b2rbnzsjn
title: Heterogeneous evolution of sex chromosomes in the torrent frog genus Amolops

description: 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. 
description: Genomic DNA of the 182 samples from nine species of
 <em>Amolops</em> was subjected to GBS and RAD-seq by the
 Novogene. Clean Illumina reads were demultiplexed, trimmed, and filtered
 by the <em>process_radtags</em> function in STACKS [v 2.4]. 
 We used RStudio 4.1.0 to generate candidate sex-linked markers for each
 individual. 
description: <strong>catalog.fa.zip
 and catalog.tags.tsv.zip</strong> Stacks2.41
 <em>denovo_map.pl</em> 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 <em>Amolops</em>' GBS data.  At the same
 time, we used the [M2n1m2] parameter to construct catalogs of loci from
 two species of <em>Amolops</em>' RAD-seq data. In
 addition, to test whether there were shared sex-linked markers among the
 species of <em>Amolops</em>, data from nine species of
 <em>Amolops</em> were obtained, and a unified component
 analysis of <em>denovo_map.pl</em> pipeline was performed
 (parameters: -M = 2, -n = 1, and -m = 2). <strong>matches.zip</strong> Samples' stacks are matched to the catalog to create a <em>matches.tsv</em> file for each sample. We counted the matches to the catalog to find the depth of each locus in each individual sample.  <strong>populations.sumstats.zip</strong> Stacks2.41 <em>populations</em> 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.  <strong>Putative sex-linked marker.zip</strong> Our goal was to isolate single nucleotide polymorphisms (SNP) and sex-limited markers. The <em>popations.sumstats.tsv</em> file was used to identify the putative sex-linked SNP loci. Similarly, the match between each individual and catalog was calculated in the <em>matches.tsv</em> 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. <strong>Sequence.zip</strong> After amplification in additional samples (10 male,10 female) respectively from nine species of <em>Amolops</em>, 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. <strong>Unified component analysis.zip</strong> With the SNP-based analysis of the nine species unified dataset, one shared sex-linked locus was found between only a species pair of <em>A. xinduqiao</em> and <em>A</em>. 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).