Hyla GBS genotypes in gzip-compressed VCF formatThe VCF file contains genotypes for 4 male and 4 female frogs of 4 species, H. arborea, H. intermedia, H. orientalis, and H. molleri. H. arborea sample names begin with "Ha", H. intermedia with "Hi", H. orientalis with "Ho", and H. molleri with "Hm". Samples with names containing "F" are female, samples with names containing "M" are male. Genotyping-by-sequencing libraries were constructed with restriction enzymes EcoRI and MseI, with a selective nucleotide on the MseI PCR primer. Single-end Illumina reads were aligned to a low-coverage draft genome of H. arborea, and SNPs called with SAMtools. Raw genotypes were filtered based on genotype quality, missing data, and minor allele frequency.HylaDmrt1.variants.vcf.gzHyla Dmrt1 exon 1 phased alignmentAlignment of Dmrt1 exon 1 and upstream sequences of five Hyla species. Phased X and Y haplotypes were determined with PHASE software. All sites containing indel variation within or between species were removed. All sequences ending in "X" or "Y" could be assigned to the X or Y chromosome. See readme file for key to sample names.dmrt1ex1PHASE.fasHyla Dmrt1 exon 3 phased alignmentAlignment of Dmrt1 exon 3 and adjacent partial intron sequences of five Hyla species. Phased X and Y haplotypes were determined with PHASE software. All sites containing indel variation within or between species were removed. All sequences ending in "X" or "Y" could be assigned to the X or Y chromosome. See readme file for key to sample names.Dmrt1Ex3PHASE.fasHyla Kank1 intron 10 phased alignmentAlignment of Kank1 intron 10 sequences of five Hyla species. Phased X and Y haplotypes were determined with PHASE software. All sites containing indel variation within or between species were removed. All sequences ending in "X" or "Y" could be assigned to the X or Y chromosome. See readme file for key to sample names.kank1intron10.PHASE.fas

Empirical studies on the relative roles of occasional XY recombination versus sex-chromosome turnover in preventing sex-chromosome differentiation may shed light on the evolutionary forces acting on sex-determination systems. Signatures of XY recombination are difficult to distinguish from those of homologous transitions (i.e., transitions in sex-determination systems that keep sex-chromosome identity): both models predict X and Y alleles at sex-linked genes to cluster by species. However, the XY-recombination model specifically predicts the reverse pattern (clustering by gametologs) for those genes that are directly involved in sex determination. Hence, the latter model can only be validated by identification of an ancestral sex-determining region (SDR) with trans-species polymorphism associated to sex. Here we combine a candidate-gene approach with a genome scan to identify a small SDR shared by four species of a monophyletic clade of European tree frogs. This SDR encompasses at least the N-terminal part of Dmrt1 and immediate upstream sequences. Our findings provide definitive evidence that sex-chromosome homomorphy in this clade results only from XY recombination, and take an important step towards the identification of the sex-determining locus. Moreover, the sex-diagnostic markers we identify will enable research on environmental sex reversal in a wider range of frog species.