Novel sex markers
Marker development yielded 3 agile frog loci with sex-linked SNPs in 10 individuals (Table 2). Rds1 and Rds3 contained one sex-linked SNP each, while Rds2 contained 3 sex-linked SNPs. Accordingly, Y-SNPs were present in all 5 males at each locus, and absent from all 5 females at Rds3, but at Rds1 and Rds2, they were also present in one female each (different individuals). X-SNPs were present in all 5 females and 5 males at Rds1, and were absent from two males at Rds2 and one male at Rds3. Based on common frog genome data, SNPs of Rds1 and Rds3 are located more than 112 million nucleotides away from each other (positions 199806348 and 312650318, respectively), and even the distance between SNPs of the two closest markers Rds2 and Rds3 is more than 6 million nucleotides (Rds2 SNP position closest to Rds3 is 306051765).
All of the 125 laboratory-raised froglets were successfully genotyped with all three markers. The strongest sex-linkage was shown by Rds3 (95% match between phenotypic sex and genotype at the locus), followed by Rds1 (89% match) and finally Rds2 (70% match) (Table 3). Because we had not exposed the laboratory-raised froglets to sex-reversing effects, we concluded that Rds2 is not suitable for genetic sexing in our populations, as the 30% mismatch rate is much higher than the sex-reversal rates reported from natural populations of other species (Alho et al., 2010; Lambert et al., 2019). When we used Method 1 for identifying sex reversals based on Rds3 and Rds1, six out of the 125 froglets qualified as sex-reversed (all XX males), yielding a female-to-male sex-reversal rate of 8%, and an XX/male ratio of 10% (Table 1). Four out of these 6 sex-reversed animals had both XX and XY siblings (making it unlikely that they were identified as XX due to the presence of null alleles or as an outcome of recombination or mutation, i.e. X-SNPs on Y), whereas two of them came from a family in which we found only XX individuals (N=12), suggesting that the latter might have been fathered by an XX male. Due to discrepancies between Rds1 and Rds3 (Table 3), Method 2 failed to assign genetic sex to 8 individuals (6.4% of all froglets). These failures resulted in slightly higher estimates of both the female-to-male sex-reversal rate (9%) and the XX/male ratio (11%) for Method 2. These discrepancies occurred in 4 families from 3 ponds (not in the same families that contained sex-reversed individuals), the discrepant genotypes being XY with Rds1 and XX with Rds3 in all but one family, with the Rds3 genotype being always concordant with the phenotype. Because Rds3 showed the highest sex-linkage, and Rds1 results matched the Rds3 results for all putative sex-reversed individuals based on Rds3, we concluded that Method 1 allows reliable genetic sexing while also keeping sex-identification failures at minimum, whereas cases where phenotypic sex matches Rds3 but not Rds1 are likely to result from recombination rather than from sex reversal.