We demonstrate a genotyping-by-sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild-caught individuals, in the moor frog Rana arvalis. Double-digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex-limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex-linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co-opted for sex determination among amphibians. The most efficient mapping strategy was a three-step hierarchical approach, where R. arvalis reads were first mapped to a low-coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex-diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.

Female mate choice promotes the development of male secondary sexual traits such as nuptial colouration, whereas scramble competition favours male traits which enhance their ability for access to females. In the explosively breeding moor frog (Rana arvalis), males express a conspicuous blue colouration during a short reproductive period characterised by scramble competition. In the present study we used controlled mating experiments and genetic markers to disentangle the effects of colouration, body size and pairwise genetic relatedness in determining paternity success. Males observed in amplexus with a female prior to spawning were larger than their competitors but did not differ from them in colouration. Polyandry occurred in 67% of the 18 analysed egg clutches, and amplecting males did not always achieve the highest siring success, most likely due to stray sperm. Successful mating pairs were characterised by higher genetic divergence between them than expected by chance. We confirm previous evidence that male nuptial colouration is not a trait selected by females, and provide evidence that male reproductive success is influenced by male size as well as genetic attributes.