Non-canonical sex chromosome evolution revealed by extreme heterogeneity in homomorphic Y chromosome differentiation in the common frog

Sex chromosome evolution is traditionally viewed as a unidirectional process of recombination suppression followed by progressive Y degeneration, yet many vertebrates deviate from this canonical trajectory. Frogs represent a striking exception: they retain homomorphic sex chromosomes, exhibit extreme heterochiasmy, and undergo X-Y recombination in sex-reversed females, providing a powerful system to explore alternative evolutionary pathways. Here, we investigate a Swiss Alpine population of the European common frog (Rana temporaria) with homomorphic XY chromosomes and three coexisting male genotypes, using whole genome sequencing of pooled samples carrying distinct Y haplotypes, whole genome sequencing of doubled haploid YY individuals, and transcriptomic (RNA-seq) data. We uncover extreme heterogeneity in X-Y divergence: two fully differentiated Y haplotypes span nearly 90% of the sex chromosome ([~]618-625 Mb), forming the largest non-recombining region (large NRR) described in a vertebrate to date, whereas a semi-differentiated Y harbors only a NRR of 4.64 Mb (small NRR), and XX males display no detectable X-Y divergence. Despite extensive recombination suppression, Y degeneration is minimal even within the large NRR: we detect elevated transposable element insertions but no gene loss, Y-linked gene copy decay, faster-X effects, or enrichment of sex-biased genes. All Y haplotypes share a small NRR encompassing the candidate master sex-determining gene Dmrt1, defining a common sex-determining locus. Our data support a dynamic model in which extreme heterochiasmy and recurrent X-Y recombination via sex reversal repeatedly reset X-Y divergence, generating and potentially maintaining multiple Y haplotypes with distinct evolutionary histories. The long-term coexistence of these haplotypes across the species range is compatible with a dynamic equilibrium between drift-driven loss and mutation- and recombination-driven renewal of Y chromosome variation. Together, our findings reveal a non-canonical, reversible pathway of sex chromosome evolution shaped by sex-specific recombination patterns and sex reversal, challenging the universality of classical models and highlighting the value of non-model vertebrates for understanding sex chromosome diversity.