The growing interest in the lability of sex determination in non‐model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex‐determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome‐wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of B. spinosus across B. bufo’s former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto‐nuclear discordances (B. spinosus backrosses carrying B. bufo mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross‐breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation.

Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog P. esculentus (diploid RL and triploids RLL and RRL), a natural hybrid between P. lessonae (LL) and P. ridibundus (RR). Hybridogenesis relies on elimination of one genome (L or R) from gonocytes (G) in tadpole gonads during prespermatogenesis, but not from spermatogonial stem cells (SSCs) in adults. Here we provide the first comprehensive study of testis morphology combined with chromosome composition in the full spectrum of spermatogenic cells. Using genomic in situ hybridization (GISH) and FISH we determined genomes in metaphase plates and interphase nuclei in Gs and SSCs. We traced genomic composition of SSCs, spermatocytes and spermatozoa in individual adult males that were crossed with females of the parental species and gave progeny. Degenerating gonocytes (24%–39%) and SSCs (18%–20%) led to partial sterility of juvenile and adult gonads. We conclude that elimination and endoreplication not properly completed during prespermatogenesis may be halted when gonocytes become dormant in juveniles. After resumption of mitotic divisions by SSCs in adults, these 20% of cells with successful genome elimination and endoreplication continue spermatogenesis, while in about 80% spermatogenesis is deficient. Majority of abnormal cells are eliminated by cell death, however some of them give rise to aneuploid spermatocytes and spermatozoa which shows that hybridogenesis is a wasteful process.

Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a flow-through-system, we tested the effects of 17β-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in three environmentally and/or physiologically relevant concentrations (0.027 µg/L (10⁻¹⁰ M), 0.27 µg/L (10⁻⁹ M), 2.7 µg/L (10⁻⁸ M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10⁻⁹ M) and 2.7 µg/L (10⁻⁸ M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2).