Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable, since their sexual development can be disrupted by environmental changes, which can cause sex reversal, a mismatch between genetic and phenotypic sex, potentially leading to sex-ratio distortion and population decline. Despite these implications, we have scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may experience sex reversal more frequently, or alternatively, they may adapt to resist sex reversal. To test these alternative hypotheses, we developed PCR-based genetic sex markers for the common toad (Bufo bufo) . We assessed the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous estrogenic pollutants in a common-garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg/L 17α- ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng/L EE2 and a glyphosate-based herbicide with 3 µg/L or 3 mg/L glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the surprisingly low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.