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

Abstract

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex‐ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex‐reversing stimuli more frequently, which may lead to higher sex‐reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR‐based genetic sex markers for the common toad (Bufo bufo) to assess 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 oestrogenic 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−1 17α‐ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L−1 EE2 and a glyphosate‐based herbicide with 3 µg L−1 or 3 mg L−1 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 extremely low sex‐reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species‐specific resistance to sex reversal.

Asymmetrical sex reversal: Does the type of heterogamety predict propensity for sex reversal?

“…Local or species‐specific adaptations in various traits may be developed to better survive and exploit conditions that vary across habitats, such as temperature [ 74 ] or anthropogenic chemical pollution [ 77 ] ; similar adaptations might also increase or decrease the likelihood of sex reversal. [ 41 ] Therefore, the source of the experimental animals, such as the climatic and land‐use conditions of the collection sites or the specificities of the used breeding stocks (e.g., in Xenopus ), should be clearly described in sex‐reversal studies. When the experiments include both ZW/ZZ and XX/XY species, ideally these should be collected from the same sources or from similar habitats in order to improve the comparability of the two systems’ response to specific sex‐reversing conditions.…”

“…This is supported by the currently available, limited data on wild populations: genetically proven female‐to‐male sex reversal was frequent in four anuran species with XX/XY systems (and only rare male‐to‐female sex reversal occurred in one of them) [ 4–7 ] , while sex‐reversal frequency was negligible in Bufo bufo , the only ZW/ZZ anuran with such data to our knowledge. [ 41 ] This latter finding is especially interesting because female‐to‐male sex reversal was frequent in Rana dalmatina (XX/XY) in the same habitats, so the lack of sex reversal in these Bufo bufo populations are not attributable to lack of female‐to‐male sex‐reversing stimuli. [ 41 ] The pattern is similar in the two reptile species for which genetically confirmed sex reversal has been studied in the wild: XX males were found in Bassiana duperreyi ( = Acritoscincus duperreyi ; XX/XY) and ZZ females in Pogona vitticeps (ZW/ZZ) across several free‐living populations.…”

“…[ 41 ] This latter finding is especially interesting because female‐to‐male sex reversal was frequent in Rana dalmatina (XX/XY) in the same habitats, so the lack of sex reversal in these Bufo bufo populations are not attributable to lack of female‐to‐male sex‐reversing stimuli. [ 41 ] The pattern is similar in the two reptile species for which genetically confirmed sex reversal has been studied in the wild: XX males were found in Bassiana duperreyi ( = Acritoscincus duperreyi ; XX/XY) and ZZ females in Pogona vitticeps (ZW/ZZ) across several free‐living populations. [ 40,42,43 ] The picture is less clear in fishes [ 8 ] ; however, sex‐reversal research in fish has so far focused on its aquaculture aspects, and the genetic sex markers developed for captive populations (i.e., altered by artificial selection or genetic drift) are not always reliable in wild populations.…”

“…Experimental methods differed greatly across studies, including the applied concentrations of the same compounds as well as water temperature. Because genetic sex markers have been established for only a handful of amphibian species so far (e.g., [4][5][6][7]41] ), in the vast majority of studies sex reversal was inferred based on biased phenotypic sex ratios produced by specific treatments. Several relevant experimental conditions, such as treatment duration or mortality rates, were unclear in numerous instances, especially among broadly cited publications from the previous century.…”

Does stress make males? An experiment on the role of glucocorticoids in anuran sex reversal

“…Although environmentally sensitive sex determination (ESD) may have evolved as an adaptive sex-allocation strategy (Geffroy & Douhard, 2019;Schwanz & Georges, 2021), in our era of rapid human-induced environmental changes ESD increases vulnerability to climate change, chemical pollution, the urban heat island effect, and other habitat alterations such as microclimatic changes due to invasive species (Leslie & Spotila, 2001;Mitchell & Janzen, 2010;Nemesházi et al, 2020). The potential consequences are diverse, including the evolution of sexdetermination systems and mating preferences, and skewed adult sex ratios (Bókony et al, 2017;Grossen et al, 2011;Nemesházi et al, 2021;Schwanz et al, 2020) which can affect a wide range of social behaviors, life histories, reproductive systems, population viability and adaptive potential (Mitchell & Janzen, 2010;Schacht et al, 2022). To understand and forecast these evolutionaryecological outcomes, we need to find out what proximate mechanisms translate environmental stimuli into phenotypic sex.…”