Biology session

Environmental conditions, such as extreme temperatures and chemical pollutants can cause sex reversal in various amphibian species, resulting in a phenotypic sex that is not concordant with the genetic sex encoded by the sex chromosomes. Sex reversal may lead to sex-ratio bias, population decline and even extinction. However, identification of sex-reversed individuals is challenging, due to the lack of available genetic sex markers for most species. The common toad Bufo bufo is an amphibian species common across Europe and in Hungary, which, due to its widespread distribution could be a suitable subject for studying relationships between different environmental effects and sex reversal.

In the present study, we first developed genetic sex markers for the common toad to enable the identification of sex reversal. Second, we assessed sex-reversal frequency in wild common toad populations living in either natural or anthropogenic habitats in Northern Hungary.

For development and testing of genetic sex markers, we used non-sibling juveniles that were raised under non-sex-reversing laboratory conditions. We designed primers for 11 potentially female-specific DNA fragments identified by RADseq analysis of 24 individuals. After specific Sanger sequencing, we found four markers to be adequate for genetic sexing. PCR-based genotyping proved that all four markers are 100% sex-linked based on 26 male and 28 female toadlets.

Among 100 adults caught in n=13 wild populations, we detected sex reversal in only one individual. The sex-reversed, phenotypically male toad was captured from an agricultural habitat and was found to be genetically female based on three of the four markers, while the fourth marker gave ambiguous result.

The developed sex markers confirmed that the studied Hungarian common toad populations are female heterogametic, where females are ZW, and males are ZZ genotype. These markers provide an opportunity to study sex reversal in both natural common toad populations and laboratory experiments in the future. The sex-reversal frequency found in our study is strikingly low compared to previous findings in other anurans, which is surprising because several sex-reversing environmental effects are known to be present in the habitats we sampled. As a potential explanation for our results, we speculate that the common toad may be relatively resistant to sex reversal.