Identification of sex reversal
Sex linkage of the 3 markers was tested on 125 laboratory-raised
froglets. Individual molecular sexing was performed independently with
each marker, and we subsequently checked if the identified genotype
matched with the phenotypic sex. For each marker, we calculated the
rates of female-to-male and male-to-female sex reversal, i.e. the
proportion of phenotypic males among genetic females (XX) and the
proportion of phenotypic females among genetic males (XY), respectively,
as well as the proportion of sex-reversed individuals within each
phenotypic sex. If a marker indicated sex reversal for a
laboratory-raised individual, we accepted the result only if a second
DNA sample extracted from the other stored foot of that individual gave
the same result as the first one (i.e. to avoid false identifications of
sex reversals due to human error during the molecular laboratory work).
Because the second DNA sample always confirmed the assumptions from the
first one, we found these genotypes to be unambiguous. Note that the 125
froglets came from an experiment in which their siblings were exposed to
various treatments (Bókony et al., 2020); here we used some of the
genetic sex data of those treated siblings to evaluate whether our
findings of sex-reversed froglets may have been due to null alleles,
sex-chromosome recombination or mutation, or being sired by a
sex-reversed parent.
Because the above analyses showed that one of our three markers (Rds2)
would not be suitable for sexing in our populations (see Results), we
used the other two markers to evaluate two sexing methods. In Method 1,
we screened all laboratory-raised individuals for the marker with the
highest sex linkage (Rds3) and we accepted an individual to be normal
male or female if its Rds3 genotype was in accordance with its
phenotypic sex. Those individuals that seemed to be sex-reversed by Rds3
were screened for the marker with the second highest sex linkage (Rds1)
as well and were accepted to be sex-reversed only if both markers
confirmed sex reversal. In Method 2, all laboratory-raised individuals
were screened for both Rds1 and Rds3 and genotyping was considered to be
successful only if both markers gave the same result. In both methods,
individuals with discrepant genotyping results were considered to be of
unknown genetic sex.
We estimated sex-reversal frequency in the wild-captured adults using
Method 1, because this sexing method performed best in the
laboratory-raised individuals (see Results). As females are more
difficult to find and capture than males, the majority of the
investigated adults were males, so we had too few females to provide a
reliable estimate of female-to-male sex-reversal rate in adults.
Therefore, we report the proportion of sex-reversed individuals (XX
males) among the phenotypic males (hereafter referred to as XX/male
ratio) as a measure of female-to-male sex-reversal frequency.