Phenotypic correlates of sex reversal
In the laboratory-raised froglets, we compared the following indices of health and fitness between sex-reversed individuals (XX males) and normal individuals (XY males and XX females): duration of larval development, body mass at metamorphosis and at dissection, size of the fat bodies, size and pigmentation of the spleen, and the mean size of the two testes. We also recorded any abnormality observed during dissection. A detailed description of the biological relevance of these traits, the methods of their measurement and statistical analysis is available in the Supporting Information (pages 11-17).
Histological analysis of the sex-reversed froglets was performed to examine if sex reversal was accompanied by intersex, a condition where both male and female tissue elements are present in the gonads (Lambert et al., 2019). Our preliminary study showed that sex categorized by gonadal anatomy matched sex categorized by histology in 100% of 32 agile frogs (17 males, 15 females) that had been raised without any chemical treatment in 2016, using the same lab protocol as in 2018. Therefore, to minimize the costs of histological analysis, we chose to analyze gonad histology only in those lab-raised froglets from 2018 for which the identified genetic sex did not match the phenotypic sex categorized by gonad anatomy (i.e. to check if the mismatch was due to erroneous categorization of phenotypic sex). For histology, the gonads were placed in embedding cassettes and dehydrated through graded ethanol, cleared in xylene and infiltrated with paraffin wax in an Excelsior ES Tissue Processor (Thermo Fisher Scientific). Processed gonads were embedded in paraffin, sectioned into 3-4 μm longitudinal slices using a Reichert type microtome, stained with haematoxylin and eosin, and mounted on glass slides. The slides were examined and photographed using a Nikon Eclipse E600 microscope equipped with a QImaging MicroPublisher 3.3 RTV camera. For each individual, 5-6 sections were examined; ovaries were recognized by the presence of ovarian cavities, early meiotic oocytes and/or diplotenes, and testes by spermatogonia, spermatocytes and/or seminiferous cords or tubules (Figure S3).
In the adult frogs, we compared body mass between sex-reversed individuals (XX males) and normal (XY) males using a linear mixed-effects (LME) model with capture site as a random factor. Because most of the captured females were gravid, we did not include them in the analysis of adult body mass. All statistical analyses were run in R 3.5.2. environment (R Core Team, 2019), using the nlme package for mixed models (Pinheiro & Bates, 2019).