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).