Sampling and DNA extraction
We captured 162 adult agile frogs (121 males and 41 females) from 11
ponds in North-Central Hungary at the start of the breeding season in
February-March in 2016 and 2017 (Table 1, Table S1). The capture sites
were chosen to represent the range of habitats the species occupies, on
a natural to anthropogenic scale (Table S1). Distances between capture
sites varied from 4 to 60 km. Sample size varied between sites due to
variation in capture success. The adults were sexed by secondary sexual
characteristics (nuptial pads in males) and presence of eggs (gravid
females). Buccal swab samples were taken from all wild-caught frogs for
DNA extraction. Additionally, toe clip samples were also collected from
10 individuals (5 males and 5 females, from 3 ponds) for the purpose of
marker finding and primer design (Table 1). We measured the adult frogs’
body mass (± 0.1 g) and released them at their capture sites.
We subsequently tested the sex-linkage of our markers (see below) on 125
froglets (59 males and 66 females; from 34 clutches) collected as
freshly spawned eggs in 2018 from three different ponds of the same
geographical region (Table 1). These individuals were raised in
laboratory under conditions that are unlikely to cause sex reversal,
because the animals were not exposed to endocrine-disrupting chemicals
or to extreme temperatures or to any other stressor which trigger sex
reversal to our knowledge (Castañeda Cortés, Arias Padilla, Langlois,
Somoza, & Fernandino, 2019; Eggert, 2004; Lambert, Smylie, Roman,
Freidenburg, & Skelly, 2018). Thus, we expected that among these
animals sex reversal would be absent or occur very rarely due to
sex-chromosome recombination (Ezaz et al., 2006; Perrin, 2009; Stöck et
al., 2013) or random processes affecting sex determination (Perrin,
2016). We are confident that this setup provided the best conditions for
ascertaining the baseline level of sex reversal in this species. Water
temperature during tadpole development was 18.45 ± 0.81 (mean ± SD); all
other details of animal housing and care are described in Bókony et al.
(2020). Froglets were phenotypically sexed by gonad anatomy (Figure S3)
during dissection 2 months after metamorphosis (ca. 16 weeks after
reaching the free-swimming tadpole stage) as described in Bókony et al.
(2020). At this age the gonads are well differentiated in this species
(Bernabò, Gallo, Sperone, Tripepi, & Brunelli, 2011; Ogielska &
Kotusz, 2004). To our knowledge, ”sex races” (Rodrigues, Vuille, Loman,
& Perrin, 2015) were not reported in agile frogs. From each froglet we
took a tissue sample (hind feet) that we stored in 96% ethanol until
DNA extraction. During dissection, we recorded several fitness-related
traits (see below), and we carefully removed the gonads and fixed them
in neutral-buffered 10% formalin (Sigma 1.00496) for histology. All the
above procedures were approved by the Ethical Commission of the Plant
Protection Institute and carried out according to the permits issued by
the Government Agency of Pest County (permit numbers:
PE/KTF/3596‐6/2016, PE/KTF/3596‐7/2016, PE/KTF/3596‐8/2016,
FPH061/2472-4/2017).
DNA was extracted from toe-clip samples using Geneaid Genomic DNA
Extraction Kit (Thermo Fisher Scientific) for animal tissue, following
the manufacturer’s protocol, except that digestion time was 2 hours and
4 µl RNase was added to each sample before the binding step. From buccal
swab samples, DNA was extracted either by QIAamp DNA Investigator Kit
(Qiagen) or Geneaid Genomic DNA Extraction Kit (Thermo Fisher
Scientific) for animal tissue following the manufacturers’ instructions
with a few modifications for the latter (1 hour digestion, 30 minutes
lysis).