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).