DNA extraction and PCRs

To avoid contamination, we followed a unidirectional lab flow, whereby we performed all PCR protocols, including preparation/addition of the standards and qPCR cycling, in two separate rooms (rooms 1 and 2, respectively). To prevent carry-over contamination, no equipment or samples were returned from room 2 to room 1. In both rooms, laboratory benches were decontaminated using commercial bleach.

The water samples stored at −30 °C were thawed and vacuum-filtered through glass microfiber filters (GF/F; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with 0.7 μm mesh size, which were used in other amphibian studies (Katano et al., 2017; Iwai, Yasumiba & Takahara, 2019). Filter funnels and tweezers used in the filtration treatment were sterilized with 10% commercial bleach (ca. 0.6% hypochlorous acid) for 5 min (i.e., sodium hypochlorite treatment), flushed with a large amount of tap water, and then rinsed with DNA-free distilled water between samples to avoid cross contamination. Filtering controls (i.e., 500 mL of distilled water) in laboratory experiment controls filtered on each day of sample filtration. The filter papers were wrapped in new aluminum foil (i.e., DNA-free), placed in plastic bags, and stored at −30 °C. The eDNA was extracted from the filters according to the methods of Uchii, Doi & Minamoto (2016), using a Salivette tube (Sarstedt, Nümbrecht, Germany) and a DNeasy Blood and Tissue Kit for DNA purification (Qiagen, Hilden, Germany). The filters were incubated by submersion in a mixed buffer (400 μL buffer AL and 40 μL Proteinase K; Qiagen) using a Salivette tube in a dry oven at 56 °C for 30 min. The tubes with filters were centrifuged at 5,000×g for 5 min at room temperature. Then, 220 μL of TE (Tris–EDTA) buffer (pH: 8.0; 10 mM Tris–HCl and one mM EDTA) was added to the filters, and tubes were centrifuged again at 5,000×g for 5 min. Buffer AL (200 μL) and 100% ethanol (600 μL) were then added to each filtrate and mixed by pipetting. The mixture was applied to a DNeasy Mini spin column and centrifuged at 6,000×g for 1 min. This step was repeated until the mixture was completely processed. We followed the manufacturer’s instructions for further steps, and eDNA was eluted from each sample solution with a final volume of 100 μL Buffer AE.

The eDNA samples were then used for PCR-RFLP assay to detect DNA from the three target Japanese brown frog species based on Igawa et al. (2015). This method utilizes species-specific restriction enzyme (SpeI and HphI) digestion sites in a partial nucleotide fragment of mitochondrial 16S rRNA amplified by PCR. However, the oligonucleotide primers used by Igawa et al. (2015) could amplify 16S rRNA fragments originating from other vertebrate species including humans which showed similar banding patterns. Therefore, we altered the primers to amplify a shorter region in which only the three target frog species have restriction sites. Specifically, we modified the method of Igawa et al. (2015) by changing the primers: F96 5′-GTCCAGCCTGCCCAGTGAYAAA-3′ and R19 5′-GTTGAACAAACGAACCATTGGT-3′. These new primers were designed from an internal region of the previous study and amplify shorter fragments (Rana japonica: 514–517 bp, Rana ornativentris: 516–517 bp, and Rana tagoi tagoi: 514–516 bp).

For higher efficiency of PCR amplification, we also modified the PCR protocol described by Igawa et al. (2015). In this study, PCR was conducted using KOD FX Neo (TOYOBO, Osaka, Japan). The 20 µL total volume of reaction solution included 10 µL of 2 × PCR Buffer for KOD FX Neo, 2 µL of 2 mM dNTPs, 10 pmole of each primer, 3 µL of eDNA solution and 0.4 U of KOD FX Neo. Thermal cycling was performed using two-step PCR cycling: 95 °C for 3 min followed by 35 cycles of 98 °C for 10 s and 65 °C for 30 s. Following PCR amplification, independent digestion using two restrictions enzymes (SpeI and HphI) and electrophoresis were conducted in the same manner as Igawa et al. (2015). Then, 5 µl of the original PCR product or 15 µl of each PCR product digested with the two restriction enzymes were electrophoresed on a 2% agarose gel for 30 min at 100 V and visualized. We performed the experiment several times to confirm the reproducibility.

For Rana japonica, the amplified fragments are expected to have different digestion patterns between the western (including Hiroshima) and northern part (including Fukushima) of the Japanese mainland (Igawa et al., 2015). In all Rana japonica populations, amplicons digested with SpeI result in 255 and 259 bp subfragments. However, after Hph1 digestion, Rana japonica frogs from the western part have no subfragments, while those from the northern part have 301 and 220 bp subfragments (Fig. 2A). For Rana ornativentris and Rana tagoi tagoi, amplified fragments are commonly digested only with HphI, resulting in 231, 199, and 86 bp subfragments and 313 and 200 bp subfragments, respectively (Fig. 2A). To confirm the specificity of eDNA detection by our PCR-RFLP method, PCR products of sample no. 1, 10, 11, 14, 17, 19, and 20 were directly sequenced using the same primers and BigDye Terminator ver 3.1 (Life Technologies, Carlsbad, CA, USA) after PEG precipitation. The obtained nucleotide sequences were annotated by NCBI blastn (https://blast.ncbi.nlm.nih.gov).