1. Study sites and sampling
This study was conducted in tributaries in the upper section of the Sorachi River, Hokkaido, Japan (Figure 1; Table 1). In this area, Nakajima et al. (2021, 2023) conducted population sampling of C. nozawae individuals and investigated the population structure of this species. From the downstream of riffles or runs of 21 sites whereC. nozawae had been sampled before, water samples of 0.8 L were collected from the surface and filtered through Millipore 0.45 μ m Sterivex-HV filters (Merck KGaA, Darmstadt, Germany) with 100 mL syringes (JMS, Tokyo, Japan) in June 2023. Note that the site Pop 19 (in Figure 1) was approximately 500 m downstream from the population sampling site. Each 0.8 L of water at every site could be filtered with one Sterivex filter, except for one site (two filters were needed in Pop 18). The Sterivex filters were immersed in 1.3 mL of RNAlater (Thermo Fisher Scientific, Massachusetts, USA) and transported to the laboratory under refrigeration. After the RNAlater was removed using QIAvac Vacuum Systems (QIAGEN, Hilden, Germany) and a vacuum pump, total DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN). The protocol of DNA extraction was basically followed by the “ATL-S procedure” in Fukuzawa et al. (2023), except that the volumes of the first addition of buffer ATL and proteinase K were changed to 400 µ L and 40µ L, respectively, and the final elution with buffer AE was performed in a volume of 120 µ L. The DNA sample from Pop 18 was purified using the DNeasy PowerClean Pro Cleanup Kit (QIAGEN) to remove PCR inhibitors, as amplification in subsequent PCR was difficult.