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.