Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding

Waltham, MA, USA). Water temperature and salinity were measured during sampling. The volume of water samples varied from 200 to 1000 mL depending on turbidity (Table 1). We assumed that variation in sample volume did not affect diversity as we confirmed no correla- tion between the volume and number of species (r2 = 0.0061) by all samples (n = 28). As a neg- ative control, 500 mL of pure water was filtered at each river. Filter units were frozen at -30˚C until DNA extraction. eDNA extraction Total DNA was extracted from the Sterivex filter units using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), following the procedure described by Miya et al. [25] and the manufacturer’s protocol with minor modifications. After removing RNAlater by centrifuga- tion (4,000 × g for 2 min), the filter unit was rinsed with sterilized distilled water. For the lysis of eDNA attached to the membrane, proteinase K (20 μL) and lysis buffer AL (200 μL) were applied to the filter unit and incubated inside a 56˚C preheated oven for about 20 min. The roller was turned on to enable even collection of DNA from the membrane. After the incuba- tion, the spin column was centrifuged at 4,000 × g for 2 min to collect DNA, to which 200 μL of absolute ethanol was then added and mixed well. The resulting solution was transferred to a spin column, centrifuged (6,000 × g for 1 min), and then purified twice using wash buffer (AW1 and AW2). After the purification steps, DNA was eluted with the elution buffer (110 μL) provided in the kit. Extracted DNA was stored in a LoBind tube at -30˚C.

Fig 1. Sampling stations. Location of (a) the five rivers surveyed in this study. Maps showing the location of sampling stations RM (river mouth), L1 (left 500 m), L2 (left 1 km), R1 (right 500 m), and R2 (right 1 km) of (b) Aka River, (c) Tama River, (d) Miya River, (e) Takatsu River, and (f) Sendai River. The satellite photos from (b) to (f) were provided by Geospatial Information Authority of Japan. Scale bar = 1 km. https://doi.org/10.1371/journal.pone.0231127.g001 PLOS ONE Biodiversity of five estuaries PLOS ONE | https://doi.org/10.1371/journal.pone.0231127 October 6, 2020 3 / 15