3. Molecular protocol for the eDNA samples
A first-round PCR (1st PCR) of eDNA was performed using KOD Plus Neo
(Toyobo, Tokyo, Japan), with each 20 µ L reaction containing 2.0µ L of 10×PCR Buffer, 2.0 µ L of dNTPs (each 2 mM), 1.2µ L of MgSO4 (25 mM), each 0.5 µ L of 10µ M primers, 0.4 µ L of KOD Plus Neo polymerase, and 2.0µ L of template DNA. The PCR conditions were initial denaturation
at 94 °C for 2 min and 35–40 cycles of denaturation at 94°C for 15 sec,
annealing at 63°C for 30 sec, and extension at 68°C for 30 sec. The
number of cycles was initially 35, and samples for which no target peak
was identified by electrophoresis after the second-round PCR (2nd PCR)
were analyzed again at 38 cycles and then 40 cycles (Table S1). Although
the number of PCR cycles was kept modest to minimize errors (Wakimura et
al. 2023), the haplotype accumulation curve suggested that the
sequencing coverage was sufficient to detect haplotypes in the samples
(Figure S1). The 1st PCR was conducted with eight replicates per sample
(20 µ L × 8), and individual replicates were pooled and purified
using 160 µ L of VAHTS DNA Clean Beads (Vazyme Biotech, Nanjing,
China) as templates for the 2nd PCR. The 2nd PCR was conducted using KOD
FX Neo (Toyobo) and primers with appropriate unique index sequences. For
this PCR, each 10 µ L reaction contained 5.0 µ L of 2×PCR
Buffer, 2.0 µ L of dNTPs (each 2 mM), 0.5 μ M of each
forward and reverse primer, 0.2 µ L of KOD FX Neo polymerase, and
1.0 µ L of the template. The thermal conditions were as follows:
initial denaturation at 94 °C for 2 min, 12 cycles of denaturation at
98°C for 10 sec, annealing at 60°C for 30 sec, and extension at 68°C for
30 sec, and a final extension at 68°C for 2 min. The 2nd PCR products
were purified again using VAHTS DNA Clean Beads, and positive bands of
targeted amplicons were confirmed using the Fragment Analyzer System and
the dsDNA 915 Reagent Kit (Agilent Technologies, California, USA). The
prepared libraries were sequenced on the Illumina MiSeq platform using
the MiSeq Reagent Kit v3 (2 × 300 cycles) (Illumina, California, USA).
The procedures from the purification of 1st PCR products to this MiSeq
run were performed by Bioengineering Lab. (Kanagawa, Japan). Fastq files
containing raw reads were denoised using the DADA2 1.18.0 (Callahan et
al. 2016), an algorithm known to have high accuracy in the context of
population-level inference (Tsuji et al. 2020b; Macé et al. 2022). DADA2
was performed in Qiime2 2022.11 (Bolyen et al. 2019) with the default
parameters except: –p-trunc-len-f 240,–p-trunc-len-r 180, and –p-mim-overlap 20. The
obtained sequences were annotated by a local BLAST search against a
reference database consisting of sequences of the studied species
obtained from Yokoyama and Goto (2002), Ito et al. (2018), and the
present study (Table S2). Sequences with an identity of 90.0% or more
were picked up (but no sequences were matched at 90.0%–99.4%; see
Results).