Sampling and DNA sequencing
For mitochondrial cox1 and nuclear ITS1 sequencing, a total of
680 M. virgata individuals were collected from the intertidal
and/or subtidal zone at 34 localities on the seashores of China, Japan,
Korea, and Taiwan, which covers almost the entire distribution range ofM. virgata (Fig. 1; Table 1). MtDNA cox1 and ITS1 from
four individuals of M. keenae were also sequenced and used as
outgroup sequences in the phylogenetic analyses. All samples were
deposited as voucher specimens in the Marine Mollusk Resource Bank of
Korea (MMRBK: http://mmrbk.org/). Total genomic DNA was extracted from
the adductor muscle of each individual using a DNeasy tissue kit
following the manufacturer’s instructions. A partial fragment of the
mtDNA cox1 gene was PCR-amplified using the universal primers
jgLCO1490 and jgHCO2198 (Geller, Meyer, Parker, & Hawk, 2013) in total
reaction volumes of 50 μL, which included 2 μL of template DNA, 36.75 μL
of D.W., 5 μL of 10x Taq buffer, 1 μL of each primer (10 pmol), 4 μL of
0.2mM dNTP mixture, and 0.25 μL of Taq polymerase (TaKaTa ExTaq ). The PCR amplification conditions were as follows: 1 cycle
of denaturation at 95℃ for 3 min followed by 35 cycles of denaturation
at 94℃ for 30 s, annealing at 45℃ for 30 s, elongation at 72℃ for 1 min,
and a final extension at 72℃ for 10 min. To cross-reference the mtDNAcox1 results, the nuclear ITS1 gene was PCR-amplified and
sequenced for 101 individuals from 23 populations, which were subsampled
so that mtDNA cox1 haplotype diversity was well-represented in
the ITS1 group (Table 1). The nuclear ITS1 gene fragment was
PCR-amplified using ITS1 (5´-TCC GTA GGT GAA CCT GCG G-3´) (White,
Bruns, Lee, & Taylor, 1990) and 5.8S-R (5´-TCG ATG AAG AAC GCA GC-3´)
(Vilgalys & Hester, 1990). The PCR mixtures and conditions for the ITS1
gene fragment were the same as for mtDNA cox1 , except that the
annealing temperature was 55℃. PCR products were isolated from 1%
agarose gels using a QIAquick gel extraction kit (QIAGEN Valencia, USA)
following standard protocols and sequenced directly using an ABI PRISM
3700 DNA analyzer (Applied Biosystems, Foster City, USA).