Kusaka et al.: Genetic population structure of Dipturus chinensis around Japan 
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Table 1 
Sampling location, number of specimens, total length (range and mean), sampling depth, and capture date of the polkadot skate 
(Dipturus chinensis) collected during 2010-2017 from 7 locations in the East China Sea, Sea of Japan, and Pacific Ocean around 
Japan. For populations at 2 other locations, Taiwan and the Korean Peninsula, DNA sequences were obtained through the Interna- 
tional Nucleotide Sequence Database Collaboration. The specific location for the sequences for Taiwan is unknown, and sequences 
for the Korean Peninusula are for polkadot skate from Jindo, off the southwestern coast of the peninsula, near the boundary of the 
East China Sea and the Yellow Sea. 
Total length (mm) 
No. of SEE 
specimens Range Mean 
Location 
East China Sea 
Danjo Islands 189-684 
Goto Islands 370-643 
Koshiki-jima Islands 514 
Sea of Japan 
102-638 
116-701 
Kyoto Prefecture 
Niigata Prefecture 
Pacific Ocean 
125-584 
335-775 
Taiwan - 
Kochi Prefecture 
Aomori Prefecture 
Korean Peninsula _ 
conservation efforts. On the basis of mt cyt b sequences, 
we also estimated the evolutionary history of this species, 
information that is important for delineation of managing 
units that should be managed independently to ensure 
the viability of the species (Moritz, 1994, 2002; Palsbgll 
et al., 2007). 
Materials and methods 
Sampling and DNA extraction 
A total of 212 polkadot skate (102-775 mm in total length 
[TL]; mean: 444 mm TL) were collected from 7 locations 
around Japan: 3 locations in the East China Sea, the Danjo 
Islands (number of samples [n]=48), Goto Islands (n=18), 
and Koshiki-jima Islands (n=1); 2 locations in the Sea of 
Japan, Kyoto Prefecture (n=47) and Niigata Prefecture 
(n=49); and 2 locations in the Pacific Ocean, Kochi Prefec- 
ture (n=23) and Aomori Prefecture (n=26) (Table 1, Fig. 1). 
In addition, individuals of 2 other species of Dipturus, the 
bigtail skate (D. macrocaudus) and the acutenose skate (D. 
tengu), were collected from Kochi Prefecture so that these 
species could serve as outgroups for phylogenetic analy- 
sis. Samples were obtained from depths of 110-320 m by 
using bottom or beam trawls between 2010 and 2017. 
Muscle tissue was dissected from each fish and stored, 
either frozen at —20°C or in 99.5% ethanol, until DNA 
extraction. Total genomic DNA was extracted by using the 
Sampling depth 
(m) Capture date 
130-150 April 2010—April 2015 
203 January 2014 
Unknown November 2013 
110-170 
120-160 
April 2014—July 2014 
August 2013—August 2014 
150-320 November 2015—January 2016 
160-180 February 2017—March 2017 
DNeasy Blood & Tissue Kit? (QIAGEN, Hilden, Germany) 
following the manufacturer’s protocol. 
Mitochondrial DNA analyses 
We used a polymerase chain reaction (PCR) to amplify 
approximately 1200 base pairs of the mt cyt 6b gene, with 
the following primers: GLU-L-Dipturus (5°-TCT GAA 
AAA CTA CCG TTG TTA-3’) and CB6THR-H-Dipturus 
(5-CTC CAA TCT TTG GTT TAC AAG-3’). The design 
of the primers was based on a primer pair, GLU-L and 
CB6THR-H (Palumbi et al., 2002), with reference to the 
cyt b gene sequences of skate species registered in the 
DNA Data Bank of Japan, which is part of the Interna- 
tional Nucleotide Sequence Database Collaboration: the 
polkadot skate (registered as D. kwangtungensis; acces- 
sion number KF318309; Jeong et al., 2015), the thorny 
skate (Amblyraja radiata) (accession number AF 106038; 
Rasmussen and Arnason, 1999), the ocellate spot skate 
(accession number AY525783; Kim et al., 2005), the 
Korean skate (Hongeo koreanus) (accession number 
KC914433; Jeong et al., 2014), and the longnose skate 
(Beringraja rhina) (registered as Raja rhina; accession 
number KC914434; Jeong and Lee, 2015). Polymerase 
chain reactions were conducted by using a PCR Thermal 
2 Mention of trade names or commercial companies is for identi- 
fication purposes only and does not imply endorsement by the 
National Marine Fisheries Service, NOAA. 
