26 
Fishery Bulletin 11 7(1-2) 
viduals (EC=22; YS=0; SJ=44; EK=1; OS=20; NP=26) 
for both mtCR analysis and morphological compari¬ 
sons. The individuals for both mtCR analysis and mor¬ 
phological comparisons only partly overlapped because 
some of the individuals were in poor condition and 
could not be used for either of the mtCR analysis or 
morphological comparisons. 
mtCR analysis 
Total DNA was extracted from muscle tissue preserved 
in 99.5% ethanol by using the DNeasy Tissue Kit 
(Qiagen, Japan) or Wizard Genomic DNA Purification 
Kit (Promega Corp., Madison, WI 1 ), according to the 
manufacturer’s protocols. Polymerase chain reaction 
(PCR) was performed with the primers ElDloopF (5'- 
TCC CAA AGC CAA GAT TCT GC-3') and RajinaeP7r 
(5'-AAA CTG GGA GGG CTG GAA ATC TTG A-3') 
(Valsecchi et al., 2005), which amplified 597 base pairs 
(bp) of mtCR. The fragment was amplified by using 
the Veriti Thermal Cycler (Applied Biosystems, Foster 
City, CA) with 15.1 pL PCR reaction mix, including 
8.3 pL of distilled water, 1.5 pL of lOx PCR buffer, 
1.2 pL of 2.5 mM dNTPs, 1.5 pL of 5 mM each primer, 
0.1 pL of EX-Taq polymerase (TaKaRa Bio Inc., Shiga, 
Japan) and 1 pL of DNA template or 10.2 pL PCR 
reaction mix, including 2.2 pL of distilled water, 5 
pL of KAPA2G Robust HotStart Ready Mix PCR Kit 
(KAPA Biosystems, Wilmington, MA), 1 pL of 5 mM of 
each primer and 1 pL of DNA template. The cycling 
conditions consisted of an initial denaturation cycle at 
94°C for 5 min, followed by 30 cycles at 94°C for 15 s 
(denaturation), 56°C for 15 s (annealing) and 72°C for 
30 s (extension), and a single final extension cycle at 
72°C for 7 min. Successful amplification was confirmed 
by electrophoresis of the PCR products on 1% agarose 
gel, stained with RedSafe (iNtRON Biotechnology, 
Seoul, South Korea). The PCR products were purified 
with ExoSAP-IT (Affymetrix Inc., Santa Clara, CA). 
DNA sequencing was performed with BigDye Ter¬ 
minator Cycle Sequencing Kit,vers. 1.1, and an ABI 
Prism 310 Genetic Analyzer (Applied Biosystems). The 
sequences determined here were deposited in INSDC 
(International Nucleotide Sequence Database Collabo¬ 
ration) under accession numbers LC386653-LC386846 
(Suppl. Table) (online only). 
The mtCR sequences were edited with BioEdit, vers. 
7.2.5 (Hall, 1999), and aligned with ClustalW interface, 
available in MEGA 6 (Tamura et al., 2013). A haplo- 
type network was inferred by using the median-joining 
network method with Network, vers. 5.0.0.1 (Fluxus 
Technology Ltd, Sudbury, Suffolk, UK). Haplotype di¬ 
versity ( h) and nucleotide diversity (CD) were calculated 
by using Arlequin, vers. 3.5.1.3 (Excoffier and Lischer, 
2010). The pairwise F ST values that included informa¬ 
tion on haplotype frequencies were used for indices of 
1 Mention of trade names or commercial companies is for iden¬ 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
genetic differentiation. The null hypothesis of genetic 
homogeneity was assessed by 10,000 replications with 
Arlequin and sequential Bonferroni corrections (Rice, 
1989). 
Morphological comparisons 
Morphological characters were examined after fixa¬ 
tion in 10% formalin and preservation in 70% etha¬ 
nol or 50% isopropanol. For comparisons of coloration 
among regional populations, photographs of the fresh 
color of most specimens (before fixation) were taken 
by digital photography, except for specimens from YS 
and EK. For morphological comparisons, 13 measure¬ 
ments, those of Last et al. (2008) and Ishiyama (1958), 
were taken: 1) total length (TL); 2) disc length; 3) disc 
width; 4) tail length; 5) head length; 6) dorsal snout 
length; 7) eye diameter; 8) distance between orbits; 
9) ventral head length; 10) ventral snout length; 11) 
prenasal snout length; 12) distance between nostrils; 
and 13) distance between 1st gill openings. The num¬ 
bers of nuchal thorns were also counted. Differences 
in measurements among regional populations were as¬ 
sessed for respective males and females by analysis of 
covariance (ANCOVA) by using TL as a covariate. A 
covariance matrix of arcsine-transformed morphomet¬ 
ric ratios (with TL as denominator) was prepared for 
ANCOVA. If significant differences among the regional 
populations were observed in ANCOVA (P<0.05), post- 
hoc pairwise comparisons with Holm’s adjustment were 
used to establish differences between individual region¬ 
al populations. All statistical analyses for morphologi¬ 
cal comparisons were conducted with R language, vers. 
3.1.2 (R Core Team, 2014). 
Differences in maturity size or developmental stage 
among regional populations were estimated from clasp- 
er length in males; claspers elongate with growth and 
are indicative of maturity size and developmental stage 
(Stehmann, 2002). Following Stehmann (2002), we con¬ 
sidered males to be mature if they had both alar and 
malar thorns and a hard clasper skeleton. The number 
of rows of tail thorns in females was also compared, 
the number increasing with growth to a maximum of 
5 in the genus Okamejei (Ishiyama, 1967; Ishihara, 
1987). The number of rows of tail thorns was therefore 
considered to be indicative of developmental stage. The 
estimated maturity size in males and females was also 
assessed by post-hoc pairwise comparisons with Holm’s 
adjustment. 
Results 
mtCR analyses 
The mtCR sequences of 597 base pairs revealed 11 
variable nucleotide sites with 10 transitions and 1 
transversion, without any deletions and insertions, and 
a total of 14 haplotypes (haplotype codes were defined 
as Okl-14) in 194 individuals. Among the 5 regional 
