Muto et al.: Genetic and morphological differences between Sebastes vulpes and 5. zonatus 
431 
step, 12 randomly chosen primer pairs were used (Mse 
I + Eco RI [ACA + CAA, AAG + CAG, ACA + CTT, ACA 
+ CAC, ACT + CTA, ACA + CTG, AAG + CAC, AGG 
+ CAT, ACC + CTT, ACG + CAC, AAG + CAT, AGG 
+ CTT]). Selective amplification products were ana- 
lyzed on an ABI PRISM 310 genetic analyzer (Applied 
Biosystems) together with a GeneScan-500 Rox size 
standard (Applied Biosystems). Fragment data were 
collected with Peak Scanner software, vers. 1.0 (Ap- 
plied Biosystems). Electropherograms were scored for 
the presence (1) or absence (0) of fragments between 90 
base pairs (bp) to 450 bp in size, so as to create binary 
matrices. Fragments were inferred as homologous if 
they differed by not more than 0.5 bp from the median. 
Euclidean pairwise genetic distances (Huff et ah, 1993) 
were calculated from the binary matrices in GenAlEx, 
vers. 6.41 (Peakall and Smouse, 2006). Principal coor- 
dinate analysis (PCoA) with a covariance matrix and 
the data standardization method was performed on 
the basis of the Euclidean pairwise distance matrix, 
as implemented in GenAlEx, vers. 6.41 (Peakall and 
Smouse, 2006). By means of PCoA, we explored the 
genetic population structure among all 65 specimens 
without a priori grouping information. 
The mitochondrial DNA sequence comprising 452 bp 
extending from the threonine transfer RNA (tRNA Thr ) 
gene to the middle conserved region of the control re- 
gion (mtCR) was amplified with the primers L15876 
(5'- A AG CAC TTG AAT GAG CTT G-3') (Rocha-Olivares 
et ah, 1999) and H16498 (5'-CCT GAA GTA GGA ACC 
AGA TG-3') (Meyer et al., 1990). The polymerase chain 
reaction (PCR) proceeded for 30 cycles, with denatur- 
ation at 94°C for 1 min, annealing at 54°C for 1 min 
and extension at 72°C for 2 min, PCR products being 
purified with USB® ExoSAP-IT® (Affymetrix, Santa 
Clara, CA). DNA sequencing was performed with a Big- 
Dye Terminator Cycle Sequencing Kit (Applied Biosys- 
tems) on an ABI PRISM 310 genetic analyzer (Applied 
Biosystems). The DNA sequences were edited with the 
sequence alignment editor BioEdit 7.0. 5. 3 (Hall, 1999) 
and aligned with the program CLUSTAL X, vers. 2.1 
(Larkin et al., 2007). Estimation of mitochondrial ge- 
netic structuring among specimens based on haplotype 
frequency and uncorrected genetic distances between 
haplotypes (<f> sr ) was performed by Arlequin, vers. 3.5 
(Excoffier and Lischer, 2010). The significance of the 
<J> sr value was tested by 10,000 random permutations. 
Arlequin 3.5 was also used to construct the minimum 
spanning network (MSN) of the haplotypes on the ba- 
sis of minimum sequence differences. The sequences 
determined in this study have been deposited in Gen- 
Bank (accession numbers AB614522-AB614526 and 
AB615270-AB615329). 
Morphological analysis 
Morphological characters were examined after fixa- 
tion in 10% formalin and preservation in 70% etha- 
nol. Measurements were made on 31 morphological 
characters, including standard length, which generally 
Figure 2 
Coloration of fresh specimens of Sebastes vulpes 
and S. zonatus. (A) S. vulpes, FAKU 96090, 194.0 
mm standard length; ( B I S. zonatus, FAKU 97082, 
167.9 mm standard length. Photo by N. Muto. 
followed those described by Nakabo (2002a) except for 
the following: body depth 1 and 2 as defined by Kai and 
Nakabo (2002); upper peduncle length, lower peduncle 
length, spinous dorsal-fin base length, soft dorsal-fin 
base length, prepelvic length, and gill raker length as 
defined by Chen (1971); body width was taken as the 
distance between the uppermost bases of the right and 
left pectoral fins; pelvic-to-anal-fin length was taken as 
the distance from the anteriormost base of the pelvic fin 
to the origin of the anal fin. 
Analysis of covariance (ANCOVA) of loglO trans- 
formed measurements (with standard length as a co- 
variate) was used to assess differences in morphometric 
characters between S. vulpes and S. zonatus when as- 
sumptions of normality and homogeneity of slopes were 
satisfied. The following characters met the assumptions 
required for ANCOVA: head length, snout length, orbit 
length, interorbital width, postorbital length, upper jaw 
length, body depth 1, body depth 2, body width, caudal 
peduncle depth, upper peduncle length, pectoral-fin 
length, pelvic-fin length, dorsal-fin base length, spi- 
nous dorsal-fin base length, soft dorsal-fin base length, 
preanal length, predorsal length, prepelvic length, pel- 
vic-to-anal-fin length, 2nd dorsal-fin spine length, 3rd 
dorsal-fin spine length, and gill raker length. To pro- 
vide an objectively defined score that summarizes the 
major components of variable measurements between 
the specimens, a principal component analysis (PCA) 
was conducted on the basis of all measurements. Raw 
measurement data were standardized by log transfor- 
mation before PCA. 
