SC/J09/JR29

Mitochondrial DNA analysis on stock structure in the western
North Pacific common minke whales
Mutsuo Goto1, Naohisa Kanda1, Toshiya Kishiro2, Hideyoshi Yoshida2, Hidehiro Kato3 and Luis A.
Pastene1

1 The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo, 104-0055, Japan.
2 National Research Institute of Far Seas Fisheries, 2-12-4 Fukuura, Kanazawa, Yokohama Kanazawa,
236-8648, Japan
3Tokyo University of Marine Science and Technology, 1-5-7 Konan, Minato-ku, Tokyo,
108-0075 Japan

Contact email:goto@cetacean.jp

ABSTRACT
Genetic variation at the mtDNA control region in the western North Pacific common minke whales wasanalyzed to examine the plausibility of four stock structure scenarios adopted at the final stage of the
Implementation Simulation Trials (ISTs) process under the Revised Management Procedure by the IWC
Scientific Committee in 2003. A total of 1,639 whales collected during JARPN and JARPNII surveys
from 1994 to 2007 in the area from the Japanese coast to the offshore waters (to 170°E) on the Pacificside, was used for the analyses. The samples from 2002 to 2007 (n= 1,124) were not used during theprevious ISTs process. Heterogeneity tests were based on the randomized chi-square test. The only sourceof significant mtDNA heterogeneity was found in sub-area 7W in a 2007. Additional analysis in this subarea and year suggested that the source of heterogeneity was due to the occurrence of J stock animals. Our
updated analysis based on a larger data set confirmed that only two stocks distribute in the western North
Pacific, the O and J stocks. This study provides no evidences for the occurrence of a W stock in offshore
waters or additional structure within the O stock in the Pacific side of Japan. These results are consistent
with the results found by the microsatellite and morphometric analyses. Therefore results from the genetic
and non-genetic approaches support the single O stock scenario in the Pacific side of Japan (Scenario B).

KEY WORDS: COMMON MINKE WHALE, MTDNA, STOCK STRUCTURE, JARPN, JARPNII,NORTH PACIFICINTRODUCTION
The Implementation Simulation Trials (ISTs) for North Pacific common minke whale under the Revised
Management Procedure (RMP) was completed during the 2003 International Whaling Commission
Scientific Committee (IWC SC) meeting. At the final stage of the ISTs process the SC adopted thefollowing four stock structure baseline scenarios (IWC, 2004):(1) Baseline A: three-stock scenario (‘J’, ‘O’, ‘W’) with the ‘W’ stock found only in part of sub-area 9
and only sporadically.
(2) Baseline B: two stock scenario (‘J’ and ‘O’) with no W stock as a limiting case of Baseline A.
(3) Baseline C：four-stock scenario overall, with ‘OW’, ‘OE’ and ‘W’ to the east of Japan. Boundaries are
fixed at 147°E and 157°E and there is no mixing between the stocks.
(4) Baseline D：three-stock scenario (‘J’, ‘O’, ‘W’), with ‘O’ and ‘W’ mixing over 147°E and 162°E, O
being dominant to the west and W to the east.
Unfortunately the SC did not examine plausibility of the stock structure scenarios. In order to getagreement among SC members it gave the same ‘high’ plausibility to the four scenarios (IWC, 2004).

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In this study the plausibility of the four stock structure scenarios is examined through genetic analysis
based on mitochondrial DNA (mtDNA) and samples collected by JARPN and JARPNII from 1994 to
2007. The samples from 2002 to 2007 were not used during the previous ISTs process.

MATERIALS AND METHODS
Samples
Table 1 shows the number of samples used in the present mtDNA analysis by year, sub-area and theoffshore and coastal components of JARPN II. Details of the surveys design and methodology in theoffshore and coastal components of JARPNII were described in Tamura et al. (2009) and in Kishiro et al.
(2009), respectively. During the IST specification conducted in 2003, eighteen sub-areas were set for
management purpose of the western North Pacific common minke whale (Figure.1). Sub-areas 7, 8 and 9
were divided into western and eastern strata by 147°E, 157°E and 162°E, 7W (140.01°E -147.00°E), 7E(147.01°E -150.00°E), 8W (150.01°E -153.00°E), 8E (153.01°E -157.00°E), 9W (157.01°E -162.00°E),
and 9E (162.01°E -170.00°E). The sighting position of common minke whales used in this study is shownin Figure 2. Whales were sampled by JARPN and JARPNII surveys during 1994-2007
Sequencing of the mtDNA control region
Using established protocols (Sambrook et al., 1989), total-cell DNA was extracted from skin tissue
samples. The first half of control region of the mitochondrial genome was amplified using the polymerase
chain reaction (PCR). In order to amplify an approximately 500 bp of the mtDNA control region, primers
light-strand MT4 (Árnason et al., 1993) and heavy-strand Dlp5R (5'-CCATCgAgATgTCTTATTTAAggggAAC-3'), were used. PCR products were purified by MicroSpin S-400HR columns (Pharmacia
Biotech). Cycle sequencing was performed with the same primers, using BigDye terminator cycle
sequence Kit (Applied Biosystems, Inc). The cycle sequencing products were purified by AutoSeq G-50
spin Columns (Pharmacia Biotech). The labeled sequencing fragments were resolved by electrophoresis
through a 5% denaturing polyacrylamide matrix on an ABI 377 and ABI3100 Automated DNA
Sequencer (Applied Biosystems, Inc), following the protocols of the manufacturer. For each sample bothstrands were sequenced.
Data analysis
The evolutionary distance between two nucleotide sequences was calculated according to Kimura’s two
parameters method (Kimura, 1980). The degree of genetic diversity within each locality was estimatedusing the nucleotide diversity (Nei, 1987). The randomized chi-square Test of Independence (Roff and
Bentzen, 1989) was used to investigate the temporal/spatial differentiation of mtDNA variation. In eachtest a total of 10,000 permutations of the original data were performed. A P-value smaller than 0.05 wasused as a criterion to reject the null hypothesis of panmixia.
The following steps were conducted: a) analysis of genetic differences between the coastal and offshore
samples collected in the same year from sub-area 7W; b) analysis of genetic differences among whales
collected in different years from a same sub-area; and c) analysis of genetic differences among whales
grouped according to the geographic divisions defining the four stock structure baseline scenarios used inprevious ISTs.
Assignment for J or O types
Analyses were conducted with and without suspected J stock animals in the case of sub-area 7W.
Individual assignment to O and J stocks was based on analysis of microsatellite and the computer
program STRUCTURE (Kanda et al., 2009a).
RESULTS
Genetic diversity within samples
Sequence variations in a 487bp segment of the mtDNA control region resulted in 123 unique haplotypes
in the total sample of 1,632 whales. Forty-one polymorphic sites were detected, 37 of which weretransitions, three transversion and one insertion/deletion event..

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Table 2 shows the nucleotide (π) and haplotype (H) diversities in each sample. Nucleotide diversities andits standard error of JARPN (1994-1999), JARPNII offshore component (2000-2007) and JARPNII
coastal component (2002-2007) were 0.0079 (0.0002), 0.0081(0.0002) and 0.0089 (0.0002), respectively.
Haplotype diversities were 0.9522, 0.9529 and 0.9624, respectively. For both indices these values arehigher in the JARPNII coastal whales. For nucleotide diversity the difference was statistically significant
between JARPN and JARPNII coastal component.
Genetic divergence between samples
Genetic differences between offshore and coastal samples in sub-area 7W
Table 3 shows the results of the heterogeneity test for the comparison between samples taken in 7W bycoastal and offshore components of JARPN II, by year and with and without suspected J stock animals.
None of the comparison showed significant mtDNA differences except the 2007 samples. We conducted
the heterogeneity test for this year excluding suspected J stock animals. In this case no significant
differences were found.. Therefore significant differences found were most likely due to the inclusion ofwhales from the J stock in the sample. For the subsequent analyses, we conducted the heterogeneity tests
with and without suspected J stock animals in the 7W sample.
Yearly genetic differences within sub-areas
Table 4 shows the results of the heterogeneity test for yearly differentiation in each sub-area. In some subareas, years with less than 10 individuals were omitted. No significant yearly differences were found in
the sub-areas. In subsequent analyses samples from different years were combined in the sub-areas.
Plausibility of hypotheses
Explanation of the four stock structure hypotheses used in previous ISTs was given above.
Baseline A
In order to test the heterogeneity within sub-area 9, we compared the samples collected in the western andeastern sides of sub-area 9, by year. No significant differences were found. Further there was no
significant difference between western and eastern sides of sub-area 9 using total samples (Table 5).
Baseline B.
We examined the genetic differences among all sub-areas (7W, 7E, 8W, 8E, 9W and 9E). No significant
differences were found among all sub-areas with (P= 0.0595) and without (P=0.6106) suspected J stock
animals in the sub-area 7W.
Baseline C
Table 6 shows the results of heterogeneity test for samples divided according to this scenario (e.g.
samples divided by the longitudinal boundaries at 147°E and 157°E). First heterogeneity test was
conducted among samples from 7E, 8W and 8E, and no significant differences were found. Therefore
these samples were combined into one as 7E-8E for the following analyses. No significant differenceswere found among samples from 7W, 7E-8E and 9W-9E with and without suspected J stock animals in
the 7W samples.
Baseline D
We examined the genetic differences among three groups divided by the longitudinal boundaries at 147°E
and 162°E. First heterogeneity test was conducted among samples from 7E, 8W, 8E and 9W, and no
significant differences were found. These samples were combined into one as 7E-9W for the following
analyses. We then conducted the heterogeneity tests among 7W, 7E-9W, and 9E with and withoutsuspected J stock individuals in the 7W sample (7Wx7E-9Wx9E. 7Wx7E-9Wx9E*, Table 7). There areno significant differences among these samples with and without suspected J stock individuals in the 7W
sampleDISCUSSION
Level of genetic diversity
Genetic diversity at the mtDNA control region in common minke whales used in this study was similar tothat of other large baleen whales in the North Pacific, such as Bryde’s (Kanda et al., 2009c) and sei
whales (Kanda et al., 2009d). Diversities in the common minke whale in the JARPNII coastal component
samples are slightly higher than in the other samples. This could be caused by the occurrence of J stock3

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animals distributed around the coastal area in the Pacific side of Japan descried by Kanda et al.(2009a).
Genetic divergence
Overall our study based on a larger data set than used previously failed to find any evidence of significant
genetic heterogeneity in sub-areas 7, 8 and 9. The only source of genetic heterogeneity was found in subarea 7 in 2007. Results of further analysis in this sub-area suggested that such heterogeneity could be dueto the occurrence of J stock animals.
These results based on mtDNA and a larger data set are valuable to re-evaluate the plausibility of the four
stock structure scenarios defined by the IWC SC in 2003. Results constitute a very strong demonstrationthat the underlying hypothesis of scenario C, e.g. three stocks with hard boundaries at 147°E and 157°E,
has no independent support as there are no genetic evidences for multiple O stocks as proposed by this
scenario. Genetic differentiation among stocks is facilitated by some geographical or ecological ‘barrier’,
which acts restricting the gene flow among them. Minke whales in sub-areas 7, 8 and 9 have been
described as opportunistic feeders. Distribution of these whales is related to the distribution and dynamics
of prey species and the distribution of prey species is defined by oceanographic conditions in the area,
which are variable. There is no evidence for ecological barriers at 147°E or 157°E
Common minke whales were taken by the Japan’s small-type coastal whaling in sub-area 7W for morethan 40 years. The annual catches of minke whales were in the range 200-300 (IWC, 2004). If theoccurrence of a small coastal Ow stock, as proposed by the Baseline C stock scenario, is true, such stock
could have been extinct under such catch levels. Kawahara (2002) reviewed the small-type whaling andtwo types of CPUE analyses. Judging from the corrected CPUE1 trend for about 35 years, the drastic
decline of abundance seems unlikely. And CPUE2 analysis in 1977-1987 with operation hours by area
also does not suggest that O stock has declined. Consequently no considerable decline was detected ineither CPUE series. These results are consistent with results of the present genetic analysis and show thatthe plausibility of Baseline C is low.
Regarding to the Scenario D, results of our genetic analysis are also difficult to reconcile with theunderlying hypothesis of scenario D. This hypothesis establishes that O and W stocks mix with each otherbetween 147°E and 162°E. The O stock is predominant to the west and the W stock to the east of thisrange. Under this scenario only O stock occur between the Japanese coast and 147°E and only W stockfrom 162° to the east. One of the inconsistencies is that the mtDNA haplotype frequency in samples fromsub-area 9E (scenario D assume that only W stock is distributed in this sector) is genetically similar to
those in sub-areas 7 and 8 (O stock). Furthermore analysis of microsatellite indicated no significant
departure from the Hardy-Weinberg equilibrium in the sector 147°-162°E (Kanda et al., 2009b). A
significant departure from equilibrium could indicate a situation of mixing between different stocks assuggested by the scenario D. Scenario D is therefore inconsistent with genetic data.
One of the objectives of the JARPNII was to look for any evidence of existence of the W stock. Past
mtDNA studies (e.g., Goto et al., 2000) found the genetic heterogeneity in the 9W in some years. In thepresent study, we conducted haplotypic statistics based on Chi-square test because this statistics showed
the better performance in comparison to Hst, Fst, Kst* and PHIst (Taylor and Chivers, 2000). Based on
this statistic and a large data set were unable to find significant heterogeneity in sub-area 9W as detectedin our previous study. Therefore stock structure under scenario A was not supported by this analysis.
Therefore our updated analysis based on mtDNA and a larger data set confirmed that only two stocks
distribute in the western North Pacific, the O and J stocks. This study provides no evidences for theoccurrence of a W stock in offshore waters or additional structure within the O stock in the Pacific side ofJapan. These results are consistent with the results found by the microsatellite (Kanda et al., 2009b) andmorphometric (Hakamada and Bando, 2009) analyses.
ACKNOWLEDGEMENTS
We thank researchers and crewmembers participating in the 2002 to 2007 JARPNII surveys for theireffort in collecting the samples used in this study. Our sincere gratitude to H. Oikawa and S. Azumi for
their assistance in DNA extraction, and H. Hatanaka for his valuable comments on the manuscript.

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REFERENCES
Árnason, Ú., Gullberg, A. and Widegten, B. 1993. Cetacean mitochondrial DNA control region:
Sequences of all extant baleen whales and two sperm whale species. Mol. Biol. Evol., 10, 960-970.
Goto, M., Abe, H. and Pastene, L.A. 2000. Additional analyses of mtDNA control region sequences in thewestern North Pacific minke whales using JARPN samples. Paper SC/F2K/J32 presented to the IWC
Scientific Committee Workshop to Review the Japanese Whale Research Programme under Special
Permit for North Pacific Minke Whales (JARPN), Tokyo, 7-10 February 2000. 5pp.
Hakamada, T. and Bando, T. 2009. Morphometric analysis on stock structure in the western North Pacificcommon minke whale (Balaenoptera acutorostrata). Paper SC/J09/JR27 presented to the JARPN II
Review Workshop, Tokyo, January 2009 (unpublished). 13pp
International Whaling Commission. 2004. Report of the Scientific Committee, Annex D. Report of the
sub-committee on the revised management procedure. J. Cetacean Res. Manage. 67:75-184.
Kanda, N., Goto, M., Kishiro, T., Yoshida, H., Kato, H. and Pastene, L.A. 2009a. Individual identificationand mixing of the J and O stocks around Japanese waters examined by microsatellite analysis. Paper
SC/J09/JR26 presented to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 9pp.
Kanda, N., Goto, M., Kishiro, T., Yoshida, H., Kato, H. and Pastene, L.A. 2009b. Microsatellite analysisof minke whales in the western North Pacific. Paper SC/J09/JR30 presented to the JARPN II Review
Workshop, Tokyo, January 2009 (unpublished). 14pp
Kanda, N., Goto, M. and Pastene, L.A. 2009c. Stock structure of Bryde’s whales in the western North
Pacific as revealed by microsatellite and mitochondrial DNA analyses. Paper SC/J09/JR31 presented
to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 8pp
Kanda, N., Goto, M., Yoshida, H. and Pastene, L.A. 2009d. Stock structure of sei whales in the North
Pacific as revealed by microsatellite and mitochondrial DNA analyses. Paper SC/J09/JR32 presented
to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 14pp
Kawahara, H. 2002 A review of Japanese small-type whaling and CPUE analyses to address the relativeplausibility of trials on western North Pacific minke whale. Paper SC/55/IST16 presented to the
IWC/SC, May, 2002, (unpublished). 9pp.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions throughcomparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.
Kishiro, T., Yoshida, H., Goto, M., Bando, T., and Kato, H. 2009. Methodology and survey procedure
under the JARPNII - coastal component of Sanriku and Kushiro -, with special emphasis on whale
sampling procedures. Paper SC/J09/JR3 presented to the JARPN II Review Workshop, Tokyo,
January 2009 (unpublished). 27pp
Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York.
Roff, D.A., and Bentzen, P. 1989. The statistical analysis of mtDNA polymorphisms: χ2 and the problem
of small samples. Mol. Biol. Evol., 6, 539-545
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual. 2nd Ed.,
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Taylor, B.L. and Chivers, S.J. 2000. Evaluating the performance of different statistics to detect population
subdivision. Paper SC/F2K/J5 presented to the IWC Scientific Committee Workshop to Review theJapanese Whale Research Programme under Special Permit for North Pacific Minke Whales(JARPN), Tokyo, 7-10 February 2000. 10pp.
Tamura, T., Matsuoka, K., and Fujise, Y. 2009. Methodology and survey procedure under the JARPN II offshore component- with special emphasis on whale sampling procedures. Paper SC/J09/JR4
presented to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 16pp

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Table 1. Sample sizes used in this study by year, sub-area in offshore(A) and Coastal (B) components.
A) Offshore
Y
7W
1994
1995
1996
31
1997
1
1998
25
1999
50
2000
24
2001
43
2002
60
2003
17
2004
16
2005
32
2006
36
2007
79
Total
414
(B) Coastal
Year
2002
2003
2004
2005
2006
2007
Total

7E

8W

8E

1
31

1
1
44

15
30

7
7

2
48

21

21
8
16

6
10
2
86

7
28
13
138

9W
7
78

9E
14
22

19

48

16
29
32
25
42
19
23
2
294

14
42
30
1
4
178

7W
50
50
59
119
95
107
481

6

Total
21
100
47
100
100
50
40
100
100
100
100
94
100
100
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Table 2. Nucleotide (π) and haplotype (H) diversities in each sample.
samplesN
π
SE
H
JARPN（1994-1999）

418

0.00790.00020.9522JARPNII (2000-2007)
Offshore component

734

0.00810.00020.9529

40
100
100
100
100
94
100
100

0.00780.00860.00790.00780.00870.00830.00770.00820.00070.00040.00040.00040.00040.00050.00040.00040.9321
0.94890.95350.94910.95940.95200.95820.9590480

0.00890.00020.9624

260
220
1632

0.00910.00860.00830.00030.00030.00010.9656
0.95840.9561

2000
2001
2002
2003
2004
2005
2006
2007
JARPNII (2002-2007)
Coastal component
KushiroSanrikuSum

Table 3. Statistical comparison between offshore and coastal samples in sub-area 7W
by year, and with and without suspected J stock animals.
All animals
Without suspected J stock animals
N
N
Year
P
P
Offshore
CoastalOffshore
Coastal2002
60
50
0.172656
430.28082003
1750
0.136916440.16742004
16
59
0.281313
55
0.29192005
32
1190.232629
94
0.27992006
36
95
0.38183478
0.3074
2007
79
107
75
88
0.12380.0192
Bold indicate significant difference.

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Table 4. Results of the heterogeneity test for yearly differences within each sub-area
Sub-area
Year
N
P
N*
1996
31
30
1998
25
25
1999
50
48
2000
24
21
2001
43
42
7W
0.3603
2002
110
99
2003
67
60
2004
75
68
2005
151
123
2006
131
112
2007
186
163
1998
44
2003
21
8W
0.1807
2006
10
1996
15
1997
30
2001
21
8E
0.3676
2003
16
2006
28
2007
13
1995
78
1997
19
2000
16
2001
29
2002
32
9W
0.3702
2003
24
2004
42
2005
19
2006
23
1994
14
1995
22
1997
48
9E
0.4097
2003
14
2004
42
2005
30
*suspected J stock individuals were excluded from the 7W sample.

8P*

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Table 5.Results of the statistical comparison between 9W and 9E by year andtotal samples.
Sample size
9W
9E
P
1994
7
14
0.5425
1995
78
22
0.10311997
19
48
0.8581
2003
25
14
0.06272004
42
420.34682005
1930
0.4563
Total
292*
175*
0.4194*:including 2000, 2001 2002, 2006 and 2007 samples

Table 6. Results of the heterogeneity tests for the baseline C.

Combination of samples
7E x 8W x 8E
9W x 9E

P
0.36610.4194

7W x 7E-8E x 9W-9E
0.1930
7W x 7E-8E x 9W-9E*
0.8904*suspected J stock individuals were excluded from the 7W sample.

Table 7.Results of the heterogeneity tests for the baseline D.
Combination of samples
7E x 8W x 8E x 9W

P
0.2342

7W x 9E
7W x 9E*

0.35680.5616

7W x 7E-9W x 9E
0.1648
7W x 7E-9W x 9E*
0.7805*suspected J stock individuals were excluded from the 7W sample.

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Fig. 1. The 18 sub-areas used for the Implementation Simulation Trials for North Pacific minke whales.

50N

50N

45N

45N

40N

40N

35N

35N

30N
135E

30N
140E

145E

150E

155E

160E

165E

170E

Fig.2. Geographic distribution of sighting position of common minke whale used in this study taken by
JARPN and JARPNII surveys during 1994-2007.

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