SC/J09/JR26
Do not cite without written permission from the authors

Individual identification and mixing of the J and O stocks
around Japanese waters examined by microsatellite analysis
Naohisa Kanda1, Mutsuo Goto1, 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
National Research Institute of Far Seas Fisheries, 2-12-4, Fukuura, Kanazawa-ku, Yokohama, Kanagawa,236-8648, Japan
3
Tokyo University of Marine Science and Technology, 1-5-7, Konan, Minato-ku, Tokyo 108-0075, Japan
2

Contact email:kanda@cetacean.jp

ABSTRACTIn this study, we attempted to distinguish sampled minke whales into genetically distinct stocks using a
combination of microsatellite analysis and a Bayesian clustering approach. Past studies indicated thattwo different stocks of minke whales existed around the Japanese coast: O stock in the western North
Pacific and the J stock in the Sea of Japan. Samples of 2542 minke whales were collected during theoffshore component of JARPN and JARPNII from 1994 to 2007, during the coastal component of
JARPNII from 2002 to 2007, and from bycatches in the set net fishery along the Japanese coast from2001 to 2007, and were analyzed using 16 microsatellite loci. Result of the Bayesian clustering analysis
implemented in the computer program STRUCTURE (Pritchard et al., 2000) indicated that our samples
came from two genetically differentiated groups of minke whales. Approximately 91% of theindividuals were assigned into the either stocks based on their high membership probability (>90%)
obtained from the program. Spatial distribution of these assigned individuals clearly indicated that these
two stocks were the J and O stocks. In addition, it was also found that 1) the O stock individuals
appeared to migrate, although rarely, to the Sea of Japan, 2) the J stock individuals migrated to the 7W of
the North Pacific side and very rarely to further east, and 3) the SA2 (western side of North Pacific coast)
was mainly occupied by the J stock. Temporal distribution of the assigned bycatches collected fromSA7 (eastern side of Japan, North Pacific coast) where both the J stock and O stock whales were
contained in the samples in about 50:50 indicated seasonal movement of the whales with the number of
the O stock increased in spring. This study allowed us to better understand the pattern and dynamics of
distribution of the minke whales inhabiting around Japan.
KEY WORDS: MINKE WHALE, MICROSATELLITE, O STOCK, J STOCK, JARPN, JARPNII,NORTH PACIFICINTRODUCTION
Common Minke whales, Balaenoptera acutorostrata, are the smallest and the most abundant baleen
whales inhabiting major open oceans world-wide as spatial and temporal separations of the minke whale
populations have been occurred (Wada and Numachi, 1991; Bakke et al., 1996; Martinez and Pastene,
1999; Pastene et al., 2007). They live up to 50 years in age and the adult size is, on average, 6-7m.
They feed on various prey species, such as copepods, Euphausiids, and fish. Their age at first
reproduction is five, and they are thought to reproduce every year. Minke whales undergo seasonal
movement from winter breeding grounds in low latitude to summer feeding grounds in high latitude
although their exact breeding ground is usually unknown. They feed in the fertile cold waters insummer near the poles, and spend their winter season in warmer oceans to pair, mate, and give a birth.
Around the ocean off the Japanese coast, at least two different stocks of minke whales are known toexist: one stock distributes in the western North Pacific (O stock) and the other in the Sea of Japan (Jstock) (Omura and Sakiura, 1956; Ohsumi, 1977; Kato, 1992; Wada and Numachi, 1991; Goto andPastene, 1997; Hatanaka and Miyashita, 1997; Pastene et al., 2007). Whales of the both stocks migrate
to the Okhotsk Sea in spring and stay there untill the end of summer. Although they share feeding

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ground in the Okhotsk, their temporal distribution in the area slightly differ (Goto and Pastene, 1997).
These two stocks differ from each other in body size, conception dates, allozyme allele frequencies, andmitochondrial DNA (mtDNA) haplotype frequencies, suggesting their reproductive isolation.
One of the objectives of JARPN II is a systematic monitoring of the occurrence of J stock in
sub-area 7 to determine spatial and temporal dynamics of its occurrence. In a situation of geographicaloverlap of multiple stocks, stock identification at a individual base allows us to directly estimate mixing
rates and pattern of temporal and spatial distribution of the stock. The Scientific Committee (SC) has
recommended that the J stock individuals should be excluded from the analyses of the North Pacificminke whales. However, the past genetic studies failed to detect diagnostic markers between the twostocks. In the past genetic studies, the number of the J stock individuals available had been limited andthe genetic markers used (mtDNA and allozymes) might have not been suitable for stock identification
due to their relatively low resolution power.
The objective of this study was thus to identify stocks of minke whales collected from around Japan
at an individual level and to gain an understanding of spatial and temporal distribution of the stocks. We
attempted to distinguish samples of minke whales obtained around Japan into the J and O stocks
irrespective their sampling sites by utilizing genetic variation at hypervariable microsatellites with
Bayesian clustering approach. A model based Bayesian approach has been recently developed to defineclusters of individuals based on their genotypes at multiple loci (Pritchard et al., 2000). This approachallows us to treat each individual in the samples as a single operational taxonomic unit in a stock-level
analysis, and Bayesian approach provides relative probability to estimate the number of stocks rather thanreject one of the two hypotheses. An advantage of Bayesian method implemented in the Structure(Pritchard et al., 2000) used in this study is thus that the obtained results are not affected by arbitrary
chosen sample strata that do not necessarily correspond to real biological stock boundaries.
MATERIALS AND METHODS
Sample collections
Eighteen sub-areas were set for management purpose of the western North Pacific common minke whale(Fig.1). JARPN and JARPNII surveys were conducted in Sub-areas 7, 8, 9, and 11. These sub-areas
were further divided into western and eastern strata for analyses, 7W (140-147°E), 7E (147-150°E), 8W(150-153°E), 8E (153-157°E), 9W (157-162°E), and 9E (162-170°E).
Offshore samples of minke whales from the western North Pacific were JARPN and JARPNII
samples collected from 1994 to 2007at SA7, SA8, SA9, and SA11 (Table 1). Each year up to 100 minke
whales were collected. The number of individuals from the SA11 was total 80 collected in 1995 and1997. Sampling dates of scientific surveys expand from May to September depending on the sampling
plan of a given year. Because of other scientific purposes of the survey (e.g., feeding ecology of minke
whales), the sampling locations differed from year by year. Details of offshore component of JARPNII
survey can be found in Tamura et al. (2009). Minke whales obtained from coastal component of
JARPNII were also used in this study (Table 1). Coastal JARPNII surveys were conducted at Sanriku
region in spring of 2003, 2004, 2005, 2006, and 2007, and at Kushiro region in fall of 2002, 2004, 2005,
2006, and 2007. Sample size per survey was maximum 60 minke whales. Details of the coastal
component of JARPNII survey can be found in Kishiro et al. (2009). Minke whales that were bycaught
on set net fishery conducted along the Japanese coast from 2001 to 2007 were also used (bycatches)(Table 1). As of July 1st 2001, the new regulation governed by the Japanese government has allowed
the set net fishermen to harvest whales found in their set net and to sell these on to the market after DNA
registration of these for individual identification. The bycathes used were obtained from the SA2, SA6,
SA7, SA10, and SA11 year-round.
Microsatellites analysis
Genomic DNA was extracted from 0.05g each of the skin or muscle tissues using standard proteinase K,
phenol-chloroform procedure described by Sambrook et al. (1989). Extracted DNA was stored in the TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Microsatellite polymorphisms were analyzed using 16 sets of primers: EV1, EV14, EV21, EV37,
EV94, (Valsecchi & Amos, 1996), GT23, GT195, GT211, GT310, GT509, GT575 (Bérubé et al., 2000),
GATA28, GATA98, GATA417, TAA31 (Palsbøll et al., 1997), DlrFCB14 (Buchanan et al., 1996). EV1,
EV14, EV21 were developed from sperm whale (Physeter macrocephalus), EV37, EV94, GT23, GT310,
GT575, GATA28, GATA98, GATA417, TAA31 were from humpback whale (Megaptera novaeanglia),
and DlrFCB14 from beluga whale (Delphinapterus leucas). All GT, EV, and DlrFCB primers are
dinucleotide repeat, TAA31 trinucleotide repeat, and all GATA primers tetranucleotide repeat. Most of2

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the primers used here were already tested for amplification on minke whales by these authors. Primer
sequences and PCR profiles follows those of the original authors with slight modifications.
PCR amplifications were performed in 15µl reaction mixtures containing 10-100ng of DNA, 5pmole of each primer, 0.625 units of Ex Taq DNA polymerase (Takara Shuzo), and 2mM of each dNTP,
and 10x reaction buffer containing 20mM MgCl2 (Takara Shuzo). PCR amplifications followed the
manufacture’s instructions for the use of Ex Taq DNA polymerase (Takara Shuzo). Amplified productswith internal size standard (GENESCAN400HD, Applied Biosystems Japan) were run on a 6%
polyacrylamide denaturating gel (Long RangerTM) using an BaseStationTM 100 DNA fragment analyzer(Bio-Rad). Although alleles were visualized using CartographerTM software specifically designed for
the BaseStation, allelic sizes were determined manually in relation to the internal size standard and minke
whale DNA of known size that were rerun on each gel.
Data analysis
The number of alleles per locus and expected heterozygosity per locus were calculated using the FSTAT
2.9.3 (Goudet, 1995). Statistical tests for the deviations from expected Hardy-Weinberg genotypic
proportions were conducted using the GENEPOP 4.0 (Rousset, 2008). When simultaneous multiple
tests were conducted, Rice (1989) correction for the multiple tests was performed.
The Bayesian clustering approach was implemented with the microsatellite data in the
STRUCTURE version 2.0 (Pritchard et al., 2000) to determine the most likely number of geneticallydistinct stocks present in our samples. The program is a model-based clustering method for inferring
stock structure (K, the number of stocks in the model) using multilocus genotype data with and without
information on sampling locations. STRUCTURE allowed us to analyze the samples without choosing
sample units that did not necessarily correspond to real biological stock boundaries. Posterior
probabilities for K were estimating from three independent runs for each value of K from one to five with
only genetic information. These data were calculated based on burn-in period of 10,000 iterations andruns of 100,000 iterations. Individual assignment was then conducted for the most plausible K using
estimated individual proportion of membership probability. The ancestry model we used for thesimulation was the admixture model, which assumes individuals may have mixed ancestry. The allele
frequency model used was the correlated allele frequencies model, which assumes frequencies in the
different stocks are likely to be similar due to migration or shared ancestry.
RESULTS
Multilocus genetic variations for stock numbers and individual assignment
All 16 loci analyzed were polymorphic in overall samples of minke whales we used (Table 2). Total
number of alleles per locus ranged from two at the EV21 to 30 at the EV1 with an average of 13.0.
Expected heterozygosity at each of the loci ranged from 0.330 at EV21 to 0.855 at GT23 with an averageof 0.692. Eleven out of 16 loci showed significant deviation from the expected Hardy-Weinberg
genotypic proportions even after correction for the multiple tests. This deviation was strong indicationof existence of individuals from multiple stocks.
Bayesian clustering analyses conducted on the total samples (2542 individuals) without informationon their geographic origins presented the highest likelihood probability at K=2 (Table 3), and thus it wasstrongly indicated that our samples came from two genetically distinct groups of minke whales. We thenassigned each individual into three categories on the basis of the membership probability at arbitrary
chosen level. The individuals with the membership probability of over 90% for either of the two groups
were assigned as pure individual from either stock 1 or stock 2. All other individuals with themembership probability less than 90% to the either groups were assigned as individuals of unknown
origin. Even with this relatively strict criterion, 2302 individuals (91%) were assigned as the pureindividuals to the either stocks (770 to stock 1 and 1532 to stock 2). For the purpose of this study, weused only these pure individuals in the subsequent analyses.
Spatial distribution of the two genetically different stocks along the Japanese coast
The pure individuals were grouped based on their sampling origins (offshore, coastal, and bycatch) andlocations (IWC sub-areas) (Fig. 2). In this way, distribution of the pure individuals that were geneticallyassigned to the different stock was clearly separated geographically. Almost all of the individuals
collected from the Sea of Japan side belonged to the Stock 1, whereas almost all of individuals from theoffshore North Pacific (east of SA7E) belonged to the stock 2. Intermediate areas (SA7W and SA11)
contained individuals from the both stocks. Fig. 2 indicated that the Stock 1 and Stock 2 were the J andO stock. Hereafter, we named the Stock 1 and 2 as the J and O stock, respectively. SA2 was mainly

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occupied by the J stock, and SA7W was by the two stocks. Two individuals collected from the 9W(N=260) were assigned as the J sock individual, while four individuals from the SA6 (N=387) was
assigned as the O stock individual.
Temporal distribution of the two genetically different stocks along the Japanese coast of North
Pacific side of Japan
The proportion of the J stock and O stock individuals in the bycatches from the SA7 were approximately
50:50 (93 J and 90 O). In addition to that, the sampling dates of the bycatches expand year around.
These things allowed us to depict temporal distribution of the J and O stocks along the Japanese coast of
the North Pacific side. Because the sample sizes were relatively small in a single month base, we
calculated the proportion of the J and O stock from a three months moving average (Fig. 3). The fig. 3
revealed that the proportion of the O stock increased in spring and decreased toward winter season
although both the J and O stock individuals stayed year round. At the SA2, total of 168 individuals was
assigned to 114 J and 24 O individuals, and temporal distribution of these individuals showed similar, butless clear, pattern to that observed at the 7W.
DISCUSSION
Delineation of the samples into geographic groups for analyses is often arbitrary based on cultural,political jurisdictions, or simply sampling locations. The arbitrary predefined groups, however, may notnecessarily correspond to real biological stock boundaries. This is especially the case for thegeographically widely distributed species that are capable of long distance migration. They may nothave clear stock boundaries or may be genetically structured through unidentified barriers of gene flow.
In this study, combined use of the hypervariable microsatellite markers and Bayesian clustering
approach successfully distinguished most of the individuals in the samples into the J and O stocks. One
notable aspect of this study was that the number of samples used, especially from the Sea of Japan side,
was much larger than the previous genetic studies. Past genetic studies dealing with the J and O stocks
used only small amount of the samples taken from past commercial whaling operated in coastal areas of
Korea in 1982 as the J sock (Wada and Numachi, 1991; Goto and Pastene, 1997). In addition to that,
this paper used the samples of minke whales collected from along the Japanese coast of both sides ofJapan and offshore North Pacific at a similar time frame. This allowed us to minimize potential large
temporal influence, if it exists, on describing present day stock structure, although no samples had beenobtained in winter in the western North Pacific due to logistic constraints.
According to the probability for each of K, our data was not supported at all by K=1, 3, 4, and 5.
Very high membership probabilities and great geographic concordance indicated that K=2 wasappropriate choice for our data. When we lower our membership probability criteria from 90% to 75%,
97% of the individuals in the samples are assigned to either the two stocks and the additional assigned
individuals does not change the general picture of spatial distribution of the two stocks. Status of theunassigned individuals especially those with the membership probability of less than 75% (82individuals) are difficult to be identified at this moment. They could be either individuals ofinterbreeding origins or pure individuals simply with low power to be assigned by the current markers
and analyses. The clear genetic difference between the two stocks and the observation from the past
ecological and biological studies of minke whales suggests that the interbreeding between them isprobably a rare event.
The individual assignments by the microsatellites had good agreements with that by mtDNA
haplotype analysis. It has been known that some of the mtDNA haplotypes were unshared by theindividuals from the Sea of Japan and North Pacific and these unshared haplotypes with a few shared
ones made an independent cluster in a phylogenetic tree (Goto et al., 2000; see also Baker et al., 2000).
Based on the specific nucleotide sequences common to the haplotypes in that cluster, Goto et al. (2000)
identified 25 of the 80 minke whales from the SA11 as the J stock individuals. Our microsatellite
assignments of the same SA 11 individuals were very well consistent to those in Goto et al. (2000). By
our method, 23 of the 80 were assigned as the pure J stock, and 22 of them were identified as the J stockby the method of Goto et al. (2000). In the remaining three J stock individuals from the mtDNA
identification, two were the J stock individuals with the membership probability over 75%, and one was
the O stock individual with the membership probability over 90%. Although the microsatellite markers
we used are not complete diagnostic in the minke whales samples, all of these findings suggest thatassignments of the individuals is very reliable.
Spatial and temporal distributions of the assigned individuals depicted a pattern and dynamics ofgeographic overlap of the two stocks around Japan. As predicted, main distribution areas of the J and O

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stock were clearly separated. The J stock occupied the Sea of Japan and the O stock offshore did theNorth Pacific. Other important findings that should be raised are 1) the O stock individuals appeared tooccasionally, but rarely, migrate to the Sea of Japan, 2) the J stock individuals migrated to the 7W of theNorth Pacific side and very rarely to further east, and 3) the SA2 was mainly occupied by the J stock.
The third finding suggests that the Kuroshio Current, which is one of the strongest west-boundary
currents of the subtropical gyre, is working as the stock boundary between the two stocks. Temporal
distribution of the assigned whales indicated seasonal differences in the movement of the two stocks
along the Japanese coast of North Pacific (SA7 and SA2). The number of the O stocks increased inspring although both stocks appeared to distribute there year around.
It is important to note that the individuals from the JARPN/JARPNII and those from the bycatchsamples differ in their body length. Average body length of the all JARPN and JARPNII samples
including both the offshore and coastal components was 6.95 m (s.d.=0.992) and that of the all bycatchsample was 4.92 m (s.d.=0.942). Kato (1992) estimated mean body length at the sexual maturity of theNorth Pacific minke whales to be 6.3 m for males and 7.1 m for females, so that the bycatch sample inthis paper consisted of mostly, if not all, immature whales. The observation that the number of theimmature O stock individuals increased in spring along the Japanese coast of the North Pacific side wasthus well consistent to that illustrated by Hatanaka and Miyashita (1997). The observed difference in thematurity status between the individuals from the bycatch and JARPN/JARPNII samples, however, could
indicate that the patterns of the temporal and spatial distributions we illustrated with the bycatches for theSA2 and SA7 may be different at some extent from those of adults. In regard to the SA2, minke whales
from the offshore area have not been available yet. Related concern can be also seen in the SA11. The
number of the J stock individuals in the SA11 differed between the bycatch and JARPN samples (Fig. 2).
Average body length of the 14 bycatches from the SA11 was 4.5 m and 13 of the 14 were collected fromlate fall (September to November). This difference we observed between the bycatch and JARPN
samples could be due to the immature/mature, temporal, or both factors, but we were not able todistinguish which one accounted for at this moment. Although we definitely gained our understandingof minke whales’ distribution around the Japanese water substantially from this study, our samples arestill missing some pieces to depict whole picture of it.
The IWC Scientific Committee (SC) completed the RMP Implementation for the western North
Pacific common minke whales during the 2003 Annual Meeting and adopted four stock scenarios in thewestern North Pacific at the final stage of the Implementation process (IWC, 2004). Three of the fourbaselines assumed the third stock other than the J and O in the SA7, 8, and 9. This study denied thatpossibility because it showed that the North Pacific side of Japan was occupied by the O stock with someJ stock individuals migrated into near coastal line area. Detailed analyses on this matter can be seen in
Kanda et al. (2009).
This study is the first one that shed the light on the dynamics of geographic overlap between the twostocks at the individual base. We believe that the results of this study are also quite useful for theeffective management of the two stocks. Another usefulness of the individual identification by thegenetic markers is it can be used to look for stock differences in other traits, such as morphometry,pollutant levels, and biological parameters.
ACKNOWLEDGEMENTS
We thank researchers and crewmembers participating in the JARPN and JARPNII surveys for their effort
in collecting the samples used in this study. Our sincere gratitude also goes to H. Oikawa and S. Azumi
for their assistance in DNA extraction, and H. Hatanaka for valuable comments on the manuscript.
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Kanda, N., Goto, M., Kato, H., McPhee, M.V., and Pastene, L.A. 2007. Population genetic structureof Bryde’s whales (Balaenoptera brydei) at the inter-oceanic and trans-equatorial levels. Cons.
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Kanda, N., Goto, M., Kishiro, T., Yoshida, H., Kato, H., and Pastene, L.A. 2009. Microsatellite
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Table 1. Number of individuals used in this study.
Survey area
Total
JARPN and JARPNII
Offshore (‘94-‘07)
Coastal (‘02-‘07)
Bycatch (‘01-‘07)

1231
480
831

2

183

6

7W

7E

8W

8E

9W

9E

47

86

139

291

174

411

414
480
212

A

He

HW

DlrFCB14
EV1
EV14
EV21
EV37
EV94
GATA28GATA417
GATA98
GT195
GT211
GT23
GT310
GT509
GT575
TAA31

5
30
6
2
12
8
24
13
7
13
17
16
14
23
13
5

0.405
0.785
0.543
0.330
0.709
0.638
0.830
0.737
0.610
0.857
0.874
0.885
0.833
0.881
0.809
0.349

p<0.01p<0.01p<0.001
n.s.
n.s.
n.s.
p<0.001n.s.
n.s.
p<0.001p<0.001p<0.001p<0.001p<0.001p<0.001p<0.001

Table 3. Results of Bayesian clustering method analyzed foroverall samples.
K

Log P(k/x)*

variance*Pr(k/x)**

1
2
3
4
5

-128869.8-123973.8-124376.2-125643.1-127277.0

95.3
613.1
2243.85312.39061.4

~0.0
~1.0
~0.0
~0.0
~0.0

* log likelihood of the data for different values of K and the variance.
** Probability for each of K.

7

11

80
9

Table 2. The number of alleles (A), expected heterozygosity(He), and test result for the expected Hardy-Weinberg genotypic
proportions (HW) at 16 microsatellite loci analyzed in the
JARPNII samples of minke whales. n.s. = not significant
Marker10

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Fig 1.

The 18 sub-areas designated by the IWC for North Pacific minke whales.

Stock 1

Stock 2

100%

80%

60%

40%

20%

0%
BC6

BC10

BC2

BC11

11

BC7

CK7

CS7

7W

7E

8W

8E

9W

9E

Fig. 2. Spatial distribution of the whales that were genetically assigned to the Stock 1 and Stock 2. BC2,
BC6, BC7, BC10, BC11 included the bycatches collected from the SA2, SA6, SA7, SA10, and SA11.
CK7 and CS7 included the individuals collected from coastal survey of JARPNII at Kushiro and Sanriku
region, respectively. 7W, 7E, 8W, 8E, 9W, 9E and 11 included the individuals collected from offshore
survey of JARPN and JARPNII.

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SC/J09/JR26
Do not cite without written permission from the authors

J stockO stock

100%

80%

60%

40%

20%

0%
Jan.Mar.

Feb.Apr.

MAr.May

Apr.Jun.

MayJul.

Jun.Aug.

Jul.Sep.

Aug.Oct.

Sep.Nov.

Oct.Dec.

Nov.Jan

Dec.Feb.

Fig 3. Temporal distribution of the bycatches that were genetically assigned to the J and O stocks in theSA7. Each bar was expressed as three months moving average.

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