Not to be cited without prior consent with the authors

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Preliminary microsatellite analysis of bycaught J-stock minke
whales from Japan and Korea

Naohisa Kanda1, Jung Youn Park2, Hawsun Sohn3, Zang Geun Kim4, Mutsuo Goto1, and Luis A. Pastene1
1

The Institute of Cetacean Research, 4-5, Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
Biotechnology Research Center, National Fisheries Research and Development Institute, 408-1, Sirang-ri,
Gijang-eup, Gijang-gun, Busan, 619-902, Korea
3
Research Planning Team, National Fisheries Research and Development Institute, 408-1, Sirang-ri, Gijang-eup,
Gijang-gun, Busan, 619-902, Korea
4
Cetacean Research Institute, National Fisheries Research and Development Institute, 139-29, Maeam-dong,
Nam-gu, Ulsan, 680-050, Korea
2

Key words: minke whales, bycatch, Japan, Korea, genetic difference, microsatellites

ABSTRACTWe analyzed samples of minke whales from Japan (Sub-area 6 (SA6)) and Korea (SA5 and SA6) using
nine microsatellite loci in order to describe their genetic population structure. The samples were bycaught
in set net fisheries conducted along the Japanese coast in SA6 (N = 202) from 2001 to 2004 and in coastal
fishing gears along the Korean peninsula (N = 278) in SA6 and SA5 from 1999 to 2004. The genotypesof the individuals were first standardized using reference samples in order to avoid scoring differences
between the two laboratories. We then examined if there was any evidence of genetic differences among
the samples collected on different years within Japanese and Korean samples, respectively, among thesamples from different areas of Korea, and finally between the samples from Japan and Korea. No
evidence of statistically significant temporal heterogeneity was detected in the Japanese samples (JBC).
However, the Korean sample collected from SA6 in 1999 (99KBC-6) was different from both the JBC andthe rest of the Korean samples (KBC), although we were not able to completely reject the possibility of achance effect for this heterogeneity. Finally, no evidence of statistically significant heterogeneity was
detected within the KBC as well as between the JBC and KBC. A Bayesian clustering method did notshow evidence of multiple stocks in our samples. Although we observed the heterogeneity of the
99KBC-6, no strong evidence of the existence of an additional stock in SA5 and SA6 was indicated.

INTRODUCTION
It has been believed that only one population of minke whales exists in the sub-areas (SA) 5 and 6 between
Japan and Korea (Omura and Sakiura, 1956; Ohsumi, 1977; Kato, 1992; Wada and Numachi, 1991; Goto andPastene, 1997). This is J-stock that is genetically different from widely distributed O-stock in western North
Pacific. The J- and O-stocks differ from each other in body size, conception dates, allozyme allele frequencies,
and mitochondrial DNA (mtDNA) haplotype frequencies, suggesting their reproductive isolation. Althoughboth stocks migrate to the Okhotsk Sea in spring and stay till the end of summer (Omura and Sakiura, 1956;
Hatanaka and Miyashita, 1997), their temporal distribution in the area appears not to overlap completely (Omura
and Sakiura, 1956; Goto and Pastene, 1997).
One of the tasks accomplished prior to the in-depth assessment of the J-stock minke whales recommended by theCommittee during the 2004 meeting was to analyze genetic data to describe population structure of the J-stock.
We therefore intersessionally worked together to analyze the samples from Japan and Korea using mtDNA andmicrosatellites. The objective of this study was to gain an understanding of the level of genetic variability andgenetic population structure of the J-stock minke whales from Japanese and Korean waters throughmicrosatellite data. We used recently and geographically widely collected samples of the minke whales from
SA5 and SA6 along Japanese and Korean coast, which differed from the previous genetic studies that used only
those collected from commercial whaling in a narrow area in 1982.

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MATERIALS AND METHODS
Samples
Minke whales used in this paper were bycaught individuals on set net fisheries conducted along the Japanese
coast in SA6 from 2001 to 2004 and in coastal fishing gears along the Korean peninsula in SA6 and SA5 from1999 to 2004 (Table 1 and Fig.1). The 2001 Japanese sample contained the bycaught individuals only from
July to December, whereas all the other samples included the bycaught individuals from all seasons. Koreansamples were divided into three on the basis of where they came from: eastern side of the Korean Peninsula(SA6), southern side of the Korean Peninsula (SA5 and SA6), and western side of the Korean Peninsula (SA5).
DNA extraction
Genomic DNA was extracted from muscle or skin tissues, whichever was available, using the standard
proteinase K, phenol-chloroform procedure (Sambrook et al., 1989) and then stored in the TE buffer.
Microsatellite analysis
Microsatellite polymorphisms were analyzed using nine sets of primers: EV1, EV14, EV21, EV94 (Valsecchi &Amos, 1996), GT195, GT211 (Bérubé et al., 2000), GATA98, TAA031 (Palsbøll et al., 1997), DlrFCB14(Buchanan et al., 1996). All EV, GT, and DlrFCB primers are dinucleotide repeat, TAA31 is trinucleotide
repeat, and GATA primers are tetranucleotide repeat. Primer sequences and PCR profiles follows those of theoriginal authors with slight modifications. Because Korean samples were analyzed at the National Fisheries
Research and Development Institute, Busan, and the Japanese ones at the Institute of Cetacean research, Tokyo,
we first standardized the microsatellite allele data between the two countries in order to avoid laboratory
differences in reading allele sizes generated from different fragment analyzers. This was done analyzingreference samples of known size.
Molecular genetic sexing (Abe et al., 2001) was used to determine sexes of each individual in the samples
because it was required due to lack of professional knowledge of local people. Coamplification of SRY locus
located on Y chromosome and a single microsatellite locus allowed us to determine sex as males show PCR
bands of both SRY and microsatellite loci while females only showed the microsatellite locus.
Data analysis
Genetic diversity
Calculation of allele frequencies and tests for the deviations from expected Hardy-Weinberg genotypic
proportions were conducted using GENEPOP 3.2 (Raymond and Rousset, 1995). Expected heterozygosity per
locus, Weir and Cockerham estimator of Fst, and allelic richness were calculated using FSTAT 2.9.3 (Goudet,
1995). Decision of statistical significance on the H-W tests was made using the chi-square value obtained fromsumming the negative logarithm of p values over the nine microsatellite loci (Sokal and Rohlf, 1995). Whensimultaneous multiple tests were conducted, Rice (1989) correction for the multiple tests was performed.
Genetic divergenceThree procedures were performed to illuminate population structure of minke whales in SA5 and SA6.
First, conventional hypothesis testing procedure was conducted using heterogeneity test in allele frequencies
among the samples. Our null hypothesis to be tested is if the samples came from a genetically same group ofminke whales. If a statistically significant allele frequencies differences exist, then it could indicate thesesamples came from genetically different populations of minke whales. We examined if there was any evidenceof genetic differences among the samples collected in different years within Japanese and Korean samples,
respectively, among the samples from different areas of Korea, and finally between the samples from Japan and
Korea. Probability test (or Fisher’s exact test) implemented in GENEPOP was used to conduct theheterogeneity tests. Decision of statistical significance on the heterogeneity tests was made using thechi-square value obtained from summing the negative logarithm of p values over the nine microsatellite loci
(Sokal and Rohlf, 1995). When pair-wise comparisons were conducted, the observed p-values from theheterogeneity tests were compared to the modified level of significance proposed by Rice (1989).
Second, genetic relationship among the samples was examined using unrooted neighbor-joining tree
implemented in PHYLIP (Felsenstein, 1993). Cavalli-Sforza's cord measures (Cavalli-Sforza and Edwards,
1967) were used as the genetic distances between all pairs of samples. The genetic distance matrix was thenused to create an unrooted neighbor-joining tree, and the obtained tree was visualized using TreeView PPC (Page,
1996). We chose this genetic distance measure because it needs no assumption of mutation process.
Statistical inference was made based on 1000 replicates of bootstrap analysis

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Third, a Bayesian approach was conducted using STRUCTURE (Pritchard et al., 2000). STRUCTURE is amodel-based clustering method for inferring population structure using multi-locus genotype data. Spatialdifferentiation of samples was assessed assuming that the sampled individuals belong to an unknown number ofK populations. We conducted three independent runs for each value of K between 1 and 4 with no priorinformation (i.e., only genetic information was considered). All of the simulations were based on a burn-in
period of 50,000 iterations and runs of 500,000 iterations. The ancestry model we used for the simulation 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 populations are likely to
be similar due to migration or shared ancestry.
RESULTS
Genetics diversity within samples
Genetic diversity indices for overall samples used in this study are shown in Table 2 and for each of the samples
in Appendix 1. Averaged number of alleles per locus was 6.7 and heterozygosity per locus was 0.531 with Fstof 0.007, indicating the whales analyzed had high genetic diversity. Within each of the samples, the nine
microsatellite loci analyzed were polymorphic except for one case (DlrFCB14 in the 00KBC-6). Average
number of alleles per locus per sample ranged from 3.3 to 5.6 with an average of 4.7, but allelic richness index
indicated that the differences in the number of alleles among the samples was mostly due to sample size effect.
Average heterozygosity per locus per population was 0.531. Only one (DlrFCB14 in the 04JBC) out of the 108
cases showed evidence of deviation from the expected Hardy-Weinberg genotypic proportions after thecorrection for multiple tests.
Genetics divergence between samples
We first looked for any evidence of allele frequency differences among the samples collected from the same
coast lines in different years. Among the six KBC-6s obtained from 1999 to 2004, statistically significant
heterogeneity was obtained (chi-square = 55.976, d.f. = 18, p = 0.00001). Pair-wise comparisons indicatedevidence of statistically significant heterogeneity between the 99KBC-6 and 03KBC-6 after the correction for
multiple tests. Although insignificant, p values obtained from the comparisons involving the 99KBC-6 were alllow. We, therefore, divided the KBC-6 into two groups, 99KBC-6 and 00-04KBC-6, for the next analysis. In
contrast, no evidence of statistically significant heterogeneity in allele frequencies was detected among the fourJBCs obtained from 2001 to 2004 (chi-square = 20.704, d.f. = 18, p = 0.295). We thus treated the four JBCs as
a single sample.
Next, we looked for any evidence of statistically significant differences within SA5 and 6 of the Korean waters.
In pair-wise comparisons, evidence of heterogeneity was detected only within the Korean SA6 that was alreadynoted above. We therefore divided all Korean samples into two groups for the next analysis: 99KBC-6 andKBC (00-04KBC-6 + KBC-5/6 + KBC-5).
Finally, we looked for any evidence of statistically significant differences between Japanese and Korean samples.
The JBC showed statistically significant heterogeneity only to the 99KBC-6 (chi-square = 42.010, d.f. = 18, p =0.0011).
Phylogenetic relationship
Genetic relationship among the 12 samples was described in an unrooted neighbor-joining tree obtained from thefrequencies of microsatellite alleles (Fig. 2). No geographically unique cluster was obtained, and almost all ofbootstrap values were very low.
Bayesian clustering of samples
In order to estimate the number of populations extant in the SA5 and SA6, the bayesian clustering analyses
without information on their sampling locations were conducted on the total data set. The analysis presented
the highest likelihood probability at K = 1 (Table 3), indicating no signal of existence of multiple stocks in the
study area.
DISCUSSION
Significance of this study was that we used recently and geographically widely collected samples of the minke
whales from SA5 and SA6. The levels of allelic diversity and heterozygosity of the J-stock analyzed in thispaper were much higher than predicted from most of the previous genetic studies of the same stock. In addition,3

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the genetic diversity levels of the J-stock were only slightly less than those of the O-stock minke whales (Kanda
et al., unpublished data) as well as other whales species in the North Pacific, such as Bryde’s whales (Pastene etal., 2004) and sei whales (Kanda et al., 2006). This suggests that the effective population size of the J-stock is
probably larger than we have thought based on the previous genetic studies.
Before conducting stock structure analyses, we raised three stock structure scenarios for our samples; 1) only asingle stock in the SA5 and SA6, 2) two stocks, one along the Japanese coast and the other along the Korean
coast, and 3) two stocks, one in SA6 and the other in SA5.
We found slight genetic differences within Korean SA6 that was mainly due to the heterogeneity of the
99KBC-6. Two possibilities were raised for the detected heterogeneity of the 99KBC-6. First, this could
indicate that there are genetically different stocks in this area and that somehow we only captured the evidence inthe 99KBC-6. Second, this could have resulted from the relatively small sample size of the 99KBC-6,
suggesting it was not a good representative of the whole J-stock on the basis of their high genetic diversity.
In contrast, no evidence of genetic difference was observed among the four JBCs collected in different years aswell as between the JBC and KBC (not include the 99KBC-SA6), indicating these samples came from agenetically same stock of minke whales. The result of STRUCTURE also supported the single stock scenario,
and no geographically related cluster was observed in the phylogenetic tree. No strong indication of theexistence of an additional stock in the SA5 and SA6 was therefore observed.
At this point we favor the single stock interpretation. Continued genetic monitoring of bycaught minke whales,
however, is required to confirm which one of the above two possibilities is true for the heterogeneity weobserved. In addition, it is important to note that this study is preliminary, and care is required in fullyinterpreting the data. For instance, the standardization of microsatellite allele data was more difficult than we
had anticipated. In future, therefore, we expect that all the samples would be analyzed altogether at one time ineither Japan, Korea, or both countries.
ACKNOWLEDGEMENTS
We thank all those in both Japan and Korea who were involved in sample collections and those who helped withlaboratory work.
LITERATURE CITED
Abe, H., Goto, M., Pastene, L.A., Dewa, K., and Naito, E. 2001. Practical use of multiplex fluorescent PCR
for cetacean sex identification. Mar. Mammal Sci. 17:657-664.
Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøl, P.J. 2000. Polymorphic di-nucleotide microsatellite
loci isolated from the humpback whale, Megaptera novaeanglliae. Mol. Ecol. 9:2181-2183.
Buchanan, F.C., Friesen, M.K., Littlejohn, R.P., and Clayton, J.A. 1996. Microsatellites from beluga whale
Delphinapterus leucas. Mol. Ecol. 5:571-575.
Cavalli-Sforza, L.L. and Edwards, A.W. 1967. Phylogenetic analysis: models and estimation procedures.
Amer. J. Human Genet. 19:233-257.
Felsenstein, J. 1991. PHYLIP (Phylogenetic inference package) version 3.5. Department of Genetics,
SK-50, University of Washington, Seattle, 98195, USA.
Goto, M. and Pastene, L.A. 1997. Population structure of the western North Pacific minke whale based on an
RFLP analysis of the mtDNA control region. Rep. int. Whal. Comm. 47:531-537.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered. 86:485-486.
Kanda, N, Goto, M, and Pastene, L.A. 2006. Genetic characteristics of western North Pacific sei whales,
Balaenoptera borealis, as revealed by microsatellites. Marine Biotech. 8:86-93. First published on
November 23, 2005, 10.1007/s10126-005-5130-1.
Kato, H. 1992. Body length, reproduction and stock separation of minke whales off northern Japan. Rep.
int. Whal. Comm. 42:443-453.
Ohsumi, S. 1977. Minke whales in the coastal waters of Japan. Rep. int. Whal. Comm. 27:164-166.
Omura, H. and Sakiura, H. 1956. Studies on the little piked whale from the coast of Japan. Sci. Rep. Whales
Res. Inst. Tokyo 11:1-37.
Page, R.D.M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers.
Comput. Appli. Biosci. 12:357-358.
Palsbøll, P.J., Bérubé, M., Larsen, A.H., and Jørgensen, H. 1997. Primers for the amplication of tri- andtetramer microsatellite loci in baleen whales. Mol. Ecol. 6:893-895.

4

Not to be cited without prior consent with the authors

SC/58/NPM2

Pastene, L.A., Goto, M., and Kanda, N. 2004. An update of the mitochondrial DNA and microsatellite
analyses in western North Pacific Bryde’s whale. Paper SC/56/PFI4 presented to the IWC ScientificCommittee, June 2004, Sorrento, Italy (unpublished).
Pritchard, J.K., Stephens, M., and Donnelly, P. 2000. Inference of population structure using multilocus
genotype data. Genetics 155, 945-959.
Raymond, M. and Rousset, F. 1995. GENEPOP (version 1.2): population genetics software for exact tests
and ecumenicism. J. Hered. 83:248-249.
Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution 43:223-225.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold
Spring Harbor Laboratory, New York, USA.
Sokal, R.R. and Rohlf, F.J. 1995. Biometry: the Principles of Statistics in Biological Research. Freeman
and Company, New York.
Valsecchi, E. and Amos, W. 1996. Microsatellite markers for the study of cetacean populations. Mol. Ecol.
5:151-156.
Wada, S. and Numachi, K. 1991. Allozyme analyses of genetic differentiation among the populations andspecies of the Balaenoptera. Rep. int. Whal. Comm. Special issue 13:125-154.

Table 1. Bycaught minke whales collected from sub-areas (SA) 5 and 6 in Japan and Korea used in this study.
Locality

Sub-area

Sample

Year

N

Korea

SA6

99KBC-600KBC-6
01KBC-6
02KBC-6
03KBC-6
04KBC-6

1999
2000
2001
2002
2003
2004

20
7
15
44
68
40

Korea

SA5 & 6

KBC-5/6

1999-2004

28

Korea

SA5

KBC-5

1999-2004

26

Japan

SA6

01JBC
02JBC
03JBC
04JBC

2001
2002
2003
2004

26
49
61
66

Table 2. Genetic indices at the 10 microsatellite loci for overall samples.

DlrFCB14
EV1
EV14
EV21
EV94
GATA98
GT195
GT211
TAA31
Average

A

He

Fst

3
17
4
2
4
7
10
10
3
6.7

0.359
0.668
0.407
0.372
0.572
0.609
0.799
0.761
0.236
0.531

0.007(0.002, 0.013)

5

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Table 3. Estimated posterior probability of number of populations (K) for the pooled samples computed using
STRUCTURE.
K

Log P (x/k)

Probability

1
2
3
4

-8204.5-8534.4-8948.8-9898.6

~1.0
~0.0
~0.0
~0.0

Fig 1. Approximate distribution of sampling locations in sub-area 5 and 6.

01KBC-601JBC
02KBC-603JBC
03KBC-6

100

04KBC-6

02JBC

04JBC

50.9

KBC-5/6KBC-500KBC-6

99KBC-6

Fig 2. Un-rooted neighbor-joining tree of the 12 samples based on Cavalli-Sforza's cord measures.
Bootstrapped values of over 50% were indicated.

6

0.01

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Appendix 1. Genetic diversity indices in each of the 12 J-stock minke whales samples analyzed using nine microsatellite loci.
99KBC-600KBC-6

A

AR

He

HW

A

DlrFCB14 2
EV1
9
EV14
3
EV21
2
EV94
3
GATA98
5
GT195
5
GT211
6
TAA31
2
Average 4.1

1.5
4.9
2.7
1.8
2.6
3.6
4.0
4.0
1.7
3.0

0.108
0.776
0.494
0.224
0.544
0.674
0.722
0.633
0.193
0.485

NS
NS
NS
NS
NS
NS
NS
NS
NS

1 1.0
6 4.8
3 2.8
2 2.0
3 3.0
4 3.8
5 5.0
4 4.0
2 2.0
3.3 3.2

KBC-5/6

AR

01KBC-6

He

HW

A

0.000
0.667
0.550
0.548
0.600
0.679
0.800
0.650
0.357
0.539

NS
NS
NS
NS
NS
NS
NS
NS
NS

2 1.9
7 4.3
3 2.5
2 2.0
4 2.9
4 3.4
8 5.4
6 4.5
2 1.8
4.2 3.2

KBC-5

A

AR

He

HW

A

AR

DlrFCB14 3
EV1
10
EV14
3
EV21
2
EV94
4
GATA98
4
GT195
8
GT211
8
TAA31
2
Average 4.9

2.1
4.3
2.6
1.9
2.8
3.5
4.7
4.9
1.7
3.2

0.350
0.655
0.441
0.292
0.582
0.659
0.793
0.768
0.194
0.526

NS
NS
NS
NS
NS
NS
NS
NS
NS

2 2.0
9 3.7
3 2.4
2 2.0
4 3.0
5 3.6
8 5.1
9 5.4
2 1.5
4.9 3.2

AR

02KBC-6
A

He

HW

A

AR

0.417
0.522
0.340
0.472
0.569
0.666
0.824
0.798
0.115
0.525

NS
NS
NS
NS
NS
NS
NS
NS
NS

2 2.0
8 4.5
3 2.2
2 2.0
4 2.5
4 2.8
7 4.4
8 5.1
2 1.9
4.4 3.1

He

HW

A

HW

HW

A AR

He

HW

0.253
0.624
0.393
0.451
0.558
0.700
0.777
0.739
0.198
0.521

NS 2 2.0 0.404 NS 2 2.0 0.448 NS
NS 11 4.7 0.725 NS 10 4.2 0.660 NS
NS 3 2.4 0.412 NS 3 2.3 0.320 NS
NS 2 2.0 0.394 NS 2 1.8 0.258 NS
NS 4 2.8 0.594 NS 4 2.8 0.588 NS
NS 5 3.2 0.572 NS 4 3.2 0.620 NS
NS 7 5.1 0.828 NS 9 5.0 0.809 NS
NS 8 4.3 0.731 NS 8 4.5 0.768 NS
NS 2 1.8 0.255 NS 2 1.8 0.220 NS
4.9 3.2 0.546
4.9 3.1 0.521

2 2.0
11 4.5
4 2.4
2 2.0
4 2.8
5 3.4
7 5.0
8 5.0
2 1.8
5.0 3.2

0.486
0.664
0.350
0.418
0.544
0.614
0.824
0.813
0.227
0.549

NS
NS
NS
NS
NS
NS
NS
NS
NS

He

HW

0.429
0.620
0.420
0.385
0.584
0.490
0.820
0.766
0.274
0.532

Sig.
NS
NS
NS
NS
NS
NS
NS
NS

02JBC
A

He

He

03JBC
HW

A

AR

04JBC

He

HW

0.482
0.731
0.305
0.383
0.550
0.517
0.780
0.818
0.318
0.543

NS 3 2.1 0.451 NS 2 2.0 0.449 NS
3 2.1
NS 11 4.4 0.685 NS 13 4.5 0.675 NS 14 4.0
NS 3 2.8 0.559 NS 3 2.3 0.326 NS
3 2.4
NS 2 1.9 0.340 NS 2 1.9 0.318 NS
2 2.0
NS 4 2.7 0.580 NS 4 2.7 0.544 NS
4 2.9
NS 4 3.4 0.651 NS 5 2.9 0.491 NS
4 2.8
NS 8 4.9 0.803 NS 8 4.7 0.795 NS
8 5.0
NS 10 5.2 0.818 NS 8 5.1 0.821 NS
9 4.8
NS 2 1.8 0.247 NS 2 1.8 0.230 NS
3 1.9
5.2 3.2 0.570
5.2 3.1 0.517
5.6 3.1

7

AR

AR

04KBC-6

He

01JBC

AR

03KBC-6He

HWAAR