SC/66a/IA08

Genetic study on JARPNII and IWC-POWER samples of sei
whales collected widely from the North Pacific at the same time of
the year
Naohisa Kanda, Koji Matsuoka, Mutsuo Goto and Luis A. Pastene
The Institute of Cetacean Research, 4-5, Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan

ABSTRACT
We used microsatellite DNA markers to analyze samples of sei whales collected widely from the North Pacificat the same time of the year in order to test spatial genetic heterogeneity in this ocean basin. Although we havebeen reporting results of the genetic studies on the North Pacific sei whales to previous IWC/SC meetings, thisstudy is the first to utilize temporally similar (collected at the same year), yet geographically very different,
samples (covered west-end to east-end of the North Pacific). This study used samples collected from thenorthwestern (JARPNII), northcentral (POWER), and northeastern (POWER) areas of the North Pacific in thesame summer seasons in 2010, 2011 and 2012. No evidences of significant genetic differences between thesamples from JARPNII and POWER in each of the three years, were found. Each yearly sample was then
combined as JARPNII as well as POWER samples, respectively. No significant genetic differences weredetected between these two samples. We used genotypic profiles of each whale in the POWER biopsy samples tofind any cases of matching to the individuals in the JARPNII samples, no matching was found at all. In
conclusion, this study failed to demonstrate evidence of multiple stocks of sei whales in the North Pacific.
KEYWORDS: SEI WHALE, MICROSATELLITE, STOCK STRUCTURE, POWER, JARPNII, NORTHPACIFICINTRODUCTION
The most comprehensive studies conducted so far with regard to the stock structure of the North Pacific sei
whales were those presented at the previous JARPNII Review workshop in 2009 (Kanda et al. 2009) as well as
presented at the recent IWC/SC (Kanda et al. 2013). These studies, using multiple sets of microsatellite DNAloci markers, examined the sei whales samples collected from almost the entire range of North Pacific.
Kanda et al. (2009), on the one hand, analyzed genetic variation at 17 microsatellite DNA loci and 487 bp of
mitochondrial DNA (mtDNA) control region sequences in the JARPNII samples (N=489) from 2002 to 2007 in
the area between 143°E and 170°E as well as in the commercial whaling samples (N=301) from 1972 and 1973
conducted in the area between 165°E and 139°W. The results indicated no evidence of genetic differences within
as well as between the JARPNII and commercial whaling samples. Both females and males showed same patternof the stock structure. Sequencing and phylogenetic analysis of the mtDNA control region also showed noevidence of the genetic heterogeneity in the JARPNII samples as well as no spatially or temporally unique
phylogenetic clusters.
Kanda et al. (2013), on the other hand, examined genetic variations at 14 microsatellite DNA loci in the North
Pacific sei whales’ biopsy samples obtained from the IWC-POWER (the International Whaling
Commission/Pacific Ocean Whale and Ecosystem Research; hereafter POWER) cruises that surveyed 173°E 172°W area of the central North Pacific in 2010 (N=13), 170°W - 150°W area of the central North Pacific in
2011 (N=29), and 150°W - 135°W area of the eastern North Pacific in 2012 (N=35), and these obtained data wasanalyzed with those in Kanda et al. (2009). This study allowed the authors to detect temporal (40 years apartbetween the POWER and commercial whaling data) and spatial (143°E to 135°W area divided into western,
central and eastern) genetic differences of the North Pacific sei whales. Similarly to Kanda et al. (2009), theresults showed no evidence of the temporal genetic differences between the recent POWER and past commercial
whaling samples collected from the same area and no evidence of the spatial genetic differences among thewestern, central and eastern samples.
One drawback to these two studies was that there was no direct comparison among samples collected at the sametime of the year from the different areas over the North Pacific. Considering that sei whales conduct seasonal
migration from their breeding ground to feeding ground every year, development of stock structure hypothesis

1should test the genetic differentiation in the samples collected in the same year that eliminate temporal negative
biases. If no genetic difference is found, it should strongly indicate no evidence of multiple stocks in the area.
This study thus looked at genetic variation at the microsatellite DNA loci to analyze the JARPNII and POWER
samples collected from the same time of years in 2010, 2011 and 2012, respectively.
MATERIALS AND METHODS
Samples
JARPNII samples (N=295) of sei whales from 2010 to 2012 were collected from the western North Pacific off
Japanese coast (Table 1 and Fig. 1). Although sampling dates and locations of the surveys slightly differed year
by year depending on the sampling plan of a given year, it was fixed to the northwestern North Pacific(37°N-42°N, 151°E-169°E). Skin biopsy samples of sei whales were obtained during the POWER surveys.
POWER survey covered the area north of 40°N from 173°E to 172°W in 2010 (10POWER; N=13), from 170°W
to 150°W in 2011 (11POWER; N=29), and from 150°W to 135°W in 2012 (12POWER; N=35) (Matsuoka et al.,2011, 2012, 2013). Table 1 summarizes collection information of the samples and Fig.1 shows the individual
positions.
DNA extraction
Total DNA from each of the whales was extracted from 0.05 g of skin tissue in the JARPNII samples and of skinbiopsy in the POWER samples using the protocol of Sambrook et al. (1989). Extracted DNA was stored in TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Microsatellite analysis
Genetic variation at microsatellite loci were analyzed using 16 sets of primers, none of which was designed
specifically from sei whales: EV1, EV21, EV94, EV104 (Valsecchi and Amos, 1996), GT011 (Bérubé et al.,1998), GT23, GT211, GT271, GT310, GT575 (Bérubé et al., 2000), GATA28, GATA53, GATA98, GATA417,
GGAA520 (Palsbøll et al., 1997), and DlrFCB17 (Buchanan et al., 1996). Primer sequences and PCR cycling
profiles generally followed those of the original authors.
PCR amplifications were performed in 15l reaction mixtures containing 10-100ng of DNA, 5 pmole of eachprimer, 0.625 units of Ex Taq DNA polymerase (Takara Shuzo), and 2mM of each dNTP, and 10x reaction buffer
containing 20mM MgCl2 (Takara Shuzo). Amplified products with internal size standard (GENESCAN400HD,
Applied Biosystems Japan) were run on a 6% polyacrylamide denaturating gel (Long Ranger) using
BaseStation100 DNA fragment analyzer (Bio-Rad). Although alleles were visualized using Cartographer
software specifically designed for the BaseStation, allelic sizes were determined manually in relation to theinternal size standard and sei whale’s DNA of known size that were rerun on each gel.
Data analysis
In regard to our DNA data quality control under the IWC guidelines, see Kanda et al. (2014). The number ofalleles per locus, allelic richness, and expected heterozygosity per locus was calculated using FSTAT 2.9.3(Goudet, 1995). Statistical tests for the deviations from expected Hardy-Weinberg genotypic proportions wereconducted using GENEPOP 4.0 (Rousset, 2008). When simultaneous multiple tests were conducted, Rice (1989)
correction for the multiple tests was performed.
Conventional hypothesis testing procedure was conducted using heterogeneity test in microsatellite allele
frequencies among samples. Our null hypothesis to be tested is whether or not the samples came from agenetically same group of sei whales. If statistically significant allele frequency differences exist, it could
indicate these samples came from genetically different stocks of sei whales. Probability test (or Fisher’s exacttest) implemented in GENEPOP 4.0 (Rousset, 2008) was used to conduct the heterogeneity tests. Whensimultaneous multiple tests were conducted, Rice (1989) correction for the multiple tests was performed. FST
value was calculated using FSTAT 2.9.3 (Goudet, 1995).
Matching exercises between the individuals from JARPNII and POWER samples were conducted using acomputer program CERVUS (Marshall et al., 1998).
RESULTS AND DISCUSSION
The POWER survey changed its research area from the northcentral North Pacific in 2011 to the northeastern
North Pacific in 2012 with no overlapping in contrast to the JARPNII surveys which were largely operated at thesame northwestern North Pacific area every years, so the comparison between the POWER and JARPNII

2

samples were geographically different among the three years (Table 1 and Fig.1). The comparison between thesamples collected in 2012 was geographically most apart from each other (northwestern vs northeastern).
The sampling dates of the two surveys were also different from each other with an overlap in August (Table 1).
Some may argue that this time lag cause duplicate sampling resulting in underestimation of the level of thegenetic differences between the samples from the two surveys. Such duplicate samplings were impossible ineach of the years because the samples from the early-operated JARPNII surveys were taken lethally. The
JARPNII samples may have contained individuals biopsied by the non-lethal POWER surveys in the previous
years. Although we attempted to match the whales in these samples using their genotypic profiles, we were notable to find any matching.
All of the 16 microsatellite loci were polymorphic in the POWER and JARPNII samples (Table 2). Although thenumber of the alleles per locus per samples was different among the samples, other genetic indices such as allelicrichness and expected heterozygosity were very similar to each other. The differences in the number of alleles
were most likely due to the differences in sample sizes among the samples. Therefore, no spatial difference in thelevel of the genetic diversity was observed among the POWER and JARPNII samples.
Firstly, we looked at the genetic differences between the JARPNII and POWER samples collected at the same
years. No evidence of the genetic differences was found at each of the 16 loci, as well as all the loci combined,
between the two in each of the collected years (Table 3). The values of FST at all three comparisons were notsignificantly different from zero. These results indicated that, within each year, the individuals in these two
samples came from a genetically same group of sei whales.
We thus combined the POWER and JARPNII samples collected at the same years into one as 2010, 2011 and2012 samples, respectively. Secondly, we tested for the deviation from the expected Hardy-Weinberg genotypic
distributions within each of the three samples (Table 4). None of the 16 microsatellite DNA loci as well as allloci combined showed significant p-values.
Thirdly, we conducted statistically tests among the three samples to see if these samples were originated fromgenetically different groups of sei whales. Again, no evidence of the genetic difference was detected at each of
the 16 loci, as well as all the loci combined, among the three samples (Table 5). The value of FST at thiscomparison was low (0.001) and was not significantly different from zero. In addition to that, no evidence of thedeviation from the expected Hardy-Weinberg genotypic distributions was detected at each of the loci as well as
all loci combined in the sample all three combined (Table 5).
We have been presenting the results of several genetic analyses using the temporally as well as geographicallyvariable samples of the North Pacific sei whales to the IWC/SC (e.g., Kanda et al., 2009, 2013). One task
remained is that we have never compared the samples collected from the different areas at the same time of year.
Evidence of or no evidence of genetic differences among the samples collected at the different years could be
due to the temporal genetic effects within the same stock. The analysis presented in this paper thus filled up the
empty space in the series of our genetic studies on the North Pacific sei whales. The results from the genetic
heterogeneity tests between the two spatially different samples (JARPNII and POWER) showed no evidence ofthe genetic differences between the two samples. Some may argue that such result can be obtained when two
genetically different stocks mix each other widely over the North Pacific with an equal proportion. The resultsfrom the Hardy-Weinberg tests in this study denied such possibility because no evidence of the deviation fromthe Hardy Weinberg equilibrium was detected in the combined samples. All of our genetic studies so far wereunable to demonstrate evidence of multiple stocks of sei whales in the North Pacific.
ACKNOWLEDGEMENTS
We thank captains, researchers and crewmembers participating in the IWC-POWER and JARPNII for their effort
in collecting the samples used in this study.
REFERENCES
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Urban-Ramirez, J., and Palsbøll, P.J. 1998. Population genetic structure of North Atlantic, Mediterranean
Sea and Sea of Cortez fin whales, Balaenoptera physalus (Linnaeus, 1758); analysis of mitochondrial andnuclear loci. Mol. Ecol. 7:585-599.
Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøll, P.J. 2000. Polymorphic di-nucleotide microsatellite loci3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.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered. 86:485-486.
Kanda, N., Goto, M., Yoshida, H., and Pastene, L.A. 2009. Stock structure of sei whales in the North Pacific as
revealed by microsatellites and mitochondrial DNA analyses. Paper SC/J09/JR32 presented to the JARPN II
Review Workshop, Tokyo, January 2009 (unpublished).
Kanda, N., Matsuoka, N., Yoshida, H. and Pastene, L. A. 2013. Microsatellite DNA analysis of sei whales
obtained from the 2010-2012 IWC-POWER. Paper SC/65a/IA05 presented to IWC Scientific Committee,
June 2013, Jeju Island, Republic Korea (unpublished) 6pp.
Kanda, N., Goto, M., Oikawa, H. and Pastene, L.A. 2014. Update of note on sampling and laboratory procedure
protocols of the genetic work at the Institute of Cetacean Research (SC/65b/J27Rev). Paper SC/65b/DNA01
presented to the IWC Scientific Committee, May 2014 (unpublished). 6pp.
Marshall, T.C., Slate, J., Kruuk, L., and Pemberton, J.M. 1998. Statistical confidence for likelihood-based
paternity inference in natural populations. Mol. Ecol. 7: 639-655.
Matsuoka, K., Hakala, S., Kim, H.Y., Aki, M., and Shinyashiki, Y. 2011. Cruise report for the 2010 IWC/Japan
joint cetacean sighting survey in the North Pacific. Paper SC/63/O5 presented to the IWC ScientificCommittee May 2011, Tromso, Norway (unpublished).
Matsuoka, K., Mizroch, S., and Komiya, H. 2012. Cruise report of the 2011 IWC-Pacific Ocean Whale andEcosystem Research (IWC-POWER). Paper SC/64/IA5 presented to the IWC Scientific Committee June
2012, Panama City, Panama (unpublished).
Matsuoka, K., Mizroch, S., An, Y.-R., Kumagai, S. and Hirose, K. 2013. Cruise report of the 2012 IWC-Pacific
Ocean Whale and Ecosystem Research (IWC-POWER). Paper SC/65a/IAXX presented to the 65th IWC
Scientific Committee, June 2013 (unpublished).
Palsbøll, P.J., Bérubé, M., Larsen, A.H., and Jørgensen, H. 1997. Primers for the amplification of tri-and tetramer
microsatellite loci in baleen whales. Mol. Ecol. 6:893-895.
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5:151-156.

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Table 1. Collection information of the POWER and JARPNII samples used for the current data analyses.
N=sample size.
SourceYear

Survey period

N

LatitudeLongitude

POWER
10POWER
11POWER
12POWER

2010
2011
2012

July-AugustAugustAugust13
29
35

40°N-46°N
40°N-50°N
40°N-53°N

173°E-172°W
170°W-150°W
150°W-135°W

JARPNII
10NP
11NP
12NP

2010
2011
2012

June - AugustJune - AugustJune-July

100
95
100

38°N-44°N
37°N-45°N
37°N-42°N

145°E-168°E
149°E-167°E
151°E-169°E

Table 2. The number of alleles (A), allelic richness, and expected heterozygosity (He) at the 16 microsatellite
loci in the POWER and JARPNII samples of sei whales collected in 2010 to 2012.
10POWER

Locus
EV21
GGAA520GATA98GT211
GATA53
EV1
EV94
GT23
GT575
GATA417GT310
EV104
GATA28
GT271
GT011
DlrFCB17
Average

11POWER

12POWER

A

AR

He

A

AR

He

A

AR

He

5
7
6
2
3
10
5
6
4
5
3
5
8
3
3
9
5.3

5.0
7.0
6.0
2.0
3.0
10.0
5.0
6.0
4.0
5.0
3.0
5.0
8.0
3.0
3.0
9.0
5.3

0.712
0.856
0.763
0.269
0.535
0.859
0.724
0.353
0.587
0.715
0.429
0.692
0.859
0.282
0.596
0.894
0.633

6
7
6
3
3
10
6
7
5
7
3
5
8
3
4
15
6.1

5.3
5.7
5.8
3.0
3.0
8.1
5.1
5.6
3.8
6.1
3.0
4.7
7.0
2.3
3.4
11.0
5.2

0.691
0.772
0.780
0.398
0.518
0.834
0.729
0.639
0.578
0.820
0.443
0.711
0.826
0.132
0.489
0.872
0.640

5
7
6
3
3
13
6
8
4
8
3
6
9
2
4
14
6.3

4.7
5.4
5.3
2.8
3.0
9.4
5.2
5.4
3.5
6.1
3.0
5.0
7.5
1.9
3.3
10.0
5.1

0.650
0.786
0.753
0.327
0.530
0.824
0.713
0.610
0.560
0.777
0.530
0.703
0.825
0.109
0.450
0.858
0.625

Table 2. continued.
10NP

Locus
EV21
GGAA520GATA98GT211
GATA53
EV1
EV94
GT23
GT575
GATA417GT310
EV104
GATA28
GT271
GT011
DlrFCB17
Average

11NP

12NP

A

AR

He

A

AR

He

A

AR

He

6
8
6
3
3
12
6
9
5
7
3
6
10
4
4
18
6.9

4.7
6.5
5.1
2.6
3.0
8.0
5.1
5.4
3.5
5.7
2.9
5.0
7.1
2.3
3.3
9.8
5.0

0.688
0.811
0.718
0.293
0.561
0.816
0.703
0.639
0.575
0.792
0.516
0.734
0.808
0.167
0.453
0.862
0.634

6
8
7
3
3
13
6
9
4
8
3
7
10
3
3
16
6.8

4.8
6.6
5.6
2.6
3.0
8.6
4.7
5.7
3.5
5.9
3.0
4.8
6.9
2.3
2.9
9.9
5.0

0.662
0.814
0.744
0.285
0.488
0.835
0.682
0.640
0.607
0.792
0.485
0.724
0.798
0.177
0.438
0.863
0.627

6
8
7
5
3
14
7
10
5
6
3
7
9
3
4
16
7.1

4.9
6.5
5.4
3.1
3.0
8.2
5.2
5.1
3.6
5.6
2.9
4.7
7.1
2.3
3.4
10.1
5.1

0.661
0.806
0.735
0.300
0.526
0.815
0.729
0.589
0.581
0.793
0.470
0.710
0.834
0.159
0.442
0.880
0.627

5

Table 3. Results (p-values) of the heterogeneity tests and FST between the POWER and JARPNII samples by
years.
Locus

10POWER x 10NP

11POWER x 11NP

12POWER x 12NP

EV21
GGAA520GATA98GT211
GATA53
EV1
EV94
GT23
GT575
GATA417GT310
EV104
GATA28
GT271
GT011
DlrFCB17
All loci

0.950
0.064
0.703
0.621
0.873
0.019
0.154
0.066
0.863
0.266
0.541
0.634
0.724
0.291
0.182
0.272
0.188

0.346
0.205
0.596
0.213
0.900
0.053
0.754
0.607
0.150
0.811
0.655
0.595
0.918
0.831
0.255
0.303
0.628

0.837
0.401
0.527
0.694
1.000
0.229
0.910
0.832
0.978
0.309
0.678
0.875
0.166
0.340
0.057
0.994
0.889

FST

0.011

-0.0002-0.004

Table 4. Results (p-values) of the test for the deviation from the Hardy-Weinberg expected genotypic proportionsin the POWER and JARPNII samples combined by years.
Locus
EV21
GGAA520GATA98GT211
GATA53
EV1
EV94
GT23
GT575
GATA417GT310
EV104
GATA28
GT271
GT011
DlrFCB17
All loci

2010(POWER+NP)

2011 (POWER+NP)

2012 (POWER+NP)

0.692
0.732
0.260
0.123
0.189
0.559
0.803
0.186
0.373
0.946
0.872
0.394
0.704
0.678
0.056
0.959
0.664

0.741
0.528
0.221
1.000
0.528
0.728
0.171
0.768
0.173
0.401
0.955
0.848
0.670
0.405
0.263
0.058
0.689

0.974
0.265
0.062
0.488
0.891
0.649
0.810
0.875
0.347
0.430
0.768
0.865
0.888
1.000
0.746
0.107
0.922

6

Table 5. Results (p-values) of the heterogeneity tests and FST among the three yearly samples (POWER and
JARPNII combined for each year) and of the test for the deviation from the Hardy-Weinberg expected genotypic
proportions in the sample all POWER and JARPNII combined.
Locus

2010x2011x2012

HW for all combined

EV21
GGAA520GATA98GT211
GATA53
EV1
EV94
GT23
GT575
GATA417GT310
EV104
GATA28
GT271
GT011
DlrFCB17
All loci

0.570
0.125
0.693
0.482
0.169
0.823
0.801
0.713
0.020
0.783
0.303
0.231
0.993
0.947
0.062
0.249
0.343

0.755
0.343
0.122
0.676
0.095
0.647
0.393
0.806
0.081
0.789
0.948
0.840
0.975
1.000
0.663
0.734
0.878

FST

0.001

7

Fig.1. Sampling locations of sei whales in the North Pacific. Square: JARPNII, Triangle: POWER; red:
2010, blue: 2011, green: 2012.

8