SC/65a/IA05

Microsatellite DNA analysis of sei whales obtained from the
2010-2012 IWC-POWER
Naohisa Kanda 1, Koji Matsuoka 1, Hideyoshi Yoshida 2, and Luis A. Pastene 1
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
2

Contact email:kanda@cetacean.jp

ABSTRACT
Genetic variations at 14 microsatellite loci were examined in the North Pacific sei whales’ biopsy samples
obtained from the IWC-POWER (the International Whaling Commission/Pacific Ocean Whale andEcosystem Research) 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 ofthe eastern North Pacific in 2012 (N=35). All of the areas were north of 40°N. The observed POWER
data was then analyzed with previously reported genetic data of the JARPNII samples (N=489) collectedfrom the western North Pacific between 143°E and 170°E in 2002-2007 and the commercial whaling
samples collected from the central North Pacific between 180° and 150°W in 1972-1973 (N=57) andfrom the eastern North Pacific between 150°W and 139°W in 1973 (N=64). Analyses of these samples
allowed us to detect temporal (40 years apart) and spatial (143°E to 135°W area divided into western,
central, and eastern) genetic differences of the North Pacific sei whales. Our results showed 1) very
similar level of genetic diversities among the POWER, JARPNII and commercial whaling samples, 2) noevidence of the genetic differences among the three POWER samples, 3) no evidence of the temporal
genetic differences between the recent POWER and past commercial whaling samples collected from thesame area, 4) no evidence of the spatial genetic differences among the western, central, and easternsamples. This study supports our previous view that the open waters of the North Pacific were occupiedby the individuals from a single stock of sei whales.
KEYWORDS: SEI WHALE, MICROSATELLITE, STOCK STRUCTURE, POWER, JARPNII,COMMERCIAL WHALING, NORTH PACIFICINTRODUCTION
The IWC/SC starts ‘in-depth assessment’ of sei whales, Balaenoptera borealis, from 65SC to investigate
the current status of stocks of this species in the North Pacific (e.g., IWC, 2010). Among all of therequired information, understanding of stock structure in the region is essential for successful in-depth
assessment. Due to the limited information on the North Pacific sei whales in the past, no conclusive
evidence has been presented for their stock structure (see Donovan, 1991). Recently, Kanda et al.
(2009) analyzed samples from the 2002-2007 JARPNII (143°E to 170°E) and 1972-1973 commercial
whaling (165°E to 139°W) using microsatellite as well as mitochondrial DNA markers, and indicated thatthe open water of the North Pacific was mainly occupied by the individuals from a single stock of sei
whales because no evidence of genetic differences was found among the samples.
Recently, using microsatellite DNA markers, we analyzed samples of the North Pacific sei whales
collected from the IWC-POWER (the International Whaling Commission/Pacific Ocean Whale andEcosystem Research) surveyed the area between 173°E -172°W in 2010, between 170°W -150°W in 2011,and between 150°W -135°W in 2012(Matsuoka et al., 2011; 2012; 2013). In this paper, we compared
the data from the POWER samples to those from the JARPNII and commercial whaling samples in
Kanda et al. (2009) to investigate temporal and spatial genetic differences among the North Pacific sei
whales.

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MATERIALS AND METHODS
Samples
DNA were extracted from the biopsy samples obtained from the IWC-POWER (the International Whaling
Commission/Pacific Ocean Whale and Ecosystem Research) cruises that surveyed 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). Genetic
data of the sei whales from the JARPNII and commercial whaling samples were used to test whether or
not there was any evidence of genetic differences among the whales from the North Pacific from 143°E to
139°W. The JARPNII sample (N=489) was obtained at the area between 143°E and 170°E from 2002 to
2007. The commercial whaling samples were obtained at the central area between 180° and 150°W in
1972/73 (CWh-central; N=57) and eastern area between 150°W and 139°W in 1973 (CWh-eastern;
N=64) between 150°W and 139°W. For details of these reference samples, please see Kanda et al., 2009.
Table 1 summarizes collection information of the samples and Fig.1 shows the individual positions.

Table 1. Collection information of the POWER, JARPNII, past commercial whaling samples used forthe 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

2002-2007

June - August

489

35°N -48°N

143°E - 170°E

Commercial
CWh-central
CWh-eastern

1972-1973
1973

May - August
May - August

57
64

40°N -50°N
47°N -50°N

180° -150°W
150°W -139°W

Fig. 1.
Sampling locations of sei whales in the North Pacific.
■: 2010POWER, ■: 2011POWER, ■: 2012POWER, ▲: Commercial whaling (CWh-central), ▲:Commercial whaling (CWh-eastern), +: JARPNII (Western).

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Microsatellite analysis
Total DNA from each of the whales in the POWER samples was extracted from 0.05 g of skin tissue
stored in ethanol using GENTRA PUREGENE DNA extraction kit (QIAGEN). Extracted DNA wasstored in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Genetic variation at microsatellite loci were analyzed using 14 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,
GGAA520 (Palsbøll et al., 1997), and DlrFCB17 (Buchanan et al., 1996). In regard to PCR
amplifications, microsatellite electrophoresis, and sample/data handling, please see Kanda et al. (2009).
Data analysis
In regard to our DNA data quality control under the IWC guidelines, see Kanda et al. (2010). The
number of alleles 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 were conducted 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 camefrom a genetically 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 (orFisher’s exact test) implemented in GENEPOP 4.0 (Rousset, 2008) was used to conduct the heterogeneity
tests. When simultaneous multiple tests were conducted, Rice (1989) correction for the multiple tests
was performed. FST value was calculated using FSTAT 2.9.3 (Goudet, 1995).
RESULTS AND DISCUSSION
No case of matchings between the individuals from the different years’ POWER samples was observed.
All of the 14 microsatellite loci were polymorphic in the POWER samples, and the level of thegenetic diversities (the number of alleles, allelic richness, and expected heterozygosity) of the POWER
samples was similar to that of the JARPNII and commercial whaling samples (Table 2). Therefore, no
temporal and spatial difference in the genetic diversity was observed among the POWER, JARPNII, andcommercial whaling samples.

Table 2. The number of alleles (A), allelic richness, and expected heterozygosity (He) at the 14
microsatellite loci in the POWER, JARPNII, and commercial whaling (CWh) samples of sei whales.
POWER

Locus
EV21
GGAA520
GT211
GATA53
EV1
EV94
GT23
GT575
GT310
EV104
GATA28
GT271
GT011
DlrFCB17
Average

JARPNII

CWh

A

AR

He

A

AR

He

A

AR

He

6
8
3
3
15
6
10
5
3
6
9
3
4
16
6.9

6.0
8.0
3.0
3.0
15.0
6.0
10.0
5.0
3.0
6.0
9.0
3.0
4.0
16.0
6.9

0.675
0.792
0.343
0.520
0.839
0.717
0.587
0.585
0.480
0.700
0.828
0.147
0.491
0.871
0.613

6
10
6
3
15
6
12
5
4
7
11
4
4
18
7.9

5.7
8.6
4.7
3.0
13.8
6.0
9.0
4.5
3.3
5.7
9.1
3.1
3.9
15.6
6.9

0.621
0.797
0.312
0.480
0.834
0.684
0.604
0.592
0.511
0.724
0.810
0.133
0.441
0.872
0.601

6
9
4
3
17
6
10
5
4
7
10
3
3
17
7.4

5.6
8.8
3.6
3.0
15.7
5.9
9.2
4.7
3.6
6.3
9.6
3.0
3.0
15.9
7.0

0.637
0.794
0.306
0.496
0.838
0.687
0.608
0.579
0.473
0.707
0.819
0.118
0.434
0.890
0.599

No evidence of the genetic differences was found at each of the 14 loci, as well as all the locicombined, among the three POWER samples (Table 3). FST was 0.002 among them and it was not
significantly different from zero. These three POWER samples were combined into one for the3

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comparison with the commercial whaling samples (CWh-central and CWh-eastern combined), that werecollected from the similar area to the POWER samples in 40 years ago, to see if there were any temporal
genetic differences. No evidence of the genetic differences was detected at each of the 14 loci, as well
as all the loci combined, between the POWER and commercial whaling samples (Table 3). FST was
negative between the two and it was not significantly different from zero. The result indicated that these
old and new samples collected after the elapse of 40 years were genetically similar to each other.

Table 3. Results (p-values) of the heterogeneity tests and FST among the three POWER samples andbetween the three POWER and two commercial whaling (CWh) samples.
10POWER x 11POWER x
12POWER

POWER x CWh

EV21
GGAA520
GT211
GATA53
EV1
EV94
GT23
GT575
GT310
EV104
GATA28
GT271
GT011
DlrFCB17
All loci

0.632
0.245
0.472
0.928
0.065
0.955
0.179
0.314
0.415
0.916
0.897
0.337
0.252
0.607
0.641

0.775
0.231
0.347
0.509
0.887
0.331
0.776
0.757
0.942
0.223
0.359
0.844
0.270
0.560
0.875

FST

0.002

negative

Locus

In order to examine genetic differences among samples by areas, each of the POWER andcommercial whaling samples were again treated separately as central (10POWER, 11POWER, and CWhcentral from 180° - 150°W) and eastern (12POWER and CWh-eastern from 150°W -135°W) samples.
No evidence of the genetic differences was detected at each of the 14 loci, as well as all the loci combined,
among the three central samples (Table 4). FST was 0.001 among them and it was not significantly
different from zero. These samples were combined into one as a single central sample for the further
analysis. Similarly, no evidence of the genetic differences was detected at each of the 14 loci, as well as
all the loci combined, between the two eastern samples (Table 4). FST was negative between the two andit was not significantly different from zero. These samples were combined into one as a single easternsample for the further analysis.
No evidence of genetic differences was detected at each of the 14 loci, as well as all the locicombined, among the western, central and eastern samples (Table 4). FST was almost 0 among the threesamples and it was not significantly different from zero. When all of the samples were combined into
one, none of the 14 loci showed significant deviation from the expected Hardy-Weinberg genotypic
proportions after correction for the simultaneous multiple tests (data not shown). This result indicatedthat the individuals utilizing the North Pacific from west end to east end (143°E to 139°W) as theirfeeding ground belonged to the genetically same group of sei whales. These results support our previous
conclusion that the offshore open waters of the North Pacific are occupied by the individuals of the single
stock of sei whales.

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Table 4. Results (p-values) of the heterogeneity tests and FST among the POWER (10-12POWER),JARPNII (Western), and commercial whaling (CWh-central, eastern) samples by areas.
Central North PacificEastern North PacificNorth Pacific

10POWER x 11POWER x
CWh-central

12POWER x CWh-eastern

Western x Central x
Eastern

EV21
GGAA520
GT211
GATA53
EV1
EV94
GT23
GT575
GT310
EV104
GATA28
GT271
GT011
DlrFCB17
All loci

0.585
0.161
0.396
0.887
0.317
0.833
0.535
0.878
0.537
0.630
0.926
0.761
0.469
0.230
0.918

0.965
0.808
0.531
0.643
0.646
0.392
0.392
0.806
0.819
0.662
0.195
1.000
0.125
0.946
0.959

0.178
0.723
0.558
0.741
0.434
0.652
0.496
0.088
0.197
0.240
0.621
0.084
0.588
0.586
0.424

FST

0.001

negative<0.001Locus

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. We also thank S. Ohsumi and H. Hatanaka for
their valuable comments to the manuscript.
REFERENCES (NOT YET COMPLETED)
Bérubé, M., Aguilar, A., Dendanto, D., Larsen, F., Notarbartolo-di-Sciara, G., Sears, R., Sigurjónsson, J.,
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); analysisof mitochondrial and nuclear loci. Mol. Ecol. 7:585-599.
Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøll, P.J. 2000. Polymorphic di-nucleotide
microsatellite loci isolated from the humpback whale, Megaptera novaeanglliae. Mol. Ecol. 9:21812183.
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.
Donovan, G.P. 1991. A review of IWC stock boundaries. Rep. int. Whal. Commn. (special issue 13) :39-68.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered. 86:485-486.
IWC. 2010. Report of the Scientific Committee, Annex G. Report of the sub-committee on in-depth
assessments. J. Cetacean Res. Manage. 11 (suppl2): 180-197.
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., Park, J.Y., Goto, M., An, Y.R., Choi, S.G., Moon, D.Y., Kishiro, T., Yoshida, H., Kato, H. andPastene, L.A. 2010. Genetic analysis of western North Pacific minke whales from Korea and Japan
based on microsatellite DNA. Paper SC/62/NPM11 presented to the IWC Scientific Committee, May
2010, Agadir, Morocco (unpublished).
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
Scientific Committee May 2011, Tromso, Norway (unpublished).
Matsuoka, K., Mizroch, S., and Komiya, H. 2012. Cruise report of the 2011 IWC-Pacific Ocean Whaleand Ecosystem Research (IWC-POWER). Paper SC/64/IA5 presented to the IWC ScientificCommittee June 2012, Panama City, Panama (unpublished).

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SC/65a/IA05

Matsuoka, K., Mizroch, S., An, Y.-R., Kumagai, S. and Hirose, K. 2013. Cruise report of the 2012 IWCPacific 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.
Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution 43:223-225.
Rousset, F. 2008. Genepop'007: a complete re-implementation of the genepop software for Windows andLinux. Molec. Ecol. Resources 8:103-106.
Valsecchi, E., and Amos, W. 1996. Microsatellite markers for the study of cetacean populations. Mol. Ecol.
5:151-156.

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