SC/63/IA12

Stock identity of sei whales in the central North Pacific based on
microsatellite analysis of biopsy samples obtained fromIWC/Japan joint cetacean sighting survey in 2010
Naohisa Kanda 1, Mutsuo Goto 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, Japan2

Contact email:kanda@cetacean.jp

ABSTRACT
Genetic variations at 16 microsatellite loci were analyzed for the central North Pacific sei whales (13biopsies) obtained during the 1st annual IWC/Japan joint cetacean sighting survey cruise conducted at thearea between 173°E -172°W in 2010. All of the loci analyzed were polymorphic with substantial level ofgenetic diversity in the sample. Genetic differences between this sample and the past JARPNII samples(N=489) obtained in the western North Pacific at the area between 143°E and 170°E from 2002 to 2007 as
well as commercial whaling sample (N=64) obtained in the eastern North Pacific at the area between 150°W
and 139°W in 1973 were examined. Due to the condition of the commercial whaling sample, the genetic
data of 14 of the 16 loci were used for the comparisons among the three samples. No evidence of genetic
differences was detected between the IWC/Japan survey and JARPNII samples as well as among the threesamples. This study supported our previous view that the open water of the North Pacific was occupied by
the individuals from a single stock of sei whales.
KEYWORDS: SEI WHALE, MICROSATELLITE, STOCK STRUCTURE, IWC/JAPAN JOINTCETACEAN SIGHTING SURVEY, JARPNII, COMMERCIAL WHALING, NORTH PACIFICINTRODUCTION
The IWC/SC decided to conduct ‘in-depth assessment’ of sei whales, Balaenoptera borealis, to investigate thecurrent status of stocks in the North Pacific (e.g., IWC, 2010). Among all of the required information,
understanding of stock structure in the region is essential for successful in-depth assessment. Due to thelimited information on the North Pacific sei whales in the past, no conclusive evidence has been presented fortheir stock structure (see Donovan, 1991). Recently, using microsatellite as well as mitochondrial DNAmarkers, Kanda et al. (2009) analyzed samples collected during the JARPNII surveys from 2002 to 2007
(143°E to 170°E) and during past commercial whaling operated in 1972 and 1973 (165°E to 139°W), andindicated that the open water of the North Pacific was mainly occupied by the individuals from a single stockof sei whales because no evidence of genetic differences was found among the samples. In this paper, weanalyzed sample of the central North Pacific sei whales collected during the 1st annual IWC/Japan joint
cetacean sighting survey cruise in 2010 from the area between 173°E -172°W (Matsuoka et al., 2011a) andcompared to the samples in Kanda et al. (2009) to better understand the stock structure of the species in thisregion.
MATERIALS AND METHODS
SamplesThirteen biopsies were obtained during the 1st annual IWC/Japan joint cetacean sighting survey cruise
conducted at the area between 173°E and 172°W in the summer of 2010 (Matsuoka et al., 2011). Genetic
data of the sei whales in the 2002 - 2007JARPNII survey samples obtained at the area between 143°E and170°E and the 1973 commercial whaling sample obtained at the area between 150°W and 139°W were also
used in this study for comparison (for details see Kanda et al., 2009). Table 1 summarizes collection
information of the samples.
Microsatellite analysis
Total DNA from each of the whales was extracted from 0.05 g of skin tissue stored in ethanol using GENTRA
PUREGENE DNA extraction kit (QIAGEN). Extracted DNA was stored in TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0).

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Genetic variation at microsatellite loci were analyzed using 16 sets of primers, none of which wasdesigned 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). Primersequences and PCR cycling profiles generally followed those of the original authors. PCR amplifications
were performed in 15µl reaction mixtures containing 10-100ng of DNA, 5 pmole of each primer, 0.625 unitsof 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). Allelic sizes were determined manually in relation to
the internal size standard and sei whale’s DNA of known size that were rerun on each gel. In regard to oursample/data handling under DNA data quality control, please see Kanda et al. (2010).
Data analysis
In regard to our DNA data quality control under the IWC guidelines, see Kanda et al. (2010). The computer
program MICRO-CHECKER (van Oosterhout et al., 2004) was used to check for null alleles andreading/typing errors. The number of alleles per locus and expected heterozygosity per locus was calculatedusing 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 if the samples came from a genetically samegroup of sei whales. If a statistically significant allele frequencies differences exist, then it could indicate
these samples came from genetically different stocks of sei whales. Probability test (or Fisher’s exact test)
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.
RESULTS AND DISCUSSION
All 16 microsatellite loci were successfully amplified and were polymorphic in the IWC/Japan joint cetaceansighting survey cruise samples (Table 2). The sample showed the level of genetic diversity similar to thepast JARPNII and commercial whaling samples (Table 2; Kanda et al., 2009). None of the 16 loci in thesample showed significant deviation from the expected Hardy-Weinberg genotypic proportions aftercorrection for the simultaneous multiple tests (data no shown).
No evidence of genetic differences was found between the IWC/Japan joint survey and the past JARPNIIsamples at each of the 16 loci after correction for the simultaneous multiple tests as well as all the locicombined (Table 3). FST was 0.007 between them and it was not significantly different from zero. We thenexamined the genetic differences between these samples and the 1973 commercial whaling one using thegenetic data obtained from 14 of the 16 loci (Table 3). These samples thus covered the wide area in theNorth Pacific from 35°N -50°N/143°E - 139°W (Fig. 1). No evidence of genetic differences was again
detected among the three samples at each of the loci as well as all the loci combined. FST was almost 0
among the three samples and it was not significantly different from zero. As similar to the past JARPNII
samples (Kanda et al. 2009), the most recent 2010 sample showed no evidence of genetic differences to thesample from the 150°W-139°W area. The results support our previous conclusion that the offshore open
water of the North Pacific was occupied mainly by the individuals of the single stock of sei whales.
Statistical power of the present tests might be low due to the small sample size of the IWC/Japan joint
survey sample. That should not discredit the significance of this paper, however. This paper is still very
informative because of the area covered. It was the first time to obtain the sample of sei whales in thecentral North Pacific from 173°E to 172°W after the end of commercial whaling era.
According to its future plan, the IWC/Japan joint cetacean sighting survey cruise will cover further
eastern area comparable to a principal part of the past commercial whaling grounds (Kato et al., 2011).
Additions of samples from the future IWC/Japan joint surveys as well as future JARPNII ones will thus not
only increase the statistical power of our analyses but further extend area covered. The currently planned indepth assessment of this species will not be successfully conducted without considering new information
obtained from the future surveys.
ACKNOWLEDGEMENTS
We thank captains, researchers and crewmembers participating in the 1st annual IWC/Japan joint cetaceansighting surveys in 2010 and the past JARPNII for their effort in collecting the samples used in this study.
We also thank J. Morishita, Fisheries Agency of Japan, H. Hatanaka, Institute of Cetacean Research, and H.
Kato, Tokyo University of Marine Science and Technology, for their useful comments to the manuscript.

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SC/63/IA12REFERENCES
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as revealed by microsatellites and mitochondrial DNA analyses. Paper SC/J09/JR32 presented to theJARPN II Review Workshop, Tokyo, January 2009 (unpublished).
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joint cetacean sighting survey in the North Pacific. Paper SC/63/O7 presented to the IWC ScientificCommittee May 2011, Tromso, Norway (unpublished).
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60N

60N

55N

55N

50N

50N

45N

45N

40N

40N

35N

35N

30N

30N

25N
120E

130E

140E

150E

160E

170E

180E

170W

160W

150W

Fig. 1. Sampling locations of sei whales in the North Pacific.
■:JARPNII, ▲: IWC/Japan joint cetacean sighting survey cruise, +: commercial whaling.

3140W

25N
130W

SC/63/IA12Table 1. Collection information of the samples obtained from JARPNII, 1st annual IWC/Japan joint
cetacean sighting survey cruise, and past commercial whaling. N=sample size.
SourceYearSurvey periodN
LatitudeLongitude
IWC/Japan2010
July-August
1340°N-46°N
173°E -172°W
2002JARPNII
June - August
489
35°N -48°N
143°E - 170°E
2007
Commercial
1973
May - August
6447°N -50°N
150°W -139°W

Table 2. The number of alleles (A) and expected heterozygosity (He) at the microsatellite loci in theJARPNII, 1st annual IWC/Japan joint cetacean sighting survey cruise, and commercial whaling samples of seiwhales. n.s. = not significant, n.a.= not available.
IWC/Japan
JARPNII
Commercial
Locus
AHeAHeAHe
EV21
50.71260.62160.616GGAA52070.856100.79780.792GATA98n.a.
n.a.
60.76370.733
GT211
20.26960.31230.305GATA5330.53530.48
30.520EV1100.859150.834150.847EV94
50.72460.68450.696GT23
60.353120.604
80.564
GT575
4
0.587
50.59240.572n.a.
n.a.
GATA41750.71580.774GT310
30.42940.51140.485
EV104
50.69270.72460.700
GATA2880.859110.81
90.832GT271
30.28240.13320.104GT011
30.59640.44130.415
DlrFCB1790.894
180.872160.885
Average*
5.3
0.633
7.90.620
6.60.595*16 loci for JARPNII and IWC/Japan, 14 loci for Commercial.

Table 3. Results (p-values) of the heterogeneity tests and FST among the JARPNII, 1st annual IWC/Japan
joint cetacean sighting survey cruise, and commercial whaling samples of sei whales.
Locus
JARPNII x IWC/Japan
JARPNII x IWC/Japan x Commercial
EV210.633
0.847
GGAA5200.117
0.357
GATA980.860
n.a.
GT211
0.483
0.505
GATA530.826
0.920
EV10.2500.419
EV94
0.244
0.279
GT23
0.154
0.481
GT575
0.758
0.664
GATA417
0.320
n.a.
GT310
0.545
0.247
EV104
0.863
0.469
GATA28
0.652
0.496
GT271
0.327
0.174
GT011
0.249
0.333
DlrFCB17
0.444
0.548
All
0.644
0.727
FST

0.007

0.0004