SC/J09/JR33
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Genetic characteristics of sperm whales sampled during
JAPRNII from 2000 to 2007 as revealed by mitochondrial
DNA and microsatellite analyses
Naohisa Kanda, Mutsuo Goto and Luis A. Pastene
The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
Contact email:kanda@cetacean.jp

ABSTRACTWe analyzed genetic variations at 15 microsatellite DNA loci and mitochondrial DNA (mtDNA) control
region sequences in sample of sperm whales collected during JARPNII from 2000 to 2007 in order toexamine the effectiveness of these genetic markers for stock structure study of the species. Analyses ofmtDNA and microsatellite markers in the total of 45 sperm whales demonstrated that these geneticmarkers were variable enough to explore stock structure of sperm whales. Statistical tests found noevidence of deviation from the expected Hardy-Weinberg genotypic proportion at all of the 15
microsatellite loci. At this point, we did not detect any signals of multiple stocks in the western North
Pacific off Japan from the JARPNII samples.
KEW WORDS: SPERM WHALE, MICROSATELLITE, JARPNII, NORTH PACIFICINTRODUCTION
Identification of the genetic structure of stocks over a species’ geographic range is of primary importancefor effective stock management. The objective of the stock structure part of JARPNII survey for sperm
whales is to obtain information useful for describing stock structure of sperm whales inhabiting thewestern North Pacific. The information is also surely helpful for future CA of this species in the IWC.
As an initial step to accomplish the objective, we examined genetic variation at mtDNA andmicrosatellites to examine effectiveness of these genetic markers to explore genetic characteristics of thespecies.
MATERIALS AND METHODS
Samples and DNA extraction
A total of 45 sperm whales used in this analysis were collected during JARPNII surveys from 2000 to2007 in the western North Pacific (Table 1; Fig. 1). Details of offshore component of JARPNII survey
can be found in Tamura et al. (2009).
Skin tissues of research takes were stored in vials filled with 95% ethanol in –70°C until DNA
extraction. Total DNA was extracted from 0.05 g of skin tissue 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).
mtDNA analysis
The first half of the mtDNA control region was amplified through the polymerase chain reaction (PCR).
Approximately 350bp of mtDNA region containing control region were amplified using the following
primer set: heavy-strand P2 (5'-GAAGAGGGATCCCTGCCAAGCGG-3' ; Hori et al., unpublished) andlight-strand MT4 (Árnason et al., 1993). PCR products were purified by MicroSpin S-400HR columns(Pharmacia Biotech). Cycle sequencing was performed with the same primers, using BigDye terminatorcycle sequence Kit (Applied Biosystems, Inc). The cycle sequencing products were purified by AutoSeq
G-50 spin Columns (Pharmacia Biotech). The labeled sequencing fragments were resolved byelectrophoresis through a 5% denaturing polyacrylamide matrix on an ABI 377 or ABI3100 Automated
DNA Sequencer (Applied Biosystems, Inc), following the protocols of the manufacture. For eachsample both strands were sequenced.
The number of mtDNA haplotypes and haplotype diversity were calculated following Nei (1987).
The nucleotide diversity (Nei, 1987: equation 10.5) and its standard error for population sampling andstochastic processes were calculated from the pairwise differences between the mtDNA sequences using1

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the Kimura’s 2- parameter adjustment (Kimura, 1980).
Microsatellite analysis
Microsatellite polymorphisms were analyzed using 15 sets of primers: EV1, EV5, EV94, EV104(Valsecchi & Amos 1996), GT011 (Bérubé et al. 1998), GT23, GT211, GT271, GT509, GT575 (Bérubé etal. 2000), GATA28, GATA98, GATA417 (Palsbøll et al. 1997), DlrFCB14, and DlrFCB17 (Buchanan etal. 1996). Only EV1 and EV5 were designed from a sperm whale, Physeter macrocephalus. DlrFcB14and DlrFcB17 were designed from a beluga whale, Delphinapterus leucas, while all other 11 sets ofprimers were from a humpback whale, Megaptera novaeanglia. Primer sequences and PCR cycling
profiles followed those of the original authors. PCR amplifications followed the manufacture’s
instructions for the use of Ex Taq DNA polymerase (Takara Shuzo). Amplified products wereelectrophoresed on a 6% polyacrylamide denaturating gel using a BaseStation 100 DNA fragment
analyzer (Bio-Rad), and alleles were sized manually in relation to an internal size standard (Genescan400HD, Applied Biosystems) and to sperm whale microsatellites of known size that were included oneach gel.
The number of microsatellite alleles and expected heterozygosity per locus was calculated using thesoftware FSTAT 2.9.3 (Goudet, 1995). Statistical tests for deviations from the expected Hardy-Weinberg
genotypic proportions were conducted using the software GENEPOP 4.0 (Rousset, 2008). Whensimultaneous multiple tests were conducted, Rice (1989) correction for the multiple tests was performed.
RESULTS AND DISCUSSION
The use of mtDNA and hypervariable microsatellite markers provides us great opportunity to acquireenough genetic variation to describe genetic stock structure. Especially for the microsatellite analysis in
this study, substantial level of genetic variation was observed and the number of loci analyzed was
doubled compared to seven in the previous report (Kanda et al., 2002). We believe these geneticmarkers are effective for the study of sperm whales’ stock structure in the western North Pacific.
mtDNA
Sequence variations at 347bp of the mtDNA control region in the North Pacific sperm whales resulted in
11 unique haplotypes. Haplotype diversity was 0.700 and nucleotide diversity was 0.0038 (standarderror=0.00069).
Lyrholm et al. (1996) reported that haplotype and nucleotide diversities for the 300bp of the mtDNA
control region, which was nearly homologous to our present study, among the 37 sperm whales collectedfrom the world-wide oceanic areas (North Atlantic n=10, North Pacific n=16, and Southern Hemispheren=11). Thirteen haplotypes were identified and the haplotype diversity was 0.74. Nucleotide diversity
was 0.0038 (SE=0.00059). The level of haplotype diversity in our sample of the North Pacific sperm
whales was similar to that in the sample of the sperm whales world-wide (Lyrholm et al. 1996) as well asthat reported in the samples of other baleen whales, such as common minke whales (Goto et al., 2009),
sei whales (Kanda et al., 2009a) and Bryde’s whales (Kanda et al., 2009b) in the North Pacific.
Contrary, although the level of nucleotide diversity estimated in our study was similar to that in Lyrholmet al. (1996) it was much lower than that reported from the other large baleen whales. Common minkewhales (Goto et al., 2009) and sei whales (Kanda et al., 2009a) in the North Pacific showed roughly twofold higher values and Bryde’s whales (Kanda et al., 2009b) showed approximately three-fold highervalue. Low nucleotide diversity appeared to be a characteristic of sperm whales, and Lyrholm et al.
(1996) described two alternative explanations for the low nucleotide diversity in the sperm whales: 1) the
highly biased substitution pattern, with most substitutions occurring as repeated transition at a few hotspot, and 2) genetic bottleneck in population size.
Microsatellites
All of the 15 microsatellite loci, 13 of which were designed from other whale species, were polymorphic(Table 2). The total number of alleles at each locus ranged from three to 17 with an average of 8.3.
Average expected heterozygosity at each locus ranged from 0.498 to 0.902 with an average of 0.724.
The level of microsatellite genetic diversity of the sperm whales used in this study was similar to thatof other oceanic areas. Lyrholm et al. (1999) analyzed sperm whales from the North Atlantic, North
Pacific, and Southern Hemisphere using nine sets of microsatellite primers, three (EV1, EV5, GATA28)
of which were same as those in this study. Lyrholm et al. (1999) showed that the level of the genetic
diversity of these oceanic samples were similar to each other with the average allele numbers of 10.0 andaverage heterozygosity of 0.72-0.73. The sperm whale sample from the Japanese coast had the average2SC/J09/JR33
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allele numbers of 7.0 and average heterozygosity of 0.73. Some of the loci showed low levels of genetic
diversity in our sample. Possibility of null alleles was excluded because no signal of such case wasdetected (e.g. Hardy-Weinberg genotypic proportions). Unlike one of the possible factors for themtDNA low diversity, the negative effect on genetic diversity at a population level appeared not to be areason because the low diversity was not consistent over all of the 15 loci.
All of the 15 loci showed no evidence of deviation from the expected Hardy-Weinberg genotypic
proportions (Table 2). At this point, we did not detect any signals of multiple stocks in the western
North Pacific off Japan from the JARPNII samples.
ACKNOWLEDGMENTS
We gratefully acknowledge the researchers and crew members that participated in JARPNII survey. We
also thank H. Oikawa and S. Azumi (ICR Ayukawa Laboratory) who collaborated in the process of DNA
extraction.
REFERENCES
Árnason, Ú., Gullberg, A., and Widegten, B. 1993. Cetacean mitochondrial DNA control region:
Sequences of all extant baleen whales and two sperm whale species. Mol. Biol. Evol. 10:960-970.
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
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.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered.
86:485-486.
Goto, M., Kanda, N., Kishiro, T., Yoshida, H., Kato, H. and Pastene, L.A. 2009. Mitochondrial DNA
analysis on stock structure in the western North Pacific common minke whales. Paper SC/J09/JR29
presented to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 10pp.
Kanda, N., Goto, M., and Pastene, L.A. 2002. Genetic analysis of sperm whales sampled during
JARPNII in 2000 and 2001. Paper SC/54/O17 Appendix11 presented to the Scientific Committeeof the International Whaling Commission. (unpublished).
Kanda, N., Goto, M. and Pastene, L.A. 2009a. Stock structure of Bryde’s whales in the western North
Pacific as revealed by microsatellite and mitochondrial DNA analyses. Paper SC/J09/JR31 presented
to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 8pp.
Kanda, N., Goto, M., Yoshida, H. and Pastene, L.A. 2009b. Stock structure of sei whales in the North
Pacific as revealed by microsatellite and mitochondrial DNA analyses. Paper SC/J09/JR32
presented to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished). 14pp.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions throughcomparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.
Nei, M. 1987. Molecular Evolutionary Genetics. New York: Columbia University Press.
Lyrholm, T., Leimar, O., and Gyllensten, U. 1996. Low diversity and biased substitution patterns in
the mitochondrial DNA control region of sperm whales: Implications for estimates of time sincecommon ancestry. Mol. Biol. Evol. 13:1318-1326.
Lyrholm, T., O. Leimar, B. Johanneson, and U. Gyllensten. 1999. Sex-biased dispersal in sperm
whales: contrasting mitochondrial and nuclear genetic structure of global populations. Proc. R. Soc.
Lond. B 266:347-354.
Palsbøll, P. J., Bérubé, M., Larsen, A.H. and Jørgensen, H. 1997. Primers for the amplification of triand tetramer microsatellite loci in baleen whales. Mol. Ecol. 6: 893-895.
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Ecol. 5: 151-156.

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Table 1. Sperm whales analyzed in this study.
Survey year
2000

2001

2002

2003

2004

2005

2006

2007

58

5

10

3

5

6

3

N

Table 2. Number of alleles detected (A), expected heterozygosity (He),the results of test for deviation from the expected Hardy-Weinberg
genotypic proportion at each of the 15 microsatellite loci (HW),
haplotype number, haplotype diversity, and nucleotide diversity with
its standard error at mtDNA analyzed in the samples (N=45) of sperm
whales collected during JARPNII.
Microsatellites

AHe

HW

DlrFCB14
DlrFCB17
EV1EV5EV94
EV104
GATA28GATA98GATA417
GT23GT011
GT211
GT271
GT509
GT575

11
17
98
11
5
3
93
4
11
6
14
10
3

0.838
0.902
0.562
0.708
0.762
0.674
0.635
0.849
0.575
0.498
0.867
0.776
0.883
0.776
0.559

n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.

mtDNA
Haplotype number
Haplotype diversity
Nucleotide diversity(standard error)

110.7000
0.0038(0.0009)n.s. = non significant

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Fig. 1Sighting positions of sperm whales collected during the JARPNII from 2000 to 2007.

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