SC/F16/JR49

A note on the genetic diversity of sperm whales in the western
North Pacific as revealed by mitochondrial and microsatellite
DNA
Luis A. Pastene1, Mutsuo Goto1 Mioko Taguchi2 and Toshihide Kitakado3
1

Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
Okitsunaka-cho 1347-3-302, Shimizu-ku, Shizuoka-shi, Shizuoka 424-0204, Japan
3
Tokyo University of Marine Science and Technology, 1-5-7 Konan, Minato-ku, Tokyo 108-0075, Japan
2

ABSTRACT
Genetic variation at 15 microsatellite DNA loci and mitochondrial DNA (mtDNA) control region
sequences (338bp) was examined in sperm whales collected during JARPNII from 2000 to 2013 in order
to examine the effectiveness of these genetic markers for studies of stock structure in this species.
Analyses of mtDNA and microsatellite markers in a total of 56 sperm whales (16 males; 40 females)confirmed that these genetic markers were variable enough to explore stock structure of sperm whales.
The overall heterozygosity over 15 loci was 0.730 while the nucleotide and haplotype diversity were0.0038 and 0.7188, respectively. Statistical tests found no evidence of deviation from the expected HardyWeinberg genotypic proportion at all of the 15 microsatellite loci. At this point, no signal of multiple
stocks of sperm whale in the western North Pacific off Japan was detected.
INTRODUCTION
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 helpful for future In-depth Assessment (IA) of this species
by the International Whaling Commission Scientific Committee (IWC SC).
As an initial step to accomplish the objective, microsatellite and mitochondrial (mt) DNA was used on
samples collected by JARPNII to examine genetic diversity and the effectiveness of these genetic markers
for studies on stock structure of the species. This is an update of the study started by Kanda et al. (2009).
MATERIALS AND METHODS
Samples and DNA extraction
A total of 56 sperm whales (16 males; 40 females) used in this study were collected during JARPNII
surveys from 2000 to 2013 in the western North Pacific (Figure 1).
The IWC guidelines for DNA data quality (IWC, 2009) were followed as much as possible (see Kanda etal., 2014). Skin tissues were stored in vials filled with 95% ethanol in –70°C until DNA extraction. Total
DNA was extracted from 0.05g 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).
Microsatellite analysis
Microsatellite polymorphisms were analyzed using 15 loci: EV1, EV5, EV94, EV104 (Valsecchi andAmos 1996), GT011 (Bérubé et al. 1998), GT23, GT211, GT271, GT509, GT575 (Bérubé et al. 2000),
GATA28, GATA98, GATA417 (Palsbøll et al. 1997), DlrFCB14, and DlrFCB17 (Buchanan et al. 1996).
Only EV1 and EV5 were designed from a sperm whale. DlrFcB14 and DlrFcB17 were designed from abeluga whale, while all other 11 loci were from a humpback whale. Primer sequences and PCR cycling
profiles followed those of the original authors. PCR amplifications followed the manufacturer’s
instructions for the use of Ex Taq DNA polymerase (Takara Shuzo). Amplified products were
electrophoresed on a 6% polyacrylamide denaturating gel using a BaseStation 100 DNA fragment

1analyzer (Bio-Rad), and alleles were sized manually in relation to an internal size standard (Genescan
400HD, Applied Biosystems) and to sperm whale microsatellites of known size that were included oneach gel.
The number of alleles per locus, expected heterozygosity per locus and inbreeding coefficient 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). The False
Discovery Rate (FDR) approach (Benjamini and Yekutieli, 2001) was used for adjustment of p-value incase of multiple comparisons.
mtDNA analysis
The first half of the mtDNA control region was amplified through the polymerase chain reaction (PCR)
using the following primer set: heavy-strand P2 (5'-GAAGAGGGATCCCTGCCAAGCGG-3'; Hori et al.,unpublished) and light-strand MT4 (Árnason et al., 1993). PCR products were purified by MicroSpin S400HR columns (Pharmacia Biotech). Cycle sequencing was performed with the same primers, using
BigDye terminator cycle sequence Kit (Applied Biosystems, Inc). The cycle sequencing products were
purified by AutoSeq G-50 spin Columns (Pharmacia Biotech). The labeled sequencing fragments wereresolved by electrophoresis through a 5% denaturing polyacrylamide matrix on an ABI 377 or ABI3100
Automated DNA Sequencer (Applied Biosystems, Inc), following the protocols of the manufacturers.
For each sample 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 using
the Kimura’s 2- parameter adjustment (Kimura, 1980).
RESULTS AND DISCUSSION
Given the level of genetic variation found, the genetic markers used are effective for future studies ofsperm whales’ stock structure in the western North Pacific.
Microsatellites
All the 15 microsatellite loci were polymorphic (Table 1). The total number of alleles at each locus ranged
from three at GT575, GATA41 and GATA28 to 18 at DlrFCB17, with an average of 8.7. The average
expected heterozygosity at each locus ranged from 0.480 at GT23 to 0.900 at DlrFCB17, with an averageof 0.730.
The level of microsatellite genetic diversity of the sperm whales used in this study was similar to that ofother oceanic areas. Lyrholm et al. (1999) analyzed sperm whales from the North Atlantic, North Pacific,
and Southern Hemisphere using nine microsatellite loci, three (EV1, EV5, GATA28) of which were sameas those in this study. Lyrholm et al. (1999) showed that the level of genetic diversity in these oceanic
samples were similar to each other with the average allele numbers of 10.0 and average heterozygosity of
0.720-0.730. These values were similar to those found in the present study.
Some of the loci showed low levels of genetic diversity in the JARPNII sample. Possibility of null alleles
was excluded because no signal of such case was detected (e.g. Hardy-Weinberg genotypic proportions).
The negative effect on genetic diversity of population level appeared not to be a reason because the low
diversity was not consistent over all of the 15 loci.
All of the 15 loci showed no evidence of significant deviation from the expected Hardy-Weinberg
genotypic proportions (Table 1). At this point, no signal of multiple stocks in the western North Pacificsperm whale was detected.
mtDNA
Sequence variations at 338bp of the mtDNA control region resulted in 11 unique haplotypes. Haplotype
diversity was 0.719 and nucleotide diversity was 0.0038 (SE=0.0006) (Table 2).
The levels of mtDNA diversity (nucleotide diversity and haplotype diversity) found for western North

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Pacific in the present study were similar to those reported by Lyrholm et al. (1996) for sperm whales
worldwide, using a similar segment of the mtDNA control region and similar sample size.
On the other hand the levels of mtDNA diversity was much lower than those reported for North Pacificcommon minke whale of the ‘O’ stock (Pastene et al., 2016a: SC/F16/JR40), Bryde’s whale (Pastene etal., 2016b: SC/F16/JR44) and sei whale (Pastene et al., 2016c: SC/F16/JR46). 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 hot spot, and 2) genetic
bottleneck in population size.
ACKNOWLEDGMENTS
We gratefully acknowledge the researchers and crew members that participated in JARPNII survey for thecollection of genetic samples. We thank H. Oikawa and S. Azumi (ICR) who collaborated in the process
of DNA extraction. We also thank N. Kanda (JANUS-Japan) for preparing the microsatellite data set usedin this study.
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studies relevant to IWC management advice. J. Cetacean Res. Manage. (Suppl.) 11:252-256.
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JAPRNII from 2000 to 2007 as revealed by mitochondrial DNA and microsatellite analyses. . Paper
SC/J09/JR33 presented to the JARPN II Review Workshop, Tokyo, January 2009 (unpublished).
5pp.
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/F14/J27rev). Paper
SC/65b/DNA1 presented to the IWC Scientific Committee, May 2014 (unpublished). 6pp.
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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.
Pastene, L.A., Goto, M., Taguchi, M. and Kitakado, T. 2016a. Updated genetic analyses based on
mitochondrial and microsatellite DNA indicated no sub-structure of the ‘O’ stock common minke

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whale in the western North Pacific. Paper SC/F16/JR40 presented to the JARPNII special permit
expert panel review workshop, Tokyo, February 2016 (unpublished). 40pp.
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and microsatellite DNA suggest possible stock differentiation of Bryde’s whales between management
sub-areas 1 and 2 in the North Pacific. Paper SC/F16/JR44 presented to the JARPNII special permit
expert panel review workshop, Tokyo, February 2016 (unpublished). 17pp.
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region sequencing and microsatellite DNA confirmed the occurrence of a single stock of sei whales inoceanic regions of the North Pacific. Paper SC/F16/JR46 presented to the JARPNII special permit
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Table 1. Number of alleles detected (A), expected heterozygosity (HE), the results of test for deviationfrom the expected Hardy-Weinberg genotypic proportion (HW) and inbreeding coefficient (FIS)at each of
the 15 microsatellite loci for western North Pacific sperm whale (n=56, 16 males; 40 females).

Microsatellite loci

A

HE

HW

FIS

GT509
GT271
EV1
EV5
GT211
GT575
DlrFCB14
GT23
DlrFCB17
EV94
GT011
GATA41
EV104
GATA98GATA28Overall

12
14
9
9
6
3
11
4
18
11
13
3
5
9
3
8,67

0,81
0,88
0,57
0,72
0,77
0,56
0,84
0,48
0,90
0,76
0,86
0,60
0,69
0,85
0,63
0,73

0,256
0,154
0,994
0,079
0,701
0,113
0,048
0,283
0,304
0,554
0,739
0,809
0,118
0,144
0,535
0,121

0,035
-0,052-0,191-0,036-0,070-0,256
0,063
0,142
0,024
0,015
0,007
0,042
0,101
-0,009-0,052-0,012

Table 2. Results of mtDNA diversity for western North Pacific sperm whale (n=56, 16 males; 40
females).

Number of
haplotypes
11

Nucleotide diversity(SE)
0.0038 (0.0006)

5

Haplotype diversity
0.7188

125E

135E

145E

155E

165E

50N

50N

40N

40N

30N
125E

135E

145E

155E

165E

30N

Figure 1. Positions of first sighting of sperm whales collected during the JARPNII from 2000 to 2016 (n=56, 16 males; 40 females).

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