SC/62/BRG5
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Preliminary mtDNA analysis of gray whales from Japan and Russia

Naohisa Kanda1, Mutsuo Goto1, V. Yu Ilyashenko2, and Luis A. Pastene1
1

The Institute of Cetacean Research, Tokyo, Japan

2

A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Science, Moscow, Russia

Contact email:kanda@cetacean.jp
ABSTRACT
Gray whales (Eschrichtius robustus) are divided into western and eastern populations. This study is thefirst to present the genetic data obtained from the gray whales migrating to or from the breeding groundof the western population along the Japanese coast. We conducted mitochondrial DNA analysis on thesamples of gray whales from Japan (western, N=6) and Russia (eastern, N=7) and analyzed the generated
data in comparison to those of Leduc et al. (2002) and Lang et al. (2004) to better understand the genetic
characteristics of these whales at the wider geographic area. The Japanese gray whales were those either
stranded on beach or bycaught on set net along the Japanese coast from 1995 to 2007, and the Russian
gray whales were those legally caught during the Chukotka aboriginal subsistence whaling in 2008. Allof the mtDNA haplotypes found in the Japanese (five) and Russian (six) samples matched to some of thepreviously reported haplotypes.

The level of genetic diversity of these samples described as haplotype

diversity and nucleotide diversity were surprisingly high, suggesting either gene flow between thewestern and eastern populations or retention of ancient polymorphism without gene flow. No
statistically significant difference in haplotype frequencies was detected between the JPN and RUS
samples possibly due to the small sample sizes.

The phylogenetic analysis of the mtDNA haplotypes

found in this study and the past studies detected no distinct cluster for the Japanese whales, supporting thepast observation that the western and eastern gray whales were indistinguishable at the evolutionary timescale.
KEYWORDS: GRAY WHALES, PACIFIC OCEAN, GENETICSINTRODUCTION
Gray whales (Eschrichtius robustus) distribute along the North Pacific coast from Asia and Russia to
United States and Mexico and are divided into the western (Okhotsk-Korea) and eastern(Chukotka-California) populations (Rice, 1998). The eastern gray whales breed in the Baja California
waters in winter and feed in the Bering and Chukchi Seas during summer, while the western gray whales
breed in the coastal waters near China and feed in the Okhotsk Sea off Sakhalin Island. After severe
reduction of the population size during the commercial whaling period, the eastern population recovered
its abundance to near pre-exploitation level but the western one has been believed to still remain small1SC/62/BRG5
Do not cite without written permission from the authors(IWC, 1998).

Conservation and management of this species has been of concern for the range countries.

Identification of population structure is essential for effective management.

Past genetic studies

based on the genetic variations at mitochondrial DNA (mtDNA) and microsatellites demonstrated that thewestern and eastern populations were genetically distinct at the population level but not at theevolutionary level (Leduc et al., 2002; Lang et al., 2004). The population structure of the species hasn’t
been fully resolved yet, however, partly because the sample of what they call the western population inthese past studies limited to the individuals from the narrow part of feeding ground off Sakhalin Island.
These whales could have been the mixture of whales from both populations (e.g., Lang et al., 2004) or
could have come from the eastern population (e.g., Ilyashenko, 2009). Lang et al. (2004) found highgenetic diversity within the samples and the differences in the level of genetic differentiation between
males and females, that made the authors suspect extended migration of some eastern gray whales,
especially males, to the area off Sakhalin Island. Ilyashenko (2009) proposed that the current graywhale population migrating to the area off Sakhalin Island was originated from the eastern gray whales
recolonized after the extinction of the species in this area during the commercial whaling period. Inorder to address this issue and to conduct effective management of gray whales, it is important to analyze
gray whales obtained from the entire range of the species.
This study is the first to present the genetic data obtained from the gray whales migrating to or fromthe breeding ground of the western population along the Japanese coast (Fig. 1; Kato et al., 2010). We
conducted mtDNA analysis on the samples of gray whales from Japan (western, N=6) and Russia (eastern,
N=7) and analyzed the generated data in comparison to those of Leduc et al. (2002) and Lang et al.
(2004) to better understand the genetic characteristics of these whales at the wider geographic area.
MATERIALS AND METHODS
SamplesThree stranded gray whales on beach and three bycaught ones on set net along the Japanese coast from1995 to 2007 were used for this study (JPN; Table 1). Detailed information on these six animals can beseen in Kato et al. (2010). Seven gray whales caught during the Chukotka aboriginal subsistence
whaling in 2008 were also used (RUS; Table 1).
mtDNA analysis
Total DNA from each of the whales was extracted from 0.05 g of skin or muscle 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).

The first half (486 bp) of the mtDNA control region was amplified through the polymerase chain

reaction (PCR) using the following primer set: light-strand MT4 (Árnason et al., 1993) and heavy-strand
Dlp 5R (5'-CCATCgAgATgTCTTATTTAAggggAAC-3'). PCR products were purified by MicroSpin
S-400HR 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 ABI3100™ Automated
DNA Sequencer (Applied Biosystems, Inc) following the protocols of the manufacture.
both strands were sequenced.

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For each sample

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Data analysis
Published mtDNA sequences in Leduc et al. (2002) were extracted from GenBank for haplotype
designation and data comparison.

The published data was generated from analyzing eastern gray whale

sample (N=120) collected from the several locations along the coast from Chukotka to California andwestern gray whale sample (N=45) collected from the northeastern coast of Sakhalin Island. Because
analyzed length of the sequence in our study was 37 bp shorter than that of Leduc et al. (2002), thehaplotype G and O (hereafter, G/O), L and U (L/U), W and X (W/X) in the original paper became
indistinguishable, reducing the total number of the haplotypes from 36 in the original paper to 33.
The number of haplotypes and haplotype diversity were calculated following Nei (1987). The
nucleotide diversity (Nei, 1987: equation 10.5) and its standard error (se) for population sampling andstochastic processes were calculated from the pair-wise differences between the mtDNA sequences using
the Kimura’s 2- parameter adjustment (Kimura, 1980). The randomized chi-square test of independence(Roff and Bentzen, 1989) was used to investigate the level of genetic differentiation. In each test a totalof 10,000 permutations of the original data were performed.

The level of genetic differentiation

expressed as HST was calculated based on Hudson et al. (1992).
Phylogenetic reconstruction of the mtDNA sequences was made using the neighbor-joining method
implemented in the PHYLIP version 3.5c program (Felsenstein, 1993). Genetic distance among
haplotypes were estimated using the program DNADIST of the PHYLIP based on Kimura’s 2- parametermodel (Kimura, 1980) with an observed transition:transversion ratio of 1:20. The obtained tree wasvisualized using TreeView PPC (Page, 1996).
RESULTSOn the basis of sequence variation at the 486 bp of control region, five different mtDNA haplotypes were
found from the six Japanese gray whales, while six different haplotypes were found from the seven
Russian gray whales (Table 2).

These haplotypes of the JPNs matched to haplotypes A (N = 1), B (1),

G/O (1), L/U (2), and Z (1) in Leduc et al. (2002), whereas the haplotypes of the RUSs matched to C (2),H (2), R (1), V (1), AE (1), so that total number of haplotypes found from the two studies was still 33.
Haplotype diversity and nucleotide diversity within samples was 0.933 and 0.0185 (se = 0.0058) for theJapanese sample and 0.952 and 0.0163 (se = 0.0032) for the Russian sample, respectively. The level ofgenetic differentiation between the Japanese and Russian samples expressed as HST was 0.0316 which
was not significantly different from 0 (p=0.164). Statistical test failed to detect statistically significant
difference in the haplotype frequencies between the JPN and RUS (p=0.211).

Because it was difficult to

extract the exact number of haplotypes within the western and eastern population samples from Lang et al.
(2004), no statistical comparison was conducted between the samples from this study and the past study.
The neighbour-joining tree was constructed for 33 haplotypes (Fig. 2). No geographically specificcluster was detected.
DISCUSSION
We analyzed the sample of the gray whales collected along the Japanese coast.

Therefore, significanceof this study is that these analyzed whales definitely came from the western population. We did not have3

SC/62/BRG5
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to worry about that we might have collected the individuals from the eastern population that migrated to
the feeding ground of the western population.
The results of this study were very same to those of the past genetic studies (Leduc et al., 2002; Langet al., 2004): the genetic diversity within each of the two samples was high and the eastern and western
populations were phylogenetically indistinguishable. As already presented by the past genetic studies,
the level of genetic diversity within the western population was surprisingly high as compared with atypical small population.

Four possible explanations can be raised.

Firstly, individual(s) from theeastern population could have been wrongly sampled as the western population. Secondly, there may begene flow between the two populations.

Thirdly, because of the long life and historically large

abundance, the western population may still retains considerable amount of genetic diversity after sever,
but recent, population reduction.

Finally, the population size of the western population may not be as

small as it has been estimated (e.g., Cooke et al., 2008). Among these, the first one is the most unlikely
because our sample came from migratory corridor between the feeding and breeding grounds of thewestern population. The site fidelity of gray whales during the breeding migration has been believed
strong. If all of the western gray whales feed at the area off Sakhalin Island and have been completely
covered through photo-identification, the population size of the western population is indeed small as
estimated (Cooke et al., 2008) and thus the fourth reason raised above becomes unlikely.

Among theremained two, it is hard to decide. Because of the small sample size as no statistically significant
genetic differentiation was detected between the Japanese and Russian samples, it was difficult to reliably
estimate the level of gene flow in this study. The population reduction during the commercial whaling
period was quite severe biologically but might not have been genetically, allowing the western population
to retain ancient diversity. Continued population monitoring and survey is important to estimate
population size and describe migration pattern of gray whales.
Five of the six individuals in the Japanese sample had the different haplotypes and those haplotypes
were widespread in the phylogenetic tree. Although two (L/U and Z) of the haplotypes found in theJapanese sample were referred as the eastern population types in Leduc et al. (2002), the analysis of theadditional individuals in Lang et al. (2004) detected these two in the sample collected from the area off
the Sakhalin Island. Contrary to the large differences in the numbers and frequencies of the different
haplotypes within the samples from the same region among the different studies (i.g., Leduc et al., 2002;
Lang et al., 2004; this study), the total number of the different haplotypes found from these studies did
not changed as much.

This indicated that the inference drawn from the phylogenetic analysis in thisstudy should reflect gray whales’ evolutionary history. Sharing of quite many haplotypes between thetwo populations and their positions in the phylogenetic tree indicated recent divergence of the populations
within the species.
During the last year’s scientific committee meeting (IWC, in press), population status of the graywhales feeding at the area off Sakhalin Island, whether the whales were the member of the eastern,western or mixture of both populations, was discussed (Brownell et al., 2009; Ilyashenko, 2009). On thebasis of the results from mtDNA and microsatellite analyses, Lang et al. (2004) showed that the Sakhalin
gray whale sample was genetically different from the eastern gray whale sample but raised at the sametime the possibility of extended migration of the eastern gray whales to the area off the Sakhalin Island.

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SC/62/BRG5
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With increase of the sample size, the newly analyzed individuals in the Sakhalin sample tended to havethe haplotypes originally found only in the eastern sample but not newly discovered (see Leduc et al.,2002 and Lang et al., 2004). This could reflect the level of the ancient polymorphism retained in the
Sakhalin sample or this could indicate that the number of the eastern gray whales migrating to the area off
Sakhalin Island is more than we have anticipated.

Use of our Japanese gray whales as a reference basesample is suitable to address this kind of issue. However, it is unfortunate that its sample size is small
and only mtDNA data is available at this moment. We are thus planning to analyze our samples withmore than ten microsatellite markers that should overcome the reluctance for population structure analysis
attributable to the small sample size.
ACKNOWLEDMENTS
We wish to express our sincere appreciation to the following people for their assistance and advice during
the sample collection and manuscript preparation: J. Morishita, K. Ueda, and T. Uoya from Fisheries
Agency of Japan, H. Kato from Tokyo University of Marine Science and Technology, T. Bando, Y. Fujise,
H. Hatanaka, H. Ishikawa, T. Isoda, T. Mogoe, and S. Otani from Institute of Cetacean Research, I.
Mikhno from Chukotka Government and A. Ottoy - Aboriginal whalers captain from Lorino village in
Chukotka.
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.
Brownell, R.L., Jr., Lang, A.R., Burdin, A.M., Bradford, A.B., and Weller, D.W. 2009. The westerngray whale population is distinct: a response to SC/61/BRG22. Paper SC/61/BRG30 presented to
the 61 Scientific Committee meeting.
Cooke, J.G., Weller, D.W., Bradford, A.L., Burdin, A.M., and Brownell, R.L., Jr. 2008. Populationassessment of western gray whales in 2008. Paper SC/60/BRG 11 presented to the 60 SientificCommittee meeting.
Felsenstein, J. 1993.

PHYLIP (Phylogeny Inference Package) 3.5c. University of Washington,

Seattle.
Hudson, R.R., Boos, D.D., and Kaplan, N.L. 1992. A statistical test for detecting geographic
subdivision. Mol. Biol. Evol. 9: 138-151.
Ilyashenko, V.Y.

2009. How isolated is the “western” gray whale population?

Paper SC/61/BRG22

presented to the 61 Scientific Committee meeting.
International Whaling Commission. 1998.

Report of the Scientific Committee. Annex J.

Report of

the sub-committee on aboriginal subsistence whaling. Rep. int. Whal. Commn. 48: 237-248.
International Whaling Commission.

in press.

Report of the Scientific Committee.

Annex F.

Reportof the sub-committee on bowhead, right and gray whlaes. J. Cetacean Res. Manage.
Kato, H., Miyashita, T., Kanda, N., Ishikawa, H., Furukawa, H., and Uoya, T. 2010. Status report ofconservation and researches on the western gray whales in Japan, May 2008 – April 2010. Paper
SC/62/O7 presented to the 62 Sceintific Committee meeting.

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Kimura, M. 1980.

A simple method for estimating evolutionary rates of base substitutions throughcomparative studies of nucleotide sequences.

J. Mol. Evol. 16: 111-120.

Lang, A.R., Weller, D.W., Leduc, R.G., Burdin, A.M., Hyde, J., and Brownell, R.L., Jr. 2004. Genetic
differentiation between western and eastern gray whale populations using microsatellite markers.
Paper SC/56/BRG38 presented to the 56 Scientific Committee meeting.
Leduc, R.G., Weller, D.W., Hyde, J., Burdin, A.M., Rosel, P.E., Brownell, R.L., Jr., Würsig, B., and Dizon,
A.E. 2002. Genetic differences between western and eastern gray whales (Eschrichtius robustus).
J. Cetacean Res. Manage. 4: 1-5.
Nei, M. 1987. Molecular Evolutionary Genetics.
Page, R.D.M. 1996.

Columbia University Press, New York.

TREEVIEW: An application to display phylogenetic trees on personal computers.

Compu. Appli. Biosci. 12: 357-358.
Rice, D.W. 1998. Marine Mammals of the World: Systematics and Distribution. Society for Marine
Mammalogy, Lawrence.
Roff, D.A., and Bentzen, P.

1989. The statistical analysis of mtDNA polymorphisms: χ2 and the

problem of small samples. Mol. Biol. Evol. 6: 539-545.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989.

Molecular Cloning: A Laboratory Manual, 2nd edn.

Cold Spring Harbor Laboratory, New York.

Chukchi Sea

70N

70N

Russia60N

60N

Bering Sea
Okhotsk Sea

50N

Sakhalin Is.

→
Pacific Ocean← Japan

40N

30N
120E

50N

130E

140E

150E

160E

170E

40N

180E

170W

160W

30N
150W

Fig. 1. Sample locations. Black dots indicate the sampling locations of the 6 Japanese gray whales,
and open circle indicates the approximate area of the Chukotka aboriginal subsistence whaling colletingthe 7 Russian gray whales for this study.

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LHAPA JPN-2
LHAPAD
LHAPF
LHAPG/O JPN-1
LHAPL/U JPN-3 JPN-6
LHAPM
LHAPT
LHAPN
LHAPAF
LHAPAA
LHAPAE
LHAPV
LHAPAJLHAPE
LHAPB JPN-5
LHAPZ JPN-4
LHAPC
LHAPK
LHAPD
LHAPAH
LHAPI
LHAPAB
LHAPACLHAPAG
LHAPH
LHAPR
LHAPS
LHAPW/X
LHAPY
LHAPP
LHAPAI
LHAPJ
LHAPQ
0.01

Fig. 2. Neighbour joining tree of the gray whales’ mtDNA haplotypes.
LHAPs were extracted from Leduc et al. (2002).

JPN-1 to JPN-5 was the Japanese gray whales 1 to 5 inTable 1.

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Table 1. Samples analyzed in this study.
Ocean

Area

Year

Leduc-E (N=120) *Leduc-W (N=45) *Eastern /Western
Eastern
Western

Bering/PacificOkhotsk

Chukotka - California
Sakhalin

1990th1990th

JPN-1
JPN-2
JPN-3
JPN-4
JPN-5
JPN-6

Western
Western
Western
Western
Western
Western

Pacific
Sea of Japan
Pacific
Pacific
Pacific
Pacific

Hokkaido
Hokkaido
Chiba
Miyagi
Iwate
Hokkaido

RUS-23
RUS-26
RUS-28
RUS-37
RUS-39
RUS-40
RUS-47

Eastern
Eastern
Eastern
Eastern
Eastern
Eastern
Eastern

BeringBeringBeringBeringBeringBeringBering

Chukotka
Chukotka
Chukotka
Chukotka
Chukotka
Chukotka
Chukotka

Month

Sex

Body length

1995
1996
2005
2005
2007
2007

Apr.
May
May
Jul.
Jan.
Aug.

F
--F
F
F
F

9.5
--7.8
12.8
9.2
12.3

2008
2008
2008
2008
2008
2008
2008

Jul.
Aug.
Aug.
Aug.
Sep.
Sep.
Sep.

M
M
M
F
M
F
F

8.2
8.1
8.9
12.0
11.1
12.3
13.4

* Data from Leduc et al. (2002): Leduc-E corresponds to their eastern population sample, while Leduc-W to their western population one.
Table 2. Haplotype distributions within the samples used in this study.

A B C D
Leduc-E 13 11 12 10
Leduc-W 20 15 2 2Japan
1 1 Russia
- 2 -

E
2
1
-

F G/O H
8 1
1 1 1
1
1

I
1
-

J
1
-

K L/U M
4
6 6
2
-

N
4
-

*
mtDNA haplotype
P Q R S T3
1 3 1 8- 1 -*Haplotype designation was according to Leduc et al. (2002).

8

V
2
1

W/X
2
-

Y
5
-

Z AA AB AC AD AE AF AG AH AI AJ Total
2 2 2 2 2 1 1 3 1 1 1
120
45
1 6
- 1 - 1 7