SC/F16/JR50

A note on the genetic diversity and phylogeny of western
North Pacific and southern right whales based on
mitochondrial and microsatellite DNA
Luis A. Pastene1, Mutsuo Goto1 Mioko Taguchi2, Shigetoshi Nishiwaki1 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 14 microsatellite DNA loci and mitochondrial DNA (mtDNA) control region
sequences (275bp) was examined in right whales from the western North Pacific and Antarctic Area IV.
Genetic analyses were based on biopsy samples collected during the surveys of the JARPNII in 2011 and2012 (n=15), and JARPAII in 1993/94-2009/10 (n=67). The overall heterozygosity was 0.630 and 0.650
for North Pacific and southern right whales, respectively, while the nucleotide diversity/haplotype
diversity were 0.0222/0.9048 and 0.0234/0.7743, respectively. Statistical tests found no evidence of
deviation from the expected Hardy-Weinberg genotypic proportion in each of the oceanic basins. The
Kimura’s two parameter net interpopulational distance was 0.0358 (mtDNA) while the Nei’s genetic
distance (Da) was 0.7582 (microsatellite DNA), between North Pacific and southern right whale. A
phylogenetic tree separated clearly mtDNA haplotypes of the North Pacific, North Atlantic and southern
right whales.
INTRODUCTION
Rosenbaum et al. (2000) provided mtDNA phylogenetic evidences to separate North Atlantic, North
Pacific and Southern Hemisphere right whales into three distinct lineages. Consequently, whales fromthese three oceanic basins have been considered different species Eubalaena glacialis, E. japonica and E.
australis, respectively.
There are several genetic studies on southern right whales, which indicate that this species is divided into
several genetic stocks: Argentina, South Africa, Western Australia, and the New Zealand sub-Antarctic(Baker, et al., 1999; Patenaude et al., 2007; Carrol et al., 2011). Apart from the study by Carrol et al.
(2011) that used both mtDNA and microsatellite DNA, these studies have been based largely on mtDNA.
None of the genetic studies on southern right whales have examined genetic samples from whales in the
Antarctic. Kanda et al. (2014a) examined the population genetic structure of southern right whales from
Antarctic Area IV based on mtDNA and microsatellite DNA at 14 loci, and suggested that some whales
could migrate long distance between breeding grounds and this feeding ground in the Antarctic.
In contrast, little is known about the population genetic structure of North Pacific right whales. A numberof biopsy samples were obtained by JARPNII surveys in the western North Pacific, and these were
examined genetically in this study to investigate level of genetic diversity in this endangered species. This
study also compares the levels of genetic diversity between North Pacific and southern right whales
examined by Kanda et al. (2014a). Finally this study expands the phylogenetic analysis of right whales
worldwide using all available mtDNA sequences.
MATERIALS AND METHODS
Samples
Biopsy samples were obtained during the dedicated sighting surveys under JARPNII in the western North
Pacific in 2011 and 2012, and JARPAII in Antarctic Area IV between 1993/94 and 2009/10 austral1

summer seasons. A total of 15 and 67 biopsy samples were available for North Pacific and southern rightwhales, respectively. Figure 1 shows the distribution of the samples.
DNA extraction
The IWC guidelines for DNA data quality (IWC, 2009) was followed as much as possible (see Kanda etal., 2014b). Total DNA was extracted from 0.05g skin biopsy 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
Genetic variation at microsatellite DNA was analyzed using 14 loci, none of which was designed
specifically from right whales: EV1, EV14, EV21, EV37, EV94 (Valsecchi and Amos, 1996), GT23,
GT211, GT310 (Bérubé et al., 2000), GATA28 (Palsbøll et al., 1997), DlrFCB17 (Buchanan et al., 1996),
TR2F3, TR3G2, TR2G5 and TR3F2 (Frasier et al., 2006). Primer sequences and PCR cycling profiles
generally followed those of the original authors.
PCR amplifications were performed in 15l reaction mixtures containing 10-100ng of DNA, 5 pmole ofeach primer, 0.625 units of 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). Although alleles werevisualized using Cartographer software specifically designed for the BaseStation, allelic sizes were
determined manually in relation to the internal size standard and right whale’s DNA of known size thatwere rerun on each gel.
Data analysis
Level of polymorphisms
The number of alleles per locus, expected heterozygosity per locus and inbreeding coefficient per locus
were 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).
Genetic differentiation
Conventional hypothesis testing procedure was conducted using heterogeneity test in microsatellite allele
frequencies among samples. Probability test (or Fisher’s exact test) implemented in GENEPOP 4.0(Rousset, 2008) was used to conduct the heterogeneity tests. Statistical significance was determined using
the chi-square value obtained from summing the negative logarithm of p-values over the 14 microsatellite
loci (Sokal and Rohlf, 1995). The False Discovery Rate (FDR) approach (Benjamini and Yekutieli, 2001)
was used for adjustment of p-value in case of multiple comparisons. FST value was calculated using
FSTAT 2.9.3 (Goudet, 1995).
Individual matching
Matching exercises between the individuals were conducted using a computer program CERVUS(Marshall et al., 1998).
Mitochondrial DNA
Sequencing analysis of the 275bp control region of mtDNA was conducted using the primers light-strand
MT4 (Árnason et al., 1993) and heavy-strand P2 (5'-GAAGAGGGATCCCTGCCAAGCGG-3'; Hori etal., unpublished). 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 were resolved 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 manufacturer. For each sample both strands weresequenced.

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Data analysis
Level of polymorphisms
The number of haplotypes and haplotype diversity were calculated following Nei (1987). The nucleotide
diversity (Nei, 1987: equation 10.5) and its standard error for population sampling and stochastic
processes were calculated from the pair-wise differences between the mtDNA sequences using theKimura’s 2- parameter adjustment (Kimura, 1980).
Phylogenetic analysis
The genealogy of the mtDNA haplotypes was estimated using the Neighbor-Joining method (Saitou andNei 1987) as implemented in the program PHYLIP (Felsenstein 1993). Genetic distances among
haplotypes were estimated using the program DNADIST of PHYLIP, based on Kimura’s 2-parametermodel (Kimura 1980). A transition-transversion ratio of 5:1was used. The genealogy was rooted using thehomologous sequence from bowhead whale. To estimate support for each node a total of 1,000 bootstrap
simulations were conducted and the majority-rule consensus genealogy estimated.
RESULTS AND DISCUSION
Microsatellites
Level of polymorphisms
All 14 loci analyzed were polymorphic. For total samples (North Pacific and Antarctic) the total numberof alleles per locus ranged from two at the TR2F3 to 19 at the EV1 with an average of 8.36 (Table 1).
Expected heterozygosity at each of the loci ranged from 0.22 at EV21 to 0.92 at DlrFCB17 with anaverage of 0.68. As expected, significant deviation from the expected Hardy-Weinberg genotypic
proportions was found. Consistently the magnitude of FIS was large in several loci as expected if thesamples were collected from different stock.
For North Pacific right whales, the total number of alleles per locus ranged from two at the EV21 to 7 at
the GATA28 with an average of just 4.15 (Table 2). Expected heterozygosity at each of the loci ranged
from 0.30 at TR3F2 and EV94 to 0.81 at EV37 with an average of 0.60. No significant deviation from theexpected Hardy-Weinberg genotypic proportions was observed although the magnitude of FIS was large
in several loci.
For southern right whales, the total number of alleles per locus ranged from two at the TR2F3 and TR2G5
to 14 at the EV1 with an average of 6.93 (Table 3). Expected heterozygosity at each of the loci ranged
from 0.09 at EV21 to 0.89 at DlrFCB with an average of 0.65. No significant deviation from the expected
Hardy-Weinberg genotypic proportions was observed although the magnitude of FIS was large in someloci.
Expected heterozygosities estimated for North Pacific and southern right whales were similar although
the average number of alleles per locus was smaller in the North Pacific. Values of heterozygosity andaverage number of alleles were smaller than those estimated by Carrol et al. (2011) for whales in several
localities of the Southern Hemisphere examined with 13 loci. The ranges estimated by those authors were0.80-0.82 and 6.31-12.15, respectively. These differences could be related to sample size (substantiallylarger in that study), localities (several localities in that study) and loci used (different sets between thetwo studies).
Genetic differentiation
As expected FST value between North Pacific and southern right whales was high (0.176 over all the loci)and highly significant (Table 4). Nei’s genetic distance (Da) was estimated at 0.7582.
Movement (mark-recapture)
There was no case of duplicate sampling in the western North Pacific. In the case of the Antarctic there
were two cases of re-sampling involving different years. These duplicates were confirmed by genotype
profiles as well by photo-identification (see details in Kanda et al., 2014a). These finding in the Antarctic
suggest fidelity to feeding areas in Area IV.

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mtDNA
Levels of polymorphisms
Nucleotide diversity was similar between North Pacific and southern right whales, and these wererelatively high and similar to estimates for several regions of the Southern Hemisphere. Haplotype
diversity was somewhat higher in the North Pacific (Table 5). The values of haplotype and nucleotide
diversity estimated for several localities of the Southern Hemisphere ranged from 0.595-1.000 and 0.0140.033, respectively (Patenaude et al., 2007). The estimates by Carrol et al. (2011) were 0.67-0.95 and0.017-0.028, respectively.
Genetic differentiation
The Kimura’s two parameter net interpopulational distance between North Pacific and southern rightwhales was estimated at 0.036. This value is much lower than those among recognized large whale
species and lower than common minke whales from different ocean basins, which are currently
recognized as sub-species.
Phylogenetic analysis
The phylogenetic analysis of haplotypes was based on an enlarged samples size with regard thephylogenetic analysis conducted by Rosenbaum et al. (2000). Haplotypes from North Pacific, North
Atlantic and southern right whales were placed in different clusters. Within the southern right whale
cluster, two sub-clusters were observed, which were supported by high bootstrap value. However these
sub-clusters were not geographically specific. Results confirmed that North Pacific, North Atlantic andSouthern Hemisphere right whales are different Evolutionary Significant Units.
ACKNOWLEDGEMENTS
We gratefully acknowledge the researchers and crew members that participated in JARPN/JARPNII and
JARPAII surveys for the collection of biopsy 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 used in this study.
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Table 1. The number of alleles (A), expected heterozygosity (HE), test results for expected HardyWeinberg genotypic proportion (HW) and inbreeding coefficient (FIS) in right whales from the North
Pacific and Antarctic, at 14 microsatellite loci.
Microsatellite loci
EV1
GT310
GT23
EV94
EV14
GT211
EV37
GATA28EV21
DlrFCB17
TR2F3
TR3G2
TR2G5
TR3F2
Overall

A
19
5
9
6
10
11
10
10
3
18
2
7
3
4
8,36

F IS
0,09
0,05
0,04
-0,01
0,03
0,19
0,07
0,04
0,21
0,10
0,03
-0,01
-0,04
0,23
0,07

HE
0,91
0,68
0,80
0,41
0,78
0,85
0,84
0,80
0,22
0,92
0,49
0,80
0,51
0,53
0,68

HW
<0.001
0,003
0,598
0,975
0,425
<0.001
0,082
0,535
0,179
<0.001
1,000
0,175
0,649
0,065
High. Sign.

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Table 2. The number of alleles (A), expected heterozygosity (HE), test results for expected HardyWeinberg genotypic proportion (HW) and inbreeding coefficient (FIS) in North Pacific right whale, at 13
microsatellite loci.
Microsatellite loci
EV1
GT310
GT23
EV94
EV14
GT211
EV37
GATA28EV21
DlrFCB
TR3G2
TR2G5
TR3F2
Overall

A
5
3
5
3
4
3
5
7
2
6
5
3
3
4,15

F IS
0,14
0,18
0,00
-0,11
0,06
-0,03
0,01
-0,01
-0,31
0,18
-0,15
0,21
-0,11
0,02

HE
0,78
0,65
0,73
0,30
0,57
0,45
0,81
0,79
0,51
0,74
0,70
0,51
0,30
0,60

HW
0,695
0,018
0,760
1,000
0,253
1,000
0,070
0,488
0,328
0,128
0,882
0,356
1,000
0,384

Table 3. The number of alleles (A), expected heterozygosity (HE), test results for expected HardyWeinberg genotypic proportion (HW) and inbreeding coefficient (FIS) in Antarctic right whale, at 14
microsatellite loci.
Microsatellite loci
EV1
GT310
GT23
EV94
EV14
GT211
EV37
GATA28EV21
DlrFCB
TR2F3
TR3G2
TR2G5
TR3F2
Overall

A
14
5
8
5
9
10
10
8
3
12
2
6
2
3
6,93

F IS
0,02
-0,04
0,03
-0,01
-0,08
0,10
0,04
0,02
0,31
0,01
0,03
-0,05
-0,10
0,12
0,01

HE
0,88
0,65
0,80
0,43
0,75
0,82
0,81
0,78
0,09
0,89
0,49
0,77
0,50
0,47
0,65

HW
0,390
0,094
0,797
1,000
0,934
0,145
0,691
0,661
0,115
0,438
1,000
0,545
0,468
0,620
0,768

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Table 4. Results of the microsatellite DNA heterogeneity test between North Pacific and Antarctic rightwhales. Bold indicate significant differences after FDR correction.

Microsatellite lociP -value
EV1<0.001GT310<0.001
GT23
0,001
EV94
0,006
EV14
<0.001
GT211<0.001EV37<0.001
GATA28<0.001EV21<0.001
DlrFCB17<0.001
TR3G2<0.001
0,058
TR2G5
TR3F2
<0.001
OverallHigh. Sign.

F ST
0,166
0,134
0,056
0,047
0,216
0,294
0,131
0,066
0,522
0,179
0,155
0,018
0,379
0,176

Table 5. Results of mtDNA diversity for North Pacific and Antarctic right whale.
Location
Number of haplotypes
Nucleotide diversity (SE)North Pacific (n=15)
Antarctic (n=67)

0E

20E

40E

60E

80E

8
10

100E

120E

140E

160E

0.02223 (0.00279)
0.02343 (0.00093)

180E

160W

140W

120W

100W

80W

60W

40W

20W

0.90476
0.77431

0W

65N

65N

45N

45N

25N

25N

5N

5N

15S

15S

35S

35S

55S

55S

75S

0E

20E

40E

60E

80E

100E

120E

140E

160E

180E

160W

140W

120W

100W

80W

60W

40W

20W

Haplotype diversity

0W

75S

Figure 1. Geographical distribution of right whales examined in this genetic study. Blue: biopsy samples
from JARPNII; Red: biopsy samples from JARPAII.

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Figure 2. Phylogenetic relationship among right whales mtDNA haplotypes from several ocean basins.

8