SC/F16/JR38

Temporal and spatial distribution of the ‘J’ and ‘O’ stocks ofcommon minke whale in waters around Japan based on
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
A total of 4,275 western North Pacific common minke whales were examined with a set of 16
microsatellite DNA loci and the program STRUCTURE to assign individual to either J or O stocks.
Samples were available from JARPN/JARPNII (1994-2014; n= 2,637)), and by-catches (2001-2014; n=1,638), from different management sub-areas (SA) around Japan. Results of the Bayesian clustering
analysis confirmed that the whales came from two genetically differentiated stocks, J and O stocks. The
number of unassigned individuals (‘unknown’) decreased with the increase in the number ofmicrosatellite loci used, and they were widely distributed. By using 16 loci, more than 90% of theindividual whales were assigned to either stocks. Almost all of the individuals collected from the Sea ofJapan side (SA6 and SA10E) belonged to the J Stock, whereas almost all of the individuals from theoffshore North Pacific (east of SA7WR) belonged to the O stock. Intermediate areas (SA7CN, 7CS andSA11) contained individuals from both stocks. The SA2 was mainly occupied by the J stock. In SA2 the J
stock was predominant (around 80% in proportion) around the year. In SA7CS and SA7CN the
proportion of the J stock increase in autumn/winter and decrease in spring/summer. A phylogenetic treeof mtDNA haplotypes showed several clades but none supported by high bootstrap values. There was nostock-specific clade although most of the individuals assigned to the J stock shared a same clade. Most of
the individuals assigned to the O stocks share clades where the J stock individuals were less frequent. The
unknown samples were widely distributed through the clades.

INTRODUCTION
At least two different stocks of common minke whales are known to occur around the Japanese coast: one
stock distributes in the western North Pacific (‘O’ stock) and the other in the Sea of Japan (‘J’ stock)(Omura and Sakiura, 1956; Ohsumi, 1977; Kato, 1992; Wada and Numachi, 1991; Goto and Pastene,
1997; Hatanaka and Miyashita, 1997; Pastene et al., 2007). Whales of both stocks migrate to the Okhotsk
Sea in spring and stay there until the end of summer. Although they share feeding ground in the Okhotsk,
their temporal distribution in the area slightly differ (Goto and Pastene, 1997). Recent genetic analyses
suggested that the J stock distribute in the Pacific side of Japan (Kanda et al., 2009a). These two stocks
differ from each other in body size, conception dates, allozyme allele frequencies, microsatellite allele
frequencies and mitochondrial DNA (mtDNA) haplotype frequencies, suggesting their reproductive
isolation.
One of the sub-objectives of JARPNII under Objective 3 (Stock structure) is a systematic monitoring of
the occurrence of J and O stock around the Japanese coast to determine spatial and temporal dynamics of
its occurrence. In a situation of geographical overlap of multiple stocks, stock identification at anindividual base will allows the direct estimate of mixing rates and the pattern of temporal and spatial
distribution of the stocks. The effect size between the O and J stocks is large so that Bayesian-based
program like STRUCTURE have been able to assign individuals to either stocks in the past (Kanda et al.,2009a; b).

1

The objective of this study is to update the work started by Kanda et al. (2009a) on the assignment of
individual whales to the O and J stocks, to gain further understanding of the spatial and temporal
distribution of these stocks around Japan. The study by Kanda et al. (2009a) was presented originally atthe 2009 JARPNII review workshop (IWC, 2010a), and updated in subsequent studies (Kanda et al.,2009b; 2010).
Discussions at the IWC SC have focused on the well-documented difficulty that STRUCTURE has indetecting weakly differentiated populations and on the significance of unassigned individuals (e.g. IWC,
2010b) (see Discussion section). On the first point, it should be noted here that the objective of thepresent study is not the use of STRUCTURE to resolve the number of stocks involved in the available
samples from by-catches and JARPN/JARPNII, but to use this program to assign individuals to therecognized J and O stocks in order to monitoring their distribution around the Japanese coast.
The question of whether or not additional stock structure occur within the ‘O’ stock is addressed in
Pastene et al. (2016: SC/F16/JR40) and Bando and Hakamada (2016: SC/F16/JR41) where other methods
usually used at the IWC SC for this purpose (e.g. hypothesis testing and PCA), are applied to theindividuals assigned to the ‘O’ stock by the STRUCTURE analysis in this study.
MATERIALS AND METHODS
Sample collections
A total of 22 sub-areas were set for management purpose of the western North Pacific common minke
whale during the RMP Implementation Review conducted in 2013 (Figure 1). JARPN and JARPNII
surveys were conducted in Sub-areas 7, 8, 9, and 11.
Offshore samples of common minke whales from the western North Pacific were from JARPN/JARPNII
surveys from 1994 to 2013 at SA7, SA8, SA9, and SA11 (Table 1). Common minke whale samples
obtained from the coastal JARPNII survey between 2002 and 2014 were also used in this study (Table 1),
Kushiro in sub-area SA7CN and Sanriku in sub-area SA7CS. Samples from common minke whales thatwere bycaught on set net fishery along the Japanese coast from 2001 to 2014 were also used (bycatches)(Table 1). The by-catches used were from the SA2, SA6, SA7, SA10, and SA11 year-round.
DNA extraction
We followed the IWC guidelines for DNA data quality (IWC, 2009) as much as possible (see Kanda etal., 2014). Genomic DNA was extracted from 0.05g of skin or muscle tissues using standard proteinase
K, phenol-chloroform procedure described by Sambrook et al. (1989). Extracted DNA was stored in the
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Microsatellites
Microsatellite polymorphisms were analyzed using 16 loci: EV1, EV14, EV21, EV37, EV94, (Valsecchi
and Amos, 1996), GT23, GT195, GT211, GT310, GT509, GT575 (Bérubé et al., 2000), GATA28,
GATA98, GATA417, TAA31 (Palsbøll et al., 1997), DlrFCB14 (Buchanan et al., 1996). EV1, EV14,
EV21 were developed from sperm whale, EV37, EV94, GT23, GT310, GT575, GATA28, GATA98,
GATA417, TAA31 from humpback whale, and DlrFCB14 from beluga whale. All GT, EV and DlrFCB
primers are dinucleotide repeats, TAA31 trinucleotide repeats, and all GATA primers tetranucleotide
repeats. Primer sequences and PCR profiles follows those of the original authors with slight
modifications.
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). PCR amplifications followed the
manufacturer’s instructions for the use of Ex Taq DNA polymerase (Takara Shuzo). Amplified productswith internal size standard (GENESCAN400HD, Applied Biosystems Japan) were run on a 6%
polyacrylamide denaturating gel (Long RangerTM) using a BaseStation TM100 DNA fragment analyzer(Bio-Rad). Although alleles were visualized using CartographerTM software specifically designed for the
BaseStation, allelic sizes were determined manually in relation to the internal size standard and common
minke whale DNA of known size that were rerun on each gel.

2

Data analysis
The number of alleles per locus, expected heterozygosity per locus, and inbreeding coefficient per locus
were calculated using the FSTAT 2.9.3 (Goudet, 1995). Statistical tests for the deviations from expected
Hardy-Weinberg genotypic proportions were conducted using the GENEPOP 4.0 (Rousset, 2008). The
False Discovery Rate (FDR) approach (Benjamini and Yekutieli, 2001) was used for adjustment of pvalue in case of multiple comparisons.
The Bayesian clustering approach was implemented with the microsatellite data in the STRUCTURE
version 2.0 (Pritchard et al., 2000) to determine the most likely number of genetically distinct stocks
present in our samples. The program is a model-based clustering method for inferring stock structure (K,the number of stocks in the model) using multilocus genotype data with and without information on
sampling locations. STRUCTURE allowed for the analyses of the samples without choosing sample units
that did not necessarily correspond to real biological stock boundaries. Posterior probabilities for K were
estimating from ten independent runs for each value of K from one to five with only genetic information.
These data were calculated based on burn-in period of 10,000 iterations and runs of 100,000 iterations.
Individual assignment was then conducted for the most plausible K using estimated individual proportionof membership probability (90%). The ancestry model used for the simulation was the admixture model,
which assumes individuals may have mixed ancestry. The allele frequency model used was the correlated
allele frequencies model, which assumes frequencies in the different stocks are likely to be similar due tomigration or shared ancestry.
More details of the application of STRUCTURE to western North Pacific common minke whales areavailable in Kanda et al. (2009b).
Mitochondrial DNA
Sequencing analysis of the 487bp 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 manufacture. For each sample both strands weresequenced.
Data 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 North Atlantic common and Antarctic minke whales. To estimate support foreach node a total of 1,000 bootstrap simulations were conducted and the majority-rule consensus
genealogy estimated.
RESULTS
Genetic variations
All 16 loci analyzed were polymorphic (Table 2). The total number of alleles per locus ranged from twoat the EV21 to 29 at the EV1 with an average of 12.7. Expected heterozygosity at each of the loci ranged
from 0.330 at TAA31 to 0.890 at GT23 with an average of 0.690. Twelve out of 16 loci showedsignificant deviation from the expected Hardy-Weinberg genotypic proportions even after correction forthe multiple tests. For those loci showing significant departure from the Hardy-Weinberg genotype
proportion, the FIS were all positive suggesting a homozygote excess. This deviation suggested existenceof individuals from multiple stocks in the sample.

3

Bayesian clustering analyses conducted on the total samples (4,275 individuals) without information on
their geographic origins presented the highest likelihood probability at K=2 (Table 3). These results
confirmed that the samples came from two genetically distinct stocks of common minke whales (J and O
stocks) (but see Discussion section). In this study, the individuals with the membership probability ofover 90% for either of the two stocks at each of the runs were assigned as pure individuals. All otherindividuals with the membership probability less than 90% to the either groups were assigned as
individuals of unknown origin (‘unknown’).
Spatial distribution of J and O stocks along the Japanese coast
Both of the assigned and unassigned individuals (‘unknown’) were grouped based on their sampling
origins (offshore, coastal, and bycatch) as well as locations (IWC sub-areas) (Figure 2). In this way,
distribution of the pure individuals that were genetically assigned to the different stock was clearly
separated geographically. Almost all of the individuals collected from the Sea of Japan side belonged to
the J Stock, whereas almost all of the individuals from the offshore North Pacific (east of the SA7WR)
belonged to the O stock.
Intermediate areas (SA7CN, 7CS and SA11) contained individuals from both stocks. The SA2 wasmainly occupied by the J stock. Locations of the assigned and ‘unknown’ individuals were plotted in
Figures 3a and coastal area was closed up in Figure 3b. The individuals of unknown origins distributed
widely through the sub-areas.
Temporal distribution of J and O stocks along the Pacific coast of Japan
Figure 4 shows the temporal distribution of the J and O stocks and unknown individuals in SA2, SA7CN
and SA7CS, expressed as three months moving average. In SA2 the J stock is predominant (around 80%
in proportion) around the year. In SA7CS and SA7CN the proportion of the J stock increase inautumn/winter and decrease in spring/summer. Conversely the proportion of O stock decrease inautumn/winter and increase in spring summer.
Phylogenetic relationship of mtDNA control region haplotypes
Figure 5 shows the phylogenetic relationship of mtDNA haplotypes. The figure also shows the haplotype
frequencies in the O and J stock as well in the unknown. Several clades were observed in the figure but
none was supported by high bootstrap values. There was no stock-specific clade although most of theindividuals assigned to the J stock shared a same clade. Most of the individuals assigned to the O stocks
shared clades where the J stock individuals were less frequent. The unknown samples were widelydistributed through the clades.
DISCUSSION
Issues on the program STRUCTURE
After reviewing the results of Kanda et al. (2009a) on the use of STRUCTURE to assign individuals to
the O and J stocks, the 2009 JARPNII review panel made a recommendation to provide more details on
the analyses involving this program (IWC, 2010a). Most of those details were presented in a revised
version of the document discussed at the annual meeting of the IWC SC in 2009 (Kanda et al., 2009b).
Furthermore analyses that followed recommendations by the IWC SC in 2009 were conducted andpresented to the 2010 IWC SC meeting (Kanda et al., 2010). Those details on the application of
STRUCTURE are not repeated here.
As noted previously, one of the major concerns was the well-documented difficulty that STRUCTURE
has in detecting weakly differentiated populations (IWC, 2010b). Regarding the results from JARPNII,
the IWC SC agreed that the STRUCTURE results provided clear evidence for two populations/stocks,
and that these generally conform to what have been referred to as O and J stocks (IWC, 2010b). It also
recognized the difficulty to determine under what circumstances the failure to find evidence for additional
stocks might simply be an inability to detect presence of an additional gene pool (s) that is geneticallysimilar to one of the two detected stocks (IWC, 2010b). In this context the JARPNII review workshop
recommended to conduct simulations to evaluate the power of STRUCTURE to detect various mixture
fractions of closely related stocks, although recognized this was a challenging recommendation (IWC,
2010a). Such simulations have not been conducted but it is believed that this was not relevant for the4objective of this study, which was not the investigation of the number of stocks involved but theinvestigation of the spatial and temporal distribution of what have been called J and O stocks around
Japan.
Of primary interest for management is whether or not additional structure occur within the O stock, andthis topic was treated in Pastene et al. (2016: SC/F16/JR40) and Bando and Hakamada (2016:
SC/F16/JR41), using alternative approaches. Regarding the J stock, the genetic evidence for additional
structure within this stock was considered low (IWC, 2013).
Other issue discussed in 2009 was on the significance of the unassigned (‘unknown’) individuals that
could not reliably be assigned to either J or O stocks (IWC, 2010b). Some IWC SC members have argued
that some if not all of the unknown individuals, may belong to a different stock. Alternatively, these
unknown individuals could be the product of low statistical power of the analysis. Kanda et al. (2010)
showed that the second explanation was more feasible. Here this issue is further elaborated.
The effect of using different number of microsatellite loci on the proportion of unknown individuals, wasinvestigated (Figure 6). This figure shows the proportion of unknown individuals obtained for different
sub-areas and sources of samples, for the case of six, nine, twelve and 16 loci. In all cases, the proportionof unknown individuals decrease with the increase of the number of loci used. Thus even for the case of
the J and O stocks where the effect size is considered high, the application of STRUCTURE will require alarger number of loci to minimize the number of unassigned individuals to those stocks.
These results demonstrate that the ‘unknown’ individuals are not related to the occurrence of a different
stock but they are derived by the low power of the analyses (in term of number of loci). If more loci areused then all individuals will ultimately be assigned to either J or O stocks. This result, summed to thewide and random nature in the temporal and spatial distribution of the unknown samples (Figures 3a, b),provide strong validation for the application of additional analytical approaches to investigate additional
stock structure within the O and J individuals assigned by STRUCTURE (see Pastene et al., 2016:
SC/F16/JR40).
Temporal and spatial distribution of J and O stocks
Results of the present analyses on the total available samples of common minke whale are similar to theresults provided by Kanda et al. (2009a; b). J stock animals distribute mainly in the Sea of Japan and in
SA2 in the Pacific side of Japan. O stock animals distribute mainly in the Pacific side of Japan and mix
with J stock animals in SA7CS, SA7CN and SA11. The pattern of mixing between the two stocks in theseintermediate sub-areas has a strong temporal component with J stock predominating in autumn and winterand the O stock in spring and summer.
The fact that the J stock distribute in SA2 through the year suggests that the Kuroshio Current, which isone of the strongest west-boundary currents of the subtropical gyre, is working as the stock boundary
between J and O stocks.
It is important to note that the individuals from the JARPN/JARPNII and those from the bycatch samples
differ in their body length. Average body length of the JARPN and JARPNII samples including both theoffshore and coastal components was 6.67m (SD= 1.13) and that of the all bycatch sample was 4.94m(SD=0.985). Kato (1992) estimated mean body length at the sexual maturity of the North Pacific minke
whales to be 6.3m for males and 7.1m for females, so that the bycatch sample in the present study
consisted mostly, if not all, of immature whales. The observation that the number of immature O stockindividuals increased in spring along the Pacific coast of Japan is well consistent to that illustrated by
Hatanaka and Miyashita (1997). The observed difference in the maturity status between the individuals
from the bycatch and JARPN/JARPNII samples, however, could indicate that the patterns of the temporaland spatial distributions illustrated with the bycatches for the SA2 and SA7 in this study may be different
at some extent from those of adults. In regard to SA2, common minke whales from the offshore area havenot been available yet. Related concern can be also seen in the SA11. The number of the J stockindividuals in the SA11 differed between the bycatch and JARPN samples. This difference we observed
between the bycatch and JARPN samples could be due to the immature/mature, temporal, or both factors,
but we were not able to distinguish which one accounted for at this moment. Although we definitely

5gained our understanding of common minke whales distribution around the Japanese water substantiallyfrom this study, our samples are still missing some pieces to depict the whole picture of distribution andmovement.
The series of studies on J and O stock (Kanda et al., 2009a; b; 2010; this study) are the first that shed lighton the dynamics of geographic overlap between the two stocks at the individual base. It is believed thatthe results of these studies are also quite useful for the effective management of the two stocks. Anotherusefulness of the individual identification by the genetic markers is that it can be used to look for stock
differences in other traits, such as morphometry, pollutant levels, and biological parameters (seeNakamura et al., 2016: SC/F16/JR39; Bando and Hakamada 2016: SC/F16/JR41).
ACKNOWLEDGEMENTS
We gratefully acknowledge the researchers and crew members that participated in JARPN/JARPNII
surveys for the collection 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 used in this study.
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Kanda, N., Goto, M., Kishiro, T., Yoshida, H., Kato, H., and Pastene, L.A. 2009a. Individual
identification and mixing of the J and O stocks around Japanese waters examined by microsatellite
analysis. Paper SC/J09/JR26 presented to the JARPN II Review Workshop, Tokyo, January 2009
(unpublished). 9pp.
Kanda, N., Goto, M., Kishiro, T., Yoshida, H., Kato, H., and Pastene, L.A. 2009b. Update of theanalyses on individual identification and mixing of the J and O stocks around Japanese waters

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Table 1. Sample size used in the microsatellite analyses. This involves the number of whales genotyped
for all 16 loci successfully.
By-catch(2001-2014)

JARPN/JARPNII
Coastal(2002-2014)

JARPN/JARPNII Offshore(1994-2014)

BC2

BC6

BC7CN

BC7CS

BC10

BC11

K7CN

S7CS

7CN

7CS

7E

7WR

8

9

11

487

717

90

282

13

49

656

514

320

125

49

100

252

541

80

Table 2. The number of alleles (A), expected heterozygosity (HE), test result for the expected HardyWeinberg genotypic proportions (HW) and inbreeding coefficient (FIS) at 16 microsatellite loci ofwestern North Pacific common minke whales.

Microsatellite loci
EV37
EV1
GT310
GATA28
GT575
EV94
GT23
GT509
GATA98GATA417
GT211
EV21
DlrFB14
EV14
GT195
TAA31
Overall

A
12
29
14
21
13
8
15
23
7
14
16
2
6
6
12
5
12,7

HE
HW
0,70
0,084
0,77
<0.0010,83<0.0010,83<0.0010,80<0.001
0,63
0,125
0,89
<0.0010,88<0.001
0,60
0,921
0,73
<0.0010,87<0.001
0,34
0,741
0,41
<0.0010,52<0.0010,86<0.0010,33<0.001
0,69 High. sign.

8

F IS-0,005
0,028
0,041
0,011
0,026
0,026
0,020
0,040
0,005
0,005
0,031
-0,006
0,030
0,067
0,057
0,037
0,027

Table 3. Results of the Bayesian clustering method analyzed for overall samples.

K
1
2
3
4
5

Log P(k/x) variance Pr(k/x)-210753.0
85.2 ~0.0-202085.7
873.1 ~1.0-203026.1 3693.0 ~0.0-202960.7 4761.0 ~0.0-204283.6 8075.9 ~0.0

Figure 1. Sub-areas used for the management of common minke whale under the RMP.

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Figure 2. Spatial occurrence of O and J stocks in waters around Japan. BC2, BC6, BC7CS, BC7CN,
BC10, BC11= bycatches from sub-areas 2, 6, 7CS, 7CN, 10 and 11. K7CN= coastal survey at Kushiro.
S7CS= coastal survey at Sanriku. 7CS, 7CN, 7WR, 7E, 8, 9 and 11= offshore survey of JARPN and
JARPNII. Sample size is on the top of each bar.

120E
50N

130E

140E

150E

160E

170E
50N

45N

45N

40N

40N

35N

35N

30N
120E

130E

140E

150E

160E

30N
170E

Figure 3a. Locations of the common minke whales that were assigned to O stock (green), J stock (blue),and unknown (red), based on STRUCTURE.

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140E
47N

145E
47N

42N

37N
140E

42N

37N

145E

Figure 3b. Zoomed into the coastal area of the Figure 3a. O stock (green), J stock (blue), and unknown(red), based on STRUCTURE.

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Figure 4. Monthly occurrence of O and J stocks in sub-areas 2, 7CS and 7CN. Each bar is expressed as
three months moving average. Sample size is on the top of each bar. The sampling years in SA2 was
2001-2014; in SA7CN and SA7CS was 1994-2014.

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Figure 5. Phylogenetic relationship of mtDNA control region haplotypes and frequencies of whales
assigned to O stock, J stock and unknown by the microsatellite analysis. North Atlantic common andAntarctic minke whale haplotypes are used as outgroups.

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Figure 6. Proportion of J, O and ‘unknown’ individuals in each sub-area in relation to the number ofmicrosatellite loci used in the assignment test.

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