THIS PAPER CAN NOT BE CITED WITHOUT WRITTEN PERMISSION FROM THE AUTHORS

JA/J05/JR16

Genetic analyses on stock identification in the Antarctic
humpback and fin whales based on samples collected under
the JARPA
LUIS A. PASTENE, MUTSUO GOTO, NAOHISA KANDA AND SHIGETOSHI NISHIWAKIThe Institute of Cetacean Research, Toyomi-cho 4-5, Chuo-ku, Tokyo 104-0055, Japan
ABSTRACT
Biopsy samples from 287 humpback and 23 fin whales obtained during surveys of the Japanese Whale
Research Program under Special Permit in the Antarctic (JARPA) were analyzed for mtDNA andmicrosatellite (humpback whale only) variation. In the case of the humpback whale samples weregrouped according the information of stock distribution in the feeding ground derived from the
IWC/SC comprehensive assessment of the species. Analysis of mtDNA control region sequences
discriminated clearly among Stocks C, D, E, F and G in the feeding ground. However analysis based on
six microsatellite loci, while exhibiting some degree of genetic heterogeneity, was unable todiscriminate among these stocks. Different degree of fidelity to breeding areas between females andmales are suggested to explain such result. Analysis of mtDNA suggests that the historical sector of
mixing between Stock D and E at 110-130°E is being occupied in recent years by the D stock.
Regarding fin whales, level of mtDNA diversity found is high and similar to that in the Antarctic
minke whale. No significant differences were found in the comparison between Areas III+IV and V,
corresponding approximately to Indian and western South Pacific Oceans populations, respectively.
However this result is attributed to the small number of samples used in the analysis
INTRODUCTION
JARPA has a non-lethal research component involving sighting and oceanographic surveys, skinbiopsy sampling and experiment on photo identification (photo-id). Skin biopsy sampling for genetic
analysis and photo-id experiments for individual identification have been conducted on three baleen
whale species: the humpback whale (Megaptera novaeangliae), the blue whale (Balaenoptera
musculus) and the right whale (Eubalaena australis). In recent surveys, biopsy sampling in the fin
whale (Balaenoptera physalus) has been conducted. This paper summarizes the genetic analyses
conducted under JARPA on humpback and fin whales in the Antarctic.
Background on Southern Hemisphere humpback whale
There is a considerable amount of information on the pattern of distribution and seasonal movement of
humpback whales in the Southern Hemisphere. Most of the information is derived from the analysis of‘Discover’ marks and catch distribution conducted in the past. Mackintosh (1965) showed that
humpback whales tend to gather into five or six distinct feeding concentrations in the Antarctic during
the austral summer season. He denominated these concentrations as Groups I-V (with a Group IIa andIIb). These concentrations correspond roughly to Management Areas I-VI.
Two of the areas of feeding concentrations in the Antarctic are Areas IV (70°-130°E) and V (130°E170°W), where JARPA surveys are conducted. The geographic boundary between these two Areas was
defined considering the distribution of catches and the results of mark-recapture analysis (Omura,
1953; Chittleborough, 1959).
Figure 1 shows the putative breeding grounds, feeding grounds and migratory corridors for Southern
Hemisphere humpback whales as suggested by the IWC/SC’s comprehensive assessment (IWC, 2001).
These were based mainly on information on sighting and catch distribution and mark-recapture analysis.
Seven breeding stocks are identified, labeled as A through G in Figure 1.
Genetic analyses in the Southern Hemisphere humpback whale have been based in the maternal-

1inherited mtDNA. While mtDNA in breeding areas and migratory corridors demonstrated significant
differences between Western Australia (Stock D), Eastern Australia (Stock E) and Colombia (Stock G)(Baker et al., 1998), little is known on the genetic population structure in the feeding ground.
Making use of biopsy samples collected from Areas IIIE, IV, V and VIW by the JARPA, we
conducted a genetic analysis based on the maternal-inherited mtDNA and bi-parental inherited nuclear
DNA (microsatellites) to test for genetic heterogeneity in the feeding ground. Of particular interest was
to test the feeding areas proposed in Figure 1 for Stocks C, D, E, F and G.
Background on Southern Hemisphere fin whale
Little information is currently available on the stock structure of this species in the Antarctic. As in thecase of the blue whale, earlier mark-recapture analysis showed that most whales return to the same partof the Antarctic year after year (Brown, 1954). Subsequent mark-recapture studies conducted by Brown(1962) suggested that the six whaling areas are probably more valid for blue and humpback whales
than for fin whales (see also Mackintosh, 1965). The past information suggested there was certain
segregation in the feeding ground between certain longitudes in four sectors which lie: South of theAtlantic Ocean, South of the Indian Ocean, South of Western South Pacific Ocean and South of EasternSouth Pacific Ocean (Mackintosh, 1965). South of the Indian Ocean correspond approximately to
JARPA Areas IIIE and IV and South of Western South Pacific to JARPA Areas VIW and V.
A previous study based on mtDNA used biopsy samples from JARPA to investigate the phylogenetic
relationships of fin whales from different oceanic basins (Goto et al., 2003). Although one of the cladein the tree included all the fin whales from the Antarctic, such clade also included fin whales from
North Pacific and North Atlantic (Figure 2) suggesting little time for fin whales inhabiting different
oceans since their divergence to accumulate own unique haplotypes.
No studies based on DNA have been conducted to investigate stock structure of fin whales in theSouthern Hemisphere. In this study we conducted a preliminary mtDNA analysis using biopsy samples
obtained from JARPA to test for the oceanic division in the Antarctic suggested by Mackintosh (1965).
MATERIALS AND METHOD
Samples
Biopsy samples were obtained along the sighting surveys of the JARPA, on an opportunistic basis.
These have been collected using an air gun described in Kasamatsu et al. (1991) a more recently using
Paxarm. Table 1 shows the number of biopsy samples of humpback and fin whales collected by
JARPA, by survey and Area. All biopsy samples are checked for the possibility of re-sampling byusing a set of microsatellite. For that reason the number of samples used in the analysis is less thanthose listed in Table 1. When cow-calf pairs were sampled, only one of them was used in the analysis.
Grouping of samples
Humpback whale
Samples were grouped primarily according the distribution of stocks in the feeding ground as shown in
Figure 1: Stock C (35-55°E, n=34, 9 for mtDNA and microsatellite, respectively), Stock D (80-110°E,
n=79, 62), Stock E (130°E-170°W, n=64, 54) and Stock F (170-145°W, n=36, 36). For comparison
purpose, a sample from Stock G (n= 11) was used.
Two other additional sectors were defined in the Antarctic: 55°-80°E (n= 29) for which no previous
information is available and 110°-130°E (n= 34), which has been regarded as a mixing sector between
Stocks D and E stocks (Chittleborough, 1959; Dawbin, 1966).
Fin whale
Samples were grouped into two sectors in the Antarctic: 35°-130°E (corresponding to the Indian Oceangroup) and 130°E-170°W (corresponding to the western South Pacific group). Sample sizes are small,n= 8 and n= 15, for these two sectors, respectively.
Biochemical analysis
Genomic DNA was extracted from approximately 0.05g of the outer epidermal layer of the skin biopsy.
For extracting genomic DNA, we used established protocols (Sambrook et al, 1989). Laboratory2

procedure for mtDNA control region sequencing and microsatellites was described in Pastene et al.
(2000). Sex determination was made using the method of Abe et al. (2001).
In the case of the humpback whale a segment of 323bp of the mtDNA control region was examined.
The nuclear DNA analysis involved six microsatellite loci (GATA417, GATA28, GATA98, TAA31,
GATA53 and GT23).
In the case of fin whales only mtDNA analysis was conducted. In this case a segment of 298bp of the
mtDNA control region was examined.
Statistical analysis
mtDNA
Genetic distances among haplotypes were estimated using Kimura’s two parameters method (Kimura,1980). The degree of mtDNA diversity within stock or longitudinal sectors was estimated using thenucleotide diversity.
Following recommendations from the IWC/Scientific Committee, heterogeneity tests were conductedusing the randomized chi-square test (Roff and Bentzen, 1989) with a total of 10,000 permutations ineach test. A P-value smaller than 0.05 was used as a criterion to reject the null hypothesis.
Heterogeneity tests were conducted to investigate differences between sexes within and among stocks.
Microsatellites (humpback whale only)
All the statistical tests were conducted using the computer program GENEPOP (Raymond and Rousset,
1995). Statistical tests were conducted to investigate differences between sexes within stock, test forthe deviation from Hardy-Weinberg genotypic proportion in the stocks and to investigate differencesamong stocks. Decision of statistically significance on hypothesis testing was made using the Fisher
Exact Test obtained from summing the negative logarithm of P-values over the total loci (Fisher, 1950).
RESULTS
Humpback whale
mtDNA
A total of 64 polymorphic sites defined 82 haplotypes in the total sample of 287 whales. Nucleotide
diversity for the total sample was high (0.0272). Among the stocks C-G it ranged from 0.0238 to
0.0283 (Table 2).
Table 3 shows the results of the heterogeneity test for sex differences in each stock. No significant
differences were found between sexes. Male and female samples were pooled in the subsequent
analysis.
Table 4 shows the results of pair-wise statistical comparison among stocks. The total P-value was
highly significant (P= 0.0001). Apart from the comparison between C and G, all the comparisons
resulted in significant differences.
Table 5a shows the results of the statistical comparison between Stocks C, D and sector 55°-80°E.
Results suggest that whales in that particular sector are similar to Stock C and significantly different
from Stock D. When the comparison is made at a more fine scale, sector 55°-70°E is similar to Stock C
and different from Stock D. Sector 70°-80°E is similar to both stocks.
Table 5b shows the results of the statistical comparison between Stocks D, E and mixed assemblage
sector 110°-130°E. Results suggest that whales in that particular sector are similar to Stock D andsignificantly different from Stock E.
Microsatellite
Table 6 shows the number of alleles and expected heterozygosity by locus. The average number ofalleles per locus ranged from 7.4 to 12.6. Expected heterozygosity ranged from 0.511 to 0.877.
None of the heterogeneity tests conducted to investigate differences between sexes resulted insignificant P-values (Table 7). Male and females samples were pooled in subsequent analyses.

3

Table 8 shows the results of the test for deviation from Hardy-Weinberg expectation in five stocks.
None significant deviation from expectation was observed.
Table 9 shows the results of the heterogeneity test among stocks. The result for all loci and all stocks
showed a significant P-value (0.042). Pair-wise comparisons showed little evidence of separation with
only one comparison showing a P-value smaller than 0.05 (Stocks E/F).
Fin whale
mtDNA
A total of 16 polymorphic sites identified a total of 20 haplotypes in the total sample of 23 whales(Table 10). Nucleotide diversity estimate for the total sample was 0.0121 and no significant differenceswere found in the comparison between Area IIIE+IV and Area V, due probably to the small sample
size (Table 11).
DISCUSSION
Humpback whale
Analysis of mtDNA provided support for the pattern of distribution of stocks C, D, E, F and G in the
Antarctic suggested by Figure 1. This result is important because it suggest consistency between the
non-genetic analyses conducted in the past and the mtDNA analysis conducted in this study. These
stocks seem to occupy specific longitudinal sectors in the Antarctic. This result contrasts with thatobtained using microsatellites. While some degree of heterogeneity was observed for nuclear DNA,
discrimination among stocks in the Antarctic feeding ground was much less evident using this marker
than in the case of mtDNA. The fact that microsatellites was unable to discriminate clearly amongstocks in the Antarctic does not deny the existence the multiple stocks. It is possible that female
humpback whale exhibit a strong fidelity to breeding areas than males. Genetic differences amongstocks will not be evident at the nuclear DNA if a few reproductive males interchange and breed indifferent breeding areas.
Because mtDNA discriminated clearly among stocks, this marker was used to investigate Antarctic
sectors for which no information was previously available or sector where mixing of stocks was
suspected. Results of this analysis suggest that Stock C occupy sector 55°-70°E while sector 70°-80°E
is probably occupied by both C and D stocks.
Pattern of mixing between D and E in sector 110°-130°E seems to have changed with time. Early
mark-recapture analyses suggested that the sector between 110-130°E was occupied mainly by the E
stock (Chittleborough, 1959; Dawbin, 1966). Our mtDNA analysis suggests that this sector is occupied
mainly by the D stock. An additional analysis was conducted to investigate change in the proportion of
D stock in this sector with time (Table 12). This table shows the contribution of Stock D to this mixing
area, by JARPA survey. Although the standard deviations of the estimates are large due to small
sample size, proportion of this stock in the mixed area seems to have increased with time. This isconsistent with the information that Stock D has increased its abundance in recent years.
The relation between Stocks C, D and sector 55-80°E and the pattern of mixing between Stocks D
and E (between 110° and 130°E) should be further investigated using a larger number of samples andanalyses should be conducted by year and for each sex. Changes in geographical distribution of thestocks are possible as a response to the increase in abundance. These changes should be investigated in
the future.
Fin whales
Genetic diversity, as measured by mtDNA, was examined for the first time for fin whales in the
Antarctic. Level found is higher and similar to that found for Antarctic minke whale.
Our heterogeneity test comparing Areas IIIE+IV and V (assumed to belong to Indian and westernSouth Pacific Ocean Populations, respectively) provided no evidence of structure. However, this result
is not surprising given the small number of samples used in the analysis. Probably this result reflects
the low power of the analysis.
Samples sizes available for analysis on stock structure are small and there is a need to collect more
samples for this purpose. Experiments of satellite tracking would provide valuable information on4

movement of fin whales in the feeding ground as well movement between feeding grounds and lower
latitudes.
ACKNOWLEDGEMENTS
We thank researchers and crew members of the JARPA surveys for their effort in collecting thesamples. Also we thank H. Hatanaka for useful comments. Our gratitude to H. Oikawa and S. Azumi
for their laboratory assistance.
REFERENCES
Abe, H., Goto, M., Pastene, L.A., Dewa, K. and Naito, E. 2001. Practical use of multiplex fluorescent
PCR for cetacean sex identification. Marine Mammal Science 17(3): 657-664.
Baker, C.S., Flores-Gonzalez, L., Abernethy, B., Rosenbaum, H.C., Slade, R.W., Capella, J. andBannister, J.L. 1998. Mitochondrial DNA variation and maternal gene flow among humpback whalesof the Southern Hemisphere. Marine Mammal Science 14: (4): 721-737.
Brown, S.G. 1954. Dispersal in blue and fin whales. Discovery Reports. Vol. XXVI:355-384.
Brown, S.G. 1962. The movement of fin and blue whales within the Antarctic zone. Discovery Reports.
Vol XXXIII:1-54.
Chittleborough, R.G. 1959. Australian marking of humpback whales. Norsk Hvalfangsttid 48:47-55.
Fisher, R.A. 1950. Statistical Methods for Research Workers. 11th ed. Oliver and Boy, London.
Dawbin, W.H. 1966. The seasonal migratory cycle of humpback whales. In Norris, K.S. (ed.). Whales,
dolphins and porpoises. University of California Press, Berkerley. pp. 145-171.
Goto, M., Berube, M., Kanda, N., Ishikawa, H., Nishiwaki, S. and Pastene, L.A. 2003. Phylogenetic
analysis of fin whale mtDNA control region sequences world-wide. Paper SC/55/SD6 presented to the
IWC Scientific Committee, May 2003 (unpublished). 8pp.
International Whaling Commission. 2001. Report of the Scientific Committee. J. Cetacean Res.
Manage 3 (Suppl.): 1-374
Kasamatsu, F., Iwata, S. and Nishiwaki, S. 1991. Development of biopsy skin sampling system for fastswimming whales in pelagic waters. Rep. int. Whal. Commn 41:555-557.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions throughcomparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.
Mackintosh, N.A. 1965. The stocks of whales. Fishing News (Books) Ltd.., London. 232pp.
Omura, H. 1953. Biological study on humpback whales in the Antarctic whaling areas IV and V.
Scientific Reports of the Whales Research Institute, Tokyo 8:81-102.
Pastene, L.A., Goto, M., Abe, H., Nishiwaki, S. and Palsboll, P. 2000. Genetic diversity of humpback
whales in the Antarctic feeding ground examined by mitochondrial DNA and microsatellite. Paper
SC/52/IA4 presented to the IWC Scientific Committee, June 2000 (unpublished). 16pp.
Raymond, M. and Rousset, F. 1995. GENEPOP (version 1.2): population genetics software for exact
tests and ecumenicism . J. Heredity 86:248-249.
Roff, D.A. and Bentzen, P. 1989. The statistical analysis of mtDNA polymorphisms: chi-square and the
problem of small samples. Mol. Biol. Evol. 6:539-45.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Second
Edition. Cold Spring Harbor Laboratory, New York.

5

Figure 1: Breeding areas, migratory corridors and feeding areas of southern humpback
whale stocks according the IWC/SC comprehensive assessment (after IWC, 2001)

6

Figure 2: Neighbor-Joining-based tree of mtDNA haplotypes in the fin whaleworldwide. Antarctic samples are from the JARPA surveys (After Goto et al., 2003)

7

Table 1: Summary of biopsy samples of humpback and fin whales taken during
JARPA surveys, by IWC Area.
Area
Humpback whale Fin whale
IIIE
IV
V
VIW
TOTAL

56
134
75
38
303

4
4
18
0
26

8

Table 2: Estimates of nucleotide diversity in Antarctic humpback whale stocks.
C
0.0267(0.0011)D
0.0268(0.0007)E
0.0276(0.0011)

F
0.0283(0.0013)G
0.0238(0.0040)

Total
0.0272(0.0005)

Table 3: Statistical comparison of mtDNA haplotype frequencies between sexes using
randomized chi-square test (P values derived from 10,000 simulations).
C(F:22 M:12)
0.9157.

D(F:32 M47)
0.2675

E(F:35 M:29)
0.4466

F(F:14 M:22)
0.9490G(F:7 M:4)
0.6711

Table 4: Results of statistical comparisons of mtDNA haplotype frequencies among
Stocks C, D, E and F in the Antarctic using randomized chi-square test (P values
derived from 10,000 simulations). The total P-value was 0.0001. In parenthesis is the
sample sizes.

C
D
E
F
G

C (34)-

D (79)
0.0046-E (64)
0.00010.0001-F (36)
0.00040.00010.0237-

G (11)
0.07240.00090.00240.0130-

Table 5a: Results of statistical comparisons of mtDNA haplotype frequencies between
Stock C and sector 55-80°E and between D and sector 55-80°E using randomized chisquare test (P values derived from 10,000 simulations). In parenthesis is the sample
size.

C (34)
D (79)

55-70E (16)0.57150.003470-80E (13)0.95760.171855-80E (29)0.93010.0125

Table 5b: Results of statistical comparisons of mtDNA haplotype frequencies between
Stock D and 110-130°E sector and between E and 110-130°E sector using
randomized chi-square test (P values derived from 10,000 simulations). In parenthesisis the sample size.

D (79)E (64)

110-120E (20)0.11630.0480120-130E (14)0.58140.01559110-130E (34)0.4231
0.0210Table 6: Number of samples (N), alleles (A) and expected heterozygosity (He) atseven microsatellite loci analyzed in Antarctic humpback whale.
Microsatellites
GATA417

GATA28GATA98TAA31

GATA53

GT23

Stock

N

A

He

N

A

He

N

A

He

N

A

He

N

A

He

N

A

He

C

9

8

0.839

9

6

0.530

9

6

0.641

9

10

0.864

9

6

0.778

9

7

0.808

D

62

12

0.903

62

11

0.539

62

13

0.804

62

18

0.884

62

8

0.811

62

9

0.780

E

54

14

0.883

54

11

0.630

54

9

0.759

54

16

0.898

54

12

0.804

54

9

0.765

F

36

14

0.910

36

10

0.407

36

8

0.714

36

11

0.858

36

8

0.825

36

7

0.768

G

11

9

0.851

11

5

0.447

11

7

0.802

11

8

0.851

11

6

0.736

11

5

0.690

All

172 11.4 0.877

172 8.6

0.511

172 8.6

0.744

172 12.6 0.871

172 8.0

0.791

172 7.4

0.762

Table 7: Results of the heterogeneity test between sexes in five stocks of humpback
whale in the Antarctic (figures shown are P-values).
Locus
GATA417
GATA28GATA98TAA31
GATA53
GT23

C
0.415
0.650
1.000
0.285
0.859
0.579

D
0.507
0.284
0.073
0.616
0.131
0.785

E
0.066
0.624
0.437
0.347
0.436
0.661

F
0.162
0.340
0.635
0.345
0.526
0.323

G
0.989
0.202
0.625
0.800
0.846
0.134

All loci

0.887

0.263

0.396

0.416

0.707

10

Table 8: Results of the test for deviation from Hardy-Weinberg expectation in five
humpback whale stocks in the Antarctic (figures shown are P-values).
C
GATA417
GATA28GATA98TAA31
GATA53GT23
All loci

D
0.912
1.000
0.869
0.082
0.793
0.992
0.919

E
0.460
0.440
0.155
0.685
0.340
0.816
0.594

F
0.564
0.267
0.350
0.273
0.643
0.368
0.497

G
0.253
0.460
0.647
0.261
0.781
0.984
0.754

0.969
0.613
0.873
0.963
0.685
0.053
0.783

Table 9: Results of the heterogeneity test among humpback whale stocks in the
Antarctic (Figures shown are P-values).

C
C
C
C
D
D
D
E
E
F

All stocks
D
E
F
G
E
F
G
F
G
G

Locus
GATA417GATA28 GATA98 TAA31 GATA53 GT23
All loci
0.042
0.005
0.262
0.401
0.251
0.330
0.478
0.217
0.477
0.407
0.387
0.287
0.365
0.388
0.025
0.238
0.366
0.336
0.672
0.202
0.104
0.408
0.270
0.522
0.654
0.561
0.413
0.661
0.159
0.468
0.205
0.804
0.561
0.205
0.359
0.020
0.176
0.562
0.179
0.705
0.662
0.133
0.666
0.416
0.513
0.639
0.043
0.855
0.493
0.012
0.545
0.328
0.247
0.627
0.482
0.131
0.045
0.101
0.181
0.379
0.053
0.068
0.911
0.013
0.666
0.317
0.470
0.703
0.074
0.084
0.012
0.447
0.290
0.632
0.462
0.129
0.078

11

Table 10: Variable sites defining 20 mtDNA haplotypes in the Antarctic fin whale.
The column on the left is haplotype ID. The number above lists the nucleotide
position of the polymorphic sites starting from the 5’ end of the mtDNA control
region. Haplotypes ‘2’ through ‘20’ are listed with reference to haplotype ‘1’. A dot
indicates an identical nucleotide at the position relative to haplotype ‘1’.
101222 222222
668890015 556888
6087874144 696347
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

ACACTTTCTA AGCTTT..GT...... G.....
..G......G ......
.TGT..C... ......
.TGT...T.. ....CC..GT....CG ......
..GT...T.. ....CC.TGT...T.. ..T...
.TG....... ......
.TG....T.. ......
..GTC..... ....C.
..G....T.. .A....
..G....T.. ......
..GT...T.. ......
...T...T.. ......
.TG....... .A....
.TGT...... ......
..GT...TCG ...C..
.TG..CC... .....C-..T...T.. ......

Table 11: Estimates of nucleotide diversity in Antarctic fin whale and result of theheterogeneity test comparing Areas IIIE+IV and V (column far right).
Area IIIE+IV (8)0.0133(0.0025)

Area V (15)0.0118(0.0009)

Total (23)0.0121(0.0009)

Area III+IV/Area V
P=0.8932

Table 12: Yearly change in the proportion of humpback whale Stock D in sector 110130°E, according mtDNA analysis.
Sector/Survey
Proportion of Stock D (standard deviation)
110-130°E (n=10, 93/94, 95/96)
0.6015 (0.1865)
110-130°E (n=11, 97/98, 99/00)
0.6640 (0.2166)
110-130°E (n=13, 01/02, 03/04)
0.7591 (0.1172)
110-130°E (n=34, total)
0.7422 (0.0999)

12

13