SC/F14/J32

Genetic analysis on stock structure of fin whales in the
Antarctic based on mitochondrial and microsatelliteDNA
Mutsuo Goto, Naohisa Kanda and Luis A. Pastene
Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
Contact e-mail: goto@cetacean.jp

ABSTRACT
Genetic samples (catches and biopsies) of fin whales obtained by JARPA and JARPAII were analyzed
with two genetic markers, mtDNA control region sequencing and microsatellite DNA, to investigate stock
structure of this species in the Antarctic feeding grounds. Genetic samples were available from Areas IIIE(n=6), IV (n=23), V (n=24) and VIW (n=2). No statistical significant difference in mtDNA haplotype
frequencies was found between Areas IIIE+IV and Areas V+VIW. However, because the haplotype
distributions were quite different among the areas, statistical comparison was made by re-grouping the
haplotypes into two categories: those specific to each of the areas and those shared by more than one area.
Results of the heterogeneity test showed statistically significant differences among whales in Areas IIIE,
IV and V. The microsatellite analysis showed statistically significant differences in allele frequencies of
16 loci among whales from Areas IV and V, but not IIIE and IV, as well as IIIE and V, most likely due tothe small sample size of Area IIIE. Results of the genetic analyses therefore suggested the possibility ofgenetic structuring of fin whales in the JARPAII research area, which should be further explored with theanalyses of a large number of samples in the future.
KEYWORDS: ANTARCTIC, GENETICS, FIN WHALE, SCIENTIFIC PERMITSINTRODUCTION
Little information is currently available on the stock structure of fin whales in the Antarctic. As in the caseof the blue whale, earlier mark-recapture analysis showed that most whales return to the same part of 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 of fin
whales in the feeding ground between certain longitudes in four sectors which lie: South of the Atlantic
Ocean, South of the Indian Ocean, South of Western South Pacific Ocean and South of Eastern South
Pacific Ocean (Mackintosh, 1965). South of the Indian Ocean correspond approximately to JARPAII
Areas IIIE and IV and South of Western South Pacific to JARPA and JARPAII Areas VIW and V. It is
important to investigate whether such geographical segregation is supported by genetic differences.
Wada and Numachi (1991) conducted allozyme analysis using North Pacific, Spanish coastal andAntarctic fin whales. They showed significant allele frequency differences between Hemispheres.
However they could not detect evidence of more than one stock within the Antarctic or within the North
Pacific fin whales.
Only a single study based on JARPA biopsy samples and mtDNA has been conducted to examine genetic
differences between the whales from IIIE+IV and VW (Pastene et al., 2005). Although the authors foundno evidence of the genetic differences between IIIE+IV and VW, the sample size was too small (8 and 15,
respectively) to make a firm conclusion on stock structure of this species in the Antarctic.
Additional genetic samples were obtained during the JARPAII surveys. The analysis of those samples isimportant for testing the hypothesis of segregation in the feeding grounds of the IWC management areas.
In this study we conducted mtDNA and microsatellite analyses using additional biopsy and researchsamples obtained by JARPA and JARPA II to investigate further the stock structure of fin whales in Areas

1

IIIE, IV, V and VIW.
MATERIALS AND METHOD
Samples
Genetic samples were available from fin whales caught by JARPAII surveys between 2005/06 and2010/11 and from biopsies obtained from the sighting surveys of the JARPA and JARPAII, on an
opportunistic basis. Table 1 and Fig. 1 show the number and geographical position of the genetic samples
used in this study, by year and Area.
Laboratory procedures
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).
MtDNA
The first half of the control region of the mitochondrial genome was amplified by using the polymerase
chain reaction (PCR). For the amplification of approximately 500bp of the mtDNA control region,
primers MT4 (Árnason et al., 1993) and Dlp 5R (5'-CCA TCG AGA TGT CTT ATT TAA GGG GAA
C-3'), were used. Reactions were carried out in 25 uL volumes containing 10-100ng of DNA, 2.5 pmoleof each primer, 0.5 units of Ex Taq DNA polymerase (Takara), 2mM of each dNTP, and 10x reaction
buffer. After an initial denaturation step at 95 C for 5 minutes, a PCR amplification cycle of 30 seconds
at 94C, followed by 30 seconds at 50C and 30 seconds at 72C are repeated 30 times. The
amplification is completed with a final extension step of 10 minutes at 72C. Subsequent cycle
sequencing reactions are performed with 100ng of products generated in the above PCR amplifications
using the BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems). The oligo-nucleotides used
to prime the cycle sequencing reaction were the same as employed in the initial PCR amplification listed
above. A total of 25 cycles for 10 seconds at 96C, 20 seconds at 56C and four minutes at 60C are
performed. The nucleotide sequence of each cycle sequencing reaction is determined using Applied
Biosystems 3500 Genetic Analyzer (Life Technology) under standard conditions. Both strand samples aresequenced in their entirety for all samples.
Microsatellite
Genetic variation at microsatellite loci were analyzed using 16 sets of primers as follow: EV1, EV14,
EV21, EV94, EV104 (Valsecchi and Amos, 1996), GT011 (Bérubé et al., 1998), GT23, GT211, GT271,
GT310, GT575 (Bérubé et al., 2000), GATA28, GATA53, GATA98, GATA417, GGAA520 (Palsbøll et al.,1997), and DlrFCB17 (Buchanan et al., 1996). Primer sequences and PCR cycling profiles generally
followed those of the original authors. PCR amplifications were performed in 15ml reaction mixturescontaining 10-100ng of DNA, 5 pmole of each primer, 0.625 units of Ex Taq DNA polymerase (Takara
Shuzo), and 2mM of each dNTP, and 10x reaction buffer containing 20mM MgCl 2 (Takara Shuzo).
Amplified products with internal size standard (GENESCAN400HD, Applied Biosystems Japan) wererun on a 6% polyacrylamide denaturating gel (Long Ranger) using BaseStation100 DNA fragment
analyzer (Bio-Rad). Although alleles were visualized using Cartographer software specifically designed
for the BaseStation, allelic sizes were determined manually in relation to the internal size standard and fin
whale’s DNA of known size that were rerun on each gel.
Statistical analysis
MtDNA
Sequences were aligned initially using Sequence Navigator (Applied Biosystems, Inc). The aligned
sequences were then corrected by eye for minor inconsistencies.
Haplotype and nucleotide diversity were calculated following Nei (1987). Conventional hypothesis
testing procedure was conducted based on the randomized chi-square test of independence (Roff and
Bentzen, 1989) to assess genetic differences among Areas. Fst was calculated using the AMOVA(Excoffier et al., 1992). Furthermore heterogeneity tests were conducted as described in Hudson et al.
(1992), using the Hst and the Kst* statistics. The level of statistical significance was estimated from10,000 Monte Carlo simulations as the proportion of simulations in which a similar or more extreme
value of Hst or Kst* was observed.
Microsatellites
The number of alleles per locus and expected heterozygosity per locus was calculated using FSTAT 2.9.3(Goudet, 1995). Statistical tests for the deviations from expected HardyWeinberg genotypic proportions

2

were conducted using GENEPOP 4.0 (Rousset, 2008).
Probability test (or Fisher’s exact test) implemented in GENEPOP 4.0 (Rousset, 2008) was used to
conduct the heterogeneity tests. When simultaneous multiple tests were conducted, correction for themultiple tests was performed (Rice, 1989). Statistical significance was also determined using thechi-square value obtained from summing the negative logarithm of p-values over the 16 microsatellite
loci (Sokal & Rohlf 1995). A p-value smaller than 0.05 was used as a criterion to reject the null
hypothesis of panmixia.
RESULTS AND DISCUSSION
MtDNA
Diversity
A segment of 479bp of the mtDNA control region was sequenced for all samples. A total of 32
polymorphic sites identified a total of 45 unique sequences (haplotypes) in the total sample of 55 fin
whales (Table 2). Except for two transversion sites, all substitutions were transitions.
Level of mtDNA diversity based on nucleotide and nucleon diversity is shown in Table 3. Both indices
showed a high and similar degree of mtDNA diversity through the Areas. Nucleotide diversity estimate
for the total sample was 0.0121 (Table 3).
Heterogeneity test
No significant differences were found in the statistical comparison between Areas IIIE+IV and Areas
V+VIW (Table 4). Values of Fst, Hst and Kst* were negative. It was possible that no evidence of thestatistically significant difference was because most of the haplotypes were singletons in each area.
Because the haplotype distributions were quite different among the areas, an additional statisticalcomparison was made by re-grouping haplotypes into two categories: those specific to each of the areas
and those shared by more than one area (Table 5). In this case, statistically, highly significant differenceswere observed among the areas (Table 6).
Microsatellite
Diversity
Table 7 shows the number of alleles, expected heterozygosity and test result for expected Hardy-Weinberg
genotypic proportions at 16 microsatellite loci. Total number of alleles at the loci in all the samples
combined ranged from six at EV104 to 25 at EV94 with an average of 13.7. Expected heterozygosity
ranged from 0.487 at FB14 to 0.955 at EV94 with an average of 0.843. Three loci (GAT98, EV21,
TAA31) after correction for the simultaneous multiple tests and overall value showed significant
deviation from the expected Hardy-Weinberg genotypic proportions.
Heterogeneity test
Pair-wise comparisons among the areas showed the statistically significant differences between whales
from Areas IV and V, but not between IIIE and IV, as well as IIIE and V. (Table 8).
Stock structure
Both of the genetic markers detected the genetic heterogeneity between Areas IV (Indian Ocean) and V(Pacific Ocean). In contrast to the past genetic studies (Wada and Numachi, 1991; Pastene et al., 2005),
our genetic study raised the possibility of genetic structuring in the JARPAII research area. This
corresponded to the past observation of the feeding segregation between South of the Indian Ocean (IV)and South of Western South Pacific Ocean (V) (Mackintosh, 1965).
The catch distribution of fin whales in the Antarctic based on the IWC catch database (1913-1973) isshown in Fig. 2. Fin whales were widely distributed from the middle latitudes to south of 60°S along all
longitudes. They were concentrated in Area III (between 10°E-60°E), in Area IV (between 80°E-110°E),
Area V (between 140°E-170°E) and Area VI (between 170°W-150°W). A low density zone was observed
around 130°E in the JARPAII research areas. Therefore this distribution pattern also matched well withthe results of our genetic analyses.
The mtDNA analysis also detected the difference in the haplotype distributions between Areas IIIE and IV,
although the microsatellite analysis failed to detect such differences most likely due to the small sample
size of IIIE. Our study thus suggested the possibility of an additional structuring in the JARPAII researcharea. In Area IIIE, an additional stock may be distributed or multiple stocks may be mixed. However, it

3was difficult for us at this time to distinguish between these two possibilities. A similar pattern of genetic
structure was observed in humpback whales in the JARPAII area (Pastene et al., 2013; Kanda et al.,2014). Pastene et al. (2013), by analyzing mitochondrial DNA variations on humpback whales from boththe feeding and breeding grounds, showed that IV from 80˚E to 120˚E was occupied by one stock(Western Australia stock) and V from 140˚E to 160˚E by another stock (Eastern Australia stock) and thatthe rest of the areas were mixing areas of the adjacent stocks.
Our study implies that effective management of fin whales in Antarctic should be conducted Area by Area
although it is premature yet to finalize their stock structure. In order to better understand the stock
structure of fin whales, further genetic analyses should be conducted in the future based on larger sample
sizes.
ACKNOWLEDGEMENTS
We would like to thank the researchers and crew members of the JARPA and JARPAII surveys for theirefforts in collecting the samples. Our gratitude goes to H. Oikawa and S. Azumi for their laboratory
assistance.
REFERENCES
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sequences of all extant baleen whales and two sperm whale species. J. Mol. Biol Evol. 70: 960-970.
Bérubé, M., Aguilar, A., Dendanto, D., Larsen, F., Notarbartolo-di-Sciara, G., Sears, R., Sigurjónsson,
J.,Urban-Ramirez, J., and Palsbøll, P.J. 1998. Population genetic structure of North
Atlantic,Mediterranean Sea and Sea of Cortez fin whales, Balaenoptera physalus (Linnaeus, 1758);
analysis of mitochondrial and nuclear loci. Mol. Ecol. 7:585-599.
Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøll, P.J. 2000. Polymorphic di-nucleotide
microsatellite loci isolated from the humpback whale, Megaptera novaeanglliae. Mol. Ecol.
9:2181-2183.
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.
Buchanan, F.C., Friesen, M.K., Littlejohn, R.P., and Clayton, J.A. 1996. Microsatellites from beluga
whale Delphinapterus leucas. Mol. Ecol. 5:571-575.
Excoffier, L., Smouse, P.E. and Quatro, J.M. 1992. Analysis of molecular variance inferred from metricdistances among DNA haplotypes: application to human mitochondrial DNA restriction data.
Genetics 131:479-91.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered. 86:485-486.
Hudson, R.R., Boos, D.D. and Kaplan, N.L. (1992). A statistical test for detecting geographic
subdivision. Mol Biol Evo 9:138-151.
Kanda, N., Goto, M. and Pastene, L.A. 2014. Stock structure of humpback whales in the Antarctic feeding
grounds as revealed by microsatellite DNA data. Paper SC/F14/J31 presented to the JARPAII review
meeting. February 2014. (unpublished). 5pp.
Mackintosh, N.A. 1965. The stocks of whales. Fishing News (Books) Ltd.., London. 232pp.
Matsuoka, K., Hakamada, T., Kiwada, H., Murase, H. and Nishiwaki, S. 2006. Distributions andstandardized abundance estimates for humpback, fin and blue whales in the Antarctic Areas IIIE, IV,
V and VIW (35°E -145°W), south of 60°S. Paper SC/D06/J7 submitted to the JARPA REVIEW
meeting. December 2006. (unpublished) 37pp.
Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. x+512pp.
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., Kanda, N. and Nishiwaki, S. 2005. Genetic analyses on stock identification in
the Antarctic humpback and fin whales based on samples collected under the JARPA. Paper
JA/J05/JR16 submitted to the JARPA review meeting. January 2005. (unpublished) 13pp.
Pastene, L.A., Kitakado, T., Goto, M., and Kanda, N. 2013. Mixing rates of humpback whales of Stocks
D, E and F in the Antarctic feeding grounds based on mitochondrial DNA analyses. Paper
SC/65a/SH13 presented to the IWC Scientific Committee, June 2013, Cheju Island, Korea(unpublished).
Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution 43:223-225.
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.
Rousset, F. 2008. Genepop'007: a complete re-implementation of the genepop software for Windows and4Linux. Molec. Ecol. Resources 8:103-106.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Second
Edition. Cold Spring Harbor Laboratory, New York.
Sokal, R.R., and Rohlf, F.J. 1995. Biometry: the Principles of Statistics in Biological Research. Freeman
and Company, New York.
Valsecchi, E., and Amos, W. 1996. Microsatellite markers for the study of cetacean populations. Mol.
Ecol. 5:151-156.
Wada, S. and Numachi, K. 1991. Allozyme analyses of genetic differentiation among the populations andspecies of the Balaenoptera. Rep. int. Whal. Commn (special issue 13):125-54.

5

Table 1 Number of samples used in this studies by year on Antarctic fin whales collected during JARPA and
JARPAII sueveys.
Areas
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Total
IIIE
4
2
6
IV
4
19
23
V
3
6
6
6
1
2
24
VIW
2
2

Table 2. Variable sites defining 45 mtDNA haplotypes in the Antarctic fin whale. The column on the left
are haplotype ID. The numbers above listed on the right side are the nucleotide positions of thepolymorphic sites starting from the 5’ end of the mtDNA control region. Haplotypes ‘2’ through ‘45’ arelisted with reference to haplotype ‘1’. A dot indicates an identical nucleotide at the position relative tohaplotype ‘1’. The left side of the table are the frequencies of the 45 haplotypes in the four Antarctic
Areas.

Haplotype Area IIIE
1
1
2
1
3
1
4
1
5
1
6
1
7
0
8
0
9
0
10
0
11
0
12
0
13
0
14
0
15
0
16
0
17
0
18
0
19
0
20
0
21
0
22
0
23
0
24
0
25
0
26
0
27
0
28
0
29
0
30
0
31
0
32
0
33
0
34
0
35
0
36
0
37
0
38
0
39
0
40
0
41
0
42
0
43
0
44
0
45
0
6

Area IV
0
1
0
0
0
0
1
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
23

Area V
0
0
0
0
0
0
0
0
0
0
1
0
0
2
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
2
1
1
1
1
1
1
2
1
1
1
1
1
0
0
24

10
11
1667788900
4082978748
GCAGTTTTTA.TG..C....
.TG..C....
.TG.......
..G.......
...A.C....
..G.......
.....C....
.TG..C.CC.
..........
..G..C....
..G.......
..G.....C.
.....C....
..G..C....
..G..C....
..........
ATGT.....G.TG..C....
..G....C..
.TG.......
.TG.......
..G.......
.TG.......
.TG.......
..G.......
..G.......
..G..C....
.TG.....C.
.TG.......
..G.......
..G.......
.TG..C....
.TG..C....
..G...C...
..G..C....
..G.......
.TG..C....
..G.......
..G.......
..........
.TG.......
.....C....
....C.....
.TG......G2223222222

Area VIW
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
2

6

20
1112222222
3790015555
4910144689
GCCCTTAACG....C.....
....C....A....C.....
........T.
..........
.....CG...
....C.....
....C.....
..........
..........
.........A..........
....C.....
....C.....
.TATC.....
....C.G...
....C.....
....C.....
A...C.....
..........
..........
........T.
..........
....C.....
...TC.....
....C..G..
....C.G...
....C.....
..........
....CCG...
..........
....C.....
..........
....C.....
.........A..........
....C.....
..........
..........
..........
..........
....C.....
....C.....
....C...T.
2222222222

30
2222223333 34
6888890026 90
6347811245 80
CTTTTATCAT CA......C... ..
......CT.. ..
........G. ..
.........C ..
......C... ..
.C....C... ..
........G. ..
...C...T.. ..
......C... ..
......C... ..
.....G.T.. ..
......C... ..
.......... ..
......C... ..
.C..C..... ..
......CT.. ..
.......... .G..C...CT.. ..
.......... ..
......C... .G......C... ..
.......... ..
T.....C... .G......CT.. .G.......... .G.......... .G.......... ..
......C... .G..CC..CT.. ..
......C... ..
..CC...... ..
......CT.. ..
......CT.. ..
..C...C... ..
......C... ..
......C... ..
......CT.. .G.C........ T.
.....G.T.. ..
.......... T.
..C...C..C .G.......... T.
.......... ..
.......... .G
2222222222 22

Table 3. Estimates of the nucleon and nucleotide diversities in the Antarctic fin whale.

H
IIIE+IV
V+VIW

0.99261
0.99077π
0.010760.01052SE
0.000860.00049

Table 4. Results of the heterogeneity tests using randomized chi-square, Fst, Hst and Kst* in pair-wisecomparison.

P
value

χ ２
0.413

Fst
0.524
-0.001

Hst
0.574-0.0005

Kst*
0.935-0.0058

Table 5. Frequencies of area specific haplotypes and shared common haplotype groups in areas IIIE, IV
and V.

IIIE specific
Shere between IIIE & IV
IV specific
Shere between IV & V
V specific

IIIE
5
1
0
0
0

IV
0
1
17
5
0

V
0
0
0
5
19

Table 6. Statistical comparison among areas IIIE, IV and V re-grouped in the Table 6.

IIIE vs IV
IIIE vs V
IV vs V

χ ２
<0.0001<0.0001<0.0001

Fst<0.0001<0.0001<0.0001

Value
0.599
0.659
0.599

7

Table 7. The number of alleles (A), expected heterozygosity (He), and test resultsfor expected Hardy-Weinberg genotypic proportions (HW) at 16 microsatellite loci.

A
17
24
14
17
13
25
15
9
6
20
12
8
8
16
13
8

EV37
EV1
GT310
GAT28
GT575
EV94
GT23
GAT98
EV104
GAT417
GT211
EV21
FB14
EV14
GT195
TAA31

He
0.913
0.941
0.921
0.849
0.734
0.955
0.888
0.775
0.639
0.843
0.895
0.614
0.487
0.788
0.875
0.512

HW
n.s
n.s
n.s
n.s
n.s
n.s
n.s
sign.
n.s
n.s
n.s
sign.
n.s
n.s
n.s
sign.

All

High.
Sign.

Table 8. Results of the heterogeneity test comparing allele frequencies of 16 microsatellite loci of fin
whales, among Antarctic management Areas.

P value
0.67760.99970.0136

IIIE vs IV
IIIE vs V
IV vs V

60S

60S

70S

70S

80S
30E

50E

70E

90E

110E

130E

150E

170E

170W

150W

80S

Figure 1. Geographic sampling localities of fin whales by catching and biopsy during JARPA and
JARPAII.

8

Fig.2. Distributions of fin whales from December to March (IWC catch database, noon positions (NP) of
the days they were caught along the Southern Boundary of Antarctic Circumpolar Current) (after
Matsuoka et al., 2006)9