SC/65b/SD03

Update of the genetic analysis on stock structure of fin
whales in the Antarctic based on mitochondrial andmicrosatellite DNA (SC/65b/J32rev)
Mutsuo Goto, Naohisa Kanda and Luis A. Pastene
Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan
ABSTRACTThis paper is a revised version of SC/F14/J32, which respond to some recommendations from theJARPAII review Workshop. Genetic samples (catches and biopsies) of fin whales obtained by JARPA
and JARPAII were analyzed with two genetic markers, mtDNA control region sequencing(479bp-segment) and microsatellite DNA (16 loci), to investigate stock structure of this species in theAntarctic 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. Large number of singletons and small sample sizes could havedecreased the power of the mtDNA statistical analysis. The microsatellite analysis showed a statisticallysignificant deviation from the Hardy-Weinberg equilibrium in Area V, and the heterogeneity test showedsignificant differences between Areas IV and V. Results of the genetic analyses therefore suggested thepossibility of genetic structuring of fin whales in the JARPAII research area, which should be further
explored with the analyses 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 theAntarctic 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
IIIE, IV, V and VIW.
This paper is a revised version of SC/F14/J32, which respond to the recommendations of the JARPAII

1

Review Workshop. The Workshop recommended that an updated paper be provided that:(1) omits the analysis using post-hoc groupings unless the authors can provide a robust statisticaljustification for this approach;(2) discusses in more detail the fact that three microsatellite loci showed significant departures from
Hardy-Weinberg (HW) expectations after accounting for multiple testing before the data areused in other analyses;(3) provides FIS values for each locus in table 7 of SC/F14/J32, which would indicate whether theHW departures represent an excess or deficiency of heterozygotes; (that information is essential
to understand the possible causes for the deviations);(4) shows how the samples were arranged into management Areas in fig.1; and(5) recognises that conclusions about possible differences between Areas IV and V are provisional
until the nature, cause, and influence of these HW deviations are resolved.

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 GAAC-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 listedabove. A total of 25 cycles for 10 seconds at 96C, 20 seconds at 56C and four minutes at 60C areperformed. 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 (TakaraShuzo), 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 designedfor 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 analysisMtDNA
Sequences were aligned initially using Sequence Navigator (Applied Biosystems, Inc). The aligned2

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 andBentzen, 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 Hardy-Weinberg genotypic proportions,as well as their FIS values (Weir and Cockerham, 1984) were calculated, using GENEPOP 4.0 (Rousset,2008).
Probability test (or Fisher’s exact test) implemented in GENEPOP 4.0 (Rousset, 2008) was used toconduct 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 is possible that the large number of
haplotypes represented by a single individual (singletons) summed to the small sample size had decreased
the power of the statistical analysis.
Microsatellite
Diversity
Table 5 shows the number of alleles, expected heterozygosity, test results for expected Hardy-Weinberg
genotypic proportions and inbreeding indices (FIS) at 16 microsatellite loci by the total sample and by thedifferent areas. Both average number of the alleles and average heterozygosity over the 16 loci were thehighest in Area V, so that there were alleles unique to this area. Three loci (GAT98, EV21, TAA31) after
correction for the simultaneous multiple tests and overall value showed significant deviation from theexpected Hardy-Weinberg genotypic proportions. FIS values of these three loci were all positive,indicating homozygote excess. Although these FIS values were relatively small figures, the homozygote
excess suggested existence of individuals from multiple stocks in our samples. It is important to noticethat homozygote excess was not caused by the null allele. Evidence of the deviation from the expected
Hardy-Weinberg genotypic proportions were detected only in the Area V with stronger indication of thehomozygote excess when the tests were conducted for each of the areas separately. It should be noticed
that there was a single individual in the area V sample having one or two alleles unique to only thisindividual at eight of the 16 loci. Although this fact could have caused the observed significant deviationfrom the expected Hardy-Weinberg genotypic proportions in the area, removal of this particular
individual unchanged the test result (data not shown).
Heterogeneity test
Pair-wise comparisons among the areas showed the statistically significant differences between whales

3

from Areas IV and V, but not between IIIE and IV, and between IIIE and V, most likely due to the small
sample size of Area IIIE. (Table 6).
Stock structure
One of the genetic markers (microsatellite) detected significant genetic heterogeneity between Areas IV(Indian Ocean) and V (Pacific Ocean). Significant departure from the Hardy-Weiberg equilibrium wasdetected in Area V. 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. One of theinterpretations for the results based on microsatellite is that at least two stocks distribute in the researcharea, one in Area IV and the other in Area V, which possible mixing of different stocks in Area V.
Our study implies that effective management of fin whales in Antarctic should be conducted Area by Area
although it is premature yet to conclude on their stock structure in the research area. 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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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,
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Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøll, P.J. 2000. Polymorphic di-nucleotide
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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.
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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.
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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 geographicsubdivision. Mol Biol Evo 9:138-151.
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.
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 theproblem of small samples. Mol. Biol. Evol. 6:539-45.
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Edition. Cold Spring Harbor Laboratory, New York.
Sokal, R.R., and Rohlf, F.J. 1995. Biometry: the Principles of Statistics in Biological Research. Freemanand Company, New York.
Valsecchi, E., and Amos, W. 1996. Microsatellite markers for the study of cetacean populations. Mol.
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Wada, S. and Numachi, K. 1991. Allozyme analyses of genetic differentiation among the populations and4

species of the Balaenoptera. Rep. int. Whal. Commn (special issue 13):125-54.
Weir, B.S., and Cockerham, C.C. 1984. Estimating F-statistics for the analysis of population
structure. Evolution 38: 1358-1370.

5

Table 1 Number of samples used in this studies by year on Antarctic fin whales collected during JARPA andJARPAII sueveys.
Areas
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Total
IIIE426IV41923V36661224VIW22Table 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
11213141516170809010011012013014015016017018019020021022023024025026027028029030031032
033
034
035036037038039040041042043
04404506Area IV01000012111112111111111111000000000000000000023Area V00000000001002000000000
1011111211111121111100
2410111667788900
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
00000000000000000000000000000000000
00
0
00
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.

π0.010760.01052

H
IIIE+IV
V+VIW

0.99261
0.99077SE
0.000860.00049

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

χ ２
0.413

PvalueFstHstKst*

0.524-0.001

0.574-0.0005

0.935-0.0058

Table 5. The number of alleles per locus, expected heterozygosity, test results for expected
Hardy-Weinberg genotypic proportions (p-values) and inbreeding index (FIS) at 16 microsatellite lociby total and by different areas.
Genetic index

Total

IIIEIVV

No. alleles per locus

14.4

5.5

9.9

13.1

VIW
2.8

Heterozygosity

0.816

0.799

0.789

0.834

0.875

Hardy-Weinberg / Fis
EV37

0.854

0.012

1.000-0.044

0.948-0.0480.043

0.088-EV1

0.777-0.032

1.000

0.000

0.845

-0.0630.969-0.010

1.000

0.000

GT310

0.650

-0.0731.000-0.0810.721-0.0860.783-0.0551.000-0.333

GAT28

0.993

0.005

1.000

-0.0530.883-0.076

0.622

0.068

-GT5750.801-0.066

1.000

0.172

0.897

-0.1840.568-0.006

-EV94

0.197

0.053

1.000

0.000

0.307

-0.002

0.068

0.115

-

GT23

0.102

0.045

0.164

0.059

0.528

0.021

0.336

0.053

1.000

-0.333

GAT98

0.000

0.110

1.000

-0.0320.244-0.010

0.004

0.132

0.334

1.000

EV104

0.608

-0.043

0.619

0.000

0.134

-0.0200.573-0.045-GAT417

0.041

0.004

0.622

0.086

0.445

-0.083

0.626

0.058

-

GT211

0.486

0.066

0.350

0.314

0.320

0.029

0.435

0.058

-

EV21

0.009

0.085

0.302

0.172

0.185

-0.063

0.016

0.184

-

FB14

0.032

0.186

1.000

-0.391

1.000

0.018

0.059

0.273

0.334

EV14

0.334

0.002

1.000

-0.333

0.763

0.006

0.046

0.049

-GT195

0.030

0.178

1.000

-0.1770.051

0.2550.053

0.173-TAA31

0.000

0.059

1.000

-0.1430.809-0.104

0.000

0.138

1.000

All

High. Sign.

1.000

0.753

High. Sign.

1.000-0.333

0.975

Table 6. Results of the heterogeneity test comparing allele frequencies of 16 microsatellite loci of fin
whales, among Antarctic management Areas.

IIIE vs IV
IIIE vs V
IV vs VP value
0.67760.99970.0136

7

Area III (East)

Area IV

Area V

Figure 1. Geographic sampling localities of fin whales by catching (and JARPAII.

8

Area VI (West)) and biopsy () during JARPA