THIS PAPER CAN NOT BE CITED WITHOUT WRITTEN PERMISSION FROM THE AUTHORS
SC/D06/J31

GENETIC CHARACTERISTICS AND POPULATIONSTRUCTURE OF HUMPBACK WHALES IN THE
ANTARCTIC FEEDING GROUND AS REVEALED BY
MITOCHONDRIAL DNA CONTROL REGIONSEQUENCING AND MICROSATELLITE ANALYSES
L. A. Pastene1, M. Goto1, S. Nishiwaki1, H. Yoshida2, and N. Kanda1
1

The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan

2

National Research Institute of Far Seas Fisheries, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa
236-8648, Japan
ABSTRACT
Biopsy samples from 411 humpback whales obtained during surveys of the Japanese Whale Research
Program under Special Permit in the Antarctic and International Decade for Cetacean Research/Southern
Ocean Whale and Ecosystem Research were analyzed in order to describe their genetic population
structure in the Antarctic feeding grounds. Samples were obtained from International Whaling
Commission (IWC)-designated management Areas III (n= 81), IV (n= 172), V (n= 97) and VI (n= 61),
and were examined for i) sex determination, ii) the sequence variation of the first 334bp nucleotides ofthe mtDNA control region and iii) genetic variation at the genotypes of six microsatellite loci. Duplicatedsamples were excluded from the analysis. The level of genetic diversity in the Antarctic was high for bothgenomes: the nucleotide diversity at the mtDNA was estimated at 0.0263 and the mean expected
heterozygosity at the nuclear loci at 0.7820 for the total samples. In general results based on both mtDNA
and microsatellites were similar and they suggest population structure of humpback whales in the
Antarctic feeding grounds. These genetic results are consistent with the previous view based on nongenetic data that Areas III, IV, V and VI are occupied by different populations. The most plausible patternof structure in the Southern Hemisphere therefore is multiple breeding and feeding grounds with someextent of site fidelity. Marked differences were found between whales in Areas IV and V for both mtDNA
and microsatellites, and the same pattern was found for both sexes. Results of the other pair-wisecomparisons among Areas showed more subdivisions in females than in males. An explanation for thisresult is that the difference is due to the lower sample sizes for males in these comparisons. The
possibility of intermingle of populations in bordering sectors can not be discarded yet and acomprehensive analysis that involves genetic data from low and high latitude, as recommended by the
Scientific Committee, is necessary to solve this issue.
KEYWORDS: ANTARCTIC, FEEDING GROUNDS, GENETICS, HUMPBACK WHALE
INTRODUCTION
Humpback whales, Megaptera novaeangliae, are found worldwide in all major oceans. Like other
Balaenopterid species, humpback whales migrate between summer feeding grounds in mid- and highlatitudinal waters and winter breeding grounds in tropical or subtropical waters. Animals occur primarily
in coastal and continental shelf waters.
Three oceanic populations of the humpback whale occur in the North Atlantic, North Pacific andSouthern Hemisphere, and different patterns of structure have been reported within each major oceanic
population. In the western North Atlantic, for example, whales congregate to mate and calve in a single
breeding area of the West Indies during the winter. In summer they congregate in several high latitude1feeding areas. Genetic studies suggest limited exchange among the summer feeding areas, and mixing in
the winter breeding area (Palsboll et al., 1997a). The pattern of structure here is single breeding grounds
and multiple feeding grounds. In the North Pacific the situation is much more complex. Whales show astrong fidelity to winter breeding grounds in Costa Rica, Mexico, Hawaii, Southeast Asia, and wide, butphilopatric, dispersal to feeding grounds in temperate and high latitudinal waters. Some movements ofanimals between low latitudinal breeding areas have been reported (Darling and McSweeney, 1985;
Darling and Mori, 1993). Genetic studies have shown significant differences between whales distributedin central and eastern North Pacific (Baker et al., 1998a). The pattern of structure here is single breeding
grounds and multiple feeding grounds but the case of multiple breeding grounds using a single feeding
ground has also been suggested. These phenomena seem to be related with differences in topography
among ocean basins and as a consequence, the breeding grounds in the North Atlantic are few, those in
the North Pacific are larger in number and those in the Southern Hemisphere are many.
Regarding the Southern Hemisphere, Mackintosh (1965) showed that humpback whales tend togather into five or six distinct feeding concentrations in the Antarctic during the austral summer season.
These feeding concentrations were denominated as Groups I-V (with a Group IIa and IIb) corresponding
roughly to IWC Management Areas I-VI. The Groups most documented are Groups IV and V. Omura(1953) examined the distribution of humpback whale in the feeding grounds of Areas IV and V based oncatch data. Based on catch information he suggested that two populations occur in these Areas with aboundary around 130°-142°E. He did not discard the possibility of intermingling between these two
populations in the feeding ground. He also examined the pattern of distribution by month and suggested
that for the month where more data were available (November-March) the boundary between these two
populations changed from 120°-130°E in November to eastside of 140°E in December and to 120°-140°E
in January. Dawbin (1966) summarize the distribution and seasonal migratory movement of humpback
whales from Groups IV and V, as demonstrated by mark-recapture data (Discovery-type marks). Whales
from Group IV move mainly between Antarctic Area IV and Western Australia while whales from GroupV move between Antarctic Area V and Eastern Australia and along the coast of New Zealand andsouthwest Pacific islands. Interchange of a few individuals between Groups IV and V was reported.
Dawbin (1966) also reported that the boundary of Groups IV and V in the Antarctic do not correspond to
the actual boundary between Areas IV and V and that some whales marked in Area VI were recovered ineastern Australia.
These migratory corridors between low latitudinal breeding grounds and high latitudinal feeding
grounds have been confirmed by analysis of photo-identified whales. Gill and Burton (1995) reported the
movement of a photo-identified whale between Western Australia and Antarctic Area IV and Kaufman etal. (1990) that between Eastern Australia and the boundary between Areas V and VI. More recently thematches of three individual humpback whales between Eastern Australia and Antarctic Area V, werereported (Rock et al., in press).
Genetic analyses based on mitochondrial DNA (mtDNA) and samples from low latitude waters(Baker et al., 1998b) showed significant genetic differences between Western Australia, Eastern Australia
and Colombia humpback whales, supporting the previous view that different populations occur in thoseregions (corresponding to Groups IV, V and I mentioned above). Little is known, however, on the genetic
diversity and population structure of humpback whales in the Antarctic feeding grounds. The present
paper addresses this issue by examining samples obtained in Areas IV and V and their adjacent Areas III
and VI. As noted above, previous non-genetics studies had suggested that these Areas are occupied bydifferent populations. Here the hypothesis of different populations occurring in these Areas is tested using
genetic data. The analysis is also conducted to investigate the current status of genetic variability of
humpback whales.
The analysis is based on the maternal inherited mtDNA and bi-parental inherited microsatellites.
Further the analysis is conducted for total samples as well for each sex separately. Previous studies basedon mark-recapture suggested a high fidelity of animals to feeding areas (identified approximately by theboundaries of the current Areas) and particular breeding areas. Therefore genetic differences among
Areas are expected. There is the possibility, however, of a sex-differentiated pattern of dispersal in thefeeding ground. This could be investigated by the genetic analysis conducted separately for males andfemales.

2

MATERIALS AND METHODS
Samples
Skin biopsy samples were obtained from free-ranging whales along the sighting surveys of the JARPA(Japanese Whale Research Program under Special Permit in the Antarctic) and IDCR/SOWER(International Decade for Cetacean Research/Southern Ocean Whale and Ecosystem Research) surveys in
Areas IIIE, IV, V and VI (Figure 1), on an opportunistic basis. Biopsy samples in JARPA were collectedusing an air gun described in Kasamatsu et al. (1991) and more recently using a Paxarm system. Biopsy
samples in IDCR/SOWER were collected using several methods including Paxarm system and crossbows.
At the laboratory all biopsy samples were checked for the possibility of re-sampling (two or more
samples taken from a same individual) by comparing the genotype profiles produced by a set of six
microsatellites.
Re-sampling rates were higher for IDCR/SOWER than for JARPA samples. For the former it varied
from 0% in Area V to 15.0% in Area VI (sample size in Area V was only seven). In the case of JARPA it
varied from 4.3% in Area VI to 10.0% in Area III. Taylor and Chivers (1997) demonstrated bysimulations that if duplicated samples are not recognized, results of genetic analyses on population
structure will be biased. Therefore in the present study duplicates were excluded from all analyses.
When mother/calf pairs were sampled, only the genetic information of the mother was used for the
analysis (nine cases in Area V).
Table 1 shows the actual number of samples used in the analysis by IWC management Area, sex,
and kind of surveys, and Figure 1 shows the geographical distribution of samples in the feeding groundsof Areas IIIE, IV, V and VI, by sex. A total of 411 samples were used.
Molecular genetic analysis
Extraction of DNA
Genomic DNA was extracted from approximately 0.05g of the outer epidermal layer of the skin biopsy,
by standard phenol/chloroform extractions (Sambrook et al., 1989). Extracted DNAs were stored in TE
buffer (10mM Tris-HCl, 1mM EDTA, ph8.0).
Sex determination
Sex of the whales sampled was determined following the method of Abe et al. (2001), which uses a set ofoligonucleotide primers to detect the presence/absence of the SRY gene from the Y chromosome with amicrosatellite locus (GATA 417) as an internal control.
Mitochondrial DNA control region sequencing
The first 334 nucleotides at the 5' end of the mitochondrial control region were amplified by thepolymerase chain reaction (PCR, Mullis and Faloona 1987). The oligo-nucleotides employed in the PCR
amplification were MT4 (Arnason et al. 1993) and P2R (5’-GAA GAG GGA TCC CTG CCA AGC GG3'). Reactions were carried out in 50 uL volumes containing 100 mM KCl, 20 mM Tris-HCl, 0.1 mM
EDTA, 1 mM DTT; 0.5% Tween 20, 0.5% Nonidet P-40, 200 uM dNTPs, 2.5 pM of each oligonucleotide and one unit of Taq DNA polymerase. 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
was repeated 30 times. The amplification was completed with a final extension step of 10 minutes at 72°C.
Subsequent cycle sequencing reactions were performed with 100ng of products generated in the above
PCR amplifications using the PrismTM dRhodamine Terminator Cycle Sequencing Kit (Applied
Biosystems, Inc.). 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 with 10 seconds at 96°C, 20
seconds at 56°C and four minutes at 60°C were performed. The nucleotide sequence of each cycle
sequencing reaction was determined by electrophoresis through a 5% Long RangerTM (FMC, Inc.)
denaturing polyacrylamide matrix on a DNA PrismTM 377 DNA Sequencer (Applied Biosystems, Inc.)
under standard conditions. Both strand samples were sequenced in their entirety for all samples.
Microsatellites
Six microsatellite loci were used in this study: four tetranucleotide motifs (GATA417, GATA28,
GATA98, GATA53) (Palsbøll et al. 1997b), one trinucleotide motifs (TAA31) (Palsbøll et al. 1997b),3

and one dinucleotide motifs (GT23) (Bérubé et al. 2000). PCR amplifications were performed in 15ulreaction mixtures containing 10 to 100ng of DNA, 5pmol of each primer, 0.625 units of Ex Taq DNA
polymerase (Takara Shuzo), 2mM of each dNTP, and 10x reaction buffer containing 20mM MgCl2(Takara Shuzo). Amplified products with internal size standard (GENESCAN400HD, Applied
Biosystems Japan) were run on a 6% polyacrylamide denaturing gel (Long Ranger) using BaseStation100
DNA fragment analyzer (BioRad). 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 humpback whale’s DNA of known size that were rerun on each gel.
Grouping of the samples
The samples were grouped according to the boundaries of IWC Management Areas III (0°-70°E), IV(70°-130°E), V (130°E-170°W) and VI (170°-120°W). With regard Area III, biopsy samples wereavailable only for the eastern part of this Area (34°-70°E) (Figure 1).
For the analysis samples collected in different austral summer seasons were pooled under theassumptions that the patterns of seasonal movement are the same for a given breeding population indifferent years and that lateral movement on feeding grounds and pattern of mixing are similar between
years. These assumptions are discussed later.
Data analysis
mtDNA
Genetic distances among unique sequences (haplotypes) were estimated using the Kimura’s two
parameters method (Kimura, 1980). The degree of mtDNA diversity within each Area was estimatedusing the nucleotide diversity (Nei and Li, 1979), and the nucleon diversity (Nei and Tajima, 1981).
Heterogeneity tests among Areas were conducted as described in Hudson et al. (1992), using the chisquare and the Kst* statistics. The level of statistical significance was estimated from 10,000 Monte Carlosimulations as the proportion of simulations in which a similar or more extreme value of chi-square or
Kst* was observed. Tests were conducted for all samples combined as well for males and females
separately. When multiple tests were conducted, the Rice (1989) correction for multiple tests was
performed.
Microsatellites
Allele frequencies, the number of alleles per locus, allelic richness, heterozygosity at the six microsatellite
loci and Fst values were computed using FSTAT 2.9.3 (Goudet, 1995). The program GENEPOP 3.2(Raymond and Rousset, 1995) was used to test for deviations from expected Hardy-Weinberg genotypic
proportions at each of the loci.
Heterogeneity tests among Areas were conducted using the probability test (or Fisher’s exact test)with Markov chain method implemented in the GENEPOP. Tests were conducted for all samples
combined as well for males and females separately. Statistical significance was determined using the chisquare value obtained from summing the negative logarithm of P-values over the six microsatellite loci
(Sokal and Rohlf, 1995). When multiple tests were conducted, the Rice (1989) correction for multiple
tests was performed.
The Bayesian clustering approach was conducted using STRUCTURE version 2.0 (Pritchard et al.,2000) to determine the number of genetically distinct populations present in the samples. Three
independent runs for each value of K between 1 (panmictic) and 5 with no prior information (i.e. only
genetic information was considered) were conducted. All of the simulations were based on a burn-in
period of 50,000 iterations and runs of 500,000 iterations. 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
populations are likely to be similar due to migration or shared ancestry.

4

RESULTS
Sex ratio
The male proportion by Area was as follow: Areas III: 38.3%, IV: 52.9%, V: 47.4% and VI: 59.0%.
Genetic variability
mtDNA
A segment of 334bp of the mtDNA control region was sequenced for all samples. A total of 65 variable
sites defined a total of 98 unique sequences (haplotypes) in the sample of 411 humpback whales. Level of
mtDNA diversity based on nucleotide and nucleon diversity is shown in Table 2. Both indices showed ahigh and similar degree of mtDNA diversity through the Areas. The nucleotide diversity at mtDNA for
the total samples was estimated at 0.0263.
Microsatellites
All microsatellite loci were polymorphic in all the samples (Table 3). Five of the six loci had more than
10 alleles with an overall average of 15.3 over the all samples. The number of alleles per locus per Area
varied between 10.7 and 12.7 with smaller differences in allelic richness values. Average expected
heterozygosity was 0.782 over the Areas and ranged from 0.767 to 0.799 per Area. No evidence ofsignificant deviation from expected Hardy-Weinberg expected genotypic proportions was observed ineach of the Areas for over all loci tests. The P-value smaller than 0.05 observed in the Areas V and VI
samples at GATA417 is most likely due to a chance effect. However the power of this analysis could be
low.
Genetic divergence
mtDNA
The results of the heterogeneity test are shown in Table 4. The chi-square test resulted more sensitive todetect differences than the Kst* test. Below is a summary of the results for the former test.
Males: Statistically significant difference was detected across all the loci for the overall Areas. Two of thesix pair-wise comparisons showed statistically significant differences after correction for multiple tests(Areas III/IV and Areas IV/V).
Females: Statistically significant difference was detected across all the loci for the overall Areas. Four ofthe six pair-wise comparisons showed statistically significant differences after correction for multiple
tests (Areas III/IV, Areas III/V, Areas III/VI and Areas IV/V).
Total: Statistically significant difference was detected across all the loci for the overall Areas. Five of thesix pair-wise comparisons showed statistically significant differences after correction for multiple tests.
The only exception was Areas V/VI.
Kst* values shown in Table 4 indicated low level of mtDNA divergence.
Microsatellites
The results of the heterogeneity test are shown in Table 5.
Males: Statistically significant difference was detected across the loci for the overall Areas. Only one of
the six pair-wise comparisons showed statistically significant differences after corrections for the multiple
tests (Areas IV/V).
Females: Statistically significant difference was detected across the loci for the overall Areas. Three ofthe six pair-wise comparisons showed statistically significant differences after corrections for the multiple
tests (Areas III/IV, Areas III/V and Areas IV/V).
Total: Statistically highly significant difference was detected across the loci for the overall areas. Four ofthe six pair-wise comparisons showed statistically significant differences after corrections for the multiple
tests. The only exceptions were Areas III/VI and Areas V/VI.

5

Bayesian clustering analyses conducted without information on geographic sampling origins
presented the lowest likelihood probability at K=5 (Ln probability= -11,951) and the highest likelihood
probability at K= 1 (Ln probability= -9,723).
Overall the Fst values indicated low level of genetic divergence. The overall Fst value for males was
0.008, for females 0.004 and for all samples combined 0.005. Ninety-five % confidence interval for theoverall Fst indicated it was significantly different from zero.
DISCUSSION
Genetic variability
Level of genetic variability found in humpback whales in the Antarctic feeding grounds was high, forboth mtDNA and microsatellites, and it was similar through the Areas. This is consistent with previous
results obtained for the humpback whale worldwide for both markers, mtDNA (Baker et al., 1993;
Palsboll et al., 1995) and microsatellites (Valsecchi et al., 1997).
Pattern of stock structure
In general results based on both mtDNA and microsatellites were similar and they suggest population
structure of humpback whales in the Antarctic feeding grounds. Genetic differentiation among Areas
based on both markers suggests a strong fidelity of humpback whales to migratory destinations. In
particular marked differences were found between whales in Areas IV and V for both mtDNA andmicrosatellites, and the same pattern was found for both sexes. The pattern of structure in the Southern
Hemisphere seems to be multiple breeding grounds and multiple feeding grounds as opposed to thepattern found in the North Pacific and North Atlantic.
Although the samples used in the present study were taken on an opportunistic basis along JARPA
and IDCR/SOWER surveys, sampling had covered most of the longitudinal sectors of Areas IIIE, IV, V
and VI. The exceptions are a sector in Area VI and the western part of Area V for which samples of both
sexes were limited (Figure 1). In general these samples were useful to respond the question of whether
population structure exists in those Areas. It should be noted that for both markers and total samples,
discrimination between Areas V and VI is less in evidence than the other differences. This could bereflecting the distribution of sampling in Area VI where most of the samples used from this Area wereobtained in the IWC boundary separating Areas V and VI (Figure 1).
The mtDNA analysis showed a marked degree of genetic heterogeneity suggesting population
structure of the humpback whales in the Antarctic feeding grounds. Populations appear to be structured
longitudinally. It should be noted that despite the marked mtDNA heterogeneity detected, the level ofgenetic divergence found (as suggested by Kst*) was low. Likewise, the microsatellite analysis detectedthat the individuals in the samples came from genetically different populations of humpback whales thatappeared to be distributed longitudinally. The overall Fst was low but significant, supporting theexistence of multiple populations. STRUCTURE, however, failed to detect the evidence of populationdifferentiation among the samples. Considering the low level of genetic differentiations (Fst), this could
have been caused by the low power of the analysis due to the low number of microsatellite loci used.
Comparison of results between sexes
The analyses were conducted for both sexes separately in order to check the possibility a sexdifferentiated pattern of dispersal in the feeding ground. The mtDNA analysis showed a higher degree ofdifferentiation among Areas for females (four of six pair-wise comparisons were significant statistically)
than males (two of six pair-wise comparisons resulted in significant differences). Same as in the mtDNA
analysis, the microsatellite results showed more subdivisions in females than in males, three and one
significant differences in six pair-wise comparisons, respectively. These results suggest the possibility
that males move wider than females in the Antarctic feeding grounds.
In the comparison between Areas IV and V, however, where the larger sample sizes were available,
results for both sexes were the same for both genetic markers. Then the alternative explanation thatdifferences are just due to the lower sample size available for males in the other comparisons is preferred6because the apparent differences between sexes are not consistent with populations structured
longitudinally in the feeding ground.
Yearly variation in the pattern of structure
As noted earlier for the analysis samples collected in different austral summer seasons were pooled under
the assumptions that the patterns of seasonal movement are the same for a given breeding population indifferent years and that lateral movement on feeding grounds and pattern of mixing are similar between
years. The assumptions can be examined by comparing the mtDNA composition of humpback whales
taken in different years in a same Area. A preliminary analysis of yearly variation was conducted for Area
IV, the Area supported by the larger sample size. In this Area JARPA samples from six different years
were involved. Both chi-square (P= 0.6950) and Kst* (0.431) tests failed to find significant genetic
differences among years suggesting that whales from the same populations come to this Area in each year.
Similar analyses are desirable for the other Areas.
In general these genetic results support the previous view based on non-genetic information that theAreas are occupied by different populations during the feeding season. Our analysis based on samples
from the feeding grounds can not reject yet the possibility that different populations mix to each other insome particular longitudinal sectors. A more accurate longitudinal delineation of populations in the
Antarctic feeding grounds and the identification of intermingle sectors can be accomplished by acomprehensive analysis that combines DNA data available from low latitude breeding grounds and DNA
data from high latitude feeding grounds. The IWC Scientific Committee is conducting an in-depth
assessment of southern humpback whale stocks. The Committee agreed in 2006 that assessment of Stock
D should be considered preliminary due to potential for exchange with breeding Stock E on the feeding
ground. The Committee noted that the status of Stock D should be re-evaluated in the future, but that thiswill first require analyses to clarify the structure of the E breeding sub-stocks and the extent of mixing athigh latitudes. The Committee endorsed the Hobart workshop recommendation that every effort be madefor scientists to share data and carry out such analyses under the IWC Data Availability Agreement (IWC,2006).
Factors influencing the distribution and separation of populations in feeding grounds
What factor(s) is responsible for the population separation in the feeding ground with lack of obvious
physical barriers? Humpback whales are also capable of traveling long distance. The spatial andtemporal separation of the whale populations in the feeding grounds is influenced by the ability of theirsite fidelity, the distribution of their prey species, water current relating to temperature gradient, andunderwater topography (Berta and Sumich, 1999), so that we infer one or a combinations of these factors
was responsible for the separation of the humpback whale populations in the feeding ground. Humpback
whales appeared to largely mimic the distribution of the prey species which is dependent of the highphytoplankton biomass coinciding with Antarctic Circumpolar Current (ACC; Tynan 1998). Upwellingof deep, nutrient-rich water supports very high summertime primary production. Murase et al. (2002)
described the relationship between the distribution of the prey species, euphausiids, and baleen whales in
the Antarctic from 35ºE to 145ºW. The authors found that the distributions of humpback whales werecorrelated with high euphausiid density zone, regardless of the bottom topographic features throughsimultaneous whale sighting and hydroacoustic surveys conducted during JARPA surveys in 1998/1999
an 1999/2000 austral summer. Both sighting frequency and distribution of the prey species was low in
the area between the Areas IV and V.
ACKNOWLEDGEMENTS
We thank all the researchers and crew members of JARPA and IDCR/SOWER surveys involved in thesampling of the biopsy samples used in this study. T. Tamura (ICR) assisted us by producing Figure 1.
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9

Table 1: Number of samples of the humpback whale used in the present
genetic study, by management Area and sex (M:males, F:females).
Area

M

F

Total

Area IIIE

31

50

81 (JARPA: 50; SOWER: 31)

Area IV

91

81

172 (JARPA: 126; SOWER: 46)

Area V

46

51

97 (JARPA: 90; SOWER: 7)

Area VI

36

25

61 (JARPA: 44; SOWER: 17)

Table 2: Nucleotide diversity (upper figure) and nucleon diversity(lower figures) of humpback whales in the Antarctic feeding grounds,
by management Area.
Nucleon

Nucleotide

diversity

diversity (SE)Area IIIE

0.96880.0246 (SE:0.0008)

Area IV

0.97010.0262 (SE:0.0006)

Area V

0.97120.0274 (SE: 0.0008)

Area VI

0.96700.0268 (SE: 0.0009)

10

Table 3. Number of alleles (A), allelic richness (AR), expected heterozygosity (He), and test result for expected Hardy-Weinberg genotypic proportion(HW) (p-value) at six microsatellite loci for humpback whales in the Antarctic feeding grounds, by management Area (n= number of individuals)
Area III (n=81)
A AR

He

Area IV (n=172)
HW

A AR

He

HW

Area V (n=97)
A AR He

Area VI (n=61)

HW

A AR

He

HW

All (n=411)
A AR

He

GATA417

14 13.4 0.910 0.9880

12 12.0 0.906 0.5219

17 15.5 0.901 0.0254

14 14.0 0.917 0.0340

19 13.6 0.908

GATA2810 9.7 0.477 0.8042

11 10.1 0.541 0.9245

12 11.6 0.632 0.5244

11 11.0 0.462 0.5894

14 11.4 0.528

GATA989

8.9 0.727 0.0909

13 11.1 0.794 0.1982

10 9.6 0.793 0.3135

8

8.0 0.732 0.6225

13 10.2 0.762

TAA31

14 13.2 0.874 0.4767

19 14.5 0.888 0.7771

15 14.4 0.906 0.7323

13 13.0 0.871 0.6365

24 14.5 0.885

GATA53

9 8.7 0.820 0.5894

11 9.4 0.830 0.6396

13 11.1 0.803 0.6028

10 10.0 0.837 0.9861

13 9.8 0.822

GT023

8 7.9 0.793 0.1704

9 8.3 0.797 0.1104

9 8.6 0.759 0.7344

9

9

Avg.

10.7 10.3 0.767 0.5005

12.5 10.9 0.793 0.5722

12.7 11.8 0.799 0.3537

10.8 10.8 0.770 0.5829

11

9.0 0.802 0.7133

8.3 0.788

15.3 11.3 0.782

Table 4. Results of the heterogeneity test comparing mtDNA haplotype
frequencies of humpback whales, among Antarctic management Areas(sample sizes in parenthesis). An asterisk indicates those comparisons that
resulted in significant statistically differences after Bonferroni corrections.
χ2 P-value

Kst*

P-value

III/IV (31, 91)

0.0040*-0.0009

0.5830

III/V (31, 46)

0.0310-0.0001

0.4470

III/VI (31, 36)

0.03000.00340.2180

IV/V (91, 46)

0.0000*0.00490.0360

IV/VI (91, 36)

0.05000.00210.1890

V/VI (46, 36)

0.1570-0.00020.4520

III/IV (50, 81)

0.0000*0.00580.0400

III/V (50, 51)

0.0000*0.00830.0190

III/VI (50, 25)

0.0030*0.00280.2280

IV/V (81, 51)

0.0000*0.00490.0600

IV/VI (81, 25)

0.2910-0.00270.8070

V/VI (51, 25)

0.7980-0.0030

0.7380

III/IV (81, 172)0.0000*0.00190.0920

III/V (81, 97)

0.0000*0.00710.0040*

III/VI (81, 61)

0.0000*0.00650.0190

IV/V (172, 97)

0.0000*0.00460.0040*

IV/VI (172, 61)

0.0020*0.00120.1670

V/VI (97, 61)

0.05900.00080.2800Males

Females

Males+Females

12Table 5. Results of the heterogeneity test comparing allele frequencies ofsix microsatellite loci of humpback whales, among Antarctic management
Areas (sample sizes in parenthesis). An asterisk indicates those comparisonsthat resulted in significant statistically differences after Bonferroni corrections.
Males

Females

Males+Females

All Areas

0.003690.00002High. Sig.

III/IV

0.437370.01129*0.00471*

III/V

0.04608

0.00647*0.00000*

III/VI

0.23073

0.15472

0.21129

IV/V

0.00075*

0.00621*0.00000*

IV/VI

0.026190.19756

0.00427*

V/VI

0.01843

0.36140

0.09334

13

MALESM

FEMALESF

F

Figure 1: Geographical distribution of humpback whales examined in this study (upper
figure: males; lower figure: female).

14