SC/F14/J31

Stock structure of humpback whales in the Antarctic
feeding grounds as revealed by microsatellite DNA data
Naohisa Kanda, Mutsuo Goto, and Luis A. Pastene
The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan

Contact email: kanda@cetacean.jp

ABSTRACT
A total of 581 humpback whale biopsy samples obtained from Areas III to VI during surveys of the JARPA and
JARPAII up to 2010/11 season as well as International Decade for Cetacean Research/Southern Ocean Whaleand Ecosystem Research (IDCR/SOWER) were analyzed using 14 microsatellite DNA loci in order to describe
their stock structure in the Antarctic feeding ground. The number of the loci used was increased from six in thepaper submitted to the JARPA Review workshop in 2006. In three of 37 cases of duplicate sampling, the second
samples were collected at least a day apart (1 day, 9 years, and 11 years). Paternity analysis of 13 calf andmother pairs failed to detect any potential fathers in the samples for the calves. After exclusion of some of thesesamples, 528 were used for further analyses at stock level. These individuals were divided into four groups basedon the IWC management areas: IIIE (N=93), IV (N=218), V (N=153) and VIW (N=64). Heterogeneity tests
were conducted for the samples of females only, males only, and both sexes combined, respectively. Although a
few cases of small temporal genetic differences were detected within the areas, major genetic differences wereobserved among the samples from the different areas. As similar to the previous report, stronger differentiation
was seen in females than in males. These results corresponded to those of the previous studies. Despite theincrease of the number of loci, the level of the stock differentiation (FST = 0.003) was still too low to conduct aclustering analysis at the individual level.
With substantial increases in the numbers of the analyzed microsatellite loci and the biopsy samples, ourgenetic study again showed that humpback whales from the different stocks occupied the research areas withhigher differentiation in females than in males. The level of the genetic differentiation among the areas was still
so low that further analysis would require using samples from their breeding areas to better understand the stock
structure in the feeding grounds. This study demonstrated one of the significant contributions of non-lethal partof the comprehensive large-scale JARPAII to acquire valuable information for effective management of large
whales in the Antarctic.

KEYWORDS: ANTARCTIC, BIOPSY SAMPLING, FEEDING GROUNDS, GENETICS, HUMPBACKWHALEINTRODUCTION
Humpback whales conduct seasonal migration between mid to high latitudinal waters in summer for feeding andlow latitudinal waters in winter for breeding. In the Antarctic, humpback whales appear to congregate into fiveor six distinct feeding groups during the austral summer season that roughly match to IWC Management Areas
I-VI (Mackintosh, 1965).
At the JARPA Review workshop in 2006, Pastene et al., (2006) conducted genetic analyses of humpback
whales from the JARPA research area (Areas III to VI) using mitochondrial DNA and microsatellite DNA
markers, and demonstrated that humpback whales from the different areas belonged to genetically different
groups because of the observed genetic differences among them. In addition, it showed that the level of genetic
differentiation was larger in females than in males, strongly suggesting higher female phylopatry and large maledispersion in the feeding grounds. The next question to be addressed was whether or not these feeding groups
were genetically distinct enough from each other that they could correspond to breeding stocks. Pastene et al.
(2013) conducted mtDNA analysis using samples from both the feeding and breeding grounds, and showed thegenetic differences observed among the areas were a consequence of the different proportions of the stocks fromsingle stock occupancy in some areas to multi-stock occupancy in other areas.

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This study used microsatellite DNA markers to analyze the same humpback whale samples from Areas III to
VI used in Pastene et al. (2013). The main purpose of the study is to better understand their stock structure byincreasing the number of the loci from Pastene et al. (2006). In that study, the use of the only six loci might haveresulted in the observed low level of the genetic differentiation among the samples. The estimated FST value wasabout 0.005. At that level of differentiation, it was difficult to conduct clustering analysis at the individual base.
In this paper, we increased the number of the microsatellite loci to 14. In addition, sample size was also
substantially increased from the previous study because of the effort of JARPAII.
MATERIALS AND METHODS
Samples
Table 1 shows the number of humpback whale biopsy samples used in this study separated by IWC management
areas. A total of 581 samples were obtained from the JARPA and JARPAII surveys up to 2010/11 season as well
as International Decade for Cetacean Research/Southern Ocean Whale and Ecosystem Research(IDCR/SOWER).
DNA extraction
In regard to our DNA data quality control under the IWC guidelines, see Kanda et al. (2014). Total DNA wasextracted from 0.05 g of biopsy skin tissue using either the protocol of Sambrook et al. (1989) or GENTRA
PUREGENE DNA extraction kit (QIAGEN). Extracted DNA was stored in the TE buffer (10 mM Tris-HCl, 1
mM EDTA, pH 8.0).
Microsatellite analysis
Genetic variation at microsatellite loci was analyzed using 14 sets of primers: AC137, CA234 (Bérubé et al.,2005), EV1, EV14, EV37 (Valsecchi and Amos, 1996), GT23, GT195, GT271, GT310 (Bérubé et al., 2000),
GATA28, GATA53, GATA98, GATA417, TAA31 (Palsbøll et al., 1997). All except EV1 and EV14 were
designed specifically from humpback whales. Amplified products were run on a 6% polyacrylamide
denaturating gel using a BaseStation 100 DNA fragment analyzer (Bio-Rad), and then sizes of visualized alleles
were determined manually in relation to the internal size standard (Genescan 400HD, Life technologies) as wellas the humpback whale’s microsatellites of known size that were rerun on each gel. The sex of the whales wasdetermined through co-amplification of SRY locus located on the Y chromosome and GT23, which is a slight
modification from Abe et al. (2001). With this combination of loci, males show amplified products of both SRY
and GT23 loci, while females show only GT23.
Data analysis
MICRO-CHECKER (van Oosterhout et al., 2004) was used to check for null alleles and reading/typing errors.
The number of alleles per locus, allelic richness, 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 were conducted using GENEPOP 4.0 (Rousset, 2008). Paternity analysis for assignment of motherand calf pairs to their potential fathers using genetic markers was conducted using CERVUS (Marshall et al.,1998). In order to examine genetic differences among samples, conventional hypothesis testing procedure wasconducted using heterogeneity test in microsatellite allele frequencies among samples. A probability test (orFisher’s exact test) implemented in GENEPOP 4.0 (Rousset, 2008) was used to conduct the heterogeneity tests.
Our null hypothesis to be tested is whether or not the samples came from a genetically same group of humpback
whales. If statistically significant allele frequency differences exist, it could indicate these samples came fromgenetically different stocks of humpback whales. Statistical significance was determined using the chi-squarevalue obtained from summing the negative logarithm of p-values over the 14 microsatellite loci (Sokal & Rohlf
1995). FST value was calculated using FSTAT 2.9.3 (Goudet, 1995).
RESULTS AND DISCUSSION
Among 37 cases of the duplicate sampling, the second samples of three cases were collected at least a day apart(1 day, 9 years, and 11 years; Table 2). One case was first collected in Area IV and was recollected in Area III,
while the other two cases were collected and re-collected in the same area. Because Pastene et al. (2013) showed
the migration of whales from the same stock to Areas III and IV, our results were consistent to the feeding site
fidelity of humpback whales in the Antarctic. Thirteen calf and mother pairs were used for paternity analysis, but
no potential father was detected in the samples.
After exclusion of the second individuals from the duplicated samples and calves sampled with mothers, a
total of 528 samples were used for further analyses. These individuals were divided into four groups based on the2

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management areas: IIIE (N=93), IV (N=218), V (N=153) and VIW (N=64). The levels of genetic diversity of thearea samples were represented as an average number of alleles per locus, average allelic richness, and expected
average heterozygosity (Table 3). These values were similar among them as well as to those based on the six locipreviously reported in Pastene et al. (2006).
Heterogeneity tests for genetic differences among the samples from the different areas were conducted forsamples of females+males, females only, and males only, respectively (Tables 4-9). For the females+males
samples, first, genetic differences among the different year samples within the areas were examined (Table 4).
No evidence of genetic differences was found among the samples from the different years in Areas IIIE, IV, andV, while small level of genetic differences was observed in Area VIW. Because the observed significance in
Area VI was small and none of the pair-wise comparisons among the year samples was significant, the samples
from the different years within each of the areas were combined for further analysis. Evidence of the genetic
differences was then detected among the samples from the different areas (Table 5). Pair-wise comparisons
showed evidence of statistically significant differences for all possible area-pairs (Table 5). The heterogeneity
tests for the females only and males only samples showed similar results to each other with clearer structuring infemales than in males (Tables 6-9). A yearly genetic difference was detected in both samples (Tables 6 and 8). In
females, two (94/95x98/99 and 98/99x08/09) out of the 31 pair-wise comparisons were significant with close-to
5% p-values (0.035 and 0.049). In males, one (96/97x00/01) out of three pair-wise comparisons was significant
with a 0.017 p-value. Because it was difficult to conclude whether or not the difference had a biological meaning,
we combined the year samples within the areas for the next tests. Evidence of the genetic difference among thedifferent areas was detected in both samples. In females, all of the pair-wise comparisons were significantly
different, whereas in males, all except Areas IIIxV and Areas VxVI comparisons were significantly different(Tables 7 and 9). FST was 0.003 among the different area samples for all three sample cases. The results of thisstudy were thus consistent to those previously reported based on six microsatellite loci as whale groups
occupying the different areas were genetically defferent from each other with stronger differentiation in females
than in males.
Pastene et al. (2013) analyzed mitochondrial DNA variations on humpback whales from both the feeding andbreeding grounds to better describe their stock structure, and 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).
The rest of the areas were mixing areas of the adjacent stocks. Despite the substantial increase in the number ofthe loci, the level of the stock differentiation (FST = 0.003) was still too low to conduct a clustering analysis at theindividual level. In such a situation, it was difficult for this study to further distinguish between stock mixing andstock core areas without using the data from the breeding areas. Future microsatellite study should use thesamples from the breeding areas. Nevertheless, substantial increases in the numbers of the analyzed
microsatellite loci and the biopsy samples allowed us to confirm our previous conclusion on the humpback
whale stock structure in the Antarctic. This study demonstrated one of the significant contributions of non-lethal
part of the comprehensive large-scale JARPAII to acquire valuable information for effective management oflarge whales in the Antarctic.
ACKNOWLEDGEMENTS
We would like to thank all the researchers and crew members of JARPA/JARPA II and IDCR/SOWER surveys
involved in the sampling of the Antarctic biopsy samples used in this study. We also would like to thank H.
Hatanaka for his useful comments on an earlier version of this manuscript.
REFERENCES
Abe, H., Goto, M., Pastene, L.A., Dewa, K., Naito, E. 2001. Practical use of multiplex fluorescent PCR for
cetacean sex identification. Mar. Mammal Sci. 17: 657-664.
Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøll, P.J. 2000. Polymorphic di-nucleotide microsatellite lociisolated from the humpback whale, Megaptera novaeanglliae. Mol. Ecol. 9:2181-2183.
Bérubé, M., Rew, M.B., Skaug, H., Jørgensen, H., Robbins, J., Best, P., Sears, R., and Palsbøll, P.J. 2005.
Polymorphic microsatellite loci isolated from humpback whale, Megaptera novaeanglliae and fin whale,balaenoptera physalus. Cons. Genet. 6: 631-636.
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.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered. 86:485-486.
Kanda, N., Goto, Oikawa, H., and Pastene, L.A. 2014. A note on sampling and laboratory procedure protocols of
the genetic work at the Institute of Cetacean Research. Paper SC/F14/J27 presented to the JARPAII Review
Workshop, Tokyo, February 2014.
Mackintosh, N.A. 1965. The stocks of whales. Fishing News (Books) Ltd., London. 232pp.

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Marshall, T.C., Slate, J., Kruuk, L., and Pemberton, J.M. 1998. Statistical confidence for likelihood-based
paternity inference in natural populations. Mol. Ecol. 7: 639-655.
Pastene, L.A., Goto, M., Nishiwaki, S., Yoshida, H. and Kanda, N. 2006. Genetic characteristics and population
structure of humpback whales in the Antarctic feeding ground as revealed by mitochondrial DNA control
region seqwuencing and microsatellite analyses. Paper SC/D06/J31 presented to the JARPA Review
Workshop, December 2006 (unpublished). 14pp.
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).
Palsbøll, P.J., Bérubé, M., Larsen, A.H., and Jørgensen, H. 1997. Primers for the amplification of tri-and
tetramer microsatellite loci in baleen whales. Mol. Ecol. 6:893-895.
Rousset, F. 2008. Genepop'007: a complete re-implementation of the genepop software for Windows and Linux.
Mol. Ecol. Resources 8:103-106.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual. 2nd edn. Cold
Spring Harbor Laboratory, New York, USA.
Sokal, R.R., and Rohlf, F.J. 1995. Biometry: the Principles of Statistics in Biological Research. Freeman andCompany, New York.
Valsecchi, E., and Amos, W. 1996. Microsatellite markers for the study of cetacean populations. Mol. Ecol.
5:151-156.
van Oosterhout, C., Hutchinson, W.F., Wills, D.P., and Shipley, P. 2004. MICRO-CHECKER: software for
identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4:535-538.

Table 1. The number of humpback whale biopsy samples usid in this study by surveys and areas.
Survey area
Survey
IIIE
IV
V
VIW
56 (33, 23)
132 (58, 74)
106 (56, 50)
46 (18, 28)
JARPA
14 (6, 8)
49 (26, 23)
61 (36, 25)
3 (0, 3)
JARPAII
36 (24, 12)
51 (30, 21)
7 (3, 4)20 (12, 8)
IDCR/SOWER
All
106 (63, 43)
232 (114, 118)
174 (95, 79)
69 (30, 39)
Total (female, male)Table 2. Sampling dates and lcoations of the matched samples.
Sex
Sampling
lat.
long.
1994/2/12
66.54
70.07E
Female2003/12/14
60.31S
48.57E

Cap.→recap.
IV → IIIE, 9 yrs later

Female1997/1/25
2008/12/13

61.44S
63.05S

151.42E
138.26E

V → V, 11 yrs laterMale

2002/2/12
2002/2/13

64.08S
64.06S96.58E
97.24E

IV → IV, next day

Table 3. Genetic diversity indices estimated from all samples within each of survey areas.
Survey area
Genetic indice
IIIE
IV
V
VIW
No. alleles per locus
10.6
11.9
11.9
10.1
Allelic richness per locus
10.2
10.3
10.7
10.1
Heterozygosity
0.758
0.752
0.755
0.746
n.s.
n.s.
n.s.
n.s.
Hardy-Weinberg
n.s. = no significance4SC/F14/J31

Table 4. Results (p-values) of heterogeneity tests for year
differences within areas: Females+Males.
IIIE
IV
V
VIW
0.814
0.555
0.204
0.049
Bold p-values indicate statistically significance.

Table 5. Results (p-values) of heterogeneity tests among areas: Females+Males.
Among areas
IIIExIV
IIIExV
IVxV
IIIxVIWIVxVIW
Highly sig.
0.000043
000.000380
0.000176
Bold p-values indicate statistically significance.

VxVIW
0.0471

Table 6. Results (p-values) of heterogeneity tests for year
differences within areas: Females only.
IIIE
IV
V
VIW
0.015
0.595
0.510
Bold p-values indicate statistically significance.

Table 7. Results (p-values) of heterogeneity tests among areas: Females only.
Among areas
IIIExIV
IIIExV
IVxV
IIIxVIWIVxVIW
1.28E-06
0.014
0.000118
0.000052
0.019
0.046
Bold p-values indicate statistically significance.

VxVIW
0.045

Table 8. Results (p-values) of heterogeneity tests for year
differences within areas: Males only.
IIIE
IV
V
VIW
0.020
0.414
0.641
0.284
Bold p-values indicate statistically significance.

Table 9. Results (p-values) of heterogeneity tests among areas: Males only.
Among areas
IIIExIV
IIIExV
IVxV
IIIxVIW
8.75E-05
0.009
0.007
0.022
0.067
Bold p-values indicate statistically significance.

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IVxVIW
0.001

VxVIW
0.163