SC/F14/J33rev

Long-distance longitudinal migration of southern rightwhales suspected from mtDNA and microsatellite DNA
analysis on JARPA and JARPAII biopsy samples
Naohisa Kanda, Mutsuo Goto, Shigetoshi Nishiwaki, 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 70 southern right whale biopsy samples obtained from Areas IIIE (2), IV (63), and V (5) during
JARPA and JARPAII surveys up to the 2009/10 season were genotyped at 14 microsatellite DNA loci and weresequenced for 430 bp of the mitochondrial DNA (mtDNA) control region in order to describe genetic
characteristics of the species in the Antarctic feeding ground. In two of three cases of duplicate sampling, thesecond samples were collected later year (four years, and eight years) in the same area (IV), suggesting site
fidelity to the feeding ground. Paternity analysis for a single calf-mother pair sample detected no potential fatherof the calf. After exclusion of some of these samples, 66 samples were used to estimate genetic indices. The
levels of genetic diversity observed from both markers were comparable to those reported from other southern
right whale genetic studies. A total of eight haplotypes were generated from 21 segregation sites and these
haplotypes were phylogenetically separated into two clades. On the basis of the sequence variations within the
275 bp consensus region, these eight haplotypes were then compared to the 37 haplotypes reported in Patenaude
et al. (2007) that used the samples from the Indo-Atlantic (Argentina and South Africa) and the Indo-Pacific(Southern Australia and New Zealand) basins. The comparison revealed that our two clades were same as their A
and W clades, each of the eight haplotypes matched to one of their haplotypes, and three were same as theirIndo-Atlantic specific haplotypes. These results suggest that some southern right whales operate much
longer-distance seasonal migration between their feeding and breeding grounds than we had previously thought
and thus whales from multiple stocks migrate to our research area in the Antarctic feeding ground.
Our genetic study is the first to describe the genetic characteristics of southern right whales feeding in the
Antarctic and the first to discover the possibility of southern right whales’ long-distance feeding migration fromthe Indo-Atlantic basin to the Indo-Pacific basin that results in the mixing of multiple stocks in the Antarctic.
Past genetic studies, in contrast, demonstrated whales’ restricted seasonal migration within the same basinprobably due to the use of the samples only from coastal areas. Our study implies that southern right whales
require different management strategies on an area by area basis even within the same basin for their long-term
persistence. 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 of large whales in Antarctic.

KEYWORDS: ANTARCTIC, BIOPSY SAMPLING, FEEDING GROUNDS, GENETICS, MIGRATION,
SITE FIDELITY, SOUTHERN RIGHT WHALE

INTRODUCTION
The distributions of southern right whales are restricted in the Southern Ocean (Rozenbaum et al., 2000).
Existence of genetically distinct stocks has been recognized in several areas of the ocean (Patenaude et al., 2007),and distributions of these stocks can be separated into two major basins: Indo-Atlantic, such as Argentina andSouth Africa, and Indo-Pacific, such as Australia and New Zealand. General migration patterns of southern rightwhales between winter breeding/calving and summer feeding grounds appear to be north-south (see detail
information in IWC, 2001). Such latitudinal movement patterns associated with the whales’ ability of site
fidelity result in accumulation of the regional stock differentiation between as well as within the two majorbasins as shown by the past genetic studies (e.g., Patenaude et al., 2007). In addition to that, strong structuring ina maternally inherited mitochondrial DNA (mtDNA) marker with limited differentiation in biparentally inherited
microsatellite DNA markers observed among samples from the local regions in South Pacific suggested female
philopatry and male dispersal (Carroll et al., 2011).

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Stock structure and phylogenetic relationships of southern right whales on their calving/feeding grounds wasinvestigated by Patenaude et al. (2007) using mtDNA sequencing analysis. The authors analyzed 146 individuals
sampled on two winter calving (New Zealand and Southwest Australia) and one summer feeding (south ofWestern Australia) grounds in the Indo-Pacific basin and on two winter calving (South Africa and Argentina)
and one summer feeding (South Georgia) grounds in the Indo-Atlantic basin. A total of 37 unique mtDNA
haplotypes were found and these haplotypes were clearly separated phylogenetically into two distinct clades(Crades A and W). Although the haplotypes from the A and W clades were found in the both ocean basins, theirfrequencies quite differed between as well as within the ocean basins, indicating very limited female mediatedgene flow even among the local regions. Only one of the 37 haplotypes was shared between the two ocean basins
and the other 36 were basin specific.
In this paper, we analyzed the biopsy samples obtained from southern right whales in the Antarctic Ocean(mostly from Area IV) during the JARPA and JARPAII surveys up to 2009/10 season using both mtDNA andmicrosatellite DNA markers in order to describe their genetic characteristics. We compared our results to thosein Patenaude et al. (2007) and Carroll et al. (2011) to better understand the genetic characteristics and feeding
migration pattern of the species in the Antarctic feeding ground.
MATERIALS AND METHODS
Samples
Table 1 shows the number of southern right whale biopsy samples used in this study that were separated by IWC
management areas. A total of 70 samples was obtained from JARPA and JARPAII surveys conducted from
1993/94 to 2009/10 seasons.
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 a frozen 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 were analyzed using 14 sets of primers: EV1, EV14, EV21, EV37, EV94
(Valsecchi and Amos, 1996), GT23, GT211, GT310 (Bérubé et al., 2000), GATA28 (Palsbøll et al., 1997),
DlrFCB17 (Buchanan et al., 1996), TR2F3, TR3G2, TR2G5, TR3F2 (Frasier et al., 2006). TR2F3, TR3G2,
TR2G5, and TR3F2 were designed specifically from right whales (Frasier et al., 2006). Amplified products wererun on a 6% polyacrylamide denaturating gel using a BaseStation 100 DNA fragment analyzer (Bio-Rad), andthen sizes of visualized alleles were determined manually in relation to the internal size standard (Genescan
400HD, Life Technologies) as well as the Southern right whale microsatellites of known size that were rerun oneach gel.
mtDNA analysis
Sequence of 430 bp of the mtDNA control region was amplified using a set of primers: MT4 (Árnason et al.
1993) and Dlp 5R (5'-CCA TCG AGA TGT CTT ATT TAA GGG GAA C-3'). PCR products were cycle
sequenced for purification with the same primers using the AmpliTaq FS Sequencing Kit (Perkin-Elmer). Afterthe purification, the cycle-sequenced products were direct-sequenced on either an ABI 3100 or 3500 Automated
DNA Sequencers (Life Technologies) following the manufacture’s protocols. Both light and heavy strands weresequenced for each sample. Observed sequences were aligned using the Sequence Navigator computer program(Life Technologies).
Sex determination
Sex of the whales was determined through co-amplification of SRY locus located on the Y chromosome andGT23, which is 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.
Genetic diversity analysis
For the microsatellite analysis, MICRO-CHECKER (van Oosterhout et al., 2004) was used to check for nullalleles and reading/typing errors. The number of alleles per locus and expected heterozygosity per locus wascalculated 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 was conducted

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using CERVUS (Marshall et al., 1998). For the mtDNA analysis, the number of haplotypes per sample,
haplotype diversity, and nucleotide diversity were calculated based on Nei (1987).
Phylogenetic analysis
Phylogenetic relationship of mtDNA haplotypes was resolved using neighbour-joining method in PHYLIP(Felsenstein, 1993) after 1000 bootstraps. The tree was rooted using a homologous sequence from a North
Pacific right whale obtained from JARPNII. The obtained tree was visualized using TreeView PPC (Page 1996).
RESULTS AND DISCUSSION
Among three cases of duplicate sampling, the second samples of the two cases were collected years later at thesame area the first samples were obtained (Table 2). One male was collected first in 1998 and was re-sampled
four years later, while one female was collected first in 2000 and was re-sampled eight years later in 2008. Thesematches were also ascertained during our photo-identification practice (Matsuoka and Pastene, 2014). Thesefindings indicate and support the previous knowledge that southern right whales have site fidelity to their feeding
ground. A single calf and mother pair was used for paternity analysis, but no potential father was detected in thesamples.
After exclusion of the second individuals from the three cases of the duplicated samples and one calf, a totalof 66 samples were used for further analyses at the group level. The biopsy samples from Areas III and V weregrouped with those from Area IV because their sampling sites were very close to the boundary with Area IV andthe small numbers prevented us from conducting heterogeneity tests among the different areas.
The level of genetic diversity of the samples was represented as an average number of alleles per locus and an
average expected heterozygosity in microsatellites, and the total number of haplotypes in the samples andexpected haplotype diversity in mtDNA (Table 3). Comparing these values to those reported in Patenaude et al.
(2007) and Carroll et al. (2011), only the microsatellite heterozygosity was a little lower in this study than in theprevious studies and the other genetic indices were similar to each other. It is thought that the lower
heterozygosity observed in this study is probably due to our marker selection.
Twenty one variable sites were found along the 430 bp of the control region of mtDNA, generating eight
unique haplotypes (SR1-SR8). Phylogenetic tree showed that these eight haplotypes were clearly separated into
two clades (Fig. 1). The first (SR2) and the second (SR4) most frequent haplotypes belonged to the different
clades. These eight haplotypes were then compared to the 37 haplotypes reported in Patenaude et al. (2007) to
see any haplotype matches on the basis of the sequence variations within the 275 bp consensus region (Table 4).
For this purpose, some of the 37 sequences were retrieved from Genbank, and other ones were reconstructedusing the information from their paper (Table2, page 150) for the variable sites and BakHapA (Genbank
accession number JN097593) as a template for non-variable sites. The number of segregation sites used was 33,
of which 16 were variable in both studies and 17 were variable only in Patenaude et al. (2007). All of our eight
haplotypes had matches to their study (Table 4). These matches also indicated that our two clades were the sameas their A and W clades.
Patenaude et al. (2007) described that, although the haplotypes from the A and W clades were found in thetwo different ocean basins (Indo-Atlantic and Indo-Pacific), only one haplotype (BakHapE in their study thatmatched to SR3 in this study) was shared between the two basins and all of the remaining 36 haplotypes werebasin specific. Their results thus indicated a very limited genetic exchange between the Indo-Atlantic andIndo-Pacific basins and corresponded to the field and genetic observations that demonstrated latitudinalmigration within local regions (IWC, 2001). The haplotype matches between the two studies showed thatapproximately 82% (SR2+SR4+ SR6+SR8) to 95% (SR2+SR4+SR6+SR8+SR3), depending on how we allocate
SR3, of our samples were matched to their Indo-Pacific haplotypes. This indicates that most, if not all, southern
right whales conduct north-south seasonal migration as expected from the past observations (IWC, 2001).
The results of the haplotype matches between the two studies add one more interesting and importantinterpretation. Three (SR1, SR5, and SR7) of our eight haplotypes were matched to their Indo-Atlantic
haplotypes. Two samples from Area III were the Indo-Atlantic types. Our study thus suggests that some southern
right whales migrate much longer distant than we had previously thought. Although it is difficult to determine
how frequent this type of migration is occurring, the substantial genetic differentiation between the local regions
within the same basin revealed by past studies (Patenaude et al. 2007; Carroll et al. 2011) suggests that thewhales from the Indo-Atlantic basin should migrate back to the same basin. Southern right whales might havevery strict site fidelity to the breeding and feeding grounds irrespective of its in-between distance. Furthemore,
our study discloses that whales from multiple stocks migrate to our research area at the Antarctic feeding ground.

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It is important to note that the above discussion was based on the variations at the 275bp consensus sequencesof the mtDNA control region. In addition, according to Carroll et al. (2011), their analysis of longer sequences
separated PorHap4 in Patenaude et al. et al. (2007) (SR1 in this study) into two different haplotyepes, PatHap4.1
and PatHap4.2 (Genbank accession numbers JN097601 and JN097602), and these haplotypes were actuallyfound in the Pacific. Their frequencies were quite different as PatHap4.2 (12 out of 551 individuals) was about
10 times more frequent than PatHap4.1 (1/551) was in the same subantarctic New Zealand population, and SR1
was matched to rare PatHap4.1 based on the comparison of 417 bp sequences (data not shown). Because of its
rare appearance, it may be premature to declare this haplotype (SR1) is no longer Indo-Atlantic type.
Our genetic study is the first to describe the genetic characteristics of southern right whales feeding in the
Antarctic and the first to discover the possibility of southern right whales’ long-distance feeding migration fromthe Indo-Atlantic basin to the Indo-Pacific basin that results in mixing of multiple stocks in the Antarctic. This is
in contrast to the past genetic studies that demonstrated whales’ restricted migration within the same basinprobably due to the use of the samples from coastal areas. This study implies that southern right whales requiredifferent management strategies on an area by area basis even within the same basin for their long-term
persistence. 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 of large whales in Antarctic.
ACKNOWLEDGEMENTS
We would like to thank all the researchers and crew members of JARPA/JARPAII surveys involved in thesampling 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.
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.
Carroll, E.L., Patenaude, N.J., Alexander, A.M., Steel, D., Harcourt, R., Childerhouse, S., Smith, S., Bannister,J.L., Constantine, R., and Baker, C.S. 2011. Population structure and individual movement of southern rightwhales around New Zealand and Australia. Mar. Ecol. Prog. Ser. 432:257-268.
Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) 3.5c. University of Washington, Seattle, USA.
Frasier, T.R, Rastogi, T., Brown, M.W., Hamilton, P.K., Kraus, S.D., and White, B.N. 2006. Characterization oftetranucleotide microsatellite loci and development and validation of multiplex reactions for the study ofright whale species (genus Eubalaena). Mol Ecol. Note 6: 1025-1029.
Goudet, J. 1995. FSTAT, version 1.2: a computer program to calculate F-statistics. J. Hered. 86: 485-486.
International Whaling Commission. 2001. Report of the workshop on the comprehensive assessment of rightwhales: a worldwide comparison. J. Cetacean Res. Manag. Spec. Issue. 2: 1-60.
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.
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.
Matsuoka, K., and Pastene, L.A. 2014. Summary of photo-id information of blue, southern right and humpback
whales collected by JARPA and JARPAII. Paper SC/F14/J34 presented to the JARPAII Review Workshop,
Tokyo, February 2014 (unpublished).
Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York.
Page, R.D.M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. CABIOS
12: 357-358.
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.
Patenaude, N.J., Portway, V.A., Schaeff, C.M., Bannister, J.L., Best, P.B., Payne, R.S., Rowntree, V.J., Rivarola,M., and Baker, C.S. 2007. Mitochondrial DNA Diversity and Population Structure among Southern Right
Whales (Eubalaena australis). J. Hered. 98: 147-157.
Rousset, F. 2008. Genepop'007: a complete re-implementation of the genepop software for Windows and Linux.
Mol. Ecol. Resources 8:103-106.

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Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual. 2nd edn. Cold
Spring Harbor Laboratory, New York, USA.
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 Southern right whales used in this study by
areas (Female, Male).
IIIE
IV
V
2 (1, 1)63 (28, 35)
5 (1, 4)

Table 2. Sampling dates and locations of matched individuals.
Cap.→recap.
Sampling
Lat.
Long.
1998/1/15
6255S
10028E
Male
IV→IV, 4 yrs later
2002/2/15
6436S
9215E
Female2000/2/10
2008/3/1

6435S
6454S

11431E
12617E

IV→IV, 8yrs later

Table 3. Genetic indices based on variations at microsatellite DNA and mtDNA markers.
Microsatellite DNA
mtDNA
N*
#loci
#allele
He
HW
bp
#HP
Total
66
14
6.9
0.651
n.s
430
8
Female
27
14
6.3
0.643
n.s
430
6
Male
39
14
6.5
0.655
n.s
430
6
*mtDNA:Total=65, Female=27, male=38

Table 4. Reference to Patenaude et al . (2007)
This study

h
0.767
0.795
0.754

Patenaude et al . (2007)

Haplotypen

Haplotype matchedBasin found

SR1

1

PorHap4

Indo-Atlantic

SR2

24

BakHapA

Indo-PacificSR51

PorHap11

Indo-Atlantic

SR6

12

BakHapB

Indo-PacificSR39BakHapE

SharedSR415

BackHapC

Indo-PacificSR71

PorHap16

Indo-Atlantic

SR82
BakHapD
Indo-PacificIndo-Pacific: Southwest Australia calving, New Zealand sub-Antarctic calving,
Southwest Australia feeding grounds.
Indo-Atlantic: Argentina calving, South Africa calving, South Georgia feeding
grounds.

5phylogeny

Clade A

Clade W

SC/F14/J33rev

Clade W

SR4
SR7

SR3

95.0

SR8

100.0

NR
NR
55.9

SR2 50.1

Clade A

SR5

SR1
SR6

Figure 1. Phylogenetic relationship of southern right whale mtDNA haplotypes (SR1-SR8).
The tree was rooted using a homologous sequence from a North Pacific right whales. Only bootstrap values (%)
over 50% from 1000 replicates are given. Clade A, W from Patenaude et al. (2007).

0.01

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