Technical Reports of the Ins�tute of Cetacean Research (2021)

pp. 1–6

Technical Report (not peer reviewed)

What do we know about whales and ecosystem in the Indo-Paciﬁcregion of the Antarc�c? Part 3: popula�on gene�c structureof large baleen whales other than Antarc�c minke whales
Luis A. Pastene*, Mutsuo Goto, Mioko Taguchi and Koji Matsuoka
Institute of Cetacean Research, 4–5 Toyomi-cho, Chuo-ku, Tokyo 104–0055, Japan*Contact e-mail: pastene@cetacean.jp

ABSTRACTThe Ins�tute of Cetacean Research (ICR) conducted whale research under special scien�ﬁc permit in the Antarc�c star�ng from the austral summer season 1987/88. The research was conducted systema�cally underdiﬀerent research programs such as JARPA and JARPAII, and more recently, under NEWREP-A. These research
programs employed both lethal and non-lethal methods. NEWREP-A ceased a�er the 2018/19 austral summer
season as a consequence of Japanʼs decision to withdraw from the Interna�onal Conven�on for the Regula�onof Whaling. Japanʼs whale research con�nues in the Antarc�c, using non-lethal methods only. This paper is acon�nua�on of the series of reports on research contribu�on in the Antarc�c by the ICR. This �me, the topic
is on stock structure of large baleen whale species other than Antarc�c minke whales, in the Indo-Paciﬁc sector of the Antarc�c. Gene�c analyses, which were based on the large biopsy sample collec�on of the ICR in
the Antarc�c, have provided important informa�on on the stock structure and movement of baleen whales,
including blue, ﬁn, humpback and southern right whales in the Indo-Paciﬁc sector.

INTRODUCTION
Japan conducted systema�c research on whales and theAntarc�c ecosystem for more than 30 years. The ﬁrst
research program was the Japanese Whale Research
Program under Special Permit in the Antarc�c (JARPA),
which was followed by JARPAII and subsequently by the
New Scien�ﬁc Whale Research Program in the Antarc�c
Ocean (NEWREP-A). The Ins�tute of Cetacean Research(ICR) was the ins�tu�on in charge of designing andimplemen�ng those research programs. Tamura et al.
(2017) have provided details on the objec�ves, sampling
and analy�cal methodology of the three research programs. Several interna�onal review workshops (e.g., IWC,
2015a) discussed and evaluated the large amount of data
and results from these research programs.
As a consequence of Japanʼs change in whaling policy,
the NEWREP-A ceased on 30 June 2019, the date of Japanʼs withdrawal from the Interna�onal Conven�on for
the Regula�on of Whaling (ICRW). From the 2019/20 austral summer season, Japan started whale research in the
Antarc�c using non-lethal methods. The new research
program is called the Japanese Abundance and Stockstructure Surveys in the Antarc�c program (JASS-A) (seeIsoda et al., 2020).

At this point, it was considered important to summarize the knowledge on whales and the Antarc�c ecosystem accumulated so far by Japanʼs whale research in
the Antarc�c. This paper is a con�nua�on of the series ofreports on research contribu�on in the Antarc�c by the
ICR. This �me the topic is stock structure of large baleen
whale species other than Antarc�c minke whales, in the
Indo-Paciﬁc sector of the Antarc�c. Gene�c analyses havebeen possible based on the large biopsy sample collec�on of the ICR in the Antarc�c, which is one of the largest
in the world. Biopsy samples were obtained during thesurveys of the JARPA/JARPAII and NEWREP-A but also
during the surveys of the Interna�onal Whaling Commission (IWC)ʼs Interna�onal Decade for Cetacean Research(IDCR) and Southern Ocean Whale and Ecosystem Research (SOWER) programs.
This paper is not exhaus�ve of the informa�on on stock
structure in Antarc�c large whales. More detailed results
will be presented by species in future issues of TEREP-ICR.

COLLECTION OF GENETIC SAMPLES
Biopsy sampling systems
Gene�c analyses have been carried out based on biopsy
samples collected by diﬀerent biopsy sampling systems
during systema�c sigh�ng surveys. These systems, which
1Technical Reports of the Ins�tute of Cetacean Research (2021)
Table 1
Number of biopsy samples collected by the Ins�tute of Cetacean Research in the Indo-Paciﬁc sector of the Antarc�c between 1993/94
and 2014/15 during JARPA and JARPAII surveys.
SeasonBlue

Fin

Humpback

A. minke

Total

III

IV

V

VI

III

IV

V

VI

III

IV

V

VI

III

IV

V

III

IV

V

1993/94
1994/95
1995/96
1996/97
1997/98

1
-

1

4
-

1
-

-

-

-

-

2
5

20
8
19

12
5
-

15
-

-

5
1
4

-

1

-

8
-

29
20
12
21
30

1998/99
1999/00
2000/01
2001/02
2002/03

1
-

3
1
-

2
3
-

-

-

4
-

3
9
6

-

10
12
-

32
14
-

22
14
10

1
22
-

2
-

3
14
-

2

-

10
-

1

28
59
48
47
19

2003/04
2004/05
2005/06
2006/06
2007/08

2
4

3
5
2

1
-

1
-

4
-

9
-

3
-

2
-

27
1
1

31
6
3

28
11
-

8
2
-

-

4
15
16

1
2

-

-

-

71
39
36
18
28

2008/09
2009/10
2011/122012/132014/15

-

3

-

-

1
-

9

-

-

12
3
-

26
10

13
38
1
-

1
-

-

1
39

3
-

-

-

-

14
78
4
3
61

Total

8

18

10

2

5

22

21

2

73

169

154

49

2

102

8

1

10

9

665

Figure 1.

Biopsy sampling using the Larsen system (le�) and a biopsy sample from a blue whale (right).

will be described in details in future TEREP-ICR issues,
include the ICR air gun system (Kasamatsu et al., 1991)
used between 1992/93 and 2001/02, a crossbow systemused between 2002/03 and 2013/14, and a Larsen system(Larsen, 1998) (Figure 1) used from 2014/15 to the present day.

2

Southern right

Number of biopsy samples
Surveys conducted by the ICR in the Indo-Paciﬁc sectorof the Antarc�c collected a total of 665 biopsy samples
between 1993/94 and 2014/15, 38 from Antarc�c blue
whales, 50 from ﬁn whales, 445 from humpback whales,
112 from southern right whales, and 20 from Antarc�cminke whales (Table 1). All samples collected were preserved onboard at −20°C un�l they were used at the ICR

What do we know about whales and ecosystem in the Indo-Pacific region of the Antarctic? Part 3

gene�c laboratory. An example of biopsy sample is shownin Figure 1.

OUTLINE OF THE LABORATORY WORK
The laboratory work for gene�c analyses in all casestudies involved the following steps: extrac�ons of total
genomic DNA, molecular sex determina�on, sequencing
of a por�on of the mitochondrial DNA (mtDNA) control
region and genotyping with a set of microsatellite DNA(msDNA) loci. Technical details of the laboratory work can
be found in Pastene and Goto (2016). A brief summary ispresented here based on that study.
Sampled skin �ssues were preserved frozen at −20°C
un�l use. Total genomic DNA was extracted from 0.05 g ofskin �ssue using either the standard phenol-chloroform
method or the Gentra Puregene kits (QIAGEN). Extracted
DNA was stored in TE buﬀer (10 mM Tris-HCl, 1 mM EDTA,
pH 8.0).
The SRY locus located on the Y chromosome was also
used for sex determina�on following the method of Abeet al. (2001) with a slight modiﬁca�on. With the combina�on of loci of SRY and a microsatellite locus as a posi�ve
control, males show ampliﬁed products of both SRY anda microsatellite locus, while females show only a microsatellite locus.
An approximate 500 base pairs (bp) of the mtDNA control region were ampliﬁed by the polymerase chain reac�on (PCR) using a set of primers available at the ICR. PCR
products were puriﬁed using MicroSpin S-400HR columns(Pharmacia Biotech). Cycle sequencing was performed
using BigDye terminator cycle sequence Kit (Applied Biosystems) and the PCR primers, following the protocols of
the manufacturer. The cycle sequencing products were
puriﬁed using AutoSeq G-50 spin Columns (Pharmacia
Biotech). The labeled sequencing fragments were resolved using an ABI PRISM 377 or an ABI3500 Gene�cAnalyzers (Applied Biosystems) (Figure 2).
The samples were also genotyped at a number of
msDNA loci, which varies between 14 and 17 dependingon the species, in mul�plex ﬂuorescent PCRs. PCR products were electrophoresed on an ABI3500 DNA Analyzer(Applied Biosystems) (Figure 2), and allele sizes were determined using a 600 LIZ size standard (Applied Biosystems) and GeneMapper v. 4.0 (Applied Biosystems).

ANALYTICAL PROCEDURES
The analy�cal procedures for gene�c analyses on stock
structure are those used rou�nely in these kinds of studies, and they are men�oned brieﬂy below, together withthe progress in research.

Figure 2.

ABI3500 DNA Analyzer (Applied Biosystems) available at the ICR gene�c laboratory.

PROGRESS IN RESEARCH
The summary of research progress on stock structure forsome large whale species below are based on Pastene (inpress).
Blue whales
ICR scien�sts have collaborated with foreign scien�sts inthe study of popula�on gene�cs of blue whales.
One example is the gene�c study by LeDuc et al.
(2007), who used samples from the Antarc�c (n=30 from
IDCR/SOWER cruises and n=17 from JARPA), Chile (n=16from IDCR/ SOWER), the southern and western coasts ofAustralia (n=28 from IDCR/SOWER), around the Maldives(n=6 from IDCR/SOWER) and Peruvian and Ecuadorianwaters (n=12 from US SWFSC research cruises). Analysis
focused on inves�ga�ng the pa�ern of gene�c varia�on in Southern Hemisphere blue whales and the useof an assignment test to detect mixing on the feeding
grounds. Gene�c markers used were mtDNA control
region sequences and seven microsatellite loci. Strong
gene�c diﬀerences were found among samples fromthe southeast Paciﬁc Ocean, Indian Ocean and aroundthe Antarc�c con�nent. Gene�c diﬀeren�a�on between
the geographical ranges of the nominal sub-species (i.e.,
Antarc�c vs pygmy blues in the Paciﬁc and Indian Oceans)
was not markedly greater than between popula�ons ofpygmy blue whales.
3Technical Reports of the Ins�tute of Cetacean Research (2021)

Figure 3.

Hypothe�cal stock structure for Southern Hemisphere humpback whales. The areas and sub-areas iden�ﬁed reﬂect approximate, rather than necessarily exact, boundaries. A do�ed line represents hypothe�cal
connec�on, thin lines represent a small number of documented connec�ons between areas while thicklines represent a large number of documented connec�ons between areas (from IWC, 2005).

Fin whales
Gene�c analyses on stock structure of ﬁn whales in the
Antarc�c feeding grounds are very scarce. One of the few
studies was conducted by Goto and Taguchi (2019).
These authors examined popula�on gene�c structurein IWC Management Areas III, IV, V and VIW based on
mtDNA control region sequences (478 bp) and sixteen
microsatellite loci. The analysis was based on biopsy
samples collected by the JARPA and IDCR/SOWER cruises,
and samples from takes during JARPAII. The analyses
were conducted on the basis of three arbitrary sectors:
POP1 (0°–70°E, n=39), POP2 (70°E–160°E, n=48) andPOP3 (160°E–145°W, n=18). The study showed a highmtDNA diversity, which was comparable among groups.
Gene�c analyses based on heterogeneity test (mtDNA
and msDNA) and STRUCTURE, PCA and test for Hardy
Weinberg devia�on (msDNA) failed to ﬁnd evidences ofstock structure in the research area. A phylogene�c analysis based on mtDNA haplotype showed no rela�onshipbetween clusters and par�cular geographical group. The
authors noted that the sample sizes were small and thatthe results could be due to a low power of the sta�s�cal
analyses.
Humpback whales
The IWC Scien�ﬁc Commi�ee (SC) has described hypothe�cal stock structure and migratory corridors for Southern Hemisphere humpback whales based mainly on informa�on such as Discovery marks, photo-iden�ﬁca�on,gene�cs and satellite tracks (IWC, 2005). Seven Breeding
Stocks (BS) are recognised, ʻAʼ–ʻG.ʼ Some (ʻB,ʼ ʻC,ʼ ʻEʼ andʻFʼ) were further subdivided into sub-stocks (Figure 3).
The IWC SC completed the assessment of most stocks
at its 2014 mee�ng (IWC, 2015b). For the assessment,
4

the informa�on on stock structure summarised below
contributed to the interpreta�on of stock abundance andtrend as well as for alloca�ng historical catches to thevarious stocks.
Pastene et al. (2006) examined the popula�on gene�c structure in IWC Management Areas IIIE, IV, V andVI based on mtDNA control region sequences and six microsatellite loci. The analysis was based on biopsy samples
collected by the JARPA and IDCR/SOWER cruises, namely:
n=81 for Area IIIE (JARPA: 50; IDCR/SOWER: 31); n=172
for Area IV (JARPA: 126; IDCR/SOWER: 46); n=97 for Area
V (JARPA: 90; IDCR/SOWER: 7); and n=61 for Area VI(JARPA: 44; IDCR/SOWER: 17). The analysis conﬁrmed thehigh level of gene�c diversity for both mtDNA and microsatellites. Both gene�c markers suggested diﬀeren�a�onamong these four Areas, with the diﬀerences being stronger for females than males. The authors did not reject thepossibility of some mixing of popula�ons on the bordersof the Management Areas.
Kanda et al. (2014) examined the popula�on gene�cstructure in IWC Management Areas IIIE, IV, V and VI
based on genotypes from 14 microsatellite loci. The
analysis was based on JARPA and IDCR/SOWER cruise
samples: n=93 for Area IIIE (JARPA: 62; IDCR/SOWER: 31);
n=218 for Area IV (JARPA: 172; IDCR/SOWER: 46); n=153
for Area V (JARPA: 146; IDCR/SOWER: 7); n=64 for Area VI(JARPA: 47; IDCR/SOWER: 17). Major gene�c diﬀerenceswere a�ributed to samples from diﬀerent Areas. Stronger
diﬀeren�a�on was seen in females than in males. Despite
the increase of the number of loci from six in the previous
analysis to 14, the level of stock diﬀeren�a�on was s�ll
too low for analysis at the individual level.
Pastene et al. (2013) used biopsy samples obtainedby JARPA/JARPAII and IDCR/SOWER surveys and mtDNA

What do we know about whales and ecosystem in the Indo-Pacific region of the Antarctic? Part 3

Figure 4.

Geographical distribu�on of humpback whale samples from breeding and feeding grounds analyzed in
Pastene et al. (2013).

control region sequences to study the distribu�on andmixing rates of breeding stocks BSD, BSE and BSF in Antarc�c Areas IIIE–VI (Figure 4). Breeding ground samples
were from Western Australia (n=185), Eastern Australia(n=104), New Caledonia (n=243), Tonga (n=240), Cook
Islands (n=56) and French Polynesia (n=62). They werecollected by several groups and research ins�tu�ons andwere provided for analysis under the IWC SC Data Availability Protocol. JARPA and IDCR/SOWER survey samples
were provided as outlined above (Pastene et al., 2006).
According to one of the hypotheses on baseline popula�ons, Western Australian whales were distributed mainly
in Management Areas IVW (84.5% SE: 0.043; FST: 0.0015)
and IVE (75.9% SE: 0.092; FST: −0.0015); whales from Eastern Australia in Area VW (64.7% SE: 0.074; FST: 0.0013);
whales from New Caledonia in Area VE (83.9% SE: 0.011;
FST: 0.0062), and whales from Tonga in Area VI (43.3% SE:
0.015; FST: 0.0010). Whales from Cook Islands and French
Polynesia were not represented on feeding ground Areas
IIIE–VI.
Southern right whales
Two gene�c studies based on biopsy samples collectedby the ICR have been conducted (Pastene et al., 2018; inpress). These studies were based on 157 biopsy samples
collected mainly in the Indian sector of the Antarc�c. Both
mtDNA control region sequencing (381 bp) and msDNA at
14 loci were used in the analyses. The mtDNA analysis
suggested that whales in the Antarc�c Indian sector insummer are closely related to the South West Australian
breeding grounds (Pastene et al., 2018). The msDNA
revealed eight individual matches (four males and four
females), which demonstrated that at least some males
and females returned to the same feeding grounds over
the years (Pastene et al., in press). The matching occurredin an area where visual surveys showed aggrega�ons ofwhales associated with high krill concentra�ons.

SUMMARY
The ICR, through its former whale research programs in
the Antarc�c (JARPA, JARPAII), collected a large numberof biopsy samples from diﬀerent baleen whale species.
In fact, the set of biopsy samples from the Antarc�c heldby the ICR is the largest in the world. These samples wereused in gene�c analyses on phylogeny, stock structure
and individual matching in some baleen whale species
that are key components of the Antarc�c ecosystem.
Results of these analyses have been important for theassessment of these species conducted by the IWC SC.
In the case of blue whales, the analyses contributed tounderstand the gene�c diﬀeren�a�on between Antarc�c and pygmy blue whales. The ICR started the gene�canalysis of ﬁn whales in the Antarc�c feeding grounds.
However it seems that the sample sizes are s�ll small toallow the detec�on of stocks. The gene�c analyses of biopsy samples of humpback whales contributed to understand the gene�c diversity and degree of diﬀeren�a�onof the species in the feeding grounds of Areas IIIE-VI andthe rela�onship between breeding and feeding groundsof several stocks. At least four stocks were conﬁrmed
in the Indo-Paciﬁc sector of the Antarc�c, which mix
spa�ally in some sectors. In the case of southern rightwhales, gene�c analysis suggested that whales in the
Indo-Paciﬁc sector of the Antarc�c belong to the same
stock as Western South Australia and that some whales
return to the same feeding grounds over the years. Morereﬁned analyses will be conducted on these species withaddi�onal biopsy samples collected by NEWREP-A andthe ongoing JASS-A program.

5Technical Reports of the Ins�tute of Cetacean Research (2021)

ACKNOWLEDGEMENTS
We thank the captain, crew members and researchers involved in the surveys of JARPA, JARPAII and IDCR/
SOWER for their eﬀort in collec�ng biopsy samples from
diﬀerent baleen whale species. The gene�c analyses on
stock structure summarized in this paper are based on
those samples.

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