ICES Journal of Marine Science, 56: 640–651. 1999
Article No. jmsc.1999.0511, available online at http://www.idealibrary.com on
RAPD-typing of Central and Eastern North Atlantic and
Western North Pacific minke whales, Balaenoptera acutorostrata
I. Martinez and L. A. Pastene
Martinez, I. and Pastene, L. A. 1999. RAPD-typing of Central and Eastern North
Atlantic and Western North Pacific minke whales, Balaenoptera acutorostrata. – ICES
Journal of Marine Science, 56: 640–651.
RAPD analysis was performed on 258 minke whales: 201 individuals from the North
Atlantic (NA) and 57 from the Western North Pacific (WNP). Nine 10-mer primers
generated 20 RAPD-markers. Of these, two were present only in individuals from the
WNP and one was present only in individuals from the NA. Six other markers showed
a 50% or higher difference in frequency between the two oceans. The matrix of
absence/presence of the markers was submitted to principal component analysis (PCA)
and to analysis of the molecular variance (AMOVA). The first factor of the PCA
clearly separated the minke whales from the NA from those of the WNP, but minke
whales captured in different small areas of the NA (Northeastern: EN, North Sea; EC,
Lofoten-Vesterålen; EB, Finnmark and Barents Sea; ES, Bear Island and Svalbard;
and Central: CM, Jan Mayen), and in the WNP (7, 8, 11) did not form separate
clusters. AMOVA of the data allocated over 63% (p<0.0001) of the total genetic
variability of the model to differences between the NA and the WNP, and over 36% to
within ocean individual differences. Two percent (p<0.00001) of the genetic variability
in the NA could be attributed to differences between two groups: individuals from EN,
EC, and EB (1996) and those from CM (1996) and ES (1996 and 1994), while minke
whales from EC and EB captured in 1994 were indistinguishable from any of the other
two groups. There were no significant differences among the three areas of capture in
the WNP. The present results corroborate the presence of two different populations of
minke whale, that is in the NA and in the WNP, and indicate that there may be two
closely related breeding stocks in the NA waters.
1999 International Council for the Exploration of the Sea
Key words: minke whale, Balaenoptera acutorostrata, Northeast Atlantic, Western
North Pacific, RAPD.
Received 23 November 1998; accepted 28 July 1999.
I. Martinez: Norwegian Institute of Fisheries and Aquaculture, 9291-Tromsø, Norway,
L. A. Pastene: Institute of Cetacean Research, 4-18 Toyomi-cho, Chuo-ku, Tokyo
104-0055, Japan. Correspondence to I. Martinez: tel: +47 7762 9000; fax: +47 7762
9100; e-mail: [email protected]
Introduction
Minke whales (Balaenoptera acutorostrata) are migratory animals with a worldwide and temporal distribution: they have seasonal migrations between the
temperate waters where they breed during the local
winter and the polar waters where they feed during
the local summer (Jonsgård, 1966), although minke
whales can also be found in temperate waters during
all seasons.
Minke whales from the North Atlantic and the North
Pacific do not mix because they are separated by the
polar ice and the continental masses. Neither do minke
whales from the Northern and Southern hemispheres
1054–3139/99/050640+12 $30.00/0
interbreed, because animals crossing the equator would
have to adjust to a 6-month change in their breeding
and feeding seasons (Horwood, 1990). Studies of the
population genetic structure of minke whales in the
Atlantic, North Pacific, and Antarctic has confirmed
the genetic differentiation inferred from the physical and
physiological barriers (reviewed by Danı́elsdóttir, 1998).
The population structure of the species in each ocean
is less clear. Thus, minke whales in the North Atlantic
have been divided into four management units (IWC,
1977): Canadian East coast, West Greenland, Central,
and Northeastern Atlantic, and while some authors have
found significant differences among minke whales
captured in these areas (A
u rnason and Spilliaert,
1999 International Council for the Exploration of the Sea
RAPD-typing of minke whales
1991; Danı́elsdóttir, 1998; Danı́elsdóttir et al., 1992,
1995), others have not (Bakke et al., 1996; Palsbøll,
1989). Moreover, Danı́elsdóttir et al. (1995) observed
that there may be greater differences between
animals captured at the same location in different
years, than at different locations in the same year. Minke
whales tagged during the period 1964–1985 in the
Northeast Atlantic were recovered in the same area,
with no recoveries in the Central Atlantic, which seemed
to indicate the presence of a boundary between two
stocks (Øien, 1991). However, most of the taggings on
which this hypothesis was based were carried out around
Bear Island when the animals congregate during late
summer, thus excluding the possibility of disclosing
a temporal migration pattern similar to that of the
Antarctic.
The subcommittee on North Atlantic Baleen Whales
in the International Whaling Commission (IWC)
decided in 1993 (IWC, 1993) that the so-called Small
Areas boundaries should be retained from the management of Northeast Atlantic minke whales. According to
this division, minke whales from the North Sea (EN),
Lofoten-Vesterålen (EC), Finnmark and Barents Sea
(EB), Bear Island and Svalbard (ES), and Jan Mayen
(CM), are to be considered as belonging to five different
breeding stocks (IWC, 1993). This division is based on
some of the contradictory data mentioned above, and in
spite of strong evidence of exchange found between the
small areas EB-ES-EC and some evidence of exchange
between CM-ES-EB, EB-ES-EN, and EC-EN (IWC,
1993).
The situation in the Western North Pacific is similar.
There is genetic (Goto and Pastene, 1997; Wada and
Numachi, 1991) and morphological (Ohsumi, 1983)
evidence to support the existence of two stocks, one
called ‘‘O’’ in the Pacific coast of Japan and the Sea of
Okhotsk and a second called ‘‘J’’ in the Sea of Japan and
East China Sea. However, in 1994 the IWC proposed to
apply a new stock structure in these waters, whereby the
J stock should be further subdivided into three substocks, the O stock into four sub-stocks, and an
additional stock (W stock) was proposed in the central
region (157–170E) (IWC, 1994).
Random amplification of genomic DNA (RAPD)
(Welsh and McClelland, 1990; Williams et al., 1990) is a
molecular technique that has been successfully applied
to study population structure in many organisms including plants (Huff et al., 1993), fish (Caccone et al., 1997),
shrimp (Martinez et al., 1997b), snakes (Gibbs et al.,
1994), clapper rails (Nusser et al., 1996), and woodpeckers (Haigh et al., 1996). RAPD analysis lacks the
resolving power of single locus fingerprinting, capable of
identifying single individuals and their offspring (Kirby,
1990), which is not necessarily a disadvantage when
dealing with higher hierarchical levels, such as breeding
stocks (Caccone et al., 1997; Martinez et al., 1997a, b),
641
hybrids (Elo et al., 1997; Martinez and Malmheden
Yman, 1999) and species (Dinesh et al., 1993; Martinez
et al., 1994; Martinez, 1997; Martinez and Malmheden
Yman, 1999). The major drawback of this technique,
namely that the polymorphisms generated by RAPD
analysis are scored as dominant markers and consequently heterozygotes cannot be distinguished accurately (Welsh and McClelland, 1990; Williams et al.,
1990), is efficiently counteracted by (1) the development
of statistical data treatments (Huff et al., 1993; Stewart
and Excoffier, 1996) and computer programs (Schneider
et al., 1996) able to infer population structure from
RAPD markers, (2) by the theoretically unlimited
number of markers that can be generated from any
genome without the need to know specific DNA
sequences, (3) the speed of the technique, and (4) the
large number of samples that can be processed simultaneously. Moreover, since RAPD is based on the
amplification of DNA, usually some milligrams of tissue
are sufficient to perform several thousand analyses
(Martinez et al., 1997a). In our preliminary work
(Martinez et al., 1997a) we established the conditions to
perform this analysis using minke whale DNA from 31
individuals from the small areas EC, EB, and ES from
the Northeast Atlantic. Due to the low number of
individuals, it was not possible to reach a conclusion
about their genetic structure, although the results did
not indicate that there were differences among them. In
the present work RAPD has been applied to a greater
number of minke whales from the Northeast and
Central Atlantic (NA). Minke whales from the Western
North Pacific (WNP), have been included to evaluate the
suitability of the technique to discriminate among
known different breeding stocks.
Materials and methods
Samples and location of capture
The 201 minke whales from the NA used in this work
were captured in four different areas in the Northeast
Atlantic: EN, North Sea; EC, Lofoten-Vesterålen; EB,
Finnmark and Barents Sea; ES, Bear Island and
Svalbard; and one in the Central Atlantic: CM, Jan
Mayen (Fig. 1) during the Norwegian scientific whaling
expedition under special permit in 1994 (see Haug et al.,
1996) and during the commercial whaling operation in
1996. The minke whales from the WNP were captured in
areas 7E, 7W, 8, and 11, during the 1996 Japanese
Whale Research Program under Special Permit in the
Northern West Pacific (JARPN) survey (Fig. 2). Table 1
shows the number of minke whales analysed from each
location and year and the abbreviation used to refer to
the areas of capture. Samples of muscle were excised and
stored in 96% ethanol until analysed.
642
I. Martinez and L. A. Pastene
74°N
3°E
67 1/2°N
25°W
CIC
68°N
65°N
7°E
61°N
12°W
59°W
CG
63°N
CIP
WC
EC
69°
60°
12°E
73°N
CM
70°N
EB
30°E
WG
15°W
ES
EN
52°20N
42°W
18°W
50°
40°
60°W
50°
40°
30°
20°
10°W
0°
10°E
20°
30°E
Figure 1. Map of the Northeast Atlantic showing the areas where the minke whales were captured. Reproduced with permission
from IWC (IWC, 1993).
DNA extraction
Genomic DNA extraction was carried out according
to Miller et al. (1988) with the modifications described
by Martinez et al. (1997a). The amount and quality
of the DNA was estimated by 1% agarose gel electrophoresis in 0.5TBE (Sambrook et al., 1989) buffer and
comparison with known amounts of DNA.
Selection of primers
Nine 10-mer primers were chosen after screening 160 of
them as described in our preliminary work (Martinez
et al., 1997a). The primers (Operon Technologies Inc.,
Alameda, California) were: OPH-12 (5-ACG CGC
ATGT-3); OPH-18 (5-GAA TCG GCCA-3); OPJ-10
(5-AAG CCC GAGG-3); OPL-01 (5-GGC ATG
ACCT-3); OPL-03 (5-CCA GCA GCTT-3); OPS-16
(5-AGG GGG TTCC-3); OPT-14 (5-AAT GCC
GCAG-3); OPU-11 (5-AGA CCC AGAG-3) and
OPV-20 (5-CAG CAT GGTC-3). The criteria for selection were that they should consistently, between replicate polymerase chain reactions (PCRs) and for a DNA
concentration ranging at least from about 1 to 5 ng
l 1, produce clean patterns and polymorphic bands
that should be clear to score (Martinez et al., 1997a;
Stewart and Excoffier, 1996).
DNA amplification
Arbitrarily primed amplifications (Welsh and
McClelland, 1990; Williams et al., 1990) were performed
in 30-l volumes. Initially for each sample, two dilutions
containing 1 and 5 ng l 1 DNA were amplified as
described below (Welsh and McClelland, 1990; Williams
et al., 1993). Once it was shown that both concentrations
gave exactly the same RAPD profile with one of the
primers, only one of the amounts (usually 5 ng l 1)
was used. Ten microlitres of the DNA extracts containing 10 ng and/or 50 ng of the template DNA, were added
to 20 l of a mixture containing 1KlenTaq1 DNA
polymerase buffer (supplied by the manufacturer),
100 M each dATP, dCTP, dGTP, and dTTP, 0.4 M
10-mer primer (Operon Technologies Inc., Alameda,
California), and 4 mM MgCl2 (final concentration)
(Ellsworth et al., 1993), with 0.75 units of the Klen Taq1
DNA polymerase (AB Peptides, St Louis, MO, USA).
The reaction mixtures were overlaid with 20-l of Chill
Out Wax (MJ Research Inc., Watertown, MA, USA)
and amplification was performed on a PTC-100
programable thermal controller (MJ Research Inc.,
Watertown, MA, USA). The thermal program for
amplification was 94C for 1 min, followed by 40 cycles
of 94C, 10 s (denaturation); 35C, 10 s (annealing), and
72C, 1 min (extension). The program included a final
step of 72C for 5 min and the products were maintained
at 15–4C until ready to load onto the gels.
Agarose gel electrophoresis
Twelve microlitres of the products obtained after RAPD
analysis were separated in 20–10 cm, 2% (1:3) Nusieve:
Seakem LE FMC agarose gels (three gels per chamber).
RAPD-typing of minke whales
643
70°N
Okhotsk Sea
60°
52°
50°
Sea of Japan
Yellow Sea
40°
6
I
F
H F
J
East China
Sea
G
A11
141°
B
G
13
12
10
5
C
8
9
3
4
131°
1
120°
7
D
E
30°
F
127° 130°
2
140°
150°
157° 160°
170°E
Figure 2. Map of the Western North Pacific showing the areas where the minke whales were captured. Reproduced with permission
from IWC (IWC, 1994).
Gel and electrophoresis buffers were 0.5TBE
(Sambrook et al., 1989) and electrophoresis took place
for about 2 h at 4.5 V cm 1.
After electrophoresis, the gels were stained for 20 min
in 0.5TBE buffer containing 0.5 g ml 1 ethidium
bromide, destained for another 20 min in the same
buffer without ethidium bromide and photographed
under u.v. light with a Polaroid camera using film type
55. The presence or absence of markers was visually
determined on the photographs.
Data analysis
The matrix of presence (1) or absence (0) of each
polymorphic marker for each individual was submitted
to analysis of the molecular variance (AMOVA)
(Excoffier et al., 1992; Huff et al., 1993) and to principal
component analysis (PCA) (Esbesen et al., 1994)
because, both being adequate to treat RAPD markers,
their approach is basically different. AMOVA is used to
test a defined genetic structure while principal component analysis does not require the a priori definition of
structure in the data and individuals with related haplotypes should be exposed as clusters in the scores plot
(Demeke and Adams, 1994).
AMOVA was performed as implemented in the computer program ARLEQUIN (Schneider et al., 1996).
AMOVA allocates the proportion of the genetic variation attributable to (1) individuals within the areas, (2)
areas within oceans, and (3) between the two oceans.
The significance of the variance components was tested
by a random permutation procedure available in the
program. In each trial, 10 000 randomizations of the
original data set were made. The level of significance
obtained by this procedure, and referred as ‘‘p’’, is
the probability of obtaining a more extreme variance
component by chance alone.
Principal component analysis was performed using
the program Unscramble (Esbesen et al., 1994). Each
marker was given a weight of 1 and the model was
centered and fully cross-validated.
Results
RAPD analysis
The 11 primers used produced 116 bands, of which 21
seemed to be scorable polymorphic bands. Two of the
bands generated by the same primer seemed to be
alternative alleles corresponding to the same locus. This
was suspected because these two bands dominated the
multivariate data analysis when included and divided the
minke whales from the NA into two groups (each group
had one of the bands and lacked the other), each group
comprised individuals from all the areas, and the
addition of the frequencies of these band was 1 (results
not shown). Although multiple amplifiable alleles for
one single locus seem to be rarely detected by this
644
I. Martinez and L. A. Pastene
Table 1. Ocean, year, month and area of capture of the minke whales used in this work, as well as the
abreviations used and the number of individuals (n) analysed from each area.
Ocean
Year
Month
Area
Abbreviation
n
North Atlantic
1996
1996
1996
1996
1996
1994
1994
1994
1996
1996
1996
1996
Jun
May–Jun
Jun–Aug
May–Jul
Jun–Jul
Jul/Sep
Jul/Sep
Jul/Sep
Jul–Aug
Aug
Jul
Aug–Sep
CM
ES
EN
EB
EC
ES
EB
EC
8
11
7E
7W
M
S
N
F
L
s
f
l
P
Q
T
T
26
74
22
40
8
15
8
8
16
20
1
20
Western North Pacific
technique, their presence cannot be ruled out (Lynch
and Milligan, 1994; Williams et al., 1990 and references
therein). Therefore only one marker was included in the
statistical analysis. After pruning this locus, we assumed
that the following assumptions were correctly made
for the remaining 20 RAPD markers (Welsh and
McClelland, 1990; Williams et al., 1990): (1) all RAPD
loci showed complete dominance and (2) all loci had
only two alleles, with frequencies p (dominant, band
present) and q (recessive, band absent).
The average frequency of each of the 20 markers in
each areas and year is shown in Table 2. Three of the 20
RAPD markers seemed to be population-specific (illustrated in Fig. 3): two markers were present in all the
samples from the WNP and absent in all those from the
NA (markers no. 10 and 20), while one was detected in
all minke whales from the NA and in none from the
WNP (no. 9). In addition, four RAPD markers were
monomorphic in the NA but polymorphic in the WNP
(three fixed: nos 3, 4, and 8 and one absent: no. 18), and
six markers were monomorphic in the WNP but polymorphic in the NA (one fixed: no. 7 and 5 absent: nos 1,
11, 12, 14, and 19). Accordingly, 20 markers were used
to construct the haplotype of each individual (string of
20, 0/1, one for each marker) for multivariate and
AMOVA analyses to test the population structure NA
vs. WNP; 13 markers to test the population structure in
the NA and 11 in the WNP.
The RAPD markers generated 134 different haplotypes in 201 minke whales from the NA and 38 haplotypes with the 57 minke whales from the WNP (Table 3).
In both oceans, there were full matches (individuals with
identical haplotypes) between individuals from almost
all the areas, except in areas where the number of
individuals analysed was low (Table 4).
Multivariate data analysis
The first component of the PCA analysis performed
on the 258 minke whales and 20 RAPD markers
explained 37% of the total variability in the model
and clearly divided them into two groups: individuals
from the NA, with scores values between 0.4 and
+1.2, and those from the WNP, with scores values
between 2.2 and 1.4 (Fig. 4a). The second PC of
Figure 4a seemed to divide the samples from the
WNP into three groups: one main group (scores
close to 0 on PC2) and two minor groups of scores about
0.7 and 0.5, which can be due to the low number of
samples from this ocean. No clusters were revealed
when only the samples from the WNP were analysed
(see below).
Principal component analysis performed on the NA
samples (Fig. 4b) confirmed the lack of clusters shown in
Figure 4a. The first component explained only 9% of the
total variability, and the second 10%, indicating that the
PCA is not able to model the variability, i.e. no pattern
could be found in the distribution of the 201 samples
with these 13 markers.
Principal component analysis performed on the
samples from the WNP did not reveal any structure
either (Fig. 4c): the first two PCs explained 11% of
the variability each, indicating again the lack of
ability of the PCA to model the variability found in
the samples.
Analysis of the molecular variance
The results of the AMOVA (Table 5) showed, as
expected, a high and significant (63% and p<0.0001)
genetic difference between minke whales from the NA
and the WNP. Thirty-six percent (p<0.0001) of the total
variation was ascribed to individuals within areas, and
less than 1% was attributable to among areas variation
within each ocean. The low among areas genetic variability was also demonstrated by the within ocean
AMOVA: in both case, over 95% of the total genetic
variability was attributable to differences among
individuals within areas.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Marker
0.690.47
0.960.20
1
1
0.540.51
0.960.20
0.880.33
1
1
0
0.150.37
0.500.51
0.920.27
0.650.49
0.080.27
0.620.50
0.540.51
0
0.920.27
0
M
0.840.37
0.950.23
1
1
0.500.50
1
0.910.29
1
1
0
0.310.47
0.550.50
0.970.16
0.540.50
0.070.25
0.380.49
0.610.49
0
0.910.29
0
S
0.730.46
1
1
1
0.400.51
1
1
1
1
0
0.270.46
0.400.51
0.870.35
0.670.49
0
0.600.51
0.330.49
0
0.800.41
0
s
0.640.49
0.770.43
1
1
0.320.48
1
0.950.21
1
1
0
0.410.50
0.640.49
0.950.21
0.590.50
0.230.43
0.360.49
0.680.48
0
1
0
N
0.730.45
0.980.16
1
1
0.320.47
0.980.16
0.760.43
1
1
0
0.270.45
0.730.45
1
0.710.46
0.120.33
0.460.50
0.710.46
0
1
0
F
0.630.52
1
1
1
0.630.52
0.880.35
0.880.35
1
1
0
0.130.35
0.880.35
0.880.35
0.880.35
0
0.380.52
0.250.46
0
0.750.46
0
f
1
0.710.49
1
1
0.290.49
1
1
1
1
0
0.570.53
0.710.49
0.860.38
0.710.49
0.290.49
0.290.49
0.860.38
0
1
0
L
l
0.880.35
1
1
1
0.380.52
1
1
1
1
0
0.250.46
0.380.52
0.880.35
0.630.52
0.130.35
0
0.250.46
0
1
0
Table 2. Average frequencys.d. of each of the 20 RAPD markers in each area and year. Abbreviations as in Table 1.
0
0.810.40
1
0.940.25
0.630.50
0.630.50
1
0.940.25
0
1
0
0
1
0
0
0
0.810.40
0.750.45
0
1
P
0
0.650.49
0.900.31
0.750.44
0.950.22
0.800.41
1
0.700.47
0
1
0
0
0.800.41
0
0.050.22
0.100.31
0.800.41
0.650.49
0
1
Q
0
0.670.48
0.950.22
0.670.48
0.900.30
0.520.51
1
0.760.44
0
1
0
0
0.810.40
0
0.050.22
0.050.22
0.900.30
0.810.40
0
1
T
RAPD-typing of minke whales
645
646
I. Martinez and L. A. Pastene
Figure 3. RAPD analysis with one of the primers that yielded a
population-specific marker, labelled with an arrow. Each lane is
one individual minke whale. S, individuals from the Western
North Pacific; A, individuals from the North Atlantic.
Almost 3% (p<0.005) of the total genetic variability in
NA could be attributed to among areas variability.
The assumption that the Central and Northeastern
waters are visited by two different breeding stocks
should be rejected according to AMOVA since this
division did not explain any of the registered genetic
variability (Table 5). Different genetic structures
were then tested. The hypothesis that explained
most of the genetic variability, at a significant level, was
based on the division of the six NA areas into three
groups: Group 1 comprising areas ‘‘N’’, ‘‘L’’, and
‘‘F’’; Group 2 with ‘‘M’’, ‘‘S’’, and ‘‘s’’, and Group 3
with ‘‘l’’ and ‘‘f’’. This explained 2.2% of the total
variability (p<0.05), although about 1% (p<0.005) of
the total variability still remained at the among areas
level (Table 6). Performing AMOVA for pairs of these
three groups seemed to indicate that the source
of the 2% ‘‘among groups’’ variation could be
attributed to differences between Groups 1 and 2, while
the 1% ‘‘among areas within groups’’ variation had its
source in differences between the areas ‘‘f’’ and ‘‘l’’ of
Group 3. It should be noted that Group 3 had a small
sample size.
In the WNP, individuals from Areas 7 and 11 seemed
to be more closely related than to minke whales from
Area 8, since there were eight shared haplotypes among
nine whales from these two areas vs. three haplotypes
shared between Areas 8 and 11 and three between Areas
8 and 7 (Table 4). However, the 4.66% of the total
genetic variability explained by AMOVA assuming a
structure of two groups (Group 1 comprising Areas 7
and 11 and Group 2 with Area 8) was not significant
(Table 5).
Table 3. Number of haplotypes generated by 13 RAPD markers in the North Atlantic (NA) and by 11
markers in the Western North Pacific (WNP).
NA
No. of
individuals
by haplotype
1
2
3
4
5
6
Total
WNP
Number of
haplotypes
% of total
haplotypes
Number of
haplotypes
% of total
haplotypes
99
19
6
5
4
1
134
73.88
14.18
4.48
3.73
2.99
0.75
100
27
8
1
0
1
1
38
71.05
21.05
2.63
0
2.63
2.63
100
Table 4. Number of minke whales sharing haplotypes and areas of capture. Abbreviations as in
Table 1.
No. of
minke whales
Atlantic
6
5
4
3
2
Pacific
6
5
3
2
Area of capture
M S S S F F
S S S S l, S S S f l, S S S s l, S S N F F
S S S M, S S F L, S s l l, S S N F, S S s F
S S M, S S M, S S N, S F L, S F F, s F F
M M, M N, S M, s M, S S, S S, S N, S N,
S F, S F, S F, S F, s F, s F, s F, N F, F F, F f, f f
P P P Q Q T
P P P Q T
Q T T
P P, P Q, P T, Q T, Q T, Q T, Q T, Q T
RAPD-typing of minke whales
1.5
Scores
PC2
(a)
1.0
0.5
0
–0.5
–1.0
PC
–1.5
–3
1.5
–2
–1
0
1
Scores
PC2
2
(b)
1.0
0.5
0
–0.5
–1.0
PC
–1.5
–1.5
1.0
–1.0
–0.5
0
1.0
0.5
Scores
PC2
1.5
(c)
0.5
0
647
1993; Williams et al., 1993) and amplification program
used (Yu and Pauls, 1992; Williams et al., 1993), the
composition of the amplification buffer and the DNA
polymerase used (Ellsworth et al., 1993; Meunier and
Grimont, 1993; Park and Kohel, 1994) and the amount
and quality of the DNA (Welsh and McClelland, 1990;
Williams et al., 1993; Micheli et al., 1994; Martinez
et al., 1997a). We have changed some of the parameters
used in our preliminary work (Martinez et al., 1997a):
the times of denaturation and annealing have been
reduced from 20 to 10 s and the amount of Klen Taq 1
DNA polymerase has been reduced from 1.5 to 0.75
units per 30 l reaction. For this work we have used two
thermocyclers, both are of the same brand and model
but one is slightly faster than the other. We have also
changed the electrophoresis equipment in order to be
able to run 120 samples per gel. The consequences of
these changes have been that the relative intensity of
some of the bands as well as the total number of bands
produced varied: we have obtained more bands in the
present work, which can be due to the shorter denaturation time used (Yu and Pauls, 1992). However, the
polymorphic bands identified in the previous work could
also be identified here. The reproducibility of the
markers was tested (1) by the 31 samples analysed in
Martinez et al. (1997a) which were extracted and analysed again under the new conditions, (2) some samples
have been analysed more than five times for each primer,
(3) the analysis of the samples from the WNP was
performed simultaneously with 35 samples from the NA,
and (4) a total of three different persons have been
involved in performing the RAPD analysis and obtained
exactly the same fingerprints from the samples included
as controls. Thus, we consider the markers considered in
this work to be reproducible.
Genetic variability
–0.5
PC
–1.0
–1.0
–0.5
0
0.5
1.0
Figure 4. Scores plot of the Principal Component Analysis of
(a) all the 258 minke whales, (b) only the 201 minke whales
from the Northeast Atlantic, and (c) only the 57 minke whales
from the Western North Pacific. All models were centred, full
cross-validated, and each RAPD marker was given a weight of
1. Abscissa, PC1; ordinate, PC2. Percent explained variance
was (a) 37% for PC1 and 6% for PC2; (b) 9% for PC1 and 10%
for PC2; and (c) 11% for PC1 and 11% for PC2.
Discussion
RAPD analysis
To perform RAPD analysis several variables need to be
standardized: the equipment (Meunier and Grimont,
As already noted (Martinez et al., 1997a), the percentage
of primers that produced polymorphic bands (19%) in
the minke whale was relatively low compared to values
between 25 and 100% reported in the literature (Xiong
et al., 1992; Martinez et al., 1994). Similarly, the proportion of polymorphic bands (17.24%) was also relatively low compared to the reported values of between
39.1% for the marsh wren (Bowditch et al., 1994) and
72.5% for the endangered black rat snake (Gibbs et al.,
1994), although it was over one order of magnitude
greater than the 1.2% reported for two endangered
clapper rail subspecies (Nusser et al., 1996). These low
values can be due to the fact that only minke whales
from the NA were used to select the primers. Thus, it is
very likely that primers rejected during the screening
procedure (performed with samples from the NA only)
for not producing polymorphisms, would have rendered
discriminant markers, if the screening had been
648
I. Martinez and L. A. Pastene
Table 5. Analysis of the molecular variance (AMOVA) for 258 individual minke whales using 20
RAPD markers. The minke whales were captured in two ocean basins: North Atlantic and Western
North Pacific. The Atlantic ocean contains individuals from eight ‘‘areas’’ (five areas from 1996 and
three from 1994), and the Pacific ocean contains minke whales from 3 areas. AMOVA was also
performed for the 201 (Atlantic) and 57 (Pacific) individuals from each ocean separately using 13 and
11 RAPD markers, respectively. Abbreviations are: d.f., degrees of freedom; SSD, sum of square
deviations; variance component estimates; % of total variance, percentage of the total variation
contributed by each component, and p, the probability of obtaining a more extreme component by
chance alone; n.a., not available. C: Central NA with the area CM only, and NE: Northeast Atlantic,
comprising the areas EN, EC, EB, and ES.
Variance
component
% of total
variance
Source of variation
d.f.
SSD
p
Atlantic vs. Pacific
Among areas within oceans
Individuals within areas
Atlantic
Among areas
Individuals within areas
C vs. NE Atlantic
Among areas in the NA
Individuals within areas
Pacific
Among areas
Individuals within areas
Area 8 vs. (7+11)
Among areas in the NP
Individuals within areas
1
9
247
288.50
26.04
453.24
3.21
0.05
1.83
63.05
0.95
35.99
<0.0001
<0.005
<0.0001
7
193
1
6
193
22.65
372.51
2.64
19.00
372.51
0.05
1.93
0.02
0.06
1.93
2.60
97.40
1.04
2.92
98.12
<0.005
n.a.
0.75
<0.005
<0.005
2
54
1
1
54
4.39
80.73
3.04
1.35
80.73
0.04
1.49
0.07
0.01
1.49
2.42
97.58
4.66
0.47
95.81
0.092
n.a.
0.33
0.60
0.09
Table 6. Analysis of the molecular variance (AMOVA) performed on the 201 individuals from the NA
using 13 RAPD markers. Abbreviations are as in Table 5. Group 1 comprises individuals from ‘‘N’’,
‘‘L’’, and ‘‘F’’; Group 2 individuals from ‘‘M’’, ‘‘S’’, and ‘‘s’’, and Group 3 individuals from ‘‘l’’ and
‘‘f’’ (see Table 1).
Source of variation
d.f.
SSD
Variance
component
% total
p
Group 1 vs. 2 vs. 3
Among areas within groups
Individuals within areas
Group 1 vs. Group 2
Among areas within groups
Individuals within areas
Group 1 vs. Group 3
Among areas within groups
Individuals within areas
Group 2 vs. Group 3
Among areas within groups
Individuals within areas
21
5
193
1
4
179
1
3
81
1
3
126
9.80
11.85
372.51
6.09
9.04
347.38
5.20
6.19
156.37
3.03
7.75
241.27
0.04
0.02
1.93
0.04
0.02
1.94
0.11
0.02
1.93
0.02
0.03
1.91
2.24
1.03
96.73
2.09
0.65
97.26
5.52
1.15
93.33
1.28
1.51
97.22
<0.05
<0.005
<0.005
<0.00001
n.s.
<0.01
n.s.
<0.01
<0.05
n.s.
<0.05
<0.05
performed with individuals from both the NA and
WNP.
The three RAPD markers absent in one ocean and
fixed in the other were sufficient to discriminate individuals from each ocean in the present work. However,
since RAPD markers are dominant, classification of
unknown samples based solely on these three markers
should be done cautiously. For example, for the NA
sample of 201 individuals, and assuming H-W equilibrium, in the case of apparently absent markers the
dominant phenotype (made up of dominant homozygotes and heterozygotes) would have remained undetectable if the frequency of the dominant allele (p) was
lower than 0.25%, and in the case of apparently fixed
markers, a frequency of 93% or higher of the dominant
allele would have obscured the presence of recessive
homozygotes. However, for the smaller WNP sample, a
frequency of the dominant allele of 0.88% or lower
would have made undetectable individuals with the
dominant phenotype, while a frequency of the dominant
RAPD-typing of minke whales
allele of 86% or higher would have made recessive
homozygotes undetectable in the present study. In any
case, about half of the 20 markers differed by over 50%
in their frequencies in the NA and WNP and they were
able to clearly differentiate minke whales from the two
oceans as shown in Figure 4.
The present work thus confirmed the known genetic
differentiation among minke whales from the NA and
WNP (see references in the introduction). As van Piljen
et al. (1995), we did not find shared haplotypes between
individuals from the NA and WNP and both groups
possessed clearly different marker frequencies, indicating
a high level of genetic divergence between the two
oceans. Within each ocean basin there was very low
inter-area (under a 3%) and high intra-area (over 97%)
genetic diversity.
Genetic structure in the NA
The very low percent (2.6%) of the total genetic variability allocated to differences among areas in this ocean
was significant according to the AMOVA analysis
(p<0.005) and due to differences between the 115 individuals captured in the small areas CM (1996) and ES
(1994 and 1996) on one hand, and the 70 individuals
captured in small areas EN (1996), EC (1996), and EB
(1996) on the other, while the 16 individuals captured in
areas EB and EC in 1994 were not significantly different
from any of the other two groups. This result may
indicate the presence of two groups in the Central and
Northeastern Atlantic: Group 1, preferentially distributed in the Southern part (North Sea) and along
the Norwegian coast towards the north (LofotenVesterålen, Finnmark, and Barents); and Group 2, in
the Central (Jan Mayen) and Northern area (Bear Island
and Svalbard). The lack of significant differentiation
between the 16 individuals of the third group (captured
in 1994 in Lofoten-Vesterålen and Finnmark and
Barents) from any of the other two groups is not
surprising, due to the low number of individuals of
Group 3, to the fact that the difference between Groups
1 and 2 was indeed small (2%) and to the high number of
inter-group shared haplotypes (see Table 3: 18 out of the
35 shared haplotypes included individuals from Groups
1 and 2). If this 2% value truly reflects genetic structuring of the minke whale in these waters, the spacial
distribution of the two groups can be explained if (1)
there is a temporal component whereby individuals
belonging to Group 2 would start the migration towards
the north earlier than individuals from Group 1, and
therefore leave (most of them) the Norwegian coastal
waters towards Bear Island, Svalbard, and the Central
Atlantic, while individuals of Group 1 would start the
migration later and/or if (2) the two groups merely have
different preferences for their feeding grounds. The
situation in the Arctic may consequently be analogous
649
to that in the Antarctic, where the temporal component
has been shown to be important: one stock may occupy
a certain longitudinal sector during part of the feeding
period while the same sector can be occupied by another
stock later on during the feeding season, and also,
different stocks may mix during their migration towards
their feeding grounds (Pastene et al., 1996). This hypothesis also helps to understand that the differences found
by Danı́elsdóttir et al. (1995) among individuals captured in the same waters but in different years may be
greater than those between individuals captured at different locations in the same year, since there may be a
mixing of the two groups and which of the two would be
the predominant group in a particular area may depend
on the extent of the migration, which may in turn be
effected by the weather conditions (ice distribution,
water temperature, etc.) and feed availability for each
particular year. A temporal component would also
support the observations of Øien (1991): if the minke
whales feeding in the Central area cross the Norwegian
coast earlier in the spring, they would not have been
around Bear Island at the time of tagging, which was
late summer.
Principal component analysis was performed in order
to expose possible clusters of genetically more related
individuals, since no genetic structure attributable to
either physical or temporal barriers needs to be presumed to perform this analysis. This analysis was not
able to modulate the variability that existed in the data
and did not either reveal the presence of clusters which
supports the very small difference among the minke
whales from the NA quantified by AMOVA as a 2%.
The present work therefore supports the results of
Danı́elsdóttir et al. (1992, 1995) and Øien (1991) in that
there may be more than one breeding stock in the
Northeast Atlantic and also the results of Bakke et al.
(1996), Palsbøll (1989), and van Piljen et al. (1995), since
the distribution of the individuals belonging to Group 1
or 2 does not respect the small areas boundaries. Unlike
Danı́elsdottir et al. (1995) and Øien (1991) we could not
find any significant genetic differentiation between the
minke whales captured in the Central and Northeast
Atlantic, and our sample from the Central Atlantic was
indistinguishable from that of the ES small area. However, our sample from the Central Atlantic was from the
small area CM, while the sample of Danı́elsdóttir et al.
(1995) was from Island (CIC).
Genetic structure in the WNP
No definite genetic structure was registered among the
three areas of the WNP considered in this study.
Although individuals from Areas 7 and 11 seemed to be
more closely related among themselves than to minke
whales from Area 8, the difference was not significant,
and it can be due to the low number of individuals from
650
I. Martinez and L. A. Pastene
each area included in this analysis. This lack of genetic
structure among these areas in the WNP confirms the
results obtained by RFLP analysis of the control region
of mitochondrial DNA (Goto and Pastene, 1997) and
microsatellite analysis (H. Abe pers. comm.).
Conclusion
The present work corroborates the genetic differentiation between minke whales from the NA and WNP. It
also seems to indicate that there may be two closely
related breeding stocks of minke whale in the Central
and Northeastern Atlantic waters, whose individuals
may intermix during the period of May to September,
adding evidence to support the exchange among the
small areas CM-ES-EB-EC-EN (IWC, 1993).
Acknowledgements
The authors are in debt to the crew and scientific
personnel of the whaling boats who took and preserved
the samples. To Dr Anna Kristin Danı́elsdóttir (Marine
Research Institute, Reykjavik, Island) for her helpful
comments and for allowing us to use her unpublished
material; to Dr Tore Haug (Norwegian Institute of
Fisheries and Aquaculture, Tromsø, Norway) for valuable comments on the manuscript; to the International
Whaling Commission for allowing us to reproduce the
maps shown in Figures 1 and 2 and to the Norwegian
Research Council for the financial support (project no.
111042/120).
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