Molecular Ecology (2007) 16, 2223– 2235
doi: 10.1111/j.1365-294X.2007.03287.x
Genetic variation in Holocene bowhead whales from
Svalbard
Blackwell Publishing Ltd
T . B O R G E ,* L . B A C H M A N N , G . B J Ø R N S T A D and Ø . W I I G
Natural History Museum, University of Oslo, PO Box 1172 Blindern, N-0318 Oslo, Norway
Abstract
Bowhead whales (Balaena mysticetus) are distributed in the Arctic in five putative stocks.
All stocks have been heavily depleted due to centuries of exploitation. In the present study,
nucleotide sequence variation of the mitochondrial control region was determined from
bone remains of 99 bowhead whales. The bones, 14C dated from recent to more than
50 000 BP, were collected on Svalbard (Spitsbergen) and are expected to relate to ancestors
of the today nearly extinct Spitsbergen stock. Fifty-eight haplotypes were found, a few
being frequent but many only found in one individual. The most abundant haplotypes
of the Spitsbergen stock are the same as those most abundant in the extant BeringChukchi-Beaufort (BCB) Seas stock of bowhead whales. Although FST indicates a slight
but statistically significant genetic differentiation between the Spitsbergen and the BCB
stocks this was not considered informative due to the very high levels of genetic diversity
of mitochondrial DNA haplotypes in both bowhead whale stocks. Other measures such as
KST also indicated very low genetic differentiation between the two populations. Nucleotide
diversity and haplotype diversity showed only minor differences between the Spitsbergen
and BCB stocks. The data suggest that the historic Spitsbergen stock — before the severe
bottleneck caused by whaling — did not have substantially more genetic variation than the
extant BCB stock. The similar haplotypes of the Holocene Svalbard samples and the current
BCB stock indicate significant migration between these two stocks and question the current
designation of five distinct stocks of bowhead whales in the Arctic.
Keywords: ancient DNA, bottleneck, conservation biology, genetic diversity, historical biogeography,
mitochondrial DNA
Received 7 November 2006; revision accepted 8 January 2007
Introduction
Ancient DNA methodology has opened for retrieving
genetic information from historic and prehistoric material
(Pääbo et al. 2004). Given nucleic acids have survived in the
finds even the genetic structure of prehistoric populations
can be characterized. Such ancient DNA data sets have
contributed provocative ideas about the development of
Correspondence: Øystein Wiig, Fax: +47 22 85 18 37; E-mail:
oystein.wiig@nhm.uio.no
*Present address: Center for Ecological and Evolutionary Synthesis,
Biological Institute, University of Oslo, Post-box 1066 Blindern,
0316 Oslo, Norway.
Data deposition: The sequences reported in this study have
been deposited in the GenBank database under Accession nos
DQ371698–DQ371723, DQ371725–DQ371737 and DQ371739–
DQ371798.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
population structure within species (Hedrick & Waits 2005).
Hofreiter et al. (2004), for example, have shown that the
current population structure of several terrestrial mammals
can be better explained by recent (< 10 000 years) rather
than long-term effects. Such information is important for
both the understanding of the past evolutionary history of
endangered species as well as for their future conservation
(Hedrick & Waits 2005). Due to its relatively high copy
number and a higher likelihood for DNA survival, mitochondrial DNA is among the preferred markers for ancient
DNA analyses. In the present study, we address the changes
in population structure in bowhead whales (Balaena mysticetus)
of the Spitsbergen stock during the Holocene through the
analyses of the control region of ancient mitochondrial
DNA (mtDNA).
Bowhead whales inhabit arctic and subarctic regions
of the Atlantic Ocean, and the Bering, Beaufort, Chukchi
2224 T . B O R G E E T A L .
Fig. 1 (a) Circumpolar distribution of bowhead whales. The five recognized geographical stocks (given in grey) are located in: (1) the
Bering/Chukchi/Beaufort Seas (BCB) (2) the Okhotsk Sea (3) the Davis Strait (4) Hudson Bay/Foxe Basin and (5) Svalbard (Spitsbergen).
(b) Sampling sites of 99 bowhead whale bones at Svalbard (excl. Bjørnøya). At some sites, more than one individual were sampled (see
Appendix for more details).
and Okhotsk Seas (Moore & Reeves 1993). Today, five
geographical stocks are recognized in (i) the BeringChukchi-Beaufort (BCB) Seas, (ii) the Okhotsk Sea, (iii) the
Davis Strait/Baffin Bay, (iv) the Hudson Bay/Foxe Basin,
and (v) in the areas around Svalbard (Spitsbergen) (Fig. 1a).
The Spitsbergen stock is distributed in the Greenland,
Norwegian, Barents, and Kara Seas (Moore & Reeves 1993).
However, most of the evidence for stock identification
is circumstantial and indirect, and currently the five
bowhead whale stocks are questioned and still await a
proper characterization in population genetics terms
(Moore & Reeves 1993; Rough et al. 2003; Heide-Jørgensen
et al. 2006).
The close association of bowhead whales with the sea
ice edge has caused fluctuations in the distribution and
abundance with climate changes (Dyke et al. 1996b). Some
10 000 years ago bowhead whales moved into the Arctic
Ocean via the Bering Strait (Dyke & Savelle 2001) and
seasonally colonized the eastern Beaufort Sea. They reached
maximum abundance between 10 000 and 8500 bp. The
Davis Strait stock may originate from the Bering Sea stock,
or alternatively from a palaeo-stock in the Gulf of Saint
Lawrence, or from the Spitsbergen stock (Dyke et al. 1996a).
The Hudson Bay stock might also have arisen from the
Saint Lawrence stock (Dyke et al. 1996a). The Spitsbergen
stock presumably originates from a refugium in the eastern North Atlantic (Fredén 1975). According to the data of
Dyke & Savelle (2001) migration between Pacific and
Atlantic bowhead whale stocks was unrestricted during
the period 10 000–8500 bp. Later, the M’Clintock Channel
sea-ice plug prevented migration until 5000–3000 bp
(Dyke et al. 1996a).
Due to exploitation over several centuries, the once
abundant bowhead whale went nearly extinct in most of its
former range (Moore & Reeves 1993). Prior to the commercial exploitation, the total number of bowhead whales has
been estimated to at least 53 000 animals (Woodby &
Botkin 1993). The total population now shows a trend of
an increase in number. While IWC (1997) indicated a population size of less than 12 000, today the population size is
likely to be close to 17 000. The BCB stock is estimated to be
close to 10 500 individuals (George et al. 2004) and recent
provisional estimates from the Canadian Arctic indicate
that the numbers there are close to 5000 (COSEWIC 2005).
In the northeast Atlantic, sporadic but persistent observations have been made within the assumed distribution
range of the Spitsbergen stock (Reeves 1980; Jonsgård 1981,
1982; Wiig 1991; de Korte & Belikov 1994; Gilg & Born
2005). The commercial hunt of bowhead whales around
Svalbard started in the early 17th century. The last four
specimens were caught north of Svalbard in 1932 (Jonsgård
1964). The pre-exploitation stock size in the Svalbard area
has been estimated at 25 000 whales (Mitchell 1977), i.e. it
was the largest of the bowhead whale stocks at that time.
Some recently published estimates are even higher and
considered the total number to be over 100 000 (Allen &
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
G E N E T I C V A R I A T I O N I N B O W H E A D W H A L E S 2225
Amplicon
Primer
Position*
Sequence (5′−3′)
Length (bp)
1
134F
dip3.3R
109F
351R
177F
351R
295F
534R
297F
dip5R
15439–15465
15635–15658
15581–15600
15806–15823
15650–15673
15806–15823
15767–15785
15987–16006
15769–15788
15943–15969
CCCAAAGCTGAAATTCTACATTAAACT
CGTRGTGAARATAATTGAATGCAC
TGGCCGATACTAGTCCCAAC
GCGGGTTGCTGGTTTCAC
TTCACTACGGGAAGTTAAAGCTCG
GCGGGTTGCTGGTTTCAC
GGCCGCTCCATTAGATCAC
TCAGTTATGTGTGGGCATGG
CCGCTCCATTAGATCACGAG
CCATCGWGATGTCTTATTTAAGRGGAA
169
2
3
4
5
Table 1 PCR amplicons and primer
sequences for the amplification of the
control region (CR) of the mitochondrial
DNA of bowhead whales
205
132
201
154
*Positions correspond to the complete bowhead whale mitochondrial DNA (Sasaki et al.
2005), GenBank Accession no. AP006472.
Keay 2006). After cessation of the hunt, the Spitsbergen
stock was considered exterminated (Jonsgård 1981).
It is reasonable to assume that large-scale fluctuations in
distribution and abundance of bowhead whales have affected
stock structure and genetic diversity over time. Rooney
et al. (1999, 2001) investigated the effects of Holocene sea
ice variability and the unregulated commercial whaling
at the end of the 19th century on the genetic variation of
the BCB stock. Through sequencing of the mitochondrial
control region (CR), many haplotypes were detected in
the extant BCB bowhead whale stock, indicating that a substantial amount of genetic variability was retained despite
the severe bottleneck caused by whaling around the turn
of the 20th century. In the current study, we sequenced the
same stretch of the mitochondrial CR from surviving DNA
in old bowhead whale bones and baleens that have been
collected on raised beaches at Svalbard. The intention of
the study is twofold. First, we want to test whether genetic
variation and haplotype structure within the Spitsbergen
stock has changed over time. Second, we want to compare
the genetic variation of the Holocene Spitsbergen samples
and the extant BCB population (Rooney et al. 2001) in
order to test for genetic differences between the two stocks.
Materials and methods
Samples
Bones and baleen from 105 bowhead whales were collected
at Svalbard during 1976 –1997 for the purpose of dating
raised beaches (e.g. Salvigsen & Slettemark 1995), and in
2001 specifically for this study (Fig. 1b). The material was
registered into the collections of the Natural History
Museum, University of Oslo, Norway (see Appendix for
full details). Conventional 14C dating was performed at the
Radiological Dating Laboratory, Norwegian University of
Science and Technology, Trondheim.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
DNA extraction, amplification and sequencing
The bone surface was washed with ethanol and small samples
were cut out of the compacta of each bone. Each bone piece
was ground in a mortar to fine powder under liquid nitrogen,
and 0.1–0.2 g of bone powder were subsequently transferred
into 2.0 mL screw-capped centrifuge tubes. The powder
was soaked in 10% Clorox for 20 min to remove potential
contaminants, and subsequently rinsed in double distilled
water. The samples were decalcified in 1.5 mL 0.5 EDTA
(pH 8.5) through incubation overnight at 37 °C (Hagelberg
& Clegg 1991). The supernatant was discarded after centrifugation at 10 000 g for 5 min and the samples were washed with
double distilled water. The samples were then incubated at
56 °C for up to 50 h in 0.5 mL Lifton’s buffer (0.2 m sucrose,
0.05 m EDTA, 0.1 m Tris and 0.5% SDS) and 35 µL of 20 mg/mL
Proteinase K. Subsequently, standard phenol/chloroform
purification (Sambrook & Russell 2001) was applied. The
DNA was then extracted according to the silica method
(Höss & Pääbo 1993). Next, 1.5 mL 5 m guanidine thiocyanate
and 4 µL silica solution (Boyle & Lew 1995) were added to
0.5 mL of the aqueous phase and incubated for at least
1 h with gentle rotation. The silica pellet obtained after
centrifugation was washed in ethanol. After air-drying, the
DNA was eluted by incubating each silica pellet in 60 µL of
RNAse free UV-treated ddH2O at 56 °C for at least 10 min.
The silica was removed and the DNA solution transferred
into a fresh tube. A second elution of the silica pellet with
40 µL of H2O was pooled with the first extraction.
The control region of the mitochondrial DNA (mtDNA)
was amplified in five overlapping fragments using the
primers listed in Table 1. Polymerase chain reaction (PCR)
was carried out in 10 µL reactions employing 1.5 U cloned
Pfu Turbo DNA polymerase (Stratagene) (a polymerase
with proofreading activity), 2 µL of the extracted DNA
solution, 2.5 mm MgCl2, 0.2 µm of each dNTP, 0.32 µm of
each primer, 1 µg of Bovine Serum Albumine (BSA) and
2226 T . B O R G E E T A L .
1× PCR buffer (Stratagene). The PCR protocol included
an initial denaturation at 95 °C for 2 min, 40 cycles with
95 °C for 45 s, 55 °C for 45 s and 72 °C for 90 s, and a final
5-min extension step at 72 °C.
The obtained PCR products were purified with ExoSap-IT
(USB) and cycle-sequenced directly with BigDye terminator
chemistry version 1.1 on an ABI PRISM 3100 automated
sequencer (Applied Biosystems) according to the manufacturer’s descriptions.
Authentication
Standard precautions and measures for ancient DNA analyses
demonstrating authenticity of the obtained data were
followed. Each sample represents a unique individual
because they were found in different sample sites or varied
significantly in 14C age (see Appendix). Bone samples were
stored individually in sealed plastic bags. The samples
were handled in a UV-light equipped PCR workstation in
a laboratory physically isolated from the laboratory where
PCR products were processed. No DNA from modern
bowhead whale or any other whale had been amplified in
the laboratory previously. Numerous negative controls,
blind tests, repeated extractions and amplifications were
used to test for contaminations. All nucleotide positions in
the CR sequences were determined by sequencing at least
two overlapping amplicons obtained with different primer
pairs. In order to detect sequence variation due to postmortem damage, at least 10 clones were sequenced from
each PCR products of amplicon 1 in five individuals. The
cloning was performed with the Zero Blunt TOPO PCR
Cloning Kit for Sequencing (Invitrogen) following the
manufacturer’s instructions. All 169 positions in all clones
are identical to the sequences obtained from direct sequencing
of PCR products except for a G/A substitution at position
#15575 in individual 107 (see Supporting Fig. 1). This
substitution is not likely to result from contamination or
jumping PCR (Pääbo 1989) as this particular G has not been
found in any other sample. The very low level of sequence
variation among clones further supports authenticity of
the sequences obtained from direct sequencing of PCR
products.
Sequence analyses
The obtained nucleotide sequences were aligned and edited
with sequencher 4.1 (GeneCodes) and subsequently
screened against GenBank for species identification using
blast (Altschul et al. 1990). Sequences that differed by at
least one nucleotide were considered different haplotypes.
The computer program dnasp (Rozas et al. 2003) was used
to calculate haplotype diversity (H ) and nucleotide diversity
(π) (Nei 1987). arlequin version 2.0 (Schneider et al. 2000)
and proseq2 (Filatov 2002) were used for population genetics
data analysis (FST and KST; Hudson et al. 1992), and the
construction of minimum spanning networks. treeview
1.6.6 (Page 1996) was used to depict the minimum spanning
network as a phylogram.
Results and discussion
Out of the 105 analysed bone samples, 101 yielded nucleotide
sequences of the CR of the mtDNA. From a technical/
methodological point of view, the very high amplification
success (96%) was surprising. This indicates very favourable
condition for DNA survival in Arctic samples. Low average
temperature may be the key parameter in this context
(Smith et al. 2001; Lambert et al. 2002).
Only two of the obtained mtDNA sequences were not
of bowhead whale origin. They matched with fin whale
(Balaenoptera physalus; GenBank Accession no. NC_001321)
and with North Atlantic bottlenose whale (Hyperoodon
ampullatus; GenBank Accession no. AJ554056) and were
excluded from further analyses.
Radiocarbon dates are available for all 99 bowhead
samples (see Appendix). Three samples are more than
40 000 years old (Late Pleistocene) (age class I). The remaining 96 samples were assigned to time periods (age classes
II–V) according to postglacial climate periods (Dyke et al.
1999; Dyke & Morris 1990; Dyke et al. 1996a) (Table 2).
Thirty-four samples fall into the Early Holocene period
(age class II, 10 500–8500 bp), when bowheads from the
western and the eastern stocks could potentially migrate
through the central Canadian arctic. Thirteen samples
fall into the first period of exclusion (age class III, 8500–
5000 bp), when the M’Clintock Channel sea ice plug
prevented migration between eastern and western stocks.
Eleven samples fall into the Mid-Holocene period (age
class IV, 5000–3000 bp), with possible recurrence of gene
flow between eastern and western stocks. Thirty-eight
samples fall into the Late Holocene period (age class V,
3000–0 bp), a period characterized by cooling and the
reoccurrence of the M’Clintock Channel sea ice plug.
Taking together the radiocarbon dates, the sampling sites,
and the obtained nucleotide sequences it can be safely
assumed that each bone sample relates to only one bowhead whale individual and that there was no redundant
sampling.
A total of 453 nucleotides, corresponding to position
15 473–15 925 in the complete mitochondrial genome of
the Bowhead whale (Arnason et al. 1993), GenBank Accession no. AP006472, were sequenced from all 99 bowhead
whale samples. The total alignment includes 50 variable
positions (Table 3), 46 of which are affected by transitions
and four by transversions. All variable positions were
only affected by one particular nucleotide substitution
and insertions/deletions were not observed. The average
nucleotide frequencies were: 0.31 for thymine, 0.22 for
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
G E N E T I C V A R I A T I O N I N B O W H E A D W H A L E S 2227
Table 2 Haplotype diversity (H) and nucleotide diversity (π) of the mitochondrial control region (CR) of Holocene bowhead whales from
Svalbard pooled in age classes according to biogeographical epochs described for bowhead whales; for details see text
14C Age classes
(years bp)
I
(> 10 500)
II
(10 500–8500)
III
(8500–5000)
I–III
(oldest)
IV
(5000–3000)
V
(3000–0)
VI + V
(youngest)
Number of sequences
Segregating sites
Number of haplotypes
Haplotype diversity
H (SD)
Nucleotide diversity
π (SD)
3
9
3
1
(0.272)
0.0136
(0.004)
34
35
24
0.952
(0.026)
0.0123
(0.0019)
13
23
11
0.962
(0.05)
0.0128
(0.0021)
50
41
34
0.954
(0.021)
0.0137
(0.0014)
11
24
9
0.945
(0.066)
0.0181
(0.0024)
38
28
24
0.913
(0.039)
0.0106
(0.0014)
49
34
31
0.917
(0.034)
0.013
(0.0015)
cytosine, 0.30 for adenine and 0.17 for guanine. In total,
58 different haplotypes were detected. The most common
haplotype (BWS1) was shared by 24 individuals whereas
49 haplotypes (85%) were unique (i.e. only obtained in
one individual) (see Table 3 for details).
The very high proportion of individual mtDNA haplotypes along with the inhomogeneous sampling over time
(age classes I, III, and IV are only poorly represented)
hampers a detailed analysis of temporal changes in haplotype structure in the data set of Holocene bowhead whales
from Svalbard. We therefore divided the 99 samples into
two groups consisting of the 49 youngest and the 50 oldest
samples (median age were 560 years and 9498 years,
respectively). Haplotype BWS1 is the most frequent haplotype in both groups (youngest: 0.286; oldest: 0.2) and the
two data sets do not differ in parameters such as nucleotide
and haplotype diversity (Table 2). Genetic differentiation
between both groups as expressed by FST = 0.00003
(P = 0.40541) and KST = −0.0007 was low. The calculation of
multiple FST with sequential Bonferroni correction moving
the division point between the two groups up and down
the years by one animal at a time until all individuals of age
class IV were included in the group of the youngest samples and all individuals of age class III were included in the
group of the oldest samples (in total 25 comparisons) did
not reveal any significant indication of genetic differentiation between the two groups (P > 0.18). We also compared
groups of the 25 oldest and the 25 youngest samples from
Svalbard in order to avoid that the middle-aged samples
might cloud any temporal difference. These two groups
comprise samples with 14C ages ranging from 9500 to 51 000
and 30–560 years, respectively. Again FST = −0.01210
(P = 0.96396) and KST = −0.0036 indicated very low genetic
differentiation between the two groups. Thus, the data
do not support the hypothesis of significant changes of
genetic variation and haplotype structure within the
Spitsbergen stock.
Apart from the present study, there is another mitochondrial CR sequence data set available that includes
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
98 bowhead whale individuals from the extant BCB stock
(Rooney et al. 2001). Thirteen haplotypes of the Spitsbergen
data set (BWS1, 3, 4, 12, 17, 19, 27, 34, 44, 48, 51, 53 and 55)
have also been detected earlier in the extant BCB stock,
while 45 (78%) of the Spitsbergen stock haplotypes were
not (Rooney et al. 2001). Of the 68 haplotypes observed
in the BCB stock (named A-PPP), 55 (81%) were stockspecific. Fifty segregating sites were detected in the
Svalbard data (Table 4) compared to 58 in the BCB stock.
The 36 shared segregating sites are affected by the same
particular nucleotide substitution in both the Spitsbergen
and the BCB stocks.
A minimum spanning network depicted as a phylogram
of all haplotypes observed in the Svalbard data set and the
extant BCB population is shown in Fig. 2. Several haplogroups can be identified in the network. Haplotypes of all
major haplogroups are found in both data sets of bowhead
whales from the Holocene Spitsbergen and the extant BCB
stocks.
The most common haplotype (BWS1) in the Svalbard
data set, with a frequency of 0.242, was also the most common haplotype in the BCB data set (F, frequency 0.0816).
Also, the overall genetic variation was similar in the
Spitsbergen and the BCB stocks if estimated as haplotype
diversity (H = 0.99 and 0.95, respectively) and as nucleotide diversity (π = 0.016 and 0.013, respectively) (Table 4).
Nevertheless, the analysis of molecular variance revealed
a slight genetic differentiation between the Spitsbergen
and the BCB stocks of bowhead whales (FST = 0.013,
P < 0.0001; KST = 0.0041). However, we do not consider the
detected level of genetic differentiation surprising given
the very high proportion of mtDNA haplotypes with very
low frequency (only found once) in both bowhead whale
stocks (Table 3). Furthermore, the most frequent haplotype
is the same in both stocks and there is no stock or age
class-specific group of haplotypes (Fig. 2). Another serious
argument to take with caution the detected genetic differentiation between the Spitsbergen and the BCB stocks
comes from the data set of the Spitsbergen stock. First, this
Nucleotide position
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
1
5
4
8
Haplotype 5
1
5
4
8
8
1
5
4
9
0
1
5
5
0
9
1
5
5
5
7
1
5
5
5
8
1
5
5
5
9
1
5
5
6
7
1
5
5
6
8
1
5
5
7
1
1
5
5
7
4
1
5
5
8
2
1
5
5
8
4
1
5
5
8
6
1
5
5
9
0
1
5
5
9
7
1
5
6
0
7
1
5
6
1
3
BWS1
BWS2
BWS3
BWS4
BWS5
BWS6
BWS7
BWS8
BWS9
BWS10
BWS11
BWS12
BWS13
BWS14
BWS15
BWS16
BWS17
BWS18
BWS19
BWS20
BWS21
BWS22
BWS23
BWS24
BWS25
BWS26
BWS27
BWS28
BWS29
BWS30
BWS31
BWS32
G
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.
T
.
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C
.
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T
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T
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1
5
6
1
4
1
5
6
1
6
1
5
6
2
2
1
5
6
9
4
1
5
6
9
9
1
5
7
2
2
1
5
7
2
6
1
5
7
2
7
1
5
7
3
0
1
5
7
3
3
1
5
7
3
6
1
5
7
3
7
1
5
7
3
8
1
5
7
4
3
1
5
7
4
4
1
5
7
4
7
1
5
7
5
7
1
5
7
6
2
1
5
7
6
3
1
5
7
9
2
1
5
7
9
3
1
5
8
2
5
1
5
8
5
8
1
5
8
6
2
1
5
8
7
6
1
3
8
8
3
1
5
8
8
5
1
5
8
8
8
1
5
8
9
1
1
5
9
1
5
1
5
9
2
2
1
5
9
2
3 Frequency
G
A
.
A
.
A
.
A
. A
. A
. A
. A
. A
. .
. A
. A
. .
. A
. A
C A
. A
. A
. A
. A
. A
. A
. A
. .
. A
. A
. .
. A
A
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G
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G
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A
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A
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G
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C
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T
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T
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T
T
G
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T .
T .
. .
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. .
G
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A
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A
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A
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A
A
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A
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A
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G
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T
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C
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C
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C
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C
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C
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C
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T
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C
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G
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C
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T
T
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T
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.
T
T
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A
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C
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T
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C
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C
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C
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C
.
C
C
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C
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C
C
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C
C
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C
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.
T
.
T
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T
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T
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T
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T
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T
.
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T
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C
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C
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C
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.
C
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C
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C
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G
.
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A
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A
.
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A
.
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T
C
.
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C
.
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C
.
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.
C
.
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.
C
C
.
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.
.
T
.
.
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.
A
.
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.
.
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.
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.
.
G
A
.
.
.
.
.
A
A
A
A
A
.
.
.
A
.
A
A
A
.
A
A
.
A
A
A
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
T
.
.
.
.
T
T
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
G
.
.
.
.
.
.
0.24
0.01
0.06
0.01
0.01
0.03
0.01
0.01
0.01
0.01
0.01
0.03
0.05
0.01
0.01
0.01
0.02
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.02
0.01
0.01
0.01
0.01
0.01
2228 T . B O R G E E T A L .
Table 3 Variable positions of the mitochondrial haplotypes (BWS1-58) and their frequency in the data set of the Holocene bowhead whales from Svalbard. Only differences to haplotype
BWS1 are depicted. Nucleotide position corresponds to the complete bowhead whale mtDNA genome (Sasaki et al. 2005), GenBank accession number AP006472
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Table 3 Continued
Nucleotide position
1
5
4
8
8
1
5
4
9
0
1
5
5
0
9
1
5
5
5
7
1
5
5
5
8
1
5
5
5
9
1
5
5
6
7
1
5
5
6
8
1
5
5
7
1
1
5
5
7
4
1
5
5
8
2
1
5
5
8
4
1
5
5
8
6
1
5
5
9
0
1
5
5
9
7
1
5
6
0
7
1
5
6
1
3
1
5
6
1
4
1
5
6
1
6
1
5
6
2
2
1
5
6
9
4
1
5
6
9
9
1
5
7
2
2
1
5
7
2
6
1
5
7
2
7
1
5
7
3
0
1
5
7
3
3
1
5
7
3
6
1
5
7
3
7
1
5
7
3
8
1
5
7
4
3
1
5
7
4
4
1
5
7
4
7
1
5
7
5
7
1
5
7
6
2
1
5
7
6
3
1
5
7
9
2
1
5
7
9
3
1
5
8
2
5
1
5
8
5
8
1
5
8
6
2
1
5
8
7
6
1
3
8
8
3
1
5
8
8
5
1
5
8
8
8
1
5
8
9
1
1
5
9
1
5
1
5
9
2
2
1
5
9
2
3 Frequency
BWS33
BWS34
BWS35
BWS36
BWS37
BWS38
BWS39
BWS40
BWS41
BWS42
BWS43
BWS44
BWS45
BWS46
BWS47
BWS48
BWS49
BWS50
BWS51
BWS52
BWS53
BWS54
BWS55
BWS56
BWS57
BWS58
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
C
.
.
.
.
.
C
.
.
.
C
.
C
.
.
.
.
.
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C
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C
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G
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T
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A
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A
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T
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T
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.
A
A
A
.
A
A
A
.
.
A
A
A
.
.
A
A
.
A
.
A
A
A
A
.
A
.
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.
G
.
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.
G
G
.
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G
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G
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A
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G
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A
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A
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A
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A
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A
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T
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C
.
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C
C
C
.
.
C
C
C
.
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C
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C
.
C
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T
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T
T
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T
T
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T
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C
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T
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.
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T
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T
.
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T
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T
.
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A
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C
.
.
C
C
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.
C
C
C
C
.
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.
C
.
.
C
.
C
C
.
.
.
T
.
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0.01
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.02
0.01
G E N E T I C V A R I A T I O N I N B O W H E A D W H A L E S 2229
1
5
4
8
Haplotype 5
2230 T . B O R G E E T A L .
Fig. 2 Minimum spanning network (phylogram) of the mitochondrial haplotypes
of bowhead whales detected in the data
sets of the Holocene Spitsbergen (this study,
in red) and the extant BCB (Rooney et al.
2001; in black) stocks. For the haplotypes
detected in the Spitsbergen samples the
corresponding age classes (see Appendix)
are indicated (in roman letters). The scalebar
corresponds to one nucleotide substitution.
Table 4 Sequence variation and haplotype diversity of the mitochondrial control region (CR) of bowhead whales from the Holocene
Spitsbergen (this study) and the extant Bering-Chukchi-Beaufort stock (Rooney et al. 2001)
Nucleotide substitutions
Haplotypes
Stock
N (bp/individuals)
Total
Pop. specific
Shared
π (SD)
Total
Pop. specific
Shared
H (SD)
BCB
Spitsbergen
453/98
453/99
60
50
24
14
36
0.016 (0.0009)
0.013 (0.0010)
68
58
55
45
13
0.986 (0.005)
0.935 (0.020)
data set is not a representative survey of a bowhead whale
population, but rather a compilation of mitochondrial
haplotypes over a period of approximately 50 000 years.
Second, there is currently no information on the relation-
ships of individual samples available. Third, there is no
homogeneous sampling over time; age classes I, III, and IV
are less represented than age classes II and V. Thus, it cannot
be entirely ruled out that the haplotype frequencies changed
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
G E N E T I C V A R I A T I O N I N B O W H E A D W H A L E S 2231
over time within the Spitsbergen stock of bowhead whales.
However, when we compared the 25 youngest Svalbard
samples (14C ages ranging from 30 to 560 years) to the
extant BCB stock the genetic differentiation was not
significant (FST = 0.00253, P = 0.18018; KST = 0.0023).
The large number of shared segregating sites and the
similar haplotype structure of the historic Svalbard
samples and the extant BCB stock samples supports previously published conclusions (Rooney et al. 1999, 2001)
that a high level of genetic variation survived the bottleneck
caused by commercial whaling of the BCB stock.
To summarize, we do not believe that our data support
the hypothesis of genetic differentiation of the two stocks
due to isolation. In contrast, the similarity between Holocene
samples from the North Atlantic and extant samples from
the North Pacific, together with the insignificant temporal
changes in haplotype structure in the former, indicate
circumpolar contact between Pacific and Atlantic bowhead
stocks over time. Holocene changes in Arctic sea ice distribution, such as the formation of the M’Clintock Channel
sea-ice plug, have apparently not caused an observable
genetic differentiation of the eastern and western stocks
of bowheads. It is noteworthy that bowhead whales
have been observed throughout the entire Russian Arctic
(Belikov & Boltunov 2002). We therefore suggest that eastern and western stocks of bowheads might also have been
in contact along the Siberian coast. Therefore, our results
question the current designation of five distinct stocks of
bowhead whales in the Arctic.
Whether the few bowhead whales that are observed
in the area between northeast Greenland and Franz Josef
Land today (Reeves 1980; Jonsgård 1981, 1982; Wiig 1991;
de Korte & Belikov 1994; Gilg & Born 2005; Wigg et al., in press)
are descendents of the original Spitsbergen stock, or if they
are immigrants from east or from west is currently not known.
However, according to the available data, it is unlikely that
this question can be answered on the basis of mtDNA CR
sequences. Other data, such as, nuclear microsatellite data
and/or nucleotide sequences from more slowly evolving
parts of the mtDNA that overcome the shortcomings of the
hypervariable CR, will allow more sophisticated analyses
required to address such questions.
The current study illustrates the importance of museum
collections for studying past population structure of threatened species as has been pointed out by several authors
(e.g. Rosenbaum et al. 1997). In particular, collections of
permafrost remains provide a unique source of genetic
information about palaeoclimate events during the last
major climate change (Leonard et al. 2000). The presented
study on bowhead whales increases our knowledge of the
effects of climate change on population structure of Arctic
marine mammals in the past. Such information is an
important contribution for the evaluation of possible
effects of future climate change (Laidre et al. in press).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Acknowledgements
We are grateful to Otto Salvigsen for information on the bones he
has collected at Svalbard, to the Governor of Svalbard and to
Fridtjof Mehlum for collection of additional bones. We are thankful to Howard Rosenbaum and Lianne Postma for discussions and
input. Alejandro P. Rooney kindly provided the frequencies of the
nucleotide sequences of the Bering-Chukchi-Beaufort bowhead
whales. The work was funded by the Nordisk Ministerråd (grant
no. 661045–20201) and the Research Council of Norway (grant nos
153028/S40; 146515/420), and the Strategic University Program
‘National Centre for Biosystematics’ (grant no. 146515/420), cofunded
by the Research Council of Norway and the NHM, University of
Oslo.
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The following supplementary material is available for this article:
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/
10.1111/j.1365-294X.2007.03287.x
(This link will take you to the article abstract).
Fig. 1. A test for sequence homogeneity of PCR products
generated from template DNA extracted from ancient bowhead
whale bone samples. Alignment of mtDNA sequences from
cloned PCR products (Amplicon 1, primers are listed in Table 1)
Please note: Blackwell Publishing are not responsible for the
content or functionality of any supplementary materials supplied
by the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.
Supplementary material
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
2234 T . B O R G E E T A L .
Appendix
Details on the Holocene bowhead whale bone samples from Svalbard that were used in this study. All 14C data refer to bone collagen that
is well preserved in the thermal regime of Svalbard. 14C ages are corrected for fractionation of carbon isotopes by normalizing to a δ 13C of
−25‰. Age classes correspond to biogeographical epochs described for bowhead whales; for details see text. To account for the oceanic
reservoir effect, 440 years are subtracted from the 13C-corrected date (Dyke et al. 1996b).
Sample
Locality
Latitude
Identification
no.
Mitochondrial
haplotype*
Reference†
SD
51 000
42 385
40 300
1405
3000
T-17156
T-16573
T-17476
BWS18
BWS40
BWS54
1
1
1
11.783
13.817
12.000
11.783
11.667
15.500
13.767
11.850
15.983
15.983
28.150
13.767
14.100
11.783
22.167
11.500
14.317
11.500
13.000
22.000
14.150
13.767
15.833
16.017
11.000
14.533
15.833
13.500
14.317
13.967
14.100
14.100
15.217
22.167
10 965
10 160
10 140
9 980
9 950
9 855
9 850
9 830
9 795
9 775
9 760
9 710
9 660
9 645
9 640
9 630
9 620
9 620
9 610
9 585
9 570
9 500
9 495
9 455
9 435
9 400
9 380
9 380
9 365
9 355
9 150
9 070
8 825
8 565
115
130
110
140
140
155
135
110
135
90
145
55
110
180
140
110
105
70
110
65
95
130
130
135
135
120
75
130
125
110
110
100
105
110
T-17154
T-6291
T-6586
T-17124
T-6589
T-16768
T-16465
T-6583
T-16487
T-16486
T-16474
T-16466
T-17148
T-17123
T-2502
T-6581
T-17143
T-6585
T-2918
T-9913
T-17137
T-16464
T-9010
T-9013
T-17125
T- 1829
T-9004
T-17113
T-17142
T-17138
T-17146
T-17139
T-17118
T-17126
BWS6
BWS57
BWS56
BWS19 (OO)
BWS1 (F)
BWS50
BWS1 (F)
BWS35
BWS3 (BB)
BWS13
BWS48 (FFF)
BWS51 (FF)
BWS13
BWS37
BWS1 (F)
BWS55 (B)
BWS36
BWS3 (BB)
BWS3 (BB)
BWS52
BWS27 (R)
BWS58
BWS13
BWS1 (F)
BWS31
BWS17 (E)
BWS49
BWS32
BWS1 (F)
BWS34 (LLL)
BWS1 (F)
BWS33
BWS24
BWS1 (F)
1
2
2
1
2
3
1
2
1
1
1
1
1
1
7
2
1
2
9
6
1
1
4
2
1
5
2
1
1
1
1
1
1
1
14.250
26.517
18.683
29.267
15.483
18.783
14.117
14.117
14.250
15.583
14.000
14.000
22.167
8 475
7 955
7 785
7 640
7 380
6 580
6 180
6 145
5 995
5 860
5 640
5 475
5 020
65
115
115
110
110
105
55
60
85
85
95
85
80
T-16479
T-16449
T-17128
T-3731
T-17117
T-17129
T-17134
T-17133
T-16476
T-16450
T-17131
T-17135
T-17119
BWS38
BWS44 ( JJJ)
BWS17 (E)
BWS1 (F)
BWS25
BWS1 (F)
BWS23
BWS13
BWS41
BWS43
BWS1 (F)
BWS35
BWS28
1
1
1
8
1
1
1
1
1
1
1
1
1
Longitude
14C
age class I: > 10 500 bp (three samples)
1985–70
Brucebukta
78.450
1980–76
Hidalen
78.900
1985–43
Aavatsmarkbreen
78.667
11.783
28.150
12.000
age class II: 8500 –10 500 bp (34 samples)
1985–64‡
Brucebukta
78.450
1984–13
Vardeborgsletta
78.083
1985–37
Aavatsmarkbreen
78.667
1985–75
Brucebukta
78.450
1985–78
Loveenbreen
78.900
T1
Adventdalen
78.167
1983–28
Linnéelva
78.083
1985–16
Snipeodden
78.667
1989–36
Kvartsittrabben
76.800
1989–35
Kvartsittrabben
76.800
1980–73
Hidalen
78.900
1983–42
Linnéelva
78.083
1987–38
Fløysletta
77.400
1985–74
Brucebukta
78.450
1976–29
Svartknausflya
79.400
1985–07
Arthurbreen
78.667
1987–92
Blomlidalen
77.567
1985–36
Sarsbukta
78.667
1977–39
Vulkanhamna
79.333
1987–812
Kap Ziehen
78.500
1987–64
Dyrstadflya
77.567
1983–13
Linnéelva
78.083
1989–17(18)
Breinesflya
76.833
1989–40
Tørrflya
79.767
1985–77
Kvadehuken
78.833
1974–01
Calypsostranda
77.567
1989–19
Breinesflya
76.833
1977–54
Velkomstpynten
79.833
1987–91
Blomlidalen
77.567
1987–69
Buvika
77.483
1987–96
Lognedalsflya
77.517
1987–71
Lognedalsflya
77.517
1981–90
Kapp Smith
78.667
1976–27
Svartknausflya
79.400
age class III: 5000–8500 bp (13 samples)
1982–51
Ytterdalselva
77.800
1980–12
Arnesenodden
78.867
1982–14
Væringsdalen
78.100
1979–21
Tømmerneset
78.850
1981–89
Bolleneset
78.717
1982–35
Belemnitsletta
78.050
1987–17
Flyangen
77.367
1987–16
Flyangen
77.367
1982–52
Ytterdalselva
77.800
1989–49
Ømmervatns
76.867
1987–06
Storvika
77.167
1987–36
Kapp Klaveness
77.167
1976–12
Svartknausflya
79.400
age
14C
14C
14C
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
G E N E T I C V A R I A T I O N I N B O W H E A D W H A L E S 2235
Appendix Continued
Sample
Locality
Latitude
Longitude
14C
age
Identification
no.
Mitochondrial
haplotype*
Reference†
SD
age class IV: 3000– 5000 bp (11 samples)
1987–73
Lognedalsflya
77.517
1989–01
Gjeslingbekken
76.800
1983–09
Båtodden
77.967
1989–15
Breinesflya
76.833
1987–23
Flyangen
77.367
1989–47
Ømmervatns
76.867
1981–15
Gipsdalen
78.500
1977–22
Store Måkeøya
79.617
1981–22
Kapp Ekholm
78.583
1976–6B
Svartknausflya
79.400
11068
Storøya
80.000
14.100
15.967
13.633
15.833
14.117
15.583
16.867
13.500
16.550
22.167
28.000
4 865
4 695
4 610
4 595
4 170
4 165
4 005
3 725
3 700
3 200
3 100
55
75
95
75
80
75
95
75
75
70
50
T-17140
T-16575
T-16463
T-16576
T-17147
T-16583
T-17115
T-17112
T-17116
T-17120
T-16001
BWS29
BWS39
BWS45
BWS1 (F)
BWS6
BWS1 (F)
BWS16
BWS26
BWS1 (F)
BWS27 (R)
BWS10
1
1
1
1
1
1
1
1
1
1
1
age class V: 0–3000 bp (38 samples)
11070
Svenskøya
11072
Edgeøya
11057
Ækongen
11060
Kong Ludvigøyane
11069
Svenskøya
1985–25
Aavatsmarkbreen
1997–14
Mosselbukta
1978–07
Vårfluesjøen
1982–39
Belemnitsletta
11067
Kvitøya
1987–90
Blomlidalen
1989–43
Tjuvjotjørna
11058
Økongen
1987–94
Lognedalsflya
1978–55
Hytteberget
11073
Edgeøya
1985–52
Øyrnes
11074
Mohnbukta
1985–51
Øyrnes
11076
Hornsund
11064
Edgeøya
1985–49
Øyrnes
11059
Kong Ludvigøyane
11075
Hornsund
11065
Lurøya
11063
Edgeøya
1989–16
Breinesflya
11056
Ækongen
11071
Edgeøya
11062
Edgeøya
11061
Lurøya
1977–67
Andøyane
11066
Martensøya
1997–13
Polheim
1978–25
Gråhuksletta
1977–52
Velkomstpynten
1985–44
Sarstangen
T5
Mistakodden
26.500
22.500
21.417
21.000
26.500
12.000
15.500
14.333
18.783
33.000
14.317
15.567
21.417
14.100
20.850
22.500
11.917
18.917
11.917
15.833
22.500
11.917
21.000
15.833
21.917
22.500
15.833
21.417
22.500
22.500
21.917
13.000
21.217
16.050
14.667
13.500
11.500
20.000
1 700
1 625
1 540
1 520
1 445
1 315
1 170
980
965
955
805
765
605
560
555
540
430
415
405
395
395
385
370
360
325
325
290
280
275
215
210
205
190
180
160
160
100
30
75
80
75
60
75
55
70
75
45
60
70
65
70
40
75
40
60
90
55
75
65
35
45
60
90
45
35
35
70
90
70
70
75
65
70
40
60
1
T-16003
T-16005
T-15990
T-15993
T-16002
T-17122
T-17150
T-17114
T-17130
T16000
T-17141
T-16488
T-15991
T-17144
T-17157
T-16006
T-17155
T-16007
T-17153
T-16009
T-15997
T-17151
T-15992
T-16008
T-15998
T-15996
T-16577
T-15989
T-16004
T-15995
T-15994
T-16456
T-15999
T-17149
T-16468
T-16454
T-6588
recent
BWS3 (BB)
BWS1 (F)
BWS4 (EE)
BWS8
BWS14
BWS12 (HH)
BWS1 (F)
BWS12 (HH)
BWS21
BWS1 (F)
BWS1 (F)
BWS1 (F)
BWS1 (F)
BWS30
BWS22
BWS1 (F)
BWS19 (OO)
BWS11
BWS3 (BB)
BWS15
BWS2
BWS20
BWS6
BWS3 (BB)
BWS1 (F)
BWS12 (HH)
BWS42
BWS1 (F)
BWS5
BWS13
BWS9
BWS1 (F)
BWS7
BWS57
BWS46
BWS47
BWS1 (F)
BWS53 (W)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
14C
14C
78.750
77.667
77.283
77.167
78.750
78.667
79.833
79.667
78.050
80.167
77.567
76.900
77.283
77.517
80.683
77.667
78.633
78.317
78.633
76.983
77.667
78.633
77.167
76.983
77.000
77.667
76.833
77.283
77.667
77.667
77.000
79.667
80.683
79.883
79.750
79.833
78.667
78.333
*Haplotype assignment in parenthesis according to Rooney et al. (2001).
†1 — present study; 2 — Salvigsen unpublished; 3 — Lønne 2005);
4 — Salvigsen & Elgersma (1993); 5 — Salvigsen (1977); 6 — Bondevik et al. (1995); 7 — Salvigsen (1978); 8 — Salvigsen (1981); 9 — Salvigsen
& Österholm (1982).
‡Due to the substantial difference in 14C age compared to other age class I samples, this bone was assigned to age class II although it would
fall according to its 14C age formally into age class I.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd