MARINE MAMMAL SCIENCE, 25(1): 229–238 ( January 2009)
C 2008 by the Society for Marine Mammalogy
DOI: 10.1111/j.1748-7692.2008.00248.x
Molecular species identification of historical whale remains
from South Georgia
CHARLOTTE LINDQVIST
ANJA PROBST
Natural History Museum,
University of Oslo,
P. O. Box 1172, Blindern, N-0318 Oslo, Norway
E-mail: charlotte.lindqvist@nhm.uio.no
ANTHONY R. MARTIN
British Antarctic Survey,
High Cross, Madingley Road,
Cambridge CB3 OET, United Kingdom
ØYSTEIN WIIG
LUTZ BACHMANN
Natural History Museum,
University of Oslo,
P. O. Box 1172, Blindern, N-0318 Oslo, Norway
The island of South Georgia is located at the southern extreme of the South
Atlantic Ocean, on the edge of the Southern Ocean that surrounds Antarctica.
Intensive commercial whaling at South Georgia began in 1904, when the first
land-based whaling station was built in Grytviken (54◦ 17 S, 36◦ 30 W). Five other
shore stations were eventually built: Ocean Harbour (54◦ 20 S, 36◦ 16 W), Leith Harbour (54◦ 08 S, 36◦ 41 W), Husvik Harbour (54◦ 18 S, 36◦ 71 W), Stromness Harbour
(54◦ 90 S, 36◦ 41 W), and Prince Olav Harbour (54◦ 40 S, 36◦ 90 W). Another site,
Godthul (54◦ 17 S, 36◦ 17 W), was used as a protected anchorage for floating factories. By 1965, when shore-based whaling activity ceased, over 175,000 whales
had been processed on the island (Moore et al. 1999). The once abundant stocks of
baleen whales in the Antarctic had at that time been reduced to about a third of
their former sizes (Laws 1977). When considering blue (Balaenoptera musculus), fin (B.
physalus), sei (B. borealis), and humpback (Megaptera novaeangliae) whales together, the
average population size was reduced to ca. 18% (Laws 1977). Humpback and blue
whales experienced the most severe bottlenecks, having been reduced to about 3%
and 5% of the estimated initial populations, respectively. According to more recent
estimates, even 80%–95% of the pristine populations of humpback, blue, and fin
whales have been killed (Baker and Clapham 2002). For the blue whales depletion to
even less than 1% of the pre-exploitation population size has been reported (Branch
et al. 2004, 2007). Currently, knowledge about the recovery from the bottlenecks
and current population sizes, structures, and migration patterns are important issues
229
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MARINE MAMMAL SCIENCE, VOL. 25, NO. 1, 2009
in the conservation of Southern Hemisphere baleen whales. In this context, insight
into historical population structures would be of great value.
The processing of thousands of whales rendered an enormous number of bone
remains that were dumped in the vicinity of the whaling stations on South Georgia
or in the sea. Today, these bones may be a unique source for retrieving genetic
information that would aid the study of the genetic structure of historical whale
populations prior to human exploitation. Examining the genetic variation of preand postwhaling populations of different Southern Hemisphere whale species could
offer an opportunity to elucidate the differences in their respective exploitation
histories as well as the effects of migration patterns and postwhaling recovery on the
levels and distribution of genetic variation.
Extraction of nucleic acids from historical samples has become relatively commonplace, and a number of recent studies have applied the ancient DNA methodology
for the genetic analyses of historical whale remains (e.g., Rosenbaum et al. 1997;
Pichler et al. 2001; Dalebout et al. 2003, 2004; Rastogi et al. 2004; Borge et al.
2007). However, the success rates remain unpredictable depending on the particular
environmental conditions to which a sample was exposed. Since DNA and organic
molecules are expected to survive longer in colder environments (Willerslev et al.
2004), the constant low temperatures on and around South Georgia may have been
more favorable for DNA survival (Smith et al. 2001, Lambert et al. 2002) than
is the case for, e.g., museum specimens. Maternally inherited mitochondrial DNA
(mtDNA) has been the most frequently applied genetic marker in phylogenetic and
population genetic studies of historical animal samples. It has been argued that its
relatively high copy number increases the likelihood for extracting suitable template
DNA (Handt et al. 1994). Moreover, due to the continuous increase of DNA sequence
data and widespread and continuing use of mtDNA sequence data in cetacean studies
as well, particularly of mitochondrial cytochrome oxidase I (cox I), cytochrome b (cyt
b), and control region (CR) sequences as well as complete mtDNA genomes, mtDNA
markers are highly suited for molecular diagnostics studies.
In the current study we present a proof-of-concept approach to molecular identification of historical whale remains from South Georgian whaling stations. We
attempted to extract DNA from 44 whalebones and dried muscle samples collected
at four localities on South Georgia (Table 1). Bone samples were all collected from
shore sites, and most have been exposed to sunlight, although over the years some
samples may have been buried temporarily under stones or other bones. Air-dried
meat samples were collected from a single drying frame. During the experiments,
all background information on the samples was kept blind to the experimenters.
Because the bone samples were collected randomly from beaches around several
whaling stations, and bones must have been mixed up after up to 100 years of ice,
wind, and waves moving them around on the boulder pavements it seems very unlikely that different bone fragments belong to the same individual. However, in the
case of the dried muscle tissue it is possible that the multiple samples were derived
from the same individual. In any case, redundant sampling does not pose a problem
in the current study because the aim was molecular species identification not genetic
Sample
ID
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
G14
G15
G16
G17
G18
G19
G20
G21
L1
L2
L3
L4
L5
Type of
materiala
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
DB
MDB
DB
MDB
DB
MDB
MDB
MDB
DB
DB
DB
A
x
x
x
x
x
x
–
x
x
–
x
x
x
x
x
–
x
x
x
x
x
x
x
–
–
–
B
x
x
x
x
x
x
–
–
–
–
–
–
–
–
–
–
x
x
x
x
x
–
x
–
–
–
Amplificationb
Haplotypec
G1
G1
G3
G4
G1
G6
–
G8A
G8A
–
G8A
G8A
G13A
G8A
G8A
–
G4
G1
G1
G1
G1
L1A
L2
–
–
–
Species
annotationd MP-analysis
Humpback
Humpback
Fin
Humpback
Humpback
Humpback
–
Humpback
Humpback
–
Humpback
Humpback
Fin
Humpback
Humpback
–
Humpback
Humpback
Humpback
Humpback
Humpback
Sei
Fin
–
–
–
Table 1. Samples used in the present study and positive species identifications.
NOTES
231
GenBank
accession no.
EU831245
EU831246
EU831262
EU831243
EU831247
EU831242
–
EU831248
EU831249
–
EU831250
EU831251
EU831263
EU831252
EU831253
–
EU831244
EU831254
EU831255
EU831256
EU831257
EU831266
EU831264
–
–
–
(Continued)
Type of
materiala
MDB
MDB
DB
MDB
M
M
M
M
M
M
M
DB
DB
DB
DB
MDB
MDB
MDB
A
–
–
–
x
x
x
x
x
x
–
x
x
–
x
x
x
–
x
B
–
–
–
–
–
–
–
x
x
–
–
x
–
–
–
–
–
–
Amplificationb
Haplotypec
–
–
–
L9A
L9A
L9A
L9A
M4
M4
–
L9A
G1
–
G8A
G13A
G8A
–
G8A
Species
annotationd MP-analysis
–
–
–
Sei
Sei
Sei
Sei
Sei
Sei
–
Sei
Humpback
–
Humpback
Fin
Humpback
–
Humpback
GenBank
accession no.
–
–
–
EU831267
EU831268
EU831269
EU831270
EU831271
EU831272
–
EU831273
EU831258
–
EU831259
EU831265
EU831260
–
EU831261
a
DB, dense bones such as bullae and similar material from the head; MDB, medium dense bones such as the outermost part of long bones; M, air
dried meat.
b
Fragment A was amplified with the “A2dir” and “A1rev” primers, and fragment B with the “B1dir” and “B1rev” primers (see Table 2).
c
Assigned only for the purpose of this study.
d
“Witness for the Whales” web identification tool (http://www.cebl.auckland.ac.nz:9000/page/whales/title; Ross et al. 2003) assigned the
submitted sequences to the very same species as the presented maximum parsimony analysis.
Sample
ID
L6
L7
L8
L9
M1
M2
M3
M4
M5
M6
M7
OH1
OH2
OH3
U1
U2
U3
U4
Table 1 (Continued)
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MARINE MAMMAL SCIENCE, VOL. 25, NO. 1, 2009
NOTES
233
differentiation of individuals or population genetic estimates such as, e.g., nucleotide
or haplotype diversity.
For the purpose of rapidly identifying any historical baleen whale sample with
reasonable confidence, we designed a robust assay that targets two short stretches
(180 base pairs and 131 base pairs) of the mitochondrial cyt b gene. Successful
PCR amplification and subsequent sequencing of the PCR products are in most
instances expected to permit species determination from samples with reasonable
DNA preservation. Previous studies have shown that short fragments of the cyt b
gene have a very high taxonomic power for identification to species of whale samples
(Dalebout et al. 2004 and references therein). The proper initial species identification
of historical bone samples is particularly important for selecting appropriate primers
for downstream population-level analyses that target more variable genetic markers
such as, e.g., microsatellite loci and/or the mitochondrial CR. In such downstream
analyses, more variable markers may be used to determine genetic diversity and
haplotype frequencies of modern vs. historical populations. Furthermore, such data
may also provide estimates of the historical population size (Roman and Palumbi
2003, Alter et al. 2007).
When working with historical samples, stringent DNA protocols are particularly
important (Cooper and Poinar 2000). Due to the small amounts and degraded nature
of recovered ancient DNA, controls for contamination require particular attention.
Contamination can occur at any stage in the processing of ancient bone DNA,
but it is particularly prevalent during sample preparation when the bone material is
ground into a powder fine enough for the extraction of DNA. In this study, chopping
and grinding of bone material was conducted in a different building physically
isolated from the DNA facility. Furthermore, in the DNA laboratory, no analyses of
any modern whale material of the species in question had ever taken place. All DNA
extractions and PCR reactions were set up in hoods equipped with UV-light in a
facility well separated from the labs where subsequent purifications and sequencing
of the PCR products were performed.
Small samples of bone material were chopped off with a hammer and chisel. The
bone fragments and the dried muscle samples were then ground into fine powder in
a mortar under liquid nitrogen. Mortars and pestles were cleaned with 5% deconex,
rinsed with distilled water and 70% EtOH, wrapped in aluminum foil and baked
at ca. 120◦ C overnight in order to avoid carry-over contamination between samples.
Approximately 0.1 g of bone powder was subsequently transferred into 2.0 mL screwcapped centrifuge tubes, and DNA was extracted according to Borge et al. (2007).
DNA sequences of the cyt b gene were aligned for several cetacean species retrieved
from the NCBI database in order to identify conserved areas. Based on sequences from
fin whale (GenBank acc. no. X61145; Arnason et al. 1991) and sei whale (GenBank
acc. no. X75582; Arnason and Gullberg 1994) primers were designed in conserved
areas in the 5 -end of the cyt b gene using the free online software Primer3 (Rozen
and Skaletsky 2000). Because DNA from historical samples can be highly degraded
it is necessary to design primers to amplify relatively shorter fragments than otherwise is the case. Two sets of primers were designed (Table 2): the primers “A2dir”
and “A1rev” amplified a fragment A of 180 base pairs (bp), whereas the “B1dir” and
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MARINE MAMMAL SCIENCE, VOL. 25, NO. 1, 2009
Table 2. Cytochrome b primers designed and used for species identification.
Amplicon
Primer
Positiona
Sequence (5 –3 )
A
A2dir
A1rev
B1dir
B1rev
3–25
161–182
161–182
270–291
GACCAACATCCGAAAAACACACC
GTTGTTGTGTCTGGTGTGTAGT
ACTACACACCAGACACAACAAC
GTGGGCGTAGAGGCAGATGAAG
B
a
Nucleotide positions correspond to the complete cytochrome b sequence from Balaenoptera
physalus (X61145) and B. borealis (X75582).
Figure 1. Phylogenetic analysis of 28 cytochrome b sequences, including 10 partial sequences determined for historical bone samples from South Georgia (marked in bold). Strict
consensus tree of three most-parsimonious trees with bootstrap values above 50% shown
below branches. GenBank accession numbers are shown in parenthesis after taxon name of
the sequences retrieved from the NCBI database.
“B1rev” primers amplified an adjacent fragment B of 131 bp, providing a total of 289
bp when both fragments were amplified and sequenced. Primers A1rev and B1dir are
reverse complements of each other and the contiguous 289 bp consisting of fragments
A and B are therefore invariant for 67 bp, i.e., 222 bp can be informative for species
identification, 135 bp for fragment A and 87 bp for fragment B. As expected (Dalebout et al. 2004) and illustrated in Figure 1, the targeted 222 bp of the cyt b gene
NOTES
235
provide already a high diagnostic power for species identification. PCR amplifications
were performed in 25 ␮L reaction volumes using 1U PfuTurbo DNA polymerase in
1× Reaction Buffer (Stratagene, La Jolla, CA, USA), 0.2 mmol/L of each dNTP,
0.04% bovine serum albumen (BSA), 1 ␮mol/L of each primer, and 2 ␮L of unquantified genomic template DNA. Thermal cycling conditions were optimized using
different annealing temperatures in the range 48◦ –58◦ C and the following protocol
yielded the highest number of successful amplifications for both fragments: 94◦ C
for 2 min; 35 cycles of 94◦ C for 50 s, 54◦ C for 50 s, and 72◦ C for 1 min; and a
final extension at 72◦ C for 10 min. PCR products were purified by incubation at
37◦ C for 30 min with 8 ␮L of 1:10 strength diluted exoSAP-IT (USB Corporation,
Cleveland, OH, USA) per reaction. Cycle sequencing of the purified PCR products, using the same primers as in the PCR reaction, was performed in 10 ␮L
reactions using 1 ␮L BigDye Terminator Cycle Sequencing Ready Reaction
Kit V 1.1 (Applied Biosystems), 3 ␮L 5× Sequencing Buffer, 10 pmol
primer, and 3 ␮L cleaned PCR product. Sequencing products were purified with
ethanol precipitation and analyzed using an ABI 3100 Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA). Forward and reverse sequences were assembled
and edited using Sequencher version 4.1.4 (GeneCodes, Ann Arbor, MI, USA).
Of the 44 samples, 32 samples (72.7%) were successfully amplified and sequenced
for fragment A. There was no difference in the success rate depending on the type of
material (see Table 1). Of these, 15 samples (34.1%) also gave positive amplification
and sequences for fragment B (Table 1). Thus, the primer pair targeting fragment A
(180 bp) was more suitable for degraded DNA templates than that targeting fragment
B (131 bp), which is in line with the results of the Primer3 (Rozen and Skaletsky
2000) search for suitable primer pairs. BLAST sequence similarity searches (Altschul
et al. 1990, Zhang et al. 2000) of the retrieved sequences against the nonredundant (nr)
NCBI database (http://www.ncbi.nlm.nih.gov) using the default settings confirmed
that all sequences represented baleen whales. In order to determine the species
identity of the South Georgia whale remains, a large number of cyt b sequences of
cetaceans representing a broad taxonomic range of taxa within the order were retrieved
from the NCBI database, including all baleen whale species found in Antarctic
waters. These baleen reference sequences, two odontocete sequences, Orcinus orca and
Physeter macrocephalus, which are also found in Antarctic water, and haplotypes for the
sequences generated in this study (see Table 1) were unambiguously aligned manually
using the software package BioEdit (Hall 1999). The two odontocete sequences
were used as the outgroup. The data matrix consisting of a total of 28 sequences
(18 from GenBank and 10 haplotypes determined in this study) was subjected to
parsimony analyses using the program TNT (Goloboff et al. 2003) and the traditional
search option with default settings (Wagner tree with 100 random replications
and saving 10 trees per replication). Additional tree bisection reconnection (TBR)
branch swapping was performed on trees resulting from the initial search to find
additional equally parsimonious trees. A strict consensus tree was calculated from
the three most-parsimonious trees found. To estimate support for internal branches,
parsimony bootstrapping was performed in TNT, using 1000 bootstrap replicates,
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MARINE MAMMAL SCIENCE, VOL. 25, NO. 1, 2009
each performing TBR branch swapping with 10 random entry orders and saving
1 tree per replicate.
The 32 historical samples were all successfully identified taxonomically with
reasonable confidence (bootstrap support > 80, following, e.g., Dalebout et al. 2004).
The samples were identified as derived from fin whales, humpback whales, or from
a member of the sei whale/Bryde’s whale (Balaenoptera brydei)/pygmy Bryde’s whale
(B. edeni) clade (Fig. 1, Table 1). No blue whale samples were detected, which is
not surprising since blue whales were mostly hunted after the stations were forced
to process the whole carcass, including use of the bones for extraction of oil, hence
leaving no bones at the site. The majority of the samples (four haplotypes) were
classified as humpback whales. Four samples (three haplotypes) were identified as fin
whales, and the remaining eight samples (three haplotypes) grouped in a strongly
supported clade including two reference sequences from sei whales as well as one
each from Brydes and pygmy Bryde’s whale. Trimming the sequences retrieved from
GenBank to the same length as the query sequences (i.e., 180 bp) yielded the same
species identification for the unknown samples. Recent genetic studies have indicated
some taxonomic uncertainties relating to the sei, Bryde’s, and pygmy Bryde’s whale
species complex (Wada and Numachi 1991, Yoshida and Kato 1999). Combining
SINE insertion and mitochondrial sequence data led Sasaki et al. (2005, 2006) and
Nikaido et al. (2006) to conclude that Bryde’s and pygmy Bryde’s whales constitute
a sister taxon to sei whale. Because Bryde’s and pygmy Bryde’s whales have not been
reported in Antarctic waters, the eight included samples L1, L9, M1–5 and M7 were
considered as representing sei whale.
To further test the validity of the approach presented here, the obtained cyt b
sequences were submitted to the web-based species identification tool “Witness
for the Whales” (http://www.cebl.auckland.ac.nz:9000/page/whales/title). The tool
offers species identification for submitted nucleotide sequences of the mitochondrial
cyt b and CR through maximum likelihood analysis (Ross et al. 2003) using a
purpose-compiled, specialist-curated database that includes multiple representatives
for the majority of recognized cetacean species. Using both the simple and advanced
search modes, the web tool assigned the submitted sequences to the very same species
as in the maximum parsimony analysis presented here (Table 1).
To summarize, in the present study we demonstrated that sufficient native DNA
suitable for PCR amplifications of short mtDNA fragments (at least 180 bp) can
be extracted from a large number of historical whale samples from South Georgia.
Amplification and sequencing of two stretches of the mitochondrial cyt b gene offers
an easy and rapid approach to identifying samples suitable for further genetic analyses
and to determine the species status of whale remains with reasonable confidence. At
least for humpback, fin, and sei whales, South Georgia provides suitable material for
studying historical population structures.
ACKNOWLEDGMENTS
We are grateful for funding from the Natural History Museum, University of Oslo and the
British Antarctic Survey. We thank Victor A. Albert for helpful comments. The bone samples
NOTES
237
were collected under permit from the Government of South Georgia and the South Sandwich
Islands to ARM.
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Received: 23 February 2008
Accepted: 22 July 2008