Molecular Ecology 1996,5,671-685

Molecular genetic identification of whale and dolphin

products from commercial markets in Korea and Japan
C. S. B A K E R , F. CIPRIANO’ and S. R. PALUMBI*
School of Biological Science, University of Auckland, Private Bag 92019, Auckland, New Zealand and *Kewalo Marine Laboratory,
University of Hawaii,41 Ahui Street, Honolulu, HI 96813 U S A

Abstract
We report the methods and results of molecular genetic identification of the species and,
in some cases, geographical origins of whale and dolphin products purchased from retail
markets and restaurants in Japan and South Korea. As reported previously (Baker &
Palumbi 1994),we used the polymerase chain reaction (PCR) and a portable laboratory to
amplify, puriify and later sequence a portion of the mitochondrial DNA control region
from 16 commercial products purchased in Japan. This ’spot check’ revealed a surprising
variety of species for sale, including minke, fin and humpback whales and one or two
species of dolphins sold as ‘kujira’ or whale. In the Korean survey, DNA amplifications
were conducted by two of us (C.S.B. and F.C.) working with independent equipment and
reagents. The two sets of DNA amplifications were returned to our respective laborato-
ries and sequenced independently for cross-validation. Among the total of 17 species-spe-
cific sequences we found a dolphin, a beaked whale, 13 Northern Hemisphere minke
whales (representing at least seven distinct individuals) and two whales which are close-
ly related to the recognized sei and Bryde’s whales but could not be identified as either
using available type sequences. We suggest that these two specimens represent a cur-
rently unrecognized species or subspecies of Bryde’s whale, possibly the so-called ’small-
form’ reported from the tropical waters of the Indo-Pacific.
We conclude that molecular systematic analyses of DNA sequences have tremendous
utility for the identification of whale and dolphin products. However, there are certain
constraints on the application of these techniques for monitoring whaling or trade in
whale products. First, PCR and DNA sequencing can generate misleading artefacts. These
can generally be recognized or eliminated through experimental controls. Second, phylo-
genetic reconstructions of DNA sequences can be misinterpreted if the database of type
sequences is inadequate or the taxonomy of the group is incomplete. This constraint is, at
present, a more serious obstacle to molecular monitoring of whaling. Our results high-
light uncertainties about the taxonomic status of oceanic populations and morphological
forms of two species (or species complexes) targeted by legal and illegal hunting, the
minke and Bryde’s whales. Despite these uncertainties, it is difficult to reconcile some of
the species available in Japanese and Korean commercial markets with recent catch
records made available to the International Whaling Commission. It is particularly dis-
turbing that two specimens of an unrecognized species or subspecies of baleen whale
were for sale in a restaurant in South Korea in October, 1994,8 years after the acceptance.
of an international moratorium on commercial whaling.

Keywords: CITES, forensics, IWC, mitochondria1 DNA, phylogenetic, whaling

Received 31 October 1995; revision received 24 April 19Y6; accepted 15 M a y 1396

Correspondence: Tel.: +61-9-373-7999;Fax.: 64-9-373-7417; e-mail cs. baker @ aukland. ac.nz

@ 19% Blackwell Science Ltd

672 C . S . B A K E R , F. C l P R l A N O AND S.R. PALUMBl
Introduction
For species protected by international regulations or
threatened by overexploitation, molecular genetics pro-
vides a powerful tool for conservation - the forensic iden-
tification of commercial products and verification of trade
records. Such products include ivory, horn, shell, meat,
internal organs, feathers, dried leaves and a host of other
tissues derived from animals or plants (e.g. Baskin 1991;
Milner-Gulland 8.1 Mace 1991). Although these products
may be impossible to classify on the basis of appearance,
they often contain DNA that can be amplified by the poly-
merase chain reaction (PCR) and compared to known or
'type' sampies. Research efforts to isolate DNA from
ancient tissues have led to a variety of methods for obtain-
ing genetic information from poorly preserved tissue
(Paabo 1988; Cooper rf ul. 1992; Janczewski et ul. 1992).By
targeting short sections of highly variable DNA (e.g. the
vertebrate mitochondrial DNA control region), the proba-
ble geographical origins of a specimen can be identified if
a sufficient collection of regional samples is available for
that species. Statistical methods developed for phyloge-
netic analyses can then be used to evaluate the reliability
of an identification to species or geographical region.
Previously, we used PCR to amplify and sequence the
mitochondria1 (mt) DNA control region from products of
whales and dolphins purchased in retail markets of Japan
(Baker Q Palumbi 1994). We showed that these sequences
could provide important independent evidence about the
species for sale in commercial outlets. Overall, OUT molec-
ular genetic approach has been motivated by the need to
monitor the hunting and international trade of these pro-
tected species. Are the available products for sale in com-
mercial markets derived exclusively from species hunted
legally under international agreements? Alternatively,
does the low level of scientific whaling that has persisted
since the 1986 moratorium on commercial hunting serve
as a cover for the sale of illegal whale products?
These questions are of particular relevance to whales
because a number of closely related species (e.g. the
Balaenopteridae) have been hunted to near-extinction. The
meat, skin and blubber of these species are difficult to
identify on the basis of morphological criteria alone. h
addition, the hunting of whales is managed by 'stocks'
(regional populations) which may be discriminated only
by genetic differences. Finally, products of protected or
endangered species are often processed in ways that make
visual identification difficult. Whale meat, for example, is
often smoked,dried, marinated or canned prior to sale. It
is also possible that products from other species are mis-
represented as whale meat. When sold as whale meat, a
dolphin represents a valuable commodity, worth up to
U S $ W on the wholesale markets of Korea (Anon. 1994a).
Such an economic gain is not likely to go unnoticed by
local fisherman who could hunt dolphins, porpoises and
other toothed whales opportunistically, or sell carcasses of
these animals taken as fisheries by-catch.
Our initial survey of Japanese markets represented
only a single 'spot check' of a small number of outlets at
one time of the year. We could not determine if the taxo-
nomic diversity of this survey was representative of a gen-
eral pattern or if similar results could be obtained in other
markets or at other seasons. We now report the results of
a second survey of whale and dolphin product from the
retail markets of Korea in 1994, along with details of the
methods and results of our initial survey of Japanese mar-
kets in 1993 (Baker & Palumbi 1994).In the Korean survey
we conducted independent analyses of the same samples,
using separate equipment and reagents from laboratories
in New Zealand and Hawaii, in order to cross-validate our
methods. Although the species identity and geographical
origin of products differed in Japan and Korea, the major
conclusions are similar - there is a surprising diversity of
whale and dolphin species currently for sale in commer-
cial markets. Some of the species are not consistent with
catch records reported to the International Whaling
Commission since the 1986 moratorium.
Methods
Snmple prepnrntion nnd analysis
Commercial products from whales and dolphins were col-
lected during two surveys of commercial markets in Asia.
In the first survey, commercial whale products were pur-
chased throughout the main island of Japan from February
to April, 1993, by agents under the direction of the conser-
vation organization Earthtrust (Table 1). The products
ranged in quality from dried and salted strips of meat,
marinated in sesame oil and soy sauce, to unfrozen sliced
meat sold for 'sashimi'. The products were subdivided by
field agenls and stored in 70% ethanol for several months
prior to genetic analysis. In the second survey, commercial
whale products were purchased in regions of South Korea
during September and October, 1994, by agents under the
direction of the Endangered Species Project and
Earthtrust. These products included fresh or frozen skin,
meat or blubber and one sample of partially cooked meat,
and were stored frozen for several weeks prior to genetic
analysis.
In order to comply fully with existing restrictions on
importation and exportation of whale products for scien-
tific research (CITES 1973), we conducted all extractions of
DNA from whale tissue and subsequent PCR amplifica-
tions on location, using a portable laboratory (Baker Q
Palumbi 1994). The amplified DNA from each product
was isolated from all true whale DNA or tissue and could
then be transported internationally without a permit
@ 1996 Blackwell Science Ltd, Molecular Ecology, 5, 671-685
 MOLECULAR GENETIC I N D E N T I F I C A T I O N OF CETACEAN PRODUCTS 673

Table 1 Details of purchase and genetic analysis of whale products from Japanese and Korean retail markets

Product Sequence Bootstrap value

Sample‘ description Date length Closest type sequence to species (family)

Japan
I53 red meat, frozen Mar. 1993 307 fin whale 100(100)
Js6 ‘sashimi’ Mar. 1993 353 southern hemisphere minke lOO(100)
IS9 bacon Mar. 1993 380 southem hemisphere minke 100(100)
JSl1 ‘nabe’, frozen Mar. 1993 377 fin whale loo(100)
JS13 dried, marinated Mar. 1993 378 Commerson’s dolphin 58(92)
JS15 dried, marinated Mar. 1993 313 southern hemisphere minke lOO(1OO)
JS16 bacon, frozen, ’kujira kun’ Mar. 1993 223 sperm whale/ harbor porpoise (71/ 65)
JSlS ’sashimi’, frozen Mar. 1993 310 northern hemisphere minke 98(1OO)
JS19 marinated steak strips Mar. 1993 (a) 168 a) southern hemisphere minke
(b)160 @) North Pacific humpback whale
1528 salted meat, ’shio kujira’ Apr. 1993 292 Commerson’s dolphin
JS29 salted blubber / skin Apr. 1993 352 southern hemisphere minke
1530 smoked bacon Apr. 1993 314 southern hemisphere minke
IS36 blubber and skin Apr. 1993 353 southern hemisphere minke
JS41 blubber and skin Apr, 1993 376 fin whale
JSWS3 lean meat Feb. 1993 275 southern hemisphere minke
JSWS4 lean meat Mar. 1993 215 fin whale

Korea
KN1 (a) skin Sept. 1994 (aW - (a) beaked whales (a)91(99)
(b) meat @W2 (b) sei and Bryde’s whales @)W96)
KN2 meat Sept. 1994 337 northern hemisphere minke 100(96)
KN3 meat Sept. 1994 457 sei and Bryde‘s whales lOO(96)
KN4 meat Sept. 1994 442 northern hemisphere minke 100196)
KN5 meat Sept. 1994 221 northern hemisphere minke lOO(96)
KN6 meat Sept. 1994 349 northern hemisphere minke loO(96)
KN7 meat Sept. 1991 256 northern hemisphere minke 100(96)
KN8 meat Sept. 1994 346 northern hemisphere minke loO(96)
KN9 (a) skin Sept. 1994 (a) dolphin, Genus Lagenorhynchus (a)93(93)
(b) meat (bYa @) northern hemisphere minke @)1OO(%)
KNlO meat Sept. 1994 356 northern hemisphere minke lOO(96)
KNll meat Sept. 1994 345 northern hemisphere minke lOO(96)
KNl2 meat Sept. 1994 417 northern hemisphere minke lOO(96)
KN13 meat Sept. 1991 407 northern hemisphere minke lOO(96)
KN14 meat Sept. 1994 375 northern hemisphere minke lOO(96)
KN15 meat Sept. 1994 403 northem hemisphere minke lOO(96)

* Product descriptions based on appearance or approximate translations of retail packaging. Sequence length is given in consecutive
base pairs of the mitochondria1 DNA control region. Closest type sequence and value of bootstrap association for species and family
level based on neighbour joining analysis.

(Jones 1994) for detailed laboratory analyses such as direct
sequencing.
Approximately 4 mg of tissue from each whale product
was prepared for PCR amplification using one of three
methods: cycles of heating and cooling with Genereleaser
(Bioventures, Inc.) according to manufacturer’s recom-
mendations; digestion with Pre-Tuq proteinase (Life
Technologies, Inc.) for 30 min at 94 “C in 200 pL of ddH,O
and PCRII buffer (Perkin-Elmer); or heating to 94 “C in a
5% solution of Chelex resin (BioRad Laboratories) as
described by Walsh et nl. (Walsh et nl. 1991). Amplification
of a 550-base pair (bp) fragment of the mtDNA control
region by PCR was conducted in a portable thermal cycler
(MJ Research, Inc.) using standard protocols (Saiki et al.
1988; Palumbi et nl. 1991) and the oligonucleotide primers,
light-strand tPro (5’-CTACCTCCAACTCCCAAAC-3’)
and heavy-strand DIp5 (5’-CCATCGWGATGTClTATT-
TAAGRGGAA-3’).We focused on the control region of the
mtDNA because of its high species- and population-spe-
cific variability (e.g. Arnason et al. 1993; Baker et al. 1993;
Brown et al. 1986). A 500-bp fragment of the mtDNA
cytochrome b gene (using the tGludg and Cyb2 primers,

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674 C.S. BAKER, F . C l P R I A N O AND S.R. PALUMBI
Palumtri et a / . 1991) was also amplified from most prod-
ucts as an additional source of markers for confirmation of
species identity. These data are not presented here.
Following amplification, 5-10 pL of the double-strand-
ed (ds) DNA product was run on a 1.6% agarose/TBE gel
and the resulting target band was excised to separate it
from genomic DNA, The remainder of the dsDNA was
bound to streptavidin-coated, para-magnetic beads
(Dynal Corp.) via a biotin group attached to the 5' end of
the Dlp5 primer. The bead-conjugated DNA was then
summoned with the aid of a magnet and washed to
remove unincorporated primer, nucleotides, and all tem-
plate whale DNA (Hultman et RI. 1989). The synthetic
DNA isoiated by these procedures was then returned to
our laboratories for solid-phase sequencing following
standard protocols (Palumbi et al. 1991; Baker et al. 1993;
Baker & Palumbi 1994). In most cases, the target region
was re-amplified from the excised band or the solid-phase
attached DNA using internal primers (light strand Dlp 10,
5'-CCACAGTACTATGTCCGTAIT-Y; or heavy strand
Dlp 4, S-GCGGGWRY'ITGRTITCACG-3'). When used
with its complement from the original primer pair (Dlp 1.5
-
or 5), each of the internal primers generated a fragment
450 bp in length. These indented fragments were then
resequenced to confirm the results of initial sequencing
reactions.
The field amplifications of commeraal products from
Japan in 1993 were conducted by one of us (C.S.B.) using
a single portable laboratory. To verify the consistency of
our field methods, we conducted a modified double-blind
experiment during our analysis of Korean whale products
in 1994. Two of us (C.S.B. and F.C.) assembled indepen-
dent field laboratories, including minicyclers, plasticware,
micropipettes and reagents, for travel to Korea. In a field
laboratory, all commercial whale products were subject to
one or more independent quick-preparation DNA extrac-
tions and PCR amplification. We individually purified
copied DNA from template DNA from our independent
amplifications,and returned to our respective laboratories
for sequencing. lnitial sequence analyses were conducted
independently by C.S.B.and F.C., including preliminary
alignment to a catalogue of type sequences for species
identification. The agreement of species identity and the
consistency of sequences were then evaluated by S.RP.
before the data sets were emr-checked, merged and re-
aligned to sequences in the type sequence database.
Sequence identification analysis
Identification of test sequences was based on phylogenet-
ic retonstruction methods using parsimony as implement-
ed in the computer program P A U P (Swofford 1993) and
the neighbur-joining genetic distance algorithms as
implemented in the computer program M E G A (Kumar et
al. 1993). Test and type sequences were aligned initially
using the program PILEUP,available in the GCG package
(Deveraux et al. 1984), with an initial gap penalty of 2 and
extension penalty of 0.3. The multiple sequence files were
then corrected for minor alignment inconsistenciesby eye.
For the neighbour-joining method, adjustments for multi-
ple substitutions were made using the Kimura Z-parame-
ter distance as implemented by M E G A (Kumar ef al. 1993).
Unless otherwise noted, all differences between sequence
are reported with the Kimura 2-parameter correction.
The statistical significance of test and type sequence
groupings (a measure of the confidence in an identification
of a test sequence) was estimated by bootstrap resampling
of the sequence data using both parsimony and distance
methods. For the neighbour-joininganalysis, an initial rela-
tionship tree was constructed using representative type
sequences from all available species (type sequences of
some geographical representatives were omitted to fadli-
tate computation). Test sequences were then analysed indi-
vidually to establish their placement into either of the two
suborders of cetaceans, Mysticeti or Odontoceti. Based on
this initial placement, bootstrap analyses were conducted
with all test and type sequences from each suborder to esti-
mate the confidence in the identification of the test
sequences to species or geographical region. Four test sam-
ples, represented by short sequences US19a, JS19b, JS16and
K4-1 'meat'), were added one at a time to the type
sequences, rather than as a group, and bootstraps were per-
formed only on the consensus region of the partial
sequences. All neighbour-joining phylogeny reconstruc-
tions were tested with lo00 bootstrap resamplings.
For the parsimony analysis we used methods described
by Baker & Palumbi (1994)as modified by one of us (F.C.).
An initial relationship tree was constructed using only
type sequences. Each test sequence was then added indi-
vidually to the complete set of type sequences and its
placement was tested by a bootstrap. Test sequences asso-
dated at high confidence (> 70%) with the same type
sequences were bootstrapped together to establish the
confidence level for groups of test sequences.
Relationships among all type sequences were established
with Wreplicate bootstrap resamplings and heuristic
searches. Identifications of test sequences were tested with
200 bootstrap resamplings for each individual' and for
groups of similar individuals. All parsimony and neigh-
bur-joining reconstructions agreed in the details relevant
to the identification of test products.
Results
Phylogenetic relationships of type sequcnces
Type sequences of the mtDNA control region were avail-
able from 33 individuals representing regional popula-
@ 1996 Blackwell Science Ltd, Molecular Ecology, 5, 671-685
 M O L E C U L A R G E N E T I C I N D E N T I F I C A T I O N OF C E T A C E A N P R O D U C T S 675

Table 2 Details of 'type' sequences from (a) mysticetes (b) and odontocetes used in the analysis of 'test' sequences from Japanese and
Korean products. The code refers to the labels used in Figs 1-3. An * indicates more than one sequence with a similar prefix (i.e. Bmy9201
and Bmy9202). Unpublished sequences were provided courtesy of the indicated investigators or institutions (UANZ, University of
Auckland; UH, University of Hawaii; SWFC, Southwest Fisheries Science Centre, La Jolla, CA, USA)

Common name Scientific name Code Geographical origin Reference (courtesy of)

A. Mysticetes
Bowhead whale Balaena rnysticetus Bmy' Alaskan Arctic Baker & Palumbi 1994
Northern right whale Eubalaena glacialis Egl-UA Arnason et al. 1993
Pygmy right whale Cuperea marginata CmaNZ-CSB New Zealand Baker k Palumbi 1994
Gray whale Eschrichtius robustus Ero397 Eastern Pacific Baker & Palumbi 1994
EroWS03
North Atlantic minke whale Balaenoptera acutorosfrata BacNA-UA North Atlantic Arnason et al. 1993
acutorostrata BacNA-A* Iceland/Norway Bakke & El-Gewely 1992
(I. Bakke)
North Pacific minke whale Balaenoptera acutorostrata BacNP-SWFC Eastern Pacific SWFC (C. Lux, S. Costa)
riavidsoni BacNP-JP Western Pacific Hori et al. 1994 (H. Hori)
Southern Hemisphere minke Balaenoptera acutorostrata BacSO-RH Antarctic Area V Hoeltel et al. 1991
whale bonaerensis BacAU-CSB East Australia Baker & Palumbi 1994
Southern Hemisphere minke Balaenoptera acutorostrata Bacdwarf-]I"' Hori et al. 1994 (H. Hori)
whale (dwarf form) -
Sei whale Balaenoptera borealis Bbo-UA North Atlantic Arnason et al. 1993
BboNA-CSB North Atlantic Baker & Palumbi 1994
BboNZSSB New Zealand UANZ (C.S. Baker)
Bryde's whale Balaenoptrra edeni BedSA-UA South Africa Arnason et al. 1993
BedMX-LM Pacific Mexico UANZ (L. Medrano)
BedNZCSB New Zealand UANZ (C.S. Baker)
Blue whale Balamoptera micsculus BmuMXCSB Pacific Mexico Baker & Palumbi 1994
rnuxulus BmuIC-UA Iceland Arnason 1991 (U. Amason)
Fin whale Balaenoptera pkysalits BphMD' Mediterranean UANZ (C.S. Baker)
BphIC Iceland Amason & Mitchell 1991
(v. Amason)
BphCan* Atlantic Canada Arnason & Mitchell 1991
(U. Arnason)
Humpback whale Megaptera novaeangline MnoSEA9 Alaska Baker h Palumbi 1994
MnoIC08 Iceland Arnason 1991 (U. Amason)

Common name Scientificname Code Reference (courtesy of)

B. Odontocetes
Sperm whale Phy& macrcuepkalus ha-UA Arnason et al. 1993
Pygmy sperm whale Kogia brmiccps Kbr-UA Arnason et al. 1993
Gray's beaked whale Mesoptodon FV' MgrNZ02-CSB UANZ (M. Dalebout, C.S. Baker)
Cuvier's beaked whale Ziphius cm'rostris zCaNZO2-CSl.l UANZ (M. Dalebout. C.S.Baker)
White whale, beluga Delpfrinapterus leucas Dleu-SWFC SWFC (G. WCorry-Crowe)
Harbor porpoise P~mphocoeM Pho-PR (Rose1 et ul. 1995)
Burmeister's porpoise Phoumrp spinipinnis Psinni-PR (Rosel el al. 1995)
Vaquita Phoumrp sinus Psinus-PR (Rosel et al. 1995)
Atlantic white-sided dolphin Lagetwrkynchus mtus Lac94001-FC UH (F. Cipriano)
Dusky dolphin Lagenorhynchus obscurus Lo5236-FC UH (F. Cipriano)
Pacific white-sided dolphin Lngororhynchusobli9uidm Lob-SWFC SWFC (C. Lux, S.Costa)
hb*-FC UH (F.Cipriano)
Bottlenose dolphin Tuniops tnrncntus Ttr-SWFC SWFC (B. Curry)
Atlantic spotted dolphin Sfenclla fmntalis sfr-SWFC SWFC(R LeDuc)
Short-beaked common dolphin Delphinus delphis Dde-SWFC (Rosel ct al. 1994)
DdeIRAMlO UH (F. Cipriano)
Commerson's dolphin Crphplorhynchus comrnersonii Cco-GenB Southem et al. 1988
Hector's dolphin Cephplorhynchw hectori Che06-CSB Baker & Pdumbi 1994
Killer whale, orca Orcinus mca Oor-RH Hoelzel et al. 1991
Long-finned pilot whale Gbbicephah melas Gme-NZ (M. Dalebout, C.S. Baker)
Gme-SWFC UANZ SWFC (A. Dizon)

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676 C.S.BAKER, F . C I P R I A N O A N D S.R. PALUMBI
BpbCaa-UA
humpback
MaolCOB -UA
F i g 1 The phylogenetic reconstruction of 'type' sequences of
mysticetes and odontocetes based on the neighbour-joining
method.Bootstrap values, based on lo00 resamplings, are shown
above relevant branches. Individual sequences are labelled
according to Table 2. Common names for major groups or species
of interest are shown along the right.
tions or subspecies of baleen whales ('Table 2A) and 25
individuals representing specimens of 18 species of
odontocetes (Table 2B). The phylogenetic relationships
reconstructed from these 58 type sequences (Fig. 1)agreed
in general with other published phylogenetic reconstruc-
tions based on molecular data (Amason et al. 1993;
Milinkovitch et al. 1993; Arnason & Gullberg 1994).
B e c a w the mtDNA control region evolves rapidly (e.g.
Vigdant et al. 1991)and thus accumulates an abundance of
informative characters, these sequences provide strong
evidence for forensic identification as indicated by > 99%
bootstrap support for the grouping of regional variants of
described species. Control region sequences are also ade-
quate for identifying some of the deeper divisions among
cetaceans, including those between the mysticete and
odontocete suborders and between some families (i.e.
Balaenidae, Ziphiidae, Delphinidae, Phocoenidae). As a
consequence of its rapid evolutionary rate, which
enhances the ability to distinguish reliably between
species, the control region accumulates so many differ-
ences that it is less useful for resolving the branching order
within families. This level of phylogenetic reconstruction
is not of particular relevance to the forensic identification
of species and geographical origin discussed here.
More problematic for our purposes is the taxonomic
uncertainty of some related cetacean species, particularly
the oceanic populations and morphological 'forms' (e.g.
the Southern Hemisphere common and 'dwarf' or diminu-
tive forms) of minke whales and the closely related sei and
Bryde's whales. The nature of these uncertainties and the
implications for forensic identification are discussed in
detail later.
Species identity of fesfsequences
Previously, we successfully amplified, purified and later
sequenced a portion of the mtDNA control region from 16
commercial products purchased in Japan (Baker &Z
Palumbi 1994). A single species-specific sequence was
found in 15 of the purchases. In one purchase (JS19), the
amplification yielded two distinct species-specific
sequences. This product was described by the field agents
who collected the sample as 'marinated strips of meat'. In
another purchase (JS16), the amplification yielded a
sequence with multiple bands at a number of positions,
suggesting a multiple species origin, although only a sin-
gle partial sequence could be discerned. This product was
purchased frozen and labelled as 'kujira kun'; field agents
described it as salted strips of meat. The 15 single-
sequence products and the one multiple-sequence product
yielded a total of 17 sequences.
The target region of the mtDNA control region was
amplified successfully from 15 products purchased in
Korean commercial markets. Where distinct tissue sources
were apparent in the purchases (e.g. unattached skin and
meat) multiple quick-preparation DNA extractions and
amplifications were conducted. A single species-specific
sequence was found in 13 of the purchases. In two of the
purchases (KN1 and KN9), amplification of skin and meat
yielded two distinct species-specificsequences. The 13 sin-
gle-species products and the two multiple-sequence prod-
ucts yielded a total of 17 product sequences.
Bootstrapped phylogenetic reconstructions Unambigu-
ously (> 90%) aligned 14 sequences from Japanese prod-
ucts (including two sequences from a single product) with
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 MOLECULAR GENETIC INDENTIFICATION OF CETACEAN PRODUCTS
type sequences from mysticete whales (Fig. 2). Seven
sequences grouped with Southern Hemisphere common
form minke whales, one with the Northern Hemisphere
and Southern Hemisphere dwarf form minke whales
(JSlS) and four grouped with fin whales. One of the
sequences from JS19 (JS19a) grouped with the type
Southern Hemisphere minke whales and one (JS19b)
grouped with the type humpback whales. Three samples
were placed in the suborder Odontoceti but could not be
identified to species (Fig. 3). Two samples, JS13 and 28,
were placed within the family Delphinidae (93%bootstrap
value), differing by 3.18% from each other and 6.25-8.82%
from other species of dolphins and the pilot whales.
Sample JS16 was placed within the odontocetes with cer-
tainty but did not group reliably with any available type
sequences.
Bootstrapped phylogenetic reconstructions unambigu-
ously aligned 14 of the Korean test samples with a type-
species sequence (Figs 2 and 3). Thirteen of the Korean test
sequences grouped (100% bootstrap value) with the
Northern Hemisphere populations of minke whale and
the Southern Hemisphere 'dwarf' form. Examination of
variation in the 13 minke whale sequences from Korean
products indicated that the products originated from at
least seven different individual whales. One sample, KN9,
grouped (93% bootstrap value) with the Pacific white-
sided dolphin, Lagenorhynchusobliquidens (Fig. 3).
Three of the sequences from Korean products could be
placed into groups of related taxa but could not be identi-
fied to species with certainty. One sequence, KNl(skin),
grouped closely (99% bootstrap value) with the type
beaked whales but could not be identified to species due
to the scarcity of type species sequences for this family.
Two sequences (KNlb and KN3) grouped in the branch
composed of the sei and Bryde's whale type sequences in
100% of the bootstrap replicates. However, the KNlb and
KN3 sequences connect as a basal branch relative to the
available sei and Bryde's whale type sequences and thus
cannot be identified as either sei or Bryde's whales with
certainty. Sequences from whale meat confiscated in
Russia (Dizon et ul. 199%) were made available for com-
parison to our test and type sequences (courtesy of A.
Dizon and R. Brownell, Southwest Fisheries Research
Centre, San Diego, CA). Our analysis agreed with Dizon et
al. (199%) in grouping the Russian whale meat with the
available Bryde's whale sequences but provided no evi-
dence that the confiscatedRussian products were from the
same population as the Korean products. Sequences of
KNlb and KN3 were, in turn, provided to researchers at
the Southwest Fisheries Centre for comparison to their
larger catalogue of Bryde's whale sequences. This inde-
pendent analysis agreed in placing the KNlb and KN3
sequences with the sei/Bryde's clade but failed to further
resolve the species identity of these products (personal
@ 1596 Blackwell Science Lid, Molecub Ecology, 5,671685
677
communication, A.E. Dizon, Southwest Fisheries Science
Centre, La Jolla, CA, USA).
Population identity of test sequences
The probable geographical origin of some products was
suggested by the grouping of test sequences with geo-
graphical representativesof some species. Among the nine
minke whale products from Japan, eight grouped unam-
biguously (100% bootstrap value) with h e specimens
from Australia and the Antarctic (Hoelzel & Dover 1991~)
indicating that they are of Southern Hemisphere origin.
The 13 minke whale products from Korea and one from
Japan (JSl8)grouped with type sequences from Northern
Hemisphere populations of minke whales. Bootstrap sim-
ulations support the grouping of these 14 products with
the two type sequences from the North Pacific population
(809'0 bootstrap value) rather than the North Atlantic or
dwarf form. The single Southern Hemisphere dwarf form
sequence falls outside this cluster, but we do not know if
other dwarf form sequences would form a monophyletic
clade (see Fig. 2). Even more problematic, the dwarf form
and the North Atlantic population cannot be distin-
guished reliably with available type sequences.
Of the fin whale sequences, samples JSWS4, JSO3 and
JSll differed by less than 3.0% from fin whales sampled
near Iceland (Amason et al. 1991), Canada and in the west-
em Mediterranean. Fin whale sample JS41, however, dif-
fered by 3.0-3.4% from these North Atlantic type
sequences. Without a more comprehensive genetic data-
base, we cannot exdude the possibility of origins outside
of the North Atlantic for some of these samples. The par-
tial humpback whale sequence (JS19b) was identical to
sequences from other humpback whales sampled near the
Mexican, Hawaiian and Japanese (i.e. Ogasawara Islands,
C.S.B. unpublished data) wintering grounds, suggesting a
North Pacific origin.
Experimentnl reliability and consistency
Given the power of PCR to amplify even a singIe target
molecule, it is important to consider the possibility of con-
tamination prior to and during laboratory handling as a
source of experimental error. Laboratory contamination
can be excluded for all odontocetes, all minke whales and
most fin whales, because these were not identical to type
sequences from either of our laboratories. For the two sam-
ples which were identical to sequences from our laborato-
ries (WS4, a North Atlantic fin whale, and JSlSb, a North
Pacific humpback whale) contamination is extremely
unlikely. All reagents used in the field were new, all
equipment was decontaminated, non-aspirating tips were
used for micropipetting and no contamination was appar-
ent in the negative controls. Contamination after the
678 C.S. B A K E R , F. CIPRIANO A N D S.R. PALUMBI
BrNp-JiU37

it
KNI 1
KNZ
KNS
KN4
KN6
KN7
KNlO
KNl2
KNlS
KN13
KNl4

rl

F i g 3 The phylogenetic reconstruction of 'type' and 'test'

sequencesof odontocetesbased on the neighbur-joiningmethod.
Bootstrap values, based on loo0 resamplings, are shown above
relevant branches. Arrows indicate positions of 'test' sequences.
Individual sequences i~ labelled according to Tables 1 and 2.

although field agents were trained to sample and store the

Fig. 2 The phylogenetic reconstruction of 'type' and 'test'
sequences of mysticete species based on the neighbour-joining
method.Bootstrap vafws, based on loo0 resamplings, are shown
above relevant branches. Species with matching 'test' sequences
are shown with common names. Individual sequences are
labelled according to Tables 1 and 2.
amplified samples were returned to the laboratory can be
excluded: reamplifications of the bead-bound DNA from
the origrnal amplification always gave the same results
when sequenced or analysed for restriction fragment
length polymorphisms.
Foreign contammation due to handling by field agents
prior to amplification is unlikely because agents had no
contact with cetacean material outside of Japan or Korea.
The possibility exists that cross-contamination between
products could have occurred during domestic handling,
products carefully. Domestic cross-product contamination
prior to purchase, however, cannot be excluded. Products
stored or displayed together without non-porous wrap
ping could cross-contaminate each other with blood or
small pieces of tissue. The potential for such cross-product
contamination is clearly demonstrated by the multiple-
species products purchased in Korea. In these two prod-
ucts (KN1and KN9), we were alerted to the possibility of
multiple species by the presence of different tissue types.
Because many of the purchases included more than one
piece of meat or skin, it is possible that other species- or
individual-speafic sequences could be found in a more
exhaustive analysis of these products.
The results of our double-blind analysis of the Korean
samples were unambiguous. DNA was amplified and
sequenced from 13 Korean products by both laboratories.
Each laboratory initiated sequencing from different ends
of the target region so that the combined dataset would
include a consensus of the forward and reverse sequences.
The two independent analyses were in complete agree-
ment on the species identifications of the 13 products.
Minor disagreements in the nudeotide sequences were
noted only near the primers where sequenceswere faint or

@ 1996 Blackwell Science Ltd, Moleculm Ecology, 5,671-685

 MOLECULAR GENETIC INDENTIFICATION O F CETACEAN P R O D U C T S
compressed. These disagreements were resolved and
corrected before the data sets were merged for the final
analysis.
Discussion
DNA sequences generated by PCR have tremendous
utility in the identification of species or evolutionarily
significant units. Over the past decade, the methodology
of molecular systematics has developed to allow reliable
construction and interpretation of phylogenetic trees from
DNA data. Confidence in phylogenetic reconstructions
can be established statistically using procedures like boot-
strap resampling (Felsenstein 1988) or permutation tests
(Faith 1991). Much of this methodology is widely applica-
ble in forensic studies (e.g. Hillis et nl. 1994).
However, we have encountered certain constraints in
our use of PCR and molecular systematics for the identifi-
cation of whale and dolphin products. These constraints
fall into two categories: (i) technical problems in the col-
lection of molecular data using PCR; and, (ii) limitations
on the analysis of species-level systematic relationships
using molecular data.
PCR and sequencing artefacts
The target and specificity of the PCR reaction are con-
trolled through the adjustment of reaction conditions and
through the choice of oligonucleotide primers that are
designed to anneal only to a particular target DNA strand.
Although the PCR is usually a precise and accurate tool,
there are circumstances in which it can provide unexpect-
ed results. Detailed descriptions of basic PCR methodolo-
gy can be found elsewhere (e.g. Innis et af. 1990; Palumbi
1995).Here we discuss technical issues that are important
in understanding the forensic interpretation of the prod-
ucts of a PCR reaction.
Polymerase errors occur at a low frequency when the
Tnq polymerase is used in PCR reactions (and it is proba-
bly the most widely used PCR enzyme). This is because
Tnq polymerase has no proof-reading function (i.e. no
exonuclease activity). As a result, when an incorrect
nudeotide is added to the growing DNA strand during
the extension step of PCR, it is not removed or replaced
with the correct nudeotide. These polymerase errors are
rare and random in their distribution along the DNA
strands produced with PCR When PCR products are
analysed by restriction digestion or by sequencing of the
whole product (such as the solid-phase sequencing used
here), low frequency errors at a particular position are far
outnumbered by other templates which have the correct
nucleotide. Thus,polymerase errors are usually not appar-
ent when using these techniques. However, if PCR prod-
ucts are cloned, the sequence of each clone is an exact
019% Blackwell Science Ltd, MoZecula~Ecology, 5,671-685
679
match to the single sequence that was inserted into the
vector. As a result, a polymerase error will be camed
through into the resulting sequence data. Our experience
shows that about one base in 500-1OOO is subject to poly-
merase errors from cloned PCR products. Errors of this
magnitude would not substantially alter identificationof a
sample to a particular species or population.
A more serious but less common problem is the poten-
tial to amplify a nuclear insertion of a mtDNA sequence
(e.g. a pseudogene, Lopez et ni. 1994)or to generate artifi-
cial recombinants during amplification (e.g. PCR ‘jump-
ing’, Thomas & Paabo 1993). The insertion and duplication
of mtDNA sequences into the nuclear genome can be an
aid (Zischler et d.1995)or a hindrance (Collura & Stewart
1995) to phylogenetic analyses. If unrecognized as a
pseudogene, these xenologous or paralogous sequences
could generate misleading phylogenies depending on the
evolutionary timing of the insertion event. To date, how-
ever, no mtDNA pseudogenes have been reported in the
nuclear genome of cetaceans.
A PCR recombinant can be generated if a partial copy
of the target sequence is generated during the initial exten-
sion phase of an amplification (Saiki et d.1988). This par-
tial sequence can then act as a large primer in subsequent
annealing and extension cycles. Should there be two dif-
ferent template sequences in the reaction mix (derived
from a mixture of tissues from two individuals or species),
a partial copy (acting as a large primer) could anneal to a
template of the second speaes. This partial copy will then
be extended by the polymerase, with the result that the
first part of the sequence will be from speaes ’A’ but the
second part will be from speaes ‘B. PCR recombinants
depend on an unlikely combination of two experimental
problems: multiple templates and incomplete extension
reactions. These seem unlikely to be consistent across
replicate amplifications and, if they are generated at all,
will probably not produce a clear sequence. If they do,
however, they will complicate phylogenetic analysis of the
DNA sequence data. By their nature, they are neither
species ‘A’ nor species ‘B,and have characteristics of both.
A phylogenetic reconstruction using these sequences will
typically place them at the base of branches leading to
species ‘A’ and species ‘B, with low bootstrap values for
either grouping.
Molecular sysfematics and species identificafion
Assuming that a particular DNA sequence has been
amplified from a test product without artefacts (see above
for alternatives) and is of sufficient length for phylogenet-
ic reconstruction, there are some circumstances in which
species identification remains difficult. These difficulties
are common to the use of molecular systematics for the
description of evolutionarily sigruficant units for conser-
680 C.S. BAKER, F . C I P R I A N O AND S.R. PALUMBI
A. B.
ICIC
Fig. 4 (a) A cladogram showing the incorrect identification of
a test sequence X as a result of an incomplete phylogeny of
related species, B and C. @) The correct identification of test
sequence X by inclusion of the missing taxa, A.
vation, management or taxonomic classification (e.g.
Ryder 1986;Pamilo & Nei 1988; Avise 1989; Davis & Nixon
1992; Moritz 1994; O'Brien 1994; Vogler & Desalle 1994).
First, an accurate phylogenetic reconstruction of DNA
sequences can be misinterpreted if the database for type
species is incomplete. In the hypothetical example shown
in Fig. 4(a), test sequence 'X' is compared with an incom-
plete database that includes type sequences from two
related species, ' B and 'C'. In the absence of other infor.
mation (e.g. branch length, see below), the branching
order of this tree suggests that test sequence 'X' is from the
same species as type sequence 'B. The inclusion of a type
sequence from a third related species (type' A in Fig. 4b)
could alter this conclusion. In this case, the revised phy-
logeny now supports the conclusion that sequence 'x'is
redly the same species as sequence 'A'.
It should be noted, however, that even without species
'A', it is possible to conclude that test sequence 'X' is close-
ly related to type sequence ' B . In many cases, this alone
may be an important conclusion. Information on branch
length (i.e. sequence divergence) can also be useful in
detecting incomplete taxonomic representation. Relative
to the sequences under consideration (mtDNA control
regions in our analysis), an unusually large divergence
within a grouping could be an indication that an interme-
diate species is missing from the database (see below, 7%
seilbryde's whle question). If there is doubt about the com-
pleteness of the database or the taxonomy of a group, a
conservative strategy is to identify species only when a
test sequence groups within a set of type samples from a
particular species.
Second, a problem of identification could arise if the
sequences of two related species are not reciprocally
monophyletic at the locus used for forensic identification
In this case,a DNA sequence from species ' B is more sim-
ilar to sequences from species 'A' than it is to other
sequences from species ' B (Fig. 5). The possibility of such
paraphyletic relationships is a general limitation in using
species B indiv. 1
species A indiv. 1
species A indiv. 2
species B indiv. 2
Fig. 5 Difficulties with species identification when sequences of
two species are not reciprocally monophyletic.
'gene trees' to infer 'species trees' or systematic relation-
ships at the organismal level (Pamilo & Nei 1988).
Paraphyletic relationships of mtDNA lineages among
closely related cetaceans cannot be excluded without an
adequate survey of population-level sequence variation.
The only mysticete species which has been systematically
surveyed for mtDNA variation world-wide is the hump-
back whale (Baker et nl. 1993). In this case, all mtDNA
sequences are monophyletic with respect to the fin whale
(Baker et nl. 1993) and all other balaenopterids (C. S. Baker
and S. R. Palumbi, unpublished. data). For toothed whales,
the examination of mtDNA among a few closely related
species or morphological forms has revealed both mono-
phyletic and paraphyletic relationships (Dizon et al. 1991;
Hoelzel& Dover 1991b; Rosel 1992; Rosel et nl. 1994; Rosel
et nl. 1995; Rosel et al. 1995). Whether other closely related
whales (like the minke whale complex and the sei and
Bryde's whales, see below) or dolphins show reciprocally
monophyletic mtDNA sequences is unknown.
Intraspecific variation of mtDNA sequences can also
limit identification of the geographical origin of a particu-
lar product. Paraphyletic relationships among conspecific
populations would be expected if gene flow is greater than
a few individuals per generation and if population sizes
are large (Birky ef nl. 1983; Ball et nl. 1990). Without diag-
nostic markers that are fixed or restricted to specific geo-
graphical populations, establishing the geographical ori-
gin of a particular sample may be dependent on a fine-
scale understanding of population structure (e.g. Baker et
nl. 1994) or on maximum likelihood solutions based on
'mixed' stock models (e.g. Bowen et nl. 1995)
A final, and critical, consideration in using molecular
systematics for species identification is the assumption
that the taxonomy of the group in question is complete
(e.g Daugherty et al. 1990). If this basic biological informa-
tion is lacking, question.. about the adequacy of type cata-
logues and the possibility of paraphyletic relationships
among recognized species cannot be answered.
Surprisingly, our analysis suggests that taxonomic uncer-
tainties remain a problem among the baleen whales, even
though this group includes only a handful of widely dis-
tributed, formerly abundant species.
@ 19% Blackwell Science Ltd, Molecular Ecology, 5,671485
 MOLECULAR GENETIC INDENTIFICATION O F CETACEAN PRODUCTS
The m i n k whale question
According to the IWC Schedule, minke whales
(Buluenopteruucufmostrutuand B. bonaerensis) are defined as
‘any whale known as lesser rorqual, little piked whale,
minke whale, pike-headed whale or sharp headed finner‘
(Dizon ef ul. 1992). Recent molecular analyses by others
and those presented here demonstrate that this definition
is inadequate. Four forms or subspecies of minke whales
have been described previously from morphology or geo-
graphical distribution: the North Atlantic (ucutorostrutu),
the North Pacific (dmidsoni), the Southern Hemisphere
(bonuerensis) and the dwarf form (Best 1985; Dizon et ul.
1992). Based on an analysis of mtDNA control region
sequences, Amason et ul. (1993) proposed that North
Atlantic and Southern Hemisphere minke whales should
be elevated to species status, B. ucutorostrutu and B.
bonuerensis, respectively. However, Amason et ul. (1993)
did not include samples from the Southern Hemisphere
dwarf form or the North Pacific population. A preliminary
analysis of mtDNA sequences (Hori et ul. 1994) suggests
that the dwarf form is closely related to both Northern
Hemisphere populations and these are distinct from the
Southem Hemisphere common type.
Our analysis of available type sequences agrees with
others (Hoelzel h Dover 1991c; Wada et ul. 1991; Bakke &
ElCewely 1992; Arnason et al. 1993; Hori et ul. 1994;
Pastene ef al. 1994; van Pijlen et ul. 1995) in distinguishing
the two North Hemisphere populations from the common
Southern Hemisphere type. Our analysis also agrees with
Hori et al. (1994) in suggesting a slightly closer relationship
between the dwarf form and the North Atlantic popula-
tion relative to the North Pacific population. However, the
range of differences (2.144%) among type sequences
from both Northern Hemisphere populations and
Southern dwarf form minke whales overlaps with that
found in conspeafic populations of other mysticetes (e.g.
humpback and fin) and is considerably smaller than that
found between other species of Balaenopterid.
With available type sequences, we could identify prod-
ucts derived from minke whales, as defined by the IWC
Schedule, with certainty (100% bootstrap value). Further,
we could distinguish, with certainty, the products of the
Southem Hemisphere common form from those of the
dwarf form and Northern Hemisphere populations.
Ascribing the origin of products to either the North Pacific
or North Atlantic population or the Southern Hemisphere
dwarf form,however, was frustrated by the limited num-
ber of type samples, low levels of divergence and possible
paraphyletic relationships between available sequences. A
world-wide sample of mtDNA variation in minke whales,
with adequate oceanic and regional population represen-
tation, is needed to fully resolve the relationships between
these species, subspecies or populations.
@ 19% Blackwell Science Ltd, Molecu&r Ecology, 5,671-685
681
The seilBryde’s whale question
Our analysis of type and test sequences from the sei and
Bryde’s whales suggests that these two related species,
like the minke whale complex, require further molecular
systematic examination for both stock and species level
classification W o n et ul. 1995). In pairwise comparisons,
the three type sei whale sequences differed from each
other by 1.2-3.0% and the four Bryde’s whales differed
from each other by 2.0-3.6%. Differences between the type
sei and Bryde‘s whales ranged from 7.0 to 9.2%. Although
the ranges of these i n t r a s p d c differences did not over-
lap with the interspecific differences, the bootstrap values
supporting the species-specific groupings of type
sequences were low in some cases (58% bootstrap value).
Only the bootstrap support for grouping the New Zealand
and North Pacific Bryde’s whales (analysis not shown)
approached that found in other conspecificsequences.The
previous analysis of mtDNA control region sequences by
Amason ef ul. (1993)agrees in showing a close relationshp
between the sei and Bryde’s whales, as represented by one
specimen each, but did not address the problem of
intraspecific variation within the two widely distributed
species.
Further ambiguities in the taxonomy of the sei and
Bryde‘s whale are indicated by our analysis of test
sequences from the Korean whale products. Here, we dis-
covered two test sequences (KNla and KN3) that grouped
closely (91-100% bootstrap values) with both sei and
Bryde’s whales type sequences but that could not be
asaibed to either recognized species with confidence. The
most complete of the two Korean sequences (KN3,437 bp
in length) differed by an average of 7.5% from both the sei
and Bryde’s type sequences. This is greater than the dif-
ference between some type sequences of sei and Bryde’s
whales in our catalogue (7.0-9.2%, range). There are sev-
eral alternative explanations for this pattern: (i) the anom-
alous sequences are a nuclear pseudogene of the mtDNA
control region or a PCR recombinant; (ii) the sequences
come from an anaent lineage (as yet unsampled) within
the recognized sei or Bryde’s whale species; or, (iii) the
sequences are from an unrecognized species or subspecies
related to the recognized sei and Bryde’s whales.
We tested for the possibility of amplifytng a control
region pseudogene by examining phylogenetic
reconstructions based on sequences of the mitochondria1
cytochrome b gene amplified from the KNlb and KN3
products (F.C., data not shown) and other mysticete
whales (Amason & Gullberg 1994). If the control region
sequences were in fact a pseudogene, we would expect a
different topology from the cytoduome b gene unless this
region was also included in the transposition. In fact, the
cytochrome b phylogeny was consistent with the control
region phylogeny, plaang KNla and KN3 in the
682 C.S. BAKER, F . C I P R I A N O AND S . R . PALUMBI
sei/Bryde’s clade but ancestral to both. The consistency of
these two phylogenies and the absence of pseudogene-like
sequences in other amplificaGons from test or type sam-
ples argues against the likelihood of this artefact.
A PCR recombinant could have arisen, as described
above, if our original template DNA included both sei and
Bryde’s whale DNA. This possibility warrants
consideration given that pieces of meat from the one
purchase yielded the sei/Bryde’s sequence (Kiilb) while
the s k i yielded a beaked whale sequence (KNla). To test
for the possibility of in aitro recombination, we used a
computer program (available from S.R.P.) to calculate
nucleotide differences between type and test sequences
along a moving ‘window’ of 60 bp in length. If in vitro
recombination had produced a sei/Bryde‘s hybrid
sequence, then the beginning of the test sequences should
be more similar to one species, whereas the end of the
sequences should be more similar to the other. The
observed pattern of sequence difference in the moving
‘window‘ was not consistent with the expectations of
recombination (Fig. 6). The two Korean sequences (KNlb
and KN3) showed about the same degree of difference to
both sei and Bryde’s sequences along its entire length.
Although the total degree of sequence heterogeneity
changed markedly along the length of this section of DNA,
this is normal for comparisons of control region sequences
where levels of sequence conservation shift from one func-
tionally distinct region to the next (Hoelzel & Dover
1991a).
Because we found no evidence consistent with a PCR
artefact, we suggest instead that KNlb and KN3 represent
either an ancient mtDNA lineage within the recognized sei
or Bryde’s whales, or an unrecognized species or
subspecies related to the recognized sei and Bryde’s
1.0,
I comparison
0.8
0.6
0.4
02
0.0
0 100 200
Position (bp) of window along sequence
whales. Ancient mitochondria1 lineages have been found
in other species with wide-scale distributions and distinct
subpopulation structure, e.g. humpbacks whales (Baker et
al. 1993),New Zealand fur seals (Lento et al. in press) and
black-backed jackals (Wayne et at. 1990). However, the
degree of divergence between the KNlb and KN3 test
sequences and the type sei and Bryde’s whales is large
even in comparison to the ancient lineages of these other
species. Further, the inclusion of the KNlb and KN3
sequences as either sei or Bryde’s would imply a likely
paraphyletic relationship of mtDNA among the recog-
nized sei and Bryde’s whales.
Alternatively, the report of a genetically distinct ’small-
form’ of Bryde’s whale (Best 1977; Wada & Numachi 1991)
suggests that the systematic relationships and taxonomic
status of the sei and Bryde’s whales is more complex than
the currently accepted two-species designation (Dizon et
nl. 1995). Eight small-form Bryde’s whale were killed
during Japanese scientific hunting -near the Solomon
Islands and in the eastern Indian Ocean near the Island of
Java from 1976 to 1979. Inspectors aboard the whaling
vessels recognized the whales as Bryde’s but, in the case of
the Solomon Island catches, noted that they were sexually
mature at a smaller size than expected and that their
baleen differed in coloration from ordinary Bryde‘s
whales. The whales from the eastern Indian Ocean were
not noted as distinct and were classified as small-form
only after the genetic analysis. Allozyme analysis at 45 loci
showed that the genetic distance between the small-form
Bryde’s whale and the sei/Bryde’s whale is greater than
the sei and Bryde’s are to each other (Wada & Numachi
1991).Furthermore, a U P G M A dendrogram based on the 45
allozyme loci (Wada & Numachi 1991), places the ‘small
form‘ Bryde‘s whale as an early divergent from the
to:
Fig. 6 Nudeotide sequence differences
between KN3 and three type species
sequences, in sequential 60 bp
comparisons (‘windows’) along the
length of the sequence. The
geographical origin of the type
300
sequences is indicated in the box: MX,
Mexico; NZ,New Zealand;AK, Alaska.
@ 1996 Blackwell Science Ltd, Molecular Ecology, 5,671-685
 MOLECULAR GENETIC INDENTIFICATION O F CETACEAN PRODUCTS
common sei/Bryde’s whales lineage relative to the fin and
minke whales. This placement is remarkably similar to the
relationship of sei, Bryde’s whales and the two Korean
samples in our phybgram (Fig. 2). Researchers at the
Southwest Fisheries Science Centre, National Marine
Fisheries Service, have recently analysed mtDNA control
region sequence from the skull of a small-form Bryde’s
whale collected by W.F.Perrin in the Philippines. These
sequences are consistent with our conclusion that two
samples of whale meat purchased in South Korea came
from this unrecognized species, although not necessarily
from this geographical location (personal communication,
A.E. Dizon, Southwest Fisheries Science Centre, La Jolla,
CA, USA).
Conclusions
A wide diversity of whale and dolphin products appeared
to be commonly available on the Japanese and Korean
domestic markets. Among the 34 sequences derived from
31 products we identified at least eight species represent-
ing three families and two suborders of cetaceans. Several
purchases induded products of mixed-species origin. In
some cases the possibility of more than a single species
was indicated by multiple tissue types. In other cases,
there was no obvious indication that more than one
species was involved until the DNA was amplified and
sequenced. Some of the species identified in the products
from the Japanese and Korean commeraal markets were
not consistent with recent catch records made available to
the International Whaling Commission (Baker & Palumbi
1994).Assessing the legality of speafic products has raised
a number of uncertainties in the international and
domestic management of whale products (&on et nl.
1995a; Freeman 1995; Hansen 1995; Heinze 1995).
Considerations raised by the whaling industry or the
Government of Japan include the following (Anon. 1998):
the availability in Japan of some legal products from
recent scientific hunting of Southern Hemisphere minke
whales; the extensive and largely unregulated coastal
hunting of many odontocete species; the possibility of
unregulated or undocumented sale of products from
coastal bycatch or strandings of whales; the absence of any
standardized form of commercial labelling of products;
and claims that some whale products now for sale on com-
mercial markets have been in storage for up to 10 years. A
more extensive and seasonally representative survey of
products is needed to adequately describe this apparently
active market in whale and dolphin products of question-
able legal origin.
AIso needed are worId-wide surveys of nuclear and
mtDNA variation among each of the 11recognized species
of baleen whales (e.g. Baker et ul. 1993). These would pro-
vide the basis for an objective evaluation of current dis-
0 1996 Blackwell Science Ltd, MoZecuIur Ecology, 5,671485
683
tinctions between species, subspecies, morphological
forms and populations or stocks. Of immediate manage-
ment concern are the two species (or species complexes)
currently targeted by legal and illegal hunting, the minke
and Bryde’s whales. Uncertainty in the current knowledge
of systematics and genetic population structure of these
species complicated our molecular genetic identificationof
the geographical origin of some products. The distinct
genetic differences between the subtly different morpho-
logical forms of minke and Bryde’s whales raises the pos-
sibility of ‘cryptic‘ and possibly endemic species of baleen
whales, similar to those reported among odontocetes (e.g.
Hoelzel & Dover 1991b; Rosel et nl. 1994). It is particularly
disturbing that two specimens of an unrecognized species
or subspecies of baleen whales were found for sale in a
restaurant in South Korea in October, 1994, 8 years after
the acceptance of an international moratorium on com-
mercial whaling.
Acknowledgements
For collection of ‘test‘ samples from retail markets we thank four
agents operating under the direction of Earthtrust and the
Endangered Species Project. These agents have asked to remain
anonymous. For access to ‘type’ samples or unpublished
sequences we thank: T.Albert, U. Amason, A. Baker, I. Bakke, J.
Calambokidis, S. Costa, M. Dalebout, S. Dawson, A. Dizon, H.
Hori,C. Lux, J. Mead, L. Medrano, G. Notarbartolodi-Sara, G.
OCorry-Crowe, C. Potter, L. Pastene, R. Paterson, P. Rosel, L.
Slooten, A. van Helden, G. Vequist and M. Zanardelli. For techni-
cal assistance and review we thank A. Perry, R. Brownell, 8.
Bowen, A. Dizon, M. Donoghue, R.G. LeDuc and G. Lento. MJ
Research, Inc. donated a PTC-150 portable MiniCyder for the
field analysis. Funding for this research was provided by
Earthtrust, the University of Auckland and the Medical
Foundation for the Study of Environment and Human Body
Charitable Trust, and a grant from the Whale and Dolphin
Conservation society to the Endangered Species Project. The
fieldwork in Korea was coordinated by S. Galster and S. LeBudde
and logistic support was provided by the Korean Confederation
of Environmental Movements and the Civil Institute for
Environmental Studies. The original project was conceived by S.
White and D. White of Earthtrust.
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