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1 - Molecular Genetic Identification of Whale and Dolphin Baker1996
1 - Molecular Genetic Identification of Whale and Dolphin Baker1996
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.
Table 1 Details of purchase and genetic analysis of whale products from Japanese and Korean retail markets
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 of a 550-base pair (bp) fragment of the mtDNA control
sequencing. region by PCR was conducted in a portable thermal cycler
Approximately 4 mg of tissue from each whale product (MJ Research, Inc.) using standard protocols (Saiki et al.
was prepared for PCR amplification using one of three 1988; Palumbi et nl. 1991) and the oligonucleotide primers,
methods: cycles of heating and cooling with Genereleaser light-strand tPro (5’-CTACCTCCAACTCCCAAAC-3’)
(Bioventures, Inc.) according to manufacturer’s recom- and heavy-strand DIp5 (5’-CCATCGWGATGTClTATT-
mendations; digestion with Pre-Tuq proteinase (Life TAAGRGGAA-3’).We focused on the control region of the
Technologies, Inc.) for 30 min at 94 “C in 200 pL of ddH,O mtDNA because of its high species- and population-spe-
and PCRII buffer (Perkin-Elmer); or heating to 94 “C in a cific variability (e.g. Arnason et al. 1993; Baker et al. 1993;
5% solution of Chelex resin (BioRad Laboratories) as Brown et al. 1986). A 500-bp fragment of the mtDNA
described by Walsh et nl. (Walsh et nl. 1991). Amplification cytochrome b gene (using the tGludg and Cyb2 primers,
Palumtri et a / . 1991) was also amplified from most prod- al. 1993). Test and type sequences were aligned initially
ucts as an additional source of markers for confirmation of using the program PILEUP,available in the GCG package
species identity. These data are not presented here. (Deveraux et al. 1984), with an initial gap penalty of 2 and
Following amplification, 5-10 pL of the double-strand- extension penalty of 0.3. The multiple sequence files were
ed (ds) DNA product was run on a 1.6% agarose/TBE gel then corrected for minor alignment inconsistenciesby eye.
and the resulting target band was excised to separate it For the neighbour-joining method, adjustments for multi-
from genomic DNA, The remainder of the dsDNA was ple substitutions were made using the Kimura Z-parame-
bound to streptavidin-coated, para-magnetic beads ter distance as implemented by M E G A (Kumar ef al. 1993).
(Dynal Corp.) via a biotin group attached to the 5' end of Unless otherwise noted, all differences between sequence
the Dlp5 primer. The bead-conjugated DNA was then are reported with the Kimura 2-parameter correction.
summoned with the aid of a magnet and washed to The statistical significance of test and type sequence
remove unincorporated primer, nucleotides, and all tem- groupings (a measure of the confidence in an identification
plate whale DNA (Hultman et RI. 1989). The synthetic of a test sequence) was estimated by bootstrap resampling
DNA isoiated by these procedures was then returned to of the sequence data using both parsimony and distance
our laboratories for solid-phase sequencing following methods. For the neighbour-joininganalysis, an initial rela-
standard protocols (Palumbi et al. 1991; Baker et al. 1993; tionship tree was constructed using representative type
Baker & Palumbi 1994). In most cases, the target region sequences from all available species (type sequences of
was re-amplified from the excised band or the solid-phase some geographical representatives were omitted to fadli-
attached DNA using internal primers (light strand Dlp 10, tate computation). Test sequences were then analysed indi-
5'-CCACAGTACTATGTCCGTAIT-Y; or heavy strand vidually to establish their placement into either of the two
Dlp 4, S-GCGGGWRY'ITGRTITCACG-3'). When used suborders of cetaceans, Mysticeti or Odontoceti. Based on
with its complement from the original primer pair (Dlp 1.5 this initial placement, bootstrap analyses were conducted
-
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
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-
reactions. ples, represented by short sequences US19a, JS19b, JS16and
The field amplifications of commeraal products from K4-1 'meat'), were added one at a time to the type
Japan in 1993 were conducted by one of us (C.S.B.) using sequences, rather than as a group, and bootstraps were per-
a single portable laboratory. To verify the consistency of formed only on the consensus region of the partial
our field methods, we conducted a modified double-blind sequences. All neighbour-joining phylogeny reconstruc-
experiment during our analysis of Korean whale products tions were tested with lo00 bootstrap resamplings.
in 1994. Two of us (C.S.B. and F.C.) assembled indepen- For the parsimony analysis we used methods described
dent field laboratories, including minicyclers, plasticware, by Baker & Palumbi (1994)as modified by one of us (F.C.).
micropipettes and reagents, for travel to Korea. In a field An initial relationship tree was constructed using only
laboratory, all commercial whale products were subject to type sequences. Each test sequence was then added indi-
one or more independent quick-preparation DNA extrac- vidually to the complete set of type sequences and its
tions and PCR amplification. We individually purified placement was tested by a bootstrap. Test sequences asso-
copied DNA from template DNA from our independent dated at high confidence (> 70%) with the same type
amplifications,and returned to our respective laboratories sequences were bootstrapped together to establish the
for sequencing. lnitial sequence analyses were conducted confidence level for groups of test sequences.
independently by C.S.B.and F.C., including preliminary Relationships among all type sequences were established
alignment to a catalogue of type sequences for species with Wreplicate bootstrap resamplings and heuristic
identification. The agreement of species identity and the searches. Identifications of test sequences were tested with
consistency of sequences were then evaluated by S.RP. 200 bootstrap resamplings for each individual' and for
before the data sets were emr-checked, merged and re- groups of similar individuals. All parsimony and neigh-
aligned to sequences in the type sequence database. bur-joining reconstructions agreed in the details relevant
to the identification of test products.
Sequence identification analysis
Identification of test sequences was based on phylogenet-
Results
ic retonstruction methods using parsimony as implement-
ed in the computer program P A U P (Swofford 1993) and
Phylogenetic relationships of type sequcnces
the neighbur-joining genetic distance algorithms as Type sequences of the mtDNA control region were avail-
implemented in the computer program M E G A (Kumar et able from 33 individuals representing regional popula-
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)
type sequences from mysticete whales (Fig. 2). Seven communication, A.E. Dizon, Southwest Fisheries Science
sequences grouped with Southern Hemisphere common Centre, La Jolla, CA, USA).
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
Population identity of test sequences
sequences from JS19 (JS19a) grouped with the type The probable geographical origin of some products was
Southern Hemisphere minke whales and one (JS19b) suggested by the grouping of test sequences with geo-
grouped with the type humpback whales. Three samples graphical representativesof some species. Among the nine
were placed in the suborder Odontoceti but could not be minke whale products from Japan, eight grouped unam-
identified to species (Fig. 3). Two samples, JS13 and 28, biguously (100% bootstrap value) with h e specimens
were placed within the family Delphinidae (93%bootstrap from Australia and the Antarctic (Hoelzel & Dover 1991~)
value), differing by 3.18% from each other and 6.25-8.82% indicating that they are of Southern Hemisphere origin.
from other species of dolphins and the pilot whales. The 13 minke whale products from Korea and one from
Sample JS16 was placed within the odontocetes with cer- Japan (JSl8)grouped with type sequences from Northern
tainty but did not group reliably with any available type Hemisphere populations of minke whales. Bootstrap sim-
sequences. ulations support the grouping of these 14 products with
Bootstrapped phylogenetic reconstructions unambigu- the two type sequences from the North Pacific population
ously aligned 14 of the Korean test samples with a type- (809'0 bootstrap value) rather than the North Atlantic or
species sequence (Figs 2 and 3). Thirteen of the Korean test dwarf form. The single Southern Hemisphere dwarf form
sequences grouped (100% bootstrap value) with the sequence falls outside this cluster, but we do not know if
Northern Hemisphere populations of minke whale and other dwarf form sequences would form a monophyletic
the Southern Hemisphere 'dwarf' form. Examination of clade (see Fig. 2). Even more problematic, the dwarf form
variation in the 13 minke whale sequences from Korean and the North Atlantic population cannot be distin-
products indicated that the products originated from at guished reliably with available type sequences.
least seven different individual whales. One sample, KN9, Of the fin whale sequences, samples JSWS4, JSO3 and
grouped (93% bootstrap value) with the Pacific white- JSll differed by less than 3.0% from fin whales sampled
sided dolphin, Lagenorhynchusobliquidens (Fig. 3). near Iceland (Amason et al. 1991), Canada and in the west-
Three of the sequences from Korean products could be em Mediterranean. Fin whale sample JS41, however, dif-
placed into groups of related taxa but could not be identi- fered by 3.0-3.4% from these North Atlantic type
fied to species with certainty. One sequence, KNl(skin), sequences. Without a more comprehensive genetic data-
grouped closely (99% bootstrap value) with the type base, we cannot exdude the possibility of origins outside
beaked whales but could not be identified to species due of the North Atlantic for some of these samples. The par-
to the scarcity of type species sequences for this family. tial humpback whale sequence (JS19b) was identical to
Two sequences (KNlb and KN3) grouped in the branch sequences from other humpback whales sampled near the
composed of the sei and Bryde's whale type sequences in Mexican, Hawaiian and Japanese (i.e. Ogasawara Islands,
100% of the bootstrap replicates. However, the KNlb and C.S.B. unpublished data) wintering grounds, suggesting a
KN3 sequences connect as a basal branch relative to the North Pacific origin.
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
Experimentnl reliability and consistency
Russia (Dizon et ul. 199%) were made available for com- Given the power of PCR to amplify even a singIe target
parison to our test and type sequences (courtesy of A. molecule, it is important to consider the possibility of con-
Dizon and R. Brownell, Southwest Fisheries Research tamination prior to and during laboratory handling as a
Centre, San Diego, CA). Our analysis agreed with Dizon et source of experimental error. Laboratory contamination
al. (199%) in grouping the Russian whale meat with the can be excluded for all odontocetes, all minke whales and
available Bryde's whale sequences but provided no evi- most fin whales, because these were not identical to type
dence that the confiscatedRussian products were from the sequences from either of our laboratories. For the two sam-
same population as the Korean products. Sequences of ples which were identical to sequences from our laborato-
KNlb and KN3 were, in turn, provided to researchers at ries (WS4, a North Atlantic fin whale, and JSlSb, a North
the Southwest Fisheries Centre for comparison to their Pacific humpback whale) contamination is extremely
larger catalogue of Bryde's whale sequences. This inde- unlikely. All reagents used in the field were new, all
pendent analysis agreed in placing the KNlb and KN3 equipment was decontaminated, non-aspirating tips were
sequences with the sei/Bryde's clade but failed to further used for micropipetting and no contamination was appar-
resolve the species identity of these products (personal ent in the negative controls. Contamination after the
it
KNI 1
KNZ
KNS
KN4
KN6
KN7
KNlO
KNl2
KNlS
KN13
KNl4
rl
compressed. These disagreements were resolved and match to the single sequence that was inserted into the
corrected before the data sets were merged for the final vector. As a result, a polymerase error will be camed
analysis. 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
Discussion magnitude would not substantially alter identificationof a
DNA sequences generated by PCR have tremendous sample to a particular species or population.
utility in the identification of species or evolutionarily A more serious but less common problem is the poten-
significant units. Over the past decade, the methodology tial to amplify a nuclear insertion of a mtDNA sequence
of molecular systematics has developed to allow reliable (e.g. a pseudogene, Lopez et ni. 1994)or to generate artifi-
construction and interpretation of phylogenetic trees from cial recombinants during amplification (e.g. PCR ‘jump-
DNA data. Confidence in phylogenetic reconstructions ing’, Thomas & Paabo 1993). The insertion and duplication
can be established statistically using procedures like boot- of mtDNA sequences into the nuclear genome can be an
strap resampling (Felsenstein 1988) or permutation tests aid (Zischler et d.1995)or a hindrance (Collura & Stewart
(Faith 1991). Much of this methodology is widely applica- 1995) to phylogenetic analyses. If unrecognized as a
ble in forensic studies (e.g. Hillis et nl. 1994). pseudogene, these xenologous or paralogous sequences
However, we have encountered certain constraints in could generate misleading phylogenies depending on the
our use of PCR and molecular systematics for the identifi- evolutionary timing of the insertion event. To date, how-
cation of whale and dolphin products. These constraints ever, no mtDNA pseudogenes have been reported in the
fall into two categories: (i) technical problems in the col- nuclear genome of cetaceans.
lection of molecular data using PCR; and, (ii) limitations A PCR recombinant can be generated if a partial copy
on the analysis of species-level systematic relationships of the target sequence is generated during the initial exten-
using molecular data. 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-
PCR and sequencing artefacts ferent template sequences in the reaction mix (derived
The target and specificity of the PCR reaction are con- from a mixture of tissues from two individuals or species),
trolled through the adjustment of reaction conditions and a partial copy (acting as a large primer) could anneal to a
through the choice of oligonucleotide primers that are template of the second speaes. This partial copy will then
designed to anneal only to a particular target DNA strand. be extended by the polymerase, with the result that the
Although the PCR is usually a precise and accurate tool, first part of the sequence will be from speaes ’A’ but the
there are circumstances in which it can provide unexpect- second part will be from speaes ‘B. PCR recombinants
ed results. Detailed descriptions of basic PCR methodolo- depend on an unlikely combination of two experimental
gy can be found elsewhere (e.g. Innis et af. 1990; Palumbi problems: multiple templates and incomplete extension
1995).Here we discuss technical issues that are important reactions. These seem unlikely to be consistent across
in understanding the forensic interpretation of the prod- replicate amplifications and, if they are generated at all,
ucts of a PCR reaction. will probably not produce a clear sequence. If they do,
Polymerase errors occur at a low frequency when the however, they will complicate phylogenetic analysis of the
Tnq polymerase is used in PCR reactions (and it is proba- DNA sequence data. By their nature, they are neither
bly the most widely used PCR enzyme). This is because species ‘A’ nor species ‘B,and have characteristics of both.
Tnq polymerase has no proof-reading function (i.e. no A phylogenetic reconstruction using these sequences will
exonuclease activity). As a result, when an incorrect typically place them at the base of branches leading to
nudeotide is added to the growing DNA strand during species ‘A’ and species ‘B, with low bootstrap values for
the extension step of PCR, it is not removed or replaced either grouping.
with the correct nudeotide. These polymerase errors are
rare and random in their distribution along the DNA
Molecular sysfematics and species identificafion
strands produced with PCR When PCR products are
analysed by restriction digestion or by sequencing of the Assuming that a particular DNA sequence has been
whole product (such as the solid-phase sequencing used amplified from a test product without artefacts (see above
here), low frequency errors at a particular position are far for alternatives) and is of sufficient length for phylogenet-
outnumbered by other templates which have the correct ic reconstruction, there are some circumstances in which
nucleotide. Thus,polymerase errors are usually not appar- species identification remains difficult. These difficulties
ent when using these techniques. However, if PCR prod- are common to the use of molecular systematics for the
ucts are cloned, the sequence of each clone is an exact description of evolutionarily sigruficant units for conser-
A. B. species B indiv. 1
species A indiv. 1
ICIC species A indiv. 2
species B indiv. 2
According to the IWC Schedule, minke whales Our analysis of type and test sequences from the sei and
(Buluenopteruucufmostrutuand B. bonaerensis) are defined as Bryde’s whales suggests that these two related species,
‘any whale known as lesser rorqual, little piked whale, like the minke whale complex, require further molecular
minke whale, pike-headed whale or sharp headed finner‘ systematic examination for both stock and species level
(Dizon ef ul. 1992). Recent molecular analyses by others classification W o n et ul. 1995). In pairwise comparisons,
and those presented here demonstrate that this definition the three type sei whale sequences differed from each
is inadequate. Four forms or subspecies of minke whales other by 1.2-3.0% and the four Bryde’s whales differed
have been described previously from morphology or geo- from each other by 2.0-3.6%. Differences between the type
graphical distribution: the North Atlantic (ucutorostrutu), sei and Bryde‘s whales ranged from 7.0 to 9.2%. Although
the North Pacific (dmidsoni), the Southern Hemisphere the ranges of these i n t r a s p d c differences did not over-
(bonuerensis) and the dwarf form (Best 1985; Dizon et ul. lap with the interspecific differences, the bootstrap values
1992). Based on an analysis of mtDNA control region supporting the species-specific groupings of type
sequences, Amason et ul. (1993) proposed that North sequences were low in some cases (58% bootstrap value).
Atlantic and Southern Hemisphere minke whales should Only the bootstrap support for grouping the New Zealand
be elevated to species status, B. ucutorostrutu and B. and North Pacific Bryde’s whales (analysis not shown)
bonuerensis, respectively. However, Amason et ul. (1993) approached that found in other conspecificsequences.The
did not include samples from the Southern Hemisphere previous analysis of mtDNA control region sequences by
dwarf form or the North Pacific population. A preliminary Amason ef ul. (1993)agrees in showing a close relationshp
analysis of mtDNA sequences (Hori et ul. 1994) suggests between the sei and Bryde’s whales, as represented by one
that the dwarf form is closely related to both Northern specimen each, but did not address the problem of
Hemisphere populations and these are distinct from the intraspecific variation within the two widely distributed
Southem Hemisphere common type. species.
Our analysis of available type sequences agrees with Further ambiguities in the taxonomy of the sei and
others (Hoelzel h Dover 1991c; Wada et ul. 1991; Bakke & Bryde‘s whale are indicated by our analysis of test
ElCewely 1992; Arnason et al. 1993; Hori et ul. 1994; sequences from the Korean whale products. Here, we dis-
Pastene ef al. 1994; van Pijlen et ul. 1995) in distinguishing covered two test sequences (KNla and KN3) that grouped
the two North Hemisphere populations from the common closely (91-100% bootstrap values) with both sei and
Southern Hemisphere type. Our analysis also agrees with Bryde’s whales type sequences but that could not be
Hori et al. (1994) in suggesting a slightly closer relationship asaibed to either recognized species with confidence. The
between the dwarf form and the North Atlantic popula- most complete of the two Korean sequences (KN3,437 bp
tion relative to the North Pacific population. However, the in length) differed by an average of 7.5% from both the sei
range of differences (2.144%) among type sequences and Bryde’s type sequences. This is greater than the dif-
from both Northern Hemisphere populations and ference between some type sequences of sei and Bryde’s
Southern dwarf form minke whales overlaps with that whales in our catalogue (7.0-9.2%, range). There are sev-
found in conspeafic populations of other mysticetes (e.g. eral alternative explanations for this pattern: (i) the anom-
humpback and fin) and is considerably smaller than that alous sequences are a nuclear pseudogene of the mtDNA
found between other species of Balaenopterid. control region or a PCR recombinant; (ii) the sequences
With available type sequences, we could identify prod- come from an anaent lineage (as yet unsampled) within
ucts derived from minke whales, as defined by the IWC the recognized sei or Bryde’s whale species; or, (iii) the
Schedule, with certainty (100% bootstrap value). Further, sequences are from an unrecognized species or subspecies
we could distinguish, with certainty, the products of the related to the recognized sei and Bryde’s whales.
Southem Hemisphere common form from those of the We tested for the possibility of amplifytng a control
dwarf form and Northern Hemisphere populations. region pseudogene by examining phylogenetic
Ascribing the origin of products to either the North Pacific reconstructions based on sequences of the mitochondria1
or North Atlantic population or the Southern Hemisphere cytochrome b gene amplified from the KNlb and KN3
dwarf form,however, was frustrated by the limited num- products (F.C., data not shown) and other mysticete
ber of type samples, low levels of divergence and possible whales (Amason & Gullberg 1994). If the control region
paraphyletic relationships between available sequences. A sequences were in fact a pseudogene, we would expect a
world-wide sample of mtDNA variation in minke whales, different topology from the cytoduome b gene unless this
with adequate oceanic and regional population represen- region was also included in the transposition. In fact, the
tation, is needed to fully resolve the relationships between cytochrome b phylogeny was consistent with the control
these species, subspecies or populations. region phylogeny, plaang KNla and KN3 in the
sei/Bryde’s clade but ancestral to both. The consistency of whales. Ancient mitochondria1 lineages have been found
these two phylogenies and the absence of pseudogene-like in other species with wide-scale distributions and distinct
sequences in other amplificaGons from test or type sam- subpopulation structure, e.g. humpbacks whales (Baker et
ples argues against the likelihood of this artefact. al. 1993),New Zealand fur seals (Lento et al. in press) and
A PCR recombinant could have arisen, as described black-backed jackals (Wayne et at. 1990). However, the
above, if our original template DNA included both sei and degree of divergence between the KNlb and KN3 test
Bryde’s whale DNA. This possibility warrants sequences and the type sei and Bryde’s whales is large
consideration given that pieces of meat from the one even in comparison to the ancient lineages of these other
purchase yielded the sei/Bryde’s sequence (Kiilb) while species. Further, the inclusion of the KNlb and KN3
the s k i yielded a beaked whale sequence (KNla). To test sequences as either sei or Bryde’s would imply a likely
for the possibility of in aitro recombination, we used a paraphyletic relationship of mtDNA among the recog-
computer program (available from S.R.P.) to calculate nized sei and Bryde’s whales.
nucleotide differences between type and test sequences Alternatively, the report of a genetically distinct ’small-
along a moving ‘window’ of 60 bp in length. If in vitro form’ of Bryde’s whale (Best 1977; Wada & Numachi 1991)
recombination had produced a sei/Bryde‘s hybrid suggests that the systematic relationships and taxonomic
sequence, then the beginning of the test sequences should status of the sei and Bryde’s whales is more complex than
be more similar to one species, whereas the end of the the currently accepted two-species designation (Dizon et
sequences should be more similar to the other. The nl. 1995). Eight small-form Bryde’s whale were killed
observed pattern of sequence difference in the moving during Japanese scientific hunting -near the Solomon
‘window‘ was not consistent with the expectations of Islands and in the eastern Indian Ocean near the Island of
recombination (Fig. 6). The two Korean sequences (KNlb Java from 1976 to 1979. Inspectors aboard the whaling
and KN3) showed about the same degree of difference to vessels recognized the whales as Bryde’s but, in the case of
both sei and Bryde’s sequences along its entire length. the Solomon Island catches, noted that they were sexually
Although the total degree of sequence heterogeneity mature at a smaller size than expected and that their
changed markedly along the length of this section of DNA, baleen differed in coloration from ordinary Bryde‘s
this is normal for comparisons of control region sequences whales. The whales from the eastern Indian Ocean were
where levels of sequence conservation shift from one func- not noted as distinct and were classified as small-form
tionally distinct region to the next (Hoelzel & Dover only after the genetic analysis. Allozyme analysis at 45 loci
1991a). showed that the genetic distance between the small-form
Because we found no evidence consistent with a PCR Bryde’s whale and the sei/Bryde’s whale is greater than
artefact, we suggest instead that KNlb and KN3 represent the sei and Bryde’s are to each other (Wada & Numachi
either an ancient mtDNA lineage within the recognized sei 1991).Furthermore, a U P G M A dendrogram based on the 45
or Bryde’s whales, or an unrecognized species or allozyme loci (Wada & Numachi 1991), places the ‘small
subspecies related to the recognized sei and Bryde’s form‘ Bryde‘s whale as an early divergent from the
1.0,
I comparison to:
0.8
0.6
0.4
common sei/Bryde’s whales lineage relative to the fin and tinctions between species, subspecies, morphological
minke whales. This placement is remarkably similar to the forms and populations or stocks. Of immediate manage-
relationship of sei, Bryde’s whales and the two Korean ment concern are the two species (or species complexes)
samples in our phybgram (Fig. 2). Researchers at the currently targeted by legal and illegal hunting, the minke
Southwest Fisheries Science Centre, National Marine and Bryde’s whales. Uncertainty in the current knowledge
Fisheries Service, have recently analysed mtDNA control of systematics and genetic population structure of these
region sequence from the skull of a small-form Bryde’s species complicated our molecular genetic identificationof
whale collected by W.F.Perrin in the Philippines. These the geographical origin of some products. The distinct
sequences are consistent with our conclusion that two genetic differences between the subtly different morpho-
samples of whale meat purchased in South Korea came logical forms of minke and Bryde’s whales raises the pos-
from this unrecognized species, although not necessarily sibility of ‘cryptic‘ and possibly endemic species of baleen
from this geographical location (personal communication, whales, similar to those reported among odontocetes (e.g.
A.E. Dizon, Southwest Fisheries Science Centre, La Jolla, Hoelzel & Dover 1991b; Rosel et nl. 1994). It is particularly
CA, USA). 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
Conclusions the acceptance of an international moratorium on com-
A wide diversity of whale and dolphin products appeared mercial whaling.
to be commonly available on the Japanese and Korean
domestic markets. Among the 34 sequences derived from Acknowledgements
31 products we identified at least eight species represent-
For collection of ‘test‘ samples from retail markets we thank four
ing three families and two suborders of cetaceans. Several
agents operating under the direction of Earthtrust and the
purchases induded products of mixed-species origin. In Endangered Species Project. These agents have asked to remain
some cases the possibility of more than a single species anonymous. For access to ‘type’ samples or unpublished
was indicated by multiple tissue types. In other cases, sequences we thank: T.Albert, U. Amason, A. Baker, I. Bakke, J.
there was no obvious indication that more than one Calambokidis, S. Costa, M. Dalebout, S. Dawson, A. Dizon, H.
species was involved until the DNA was amplified and Hori,C. Lux, J. Mead, L. Medrano, G. Notarbartolodi-Sara, G.
sequenced. Some of the species identified in the products OCorry-Crowe, C. Potter, L. Pastene, R. Paterson, P. Rosel, L.
Slooten, A. van Helden, G. Vequist and M. Zanardelli. For techni-
from the Japanese and Korean commeraal markets were
cal assistance and review we thank A. Perry, R. Brownell, 8.
not consistent with recent catch records made available to Bowen, A. Dizon, M. Donoghue, R.G. LeDuc and G. Lento. MJ
the International Whaling Commission (Baker & Palumbi Research, Inc. donated a PTC-150 portable MiniCyder for the
1994).Assessing the legality of speafic products has raised field analysis. Funding for this research was provided by
a number of uncertainties in the international and Earthtrust, the University of Auckland and the Medical
domestic management of whale products (&on et nl. Foundation for the Study of Environment and Human Body
1995a; Freeman 1995; Hansen 1995; Heinze 1995). Charitable Trust, and a grant from the Whale and Dolphin
Conservation society to the Endangered Species Project. The
Considerations raised by the whaling industry or the fieldwork in Korea was coordinated by S. Galster and S. LeBudde
Government of Japan include the following (Anon. 1998): and logistic support was provided by the Korean Confederation
the availability in Japan of some legal products from of Environmental Movements and the Civil Institute for
recent scientific hunting of Southern Hemisphere minke Environmental Studies. The original project was conceived by S.
whales; the extensive and largely unregulated coastal White and D. White of Earthtrust.
hunting of many odontocete species; the possibility of
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