Journal of the Science of Food and Agriculture J Sci Food Agric 80:527±533 (2000)

Identification of marine mammal species in foodproductsIciar Martinez1* and Anna K Danıelsdottir2

1Norwegian Institute of Fisheries and Aquaculture Ltd, N-9291 Tromsø, Norway2The Population Genetic Laboratory, Marine Research Institute, c/o Biotechnology House, Keldnaholt, IS-112 Reykjavik, Iceland

(Rec

* CoCont

# 2

Abstract: Reference samples and processed products (frozen, smoked, salted, dried and gravad meat

and blubber) from harp seal and minke, sei and ®n whales were tested for species-diagnostic molecular

markers by random ampli®cation of polymorphic DNA (RAPD) and single-strand conformational

polymorphism (SSCP) analysis of amplicons obtained using consensus primers for mitochondrial

cytochrome b (mt cytb). RAPD analysis produced clear ®ngerprints of the products analysed from

which the species could be easily identi®ed. The interpretation of the SSCP analysis was less clear,

probably owing to the use of consensus primers and, in ®n whales, to intraspeci®c polymorphism.

Except for one, all the commercial products were correctly labelled, and it was possible to identify the

species in all the products labelled simply as `whale': three of them contained minke whale and two ®n

whale.

# 2000 Society of Chemical Industry

Keywords: species identi®cation; RAPD; SSCP; marine mammals

INTRODUCTIONThe meat and blubber of marine mammals have

traditionally been important constituents of the diet of

countries such as Norway, Iceland, Greenland, Cana-

da and Japan. Other parts of the carcasses were used to

obtain products such as skins, cords, utensils, etc.

Although restrictions have been imposed on whaling

and sealing, this activity is legal for some species in

some countries, and the food products are still appre-

ciated by consumers. Meat and blubber are usually

purchased for human consumption in the form of

fresh, frozen or thawed products, or after preservation

by procedures such as smoking or salting. Identi®ca-

tion of the species used in the manufacture of these

products cannot be done visually, and seal meat has

occasionally been sold as whale meat, which is more

expensive. In addition, some species or stocks of

marine mammals are considered endangered.1 It is

therefore desirable for both consumers and the

authorities to have available methods that permit the

identi®cation of the species used in the manufacture of

a product to preserve consumer rights and current

legislation.

Species identi®cation can be achieved using protein-

and DNA-based methods (see reviews by Leighton

Jones,2 Mackie,3 Meyer and Candrian4 and Sotelo etal5). Protein pro®les are always tissue-dependent,

while species identi®cation using DNA-based

methods is independent of the tissues being compared.

Two DNA-based methods that have been used for

species identi®cation are single-strand conformational

eived 2 September 1999; accepted 8 November 1999)

rrespondence to: Iciar Martinez, Norwegian Institute of Fisheries andract/grant sponsor: Norwegian Research Council; contract/grant num

000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2

polymorphism (SSCP) of polymerase chain reaction

(PCR)-ampli®ed fragments6±8 and random ampli®ca-

tion of polymorphic DNA (RAPD).9±13 For processed

products, such as smoked or salted, in which the DNA

is well preserved or only partially degraded, RAPD has

been shown to produce good ®ngerprints,11 but in

heavily processed products (canning) the DNA is

usually degraded into smaller fragments. In the last

case the preferred techniques are those that target

small DNA fragments. The aim of the present work

was to test the suitability of RAPD and PCR-SSCP

analyses for species identi®cation in meat and blubber

products of marine mammals. It was not our aim to

determine the stock from which the sample originated,

since that would have required a different approach,

including a comprehensive genetic analysis of the

presumed existing stocks for each species with the

techniques used here. Determination of the stock can

be more easily undertaken once the species has been

determined.

MATERIALS AND METHODSSamplesThe samples and their descriptions are shown in Table

1. Samples 1±4, 9, 10±13 and 23±25 were reference

samples of harp seal (Phoca groenlandica), sei whale

(Balaenoptera borealis), minke whale (B acutorostrata)

and ®n whale (B physalus) respectively obtained during

Norwegian14,15 and Icelandic16 commercial and

scienti®c expeditions. Samples 8 and 14 were pur-

Aquaculture Ltd, N-9291 Tromsø, Norwayber: 113918/112

000/$17.50 527

Table 1. Samples analysed, their origin and means of identification

Sample no Processing (preservation) and tissue Species declared a

Species identi®ed by the

analyses

RAPD PCR-SSCP

1±4 Frozen muscle Harp seal (R) Harp seal Harp seal

5 Dried muscle Harp seal Harp seal Harp seal

6, 7 Smoked muscle Harp seal Harp seal Harp seal

8 Salted smoked cooked muscle Harp seal Minke whale Minke whale

9 Ethanol-preserved muscle Sei whale (R) Sei whale Sei whale

10 Ethanol-preserved muscle Minke whale (R) Minke whale Unidenti®ed

11, 12 Ethanol-preserved muscle Minke whale (R) Minke whale Minke whale

13 Dried muscleb Minke whale (R) Minke whale Minke whale

14 Dried muscle Minke whale Minke whale Minke whale

15, 16 Smoked muscle Whale Minke whale Minke whale

17 Gravad muscle Whale Minke whale Minke whale

18 Salted blubber Whale Fin whale Fin whale

19 Gravad muscle Whale Minke whale Minke whale

20 Dried muscle Fin whale Fin whale Fin whale

21, 22 Qiporaq muscle Fin whale Fin whale Fin whale

23±25 Ethanol-preserved muscle Fin whale (R) Fin whale Fin whale

a R, reference sample.b Dried for 2years at the laboratory.

Figure 1. Agarose gel electrophoresis (0.01gmlÿ1) of undiluted DNAextracts. L, standard l DNA (23kb). The sample number is indicated on topof each lane.

I Martinez, AK DanõÂelsdoÂttir

chased at local stores in Tromsù. The shop at which

sample 8 was purchased sold salted smoked cooked

products of both minke whale and harp seal. Sample 8,

which was unlabelled, was claimed to be made of harp

seal. The rest of the products were purchased at local

stores in Greenland.

DNA extractionGenomic DNA extraction was carried out according

to Miller et al17 with the modi®cations described

below. About 50±100mg of muscle samples were

weighed in 2ml Eppendorf tubes and ®nely cut with

scissors. Then 400ml of lysis buffer,17 0.4M NaCl,

10mM Tris-HCl, pH 8.0 and 2mM ethylenediamine-

tetraacetic acid (EDTA), pH 8.0 were added to each

tube, followed by 10ml of 0.2gmlÿ1 SDS and 10ml of

20mg mlÿ1 proteinaseK, which gave ®nal concentra-

tions of 5mg mlÿ1 and 0.5mg mlÿ1 respectively. The

contents of the tubes were mixed by inversion several

times and incubated at 55°C for 3h or overnight with

occasional mixing. After incubation the samples were

cooled on ice, and 360ml of 6M (saturated) NaCl was

added to each tube. The tubes were vortexed for 1min

at maximum speed and centrifuged for 20min at

16000�g in an Eppendorf 5415 C (Hamburg,

Germany) bench centrifuge. The supernatants were

recovered and transferred to new tubes, and isopro-

panol in a 2:1 (v/v, isopropanol/supernatant) ratio was

added to each tube. The tubes were inverted several

times, incubated at ÿ20°C for at least 1h and

centrifuged for 30min at 16000�g. The supernatants

were discarded and the DNA pellets were washed with

70:30 (v/v) ethanol/water. The pellets were dried at

room temperature and resuspended in 200ml of 50mM

Tris-HCl, pH 8.0. The amount and quality of the

DNA were estimated by comparison with known

528

amounts of l DNA after electrophoresis on 0.01g

mlÿ1 agarose gels in 0.5�Tris-boric-EDTA (TBE)

buffer.18 Fig 1 shows the pattern of the DNA extracted

from the samples analysed.

DNA amplificationArbitrarily primed ampli®cations19,20 were performed

in 30ml volumes. For each sample, two dilutions21,22

containing approximately 1 and 5ngmlÿ1 DNA were

ampli®ed as follows. 10ml aliquots of the DNA extracts

containing 10 and 50ng of the template DNA were

added to 20ml of a mixture to give ®nal concentrations

of 1�DNA polymerase buffer (supplied by the

manufacturer), 100mM each dATP, dCTP, dGTP

and dTTP, 0.4mM 10-mer primer (Operon Technol-

ogies Inc, Alameda, CA, USA) and 4mM MgCl2 (®nal

concentration)23 with 0.75 units of Klen Taq1 DNA

polymerase (AB Peptides, St Louis, MO, USA). The

primers used were M-01 (5'-GTT GGT GGC T-3')and T-20 (5'-GAC CAA TGC C-3'). The reaction

mixtures were overlaid with 20ml of Chill Out Wax

(MJ Research Inc, Watertown, MA, USA), and

J Sci Food Agric 80:527±533 (2000)

Species identi®cation of marine mammals

ampli®cation was performed on a PTC-100 program-

mable thermal controller (MJ Research Inc, Water-

town, MA, USA). The thermal programme for

ampli®cation was 94°C for 1min, followed by 40

cycles of 94°C, for 10s (denaturation), 35°C, for 10s

(annealing) and 72°C for 1min (extension). The

programme included a ®nal step of 72°C for 5min,

and the products were maintained at 4±15°C for a few

hours, or at ÿ20°C for longer periods, until ready to

load onto the gels.

Two sequences of the mitochondrial DNA cyto-

chrome b (mt cytb) gene were ampli®ed for SSCP

analysis: a 358bp fragment24 using primer cyt bL1 (5'-CCA TCC AAC ATC TCA GCA TGA TGA AA-3')and primer cyt bH (5'-CCC CTG AGA ATG ATA

TTT GTC CTC A-3'), and a 148bp fragment7 using

primer FB 349 (5'-GTC GAA TGA ATC TGA GGA

GGC TT-3') and primer FB 496 (5'-CCR ATT GGG

TTG TTT GAC CCT GTT TC-3'). Ampli®cations

were performed in 30ml volumes. 10ml aliquots of the

DNA extracts containing approximately 10ng of the

template DNA were added to 20ml of a mixture to give

®nal concentrations of 1�DNA polymerase buffer

(supplied by the manufacturer), 100mM each dATP,

dCTP, dGTP and dTTP and 0.5mM each primer

(Research and Development Systems, UK) with 0.75

units of Klen Taq1 DNA polymerase. The reaction

mixtures were overlaid with 20ml of Chill Out Wax,

and ampli®cation was performed on a PTC-100

programmable thermal controller. The thermal pro-

gramme for ampli®cation was 94°C for 1min,

followed by 40 cycles of 94°C for 10s (denaturation),

55°C for 10s (annealing) and 72°C, for 30s (exten-

sion). The programme included a ®nal step of 72°Cfor 5min, and the products were maintained at 4±

15°C for a few hours, or at ÿ20°C for longer periods,

until ready to load onto the gels.

Agarose gel electrophoresis12ml aliquots of the products obtained after RAPD

were separated in 20cm�10cm, 0.02gmlÿ1 Nusieve/

Seakem LE (1:3) FMC agarose gels (three gels per

chamber). Gel and electrophoresis buffers were

0.5�TBE,18 and electrophoresis took place for about

2h at 4.5Vcmÿ1. After electrophoresis the gels were

stained for 20min in 0.5�TBE buffer containing

0.5mg mlÿ1 ethidium bromide, destained for another

20min in the same buffer without ethidium bromide,

and photographed under ultraviolet light with a

Polaroid camera using ®lm type 55. The presence or

absence of RAPD markers was visually determined on

the photographs.

Single-strand conformational polymorphismanalysisThe Amersham Pharmacia Biotech (Uppsala,

Sweden) ExcelGel DNA Analysis kit was used.

According to the instructions of the manufacturers,

5ml aliquots of the products were denatured with 20ml

of 50mM NaOH and 1mM EDTA at 50°C for 15min

J Sci Food Agric 80:527±533 (2000)

and placed immediately in ice water. 7ml samples were

loaded onto 0.125gmlÿ1 polyacrylamide gels (Excel-

Gel 48S, DNA). Electrophoresis took place in a

horizontal chamber at 20°C for 2h at 600V, 50mA,

30W. The gels were silver stained according to Bassam

et al. 25

RESULTS AND DISCUSSIONEffect of processing on quality of DNAThe type of tissue and history of a sample, including

the time elapsed from the death of the individual until

processing, the processing conditions and the condi-

tions used to preserve the sample, have a major effect

on the amount and quality of DNA that can be

obtained from it. In general, and as expected, old

samples (Fig 1, no 13) and some samples stored in

96:4 (v/v) ethanol/water (Fig 1, nos 10, 24 and 25)

rendered less DNA than samples stored frozen for a

short period of time (Fig 1, nos 3 and 4). However, all

processing conditions tested here (drying, smoking,

salting, cooking and gravad) produced samples from

which high-molecular-weight DNA could be extracted

in suf®cient amounts to perform ®ngerprint analyses

(Fig 1). This means that, in principle, they would have

been suitable for analysis by a variety of DNA-based

methods and not only RAPD or PCR-SSCP.

General considerations about RAPD analysisTo perform RAPD analysis, several variables need to

be standardised. For ampli®cation, the equip-

ment,26,27 the ampli®cation programme,27±29 the

composition of the ampli®cation buffer, the DNA

polymerase used23,26,30 and the amount and quality of

the DNA19,21,27,31 need to be optimised. For electro-

phoresis, the amount and quality of agarose, the

composition of the buffer and the length of the gel28

require standardisation. Once the analysis is standar-

dised in a laboratory, the paÁtterns are usually

reproducible. In our laboratory, for example, three

different persons have carried out RAPD analysis of

over 700 minke whales, using two different thermal

cyclers (one of which is slightly faster than the other),

and the patterns were always similar. For this work,

RAPD analysis was performed using two different

DNA concentrations for each sample to ensure that

the patterns were independent of the amount of DNA

used. Some of the samples of minke and ®n whales and

the sei whale sample have been analysed more than 10

times. However, lab-to-lab variations and variations

(including batch-to-batch variations from the same

supplier) in the quality of the reagents, buffers and

DNA polymerase are to be expected. Therefore, in our

opinion, conclusive species identi®cation should be

carried out by analysing the unidenti®ed sample in the

same PCR and same gel as the reference samples.

Species identification by RAPD analysisIn the present work, and con®rming previous stu-

dies,10,11,13,19,20,32 each sample±primer combination

529

Figure 2. RAPD analysis on 0.02gmlÿ1 agarose gels with primers (a) M-01and (b) T-20. In (a), lanes 1 and 28 contain the standard 1kb ladder; lane 27contains the blank (no DNA added); and lanes 2–26 contain samples 1–7(harp seal), 9 (sei whale), 25 (fin whale), 10–12 (minke whale), 8 (declaredharp seal), 13–17, 19 (minke whale) and 18, 20–24 (fin whale) respectively.In (b), lanes 1 and 28 contain the standard 1kb ladder; lane 27 contains theblank (no DNA added); and lanes 2–26 contain samples 1–7 (harp seal), 9(sei whale), 25 (fin whale), 10–12 (minke whale), 8 (declared harp seal),13–15 (minke whale), 24 (fin whale), 17, 19 (minke whale) and 18, 20–24(fin whale) respectively.

I Martinez, AK DanõÂelsdoÂttir

produced a characteristic RAPD ®ngerprint from

which the species could be identi®ed (Fig 2). Primers

M-01 and T-20 were chosen because they seemed to

Table 2. Number of bands produced inminke, fin and sei whales with primersM-01 and T-20, and proportion ofshared bands between pairs of species

Primer

No of bands produce

Minke whale Fin wha

M-01 12 14

T-20 18 18

Minke whale Sei wha

M-01 12 11

T-20 18 19

Fin whale Sei wha

M-01 14 11

T-20 18 19

Minke whale Harp se

M-01 12 14

T-20 18 18

Fin whale Harp se

M-01 14 14

T-20 18 18

Sei whale Harp se

M-01 11 14

T-20 19 18

530

produce monomorphic patterns with minke whale

DNA.21 RAPD ampli®cation was performed with two

DNA concentrations for each sample (10 and 50ng

DNA per reaction), and it was assessed that the

®ngerprints were not concentration-dependent.19,21

Fig 2 shows the results obtained with 10ng DNA per

reaction in order to display in the same gel all the

samples of this study. Some bands can be observed in

the blank lanes (no DNA added). The presence of

these bands is not unusual in RAPD analysis, and they

have been attributed to the presence of small amounts

of DNA contaminating DNA polymerase.20,33,34

RAPD showed that sample 8 was not harp seal, but

minke whale meat, and that all the other products were

correctly labelled. Since this product was not labelled,

the shop sold similar products of minke whale, and the

minke whale product was more expensive, we consider

this to have been an honest mistake by the employee

who sold us the product.

The total number of bands generated by each

primer±DNA combination, as well as the proportion

of shared bands between pairs of species, is shown in

Table 2. Both primers generated a few polymorphic

bands using DNA from seven different harp seals, but

the ®ngerprints could be easily recognised as belong-

ing to the same species. It is not possible to assert the

number of polymorphic markers that these primers

could have produced in sei whale, since only one

sample was available for this analysis.

In general, the closer the genetic relationship

between samples, the more similar their RAPD

patterns will be,12,35 but it must be kept in mind that

the fact that two bands ampli®ed from different

samples display similar electrophoretic mobility does

not mean that they are identical, especially if the DNA

belongs to different species. Therefore the number of

shared bands as considered here is an overestimate of

d

Total different bands

Apparently shared bands

le No Proportion Average Proportion

19 7 0.368

29 7 0.241 0.305

le

17 6 0.353

30 7 0.233 0.293

le

17 8 0.471

29 8 0.276 0.373

al

25 1 0.040

35 1 0.029 0.034

al

26 2 0.077

34 2 0.059 0.068

al

23 2 0.087

36 1 0.028 0.057

J Sci Food Agric 80:527±533 (2000)

Species identi®cation of marine mammals

similarity between two samples. The discrimination

between harp seal and whale meat was clear: including

the results of both primers, less than seven in 100 of

the markers could be considered as shared bands. The

closely related whale species, on the other hand, had

more markers in common, varying between 29 in 100

between minke and sei whales and 37 in 100 between

®n and sei whales.

The degree of processing to which these samples

had been submitted (drying, smoking, salting, cooking

and gravad) did not affect the RAPD pattern (Fig 2),

con®rming our previous results with ®sh11 and

terrestrial mammalian meat products.12 In our experi-

ence the processing parameters do not per se have a

direct effect on the quality of the RAPD patterns, but

they do have a direct effect on the amount and quality

of extractable DNA. If the DNA is very degraded, it

will be unsuitable for ®ngerprinting.11,12 Thus RAPD

analysis is very likely inadequate to produce reliable

®ngerprints from products subjected to more stringent

procedures that degrade the DNA to small fragments,

such as sterilisation.11,36

Species identification by PCR-SSCP analysisThis analysis involves the ampli®cation of variable

mtDNA sequences in the cytb gene7,24 and requires

the use of consensus primers (to be able to produce

amplicons from a variety of species) that amplify a

variable or hypervariable region of mtDNA. The

ampli®cation product should be only one fragment

corresponding to the targeted region. The amplicons

produced from different species differ in sequence but

often not in their size, which demands additional

analyses such as sequencing,37,38 RFLP with several

restriction enzymes37,39 or SSCP.7,8,40±42

The amplicons obtained are shown in Fig 3.

Figure 3. Agarose gel electrophoresis (0.02gmlÿ1) of amplicons producedwith primer pairs (a) cyt bL1 and cyt bH (358bp fragment) and (b) FB 349and FB 496 (148bp fragment). Samples in (a) are from left to right: blank(no DNA added), 1kb ladder, 2, 6, 9, 10, 12, 13, 15, 18, 1kb ladder, blank(no DNA added), 1kb ladder, 17, 19, 20, 21, 22, 23, 24, 1kb ladder, 1kbladder, 1, 5, 7, 25, 11, 8, 14, 16 and 1kb ladder. Samples in (b) are: lanes 1and 26, 1kb ladder; and lanes 2–25 contain samples 1, 2, 5–7, 9, 25,10–12, 8, 13–17, 19, 18, 20–24 and blank (no DNA added).

J Sci Food Agric 80:527±533 (2000)

Although we increased the annealing temperature

from 50°C8 to 55°C, several amplicons were pro-

duced: primer pair bLl±bH produced two amplicons

in some of the samples, and primer pair FB 349±FB

496 failed to produce an amplicon with harp seal (Fig

3). Therefore only the products from the ampli®cation

obtained with primer pair bLl±bH were submitted to

SSCP analysis (Fig 4). Although it is clear from the

®gure that the conditions used here are not optimal for

the identi®cation of these species, all the minke whale

samples, except no 10, gave identical pro®les. Also, the

electrophoretic mobilities of the single strands of

DNA, as well as that of the double-stranded amplicon

obtained from sample 10, were clearly different from

all the others.

The sequences ampli®ed by this analysis are chosen

owing to their higher mutation rate, and they often

present intraspecies polymorphic diversity.39±42 In-

traspecies polymorphisms were detected here in the ®n

whale samples, some of which were indistinguishable

from minke whale (samples 18 and 21 in Fig 4).

Comparison of the two methodsRAPD ®ngerprinting appeared to be a better diag-

nostic method, since the patterns were species-speci®c

and did not require previous knowledge of DNA

sequences of the species under study.19,20 Further-

more, RAPD targets many sequences in the DNA of

the sample, producing complex patterns that allow

comparison of many loci simultaneously. Thus, after

arbitrary ampli®cation, comparisons can be carried

out on the basis of the electrophoretic mobility of

RAPD markers. SSCP-PCR of mt cytb sequences

requires some prior knowledge of speci®c DNA

sequences and is intended to target only one locus.

Because there are not species-speci®c sequences for all

possible species, consensus primers have to be used

Figure 4. SSCP analysis on 0.125gmlÿ1 polyacrylamide gels of ampliconsproduced by primer pairs cyt bL1 and cyt bH (358bp fragment). Lanes 1and 25 contain the standard 1kb ladder; and lanes 2–24 contain samples 1,2, 5–7 (harp seal), 9 (sei whale), 25 (fin whale), 10–12 (minke whale),8 (declared harp seal), 13–17, 19 (minke whale) and 18, 20–24 (fin whale)respectively.

531

I Martinez, AK DanõÂelsdoÂttir

which may have some degree of similarity to other

mitochondrial or genomic DNA sequences. This may

result in more than one amplicon, as seems to have

been the case here, requiring optimisation for each

amplicon. It is possible that some of the bands

observed in these gels may be artefacts generated

during electrophoresis. In particular, the running

temperature is critical, and smeared patterns were

obtained when electrophoresis was performed at 4, 8

and 10°C. As a consequence, SSCP analysis and

interpretation may be problematic. Thus, provided

that good-quality DNA can be obtained from a

sample, RAPD seems to be easier, faster and give

more information regarding species identi®cation than

SSCP-PCR analysis.

Based on our results with horse and donkey,12 the

work of Elo et al43 with brown trout and Atlantic

salmon, and the present results with baleen whales, it

seems that species capable of producing hybrids share

about 30 in 100 of their RAPD markers; hybrids and

each of the parental species (as well as 50:50 mixtures

of species) share about 50 in 100 of them,12 and races

or stocks from the same species have more than 50 in

100 of the total number of markers generated by this

analysis in common.12 Therefore RAPD analysis has

the advantage that in one step it is possible to establish

whether the sample corresponds to the species given

on the label (or not) by comparing the ®ngerprints of

the product and the reference samples. In the case of a

mismatch, RAPD can give an indication of whether a

similar species (or a mixture) or a totally different

species has been used.

Identi®cation of the individual species that might

have been included in mixtures in ®nely minced or

comminuted products (pateÂ-type products) would not

be feasible by RAPD, because it would not be possible

to identify which bands come from which species, or

the relative contribution of each species to the total

®ngerprint.11 In coarse mixtures, where it is possible to

take whole pieces of tissue, RAPD typing can simply

be performed on a number of pieces, since it requires

less than 50mg of starting material.

SSCP-PCR, or similar analyses targeting smaller

sequences, may be the only method available if the

product has been submitted to severe conditions such

as canning, where only degraded DNA can be

extracted. The use of consensus primers speci®cally

designed for marine mammals may make this analysis

suitable for these species, but the discriminant

capability of SSCP-PCR, at least as used here and

for the species used in this work, would appear to be

inferior to that of RAPD.

One advantage of the RAPD method is that com-

parison of the RAPD patterns produced by two

samples may immediately give an idea of how closely

related they are. A second advantage is illustrated by

sample 10. Since RAPD targets the total DNA, it

showed sample 10 to be a minke whale. In contrast,

the SSCP-PCR analysis could only indicate that this

sample was different from all the others. Since

532

mtDNA has been shown to be mostly maternally

inherited,40,44 and given the intraspecies polymorph-

isms detected in the SSCP-PCR analysis, the only

conclusion that could be made was that this particular

individual did not share its maternal ancestral line with

any of the others.

ACKNOWLEDGEMENTSWe wish to thank Drs Tore Haug, Kjell T Nilssen and

their co-workers (Norwegian Institute of Fisheries and

Aquaculture Ltd, Tromsù Norway) for organising the

infrastructure for the collection of the Norwegian

samples; Dr Alfred AÂ rnason from the Blood Bank and

the Marine Research Institute, Iceland for the collec-

tion of the Icelandic samples; and the Norwegian

Research Council for ®nancial support (project

113918/112).

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