Origin  and  genetic  variability  of  migratory  individuals  of  harp  

seals  (Pagophilus  groenlandicus)  from  Norway  

 

 

 

 

 

 

 

 

A report submitted in part fulfilment of the examination requirements for the

award of a B.Sc. (Hons) Biology awarded by the University of Lincoln, June

2014, supervised by Malgorzata Pilot

 

Cameron  Brown  

 

10238961  

Words: 5,118

Acknowledgments

Firstly, I thank Dr. Anne Kirstine Frie for providing the sample material for this

study, without her there simply wouldn’t have been a study. To my colleagues

that I worked with in the labs who provided entertainment during rest periods

and the lab technicians that were available for help whenever I needed it. But

in particular I would like to acknowledge my dissertation supervisor

Malgorzata Pilot. Malgorzata was always in touch with me and always made

me feel welcome to ask questions and organised meetings to check my

progress. Along with her reassurance and guidance when my work seemed to

get on top of me, I can honestly say that I was extremely lucky with my

chosen dissertation supervisor.

CERTIFICATE OF ORIGINALITY

This is to certify that I am responsible for the work submitted in this thesis,

that the original work is my own, except as specified in the

acknowledgements and in references, and that neither the thesis nor the

original work contained therein has been previously submitted to any

institution for a degree.

Signature: C.Brown

Name: Cameron Brown

Date: 08.04.2014

Abstract

Harp seal (Pagophilus groenlandicus) is a species of seal found in three

distinct populations; the Northwest Atlantic Ocean, Greenland Sea and

Barents Sea (which breed in the White Sea). Harp seal migrate on large

distances of up to 19,000 miles. These migrations are often seasonal e.g.

between breeding grounds and feeding areas in the Arctic Ocean, but also

can be due to climate change and food availability. An ancient population of

harp seals existed during the Atlantic and Subboreal periods in the Baltic Sea,

south of the present species range, but it became extinct, and the reasons for

this extinction are uncertain. Contemporarily migrating harp seals are

observed along the northern coastline of Norway. This study is aimed to

assess the origin of the harp seals from Norway by sequencing the control

region of mitochondrial DNA (mtDNA) and comparing genetic variability of this

region between this population, the modern White Sea population and the

ancient Baltic Sea population. The results suggest that the seals from Norway

and the ancient Baltic Sea most likely originated from the White Sea breeding

population. Each Norwegian individual analysed had a different mtDNA

haplotype, showing that the migratory groups are large and consist of

unrelated individuals. Genetic similarity between the ancient Baltic Sea

population and both Norwegian and White Sea harp seals suggests that the

Baltic population could have originated from individuals migrating from the

White Sea along the Norwegian coast that moved further south as compared

with the migrations observed at present. While this study provides support for

the hypothesis that harp seals from Norway may originate from the White

Sea, it cannot be excluded that there may be more than one source

population. Data from other harp seal populations, especially from the

Greenland breeding colony, are needed to assess whether the Norwegian

migrants originate from one or multiple sources. This study produced the first

genetic data on the Norwegian harp seals, which provide a valuable starting

point for further studies.

Introduction

Harp seals (Pagophilus groenlandicus) are thought to only be able to breed in

a habitat with ice, which they are believed to need to whelp their offspring

(Johnston et al. 2005). Therefore the findings of the harp seals off the coast of

Norway (Nilson, 1992) brings about the question of how and why some of

them migrate south outside of the breeding season. Migration could be due to

intra-specific competition, if the number of pups being born is too large to be

sustained within the regular range of this species in the Arctic (Benjaminsen,

1979). It is well documented that there is a high mortality in seal pups (Mattlin,

1978), and the work of Mattlin (1978) on New Zealand fur seals suggests that

the vast majority of pup deaths are due to starvation. It could be assumed that

the reason for the harp seals migration or ‘invasion’ as some have called it

could be the search by subadult individuals for a more sustainable area to live

in. While harp seals in the Arctic feed on mainly Arctic and Atlantic cod

(Stenson, 1997), individuals found off the cost of Norway have been feeding

on a variety of fish and even low numbers of squid (Haug et al. 1991). This

suggests that this area is habitable for them despite having different types of

prey species.

Fig. 1 - Picture of an adult harp seal courtesy of Lowry and Bluhm (2008) of Arctic Ocean of diversity.

Biology of harp seal There are three main distinct populations of harp seal that inhabit the Arctic

and North Atlantic oceans (Sergeant, 1976): the Greenland Sea population,

the White Sea population and the Northwest Atlantic population (Perry et al.,

2000). Figure 2 shows these populations, the ancient Baltic Sea population,

and also where the samples in this study where taken from. The harp seals

life cycle begins with an on-ice birth, progressing on to in-water mating then

an on-ice moult (Ronald & Dougan, 1982). However it is important to get a full

understanding of how these animals mature and mate. Pups initially have

yellow fur when they are born, which turns white after a few days. The mother

will continue to feed the pup up until around 10 days after birth when the pup

is weaned off the mother’s milk. The mother will leave to mate in water whilst

the pup begins to moult into a silvery-grey colour with irregular dark spots. As

the pup matures the dark spots become larger and the characteristic harp

shape is forming on the back of the seal (Nowak, 1999), signalling sexual

maturity between 4 and 6 years of age. Harp seals are a highly migratory

species, typically migrating northwards beginning in their breeding grounds to

the feeding areas in the Arctic Ocean. The White Sea population, for example,

migrates up into the Barents Sea and to Svalbard. This pauses briefly for the

pups to moult on pack ice (Nowak, 1999). After the moulting begins the

summer migration into the feeding grounds. Feeding is most intensive in the

Figure. 2 – Map showing locations of Greenland Sea, Northwest Atlantic, White Sea and Baltic Sea populations as well as the location on the Norwegian coast where samples for this study were gathered.

winter and summer, with spring and autumn being less intense (Sergeant,

2011). A harp seal diet is largely varied depending on age, location, season

and year, from small fish to ocean dwelling crustaceans (Würsig, 2008).

Migratory patterns of seals Migration is common in most species of seal, including the harp seal. Harp

seals appear to have seasonal migrations as implied by Sergeant (2011), who

described how pregnant females will move north during the winter months to

whelp and south during spring along with immature seals. Mating usually

occurs on pack ice were males will fight for females using their hind and fore

flippers (Nowak, 1999). Although this study took place in the Northwest

Atlantic along the Canadian Arctic coast, it does give us a good idea of the

migratory range of the harp seal. The study shows a migration from the Strait

of Belle Isle (a narrow stretch of sea between the north east of Canada and

Newfounland) to the western coast of Greenland, around 19,000 miles.

Long distance migration is also common in other species of seals. For

example, the northern elephant seal (Mirounga angustirostris) has been

shown to migrate more than 11,000 miles (Brent, et al. 1995). Both males and

females of the ringed seal have been seen to migrate a distance of 3,000km

during the summer months. It is thought that they migrate when the sea ice is

at its minimum (Martinez-Bakker, et al. 2013), suggesting that the rate of

migration of ringed seals is linked to sea ice conditions.

Threats One of the most prevalent and world-wide environmental changes seen over

the last few centuries has been climate change. Although harp seals are

adapted to deal with varying sea ice thickness, it has been shown that their

mortality increases as sea ice cover decreases, as it has been at a rate of 6

percent between 1979 and 2011 (Johnston, et al. 2012). Reduced sea ice has

also been shown to increase the stranding rate of yearling harp seals and the

relationship between stranding and ice cover was strongest during light ice

periods (Soulen, et al. 2013). Migration, therefore, is often beneficial to both

adult harp seals and pups. Comparisons between harp seal yearlings of the

Barents Sea and northern Norway showed that the seals of northern Norway

were of a significantly poorer condition to those in the Barents Sea, and

mature females followed a similar trend (Nilsen, et al. 1995). These long

distance migrations can result in the isolation of populations migrating to

different locations and not coming into contact with one another. This can lead

to genetically differentiated populations (Perry et al. 2000) that can be

identified e.g. by analysis of mitochondrial DNA.

Harp seals of the Baltic Sea Southern migration of harp seals also occurred in the past, as their

inhabitancy of the Baltic Sea during the Atlantic and Subboreal periods has

been documented based on the numerous sub-fossil remains of this species

(Forsten, 2008). Stora (2004) suggested that it was the high organic

productivity, higher salinity and warmer climate that drew the harp seal to the

area in comparison to the more northern environments they are used to

inhabit. Lepiksaar (1986) also suggested that the surrounding ocean waters

moving into the Baltic Sea increased its nutrient levels. The extinction of the

Baltic harp seal has been implied to be due to a number of reasons. Mclean

(1986) hypothesised that the warmer climate decreased the fertility of female

harp seals. As they need to cool their body, blood, and therefore heat, is

shifted to the extremities, which reduces blood flow to the embryo. Over-

hunting by Stone Age man has also been suggested to be a cause of

population decline as bone harpoons and skeletal evidence has been found to

suggest the substantial killing of the seals (Stora, 2000). Lastly, interspecific

competition between other seal species in the area may have been

responsible for the harp seals decline. We know that three other species of

seal were and still are present in the Baltic Sea, and they could have been

better adapted to cope with the warmer temperature in the area (Sergeant,

1991). The fact of the past existence of the harp seal population in the Baltic

Sea provides a good example of how the harp seal has previously migrated to

an environment largely different from what is thought to be its preferred

habitat.

Determining genetic variation using mitochondrial DNA Mitochondrial DNA (mtDNA) is often used for the comparison of distantly

related individuals of the same species. This is because the mtDNA comes

solely from a maternal source, and this inheritance pattern makes it easier for

comparison between distantly related individuals (Brown, 2001). Because of

its maternal inheritance, mtDNA does not mix with other DNA strains when

passed down to offspring, meaning that the mtDNA of the offspring is exactly

the same as its mothers, except for the rare cases when a mutation occurs in

the offspring. This gives a direct line of descent through the maternal line of

an individual’s ancestry. Therefore, mitochondrial DNA analysis is ideal for

comparing genetic variation between groups of seals. In a study on harp seals

(Perry et al. 2000), comparison of the sequence of the mitochondrial

cytochrome b gene showed the phylogenetic and population relationships

between the seals. This study showed that the large populations in the

Greenland Sea, the White Sea and the Northwest Atlantic sampled were

genetically different, but the two subpopulations in the Northwest Atlantic were

not genetically differentiated. This meant the three main populations were

genetically isolated, but the sub-populations in the Northwest Atlantic were

not. Another example of a study determining the geographical isolation of

seals comes from Boskovic et al. (1996). This study analysed restriction

fragment length polymorphisms in the mitochondrial DNA of grey seals. This

allowed them to estimate when the populations of grey seals diverged.

Findings suggested that Eastern and Western Atlantic populations of the grey

seal diverged around 1-1.2 million years ago, whereas the Baltic Sea and

Norwegian populations diverged around 0.35 million years ago. In a study on

northern elephants seals by Hoelzel et al. (1993) mitochondrial DNA was

analysed in two regions (control region and 16S RNA). This study looked into

the genetic diversity of elephant seals along the coast of the USA and Mexico.

During the 19th century northern elephant seals were heavily exploited

resulting in a genetic bottleneck. It was found this species had little diversity in

their mitochondrial DNA.

Aim of the Study A population of harp seals is now present of the coast of Norway. Using DNA

extraction, amplification and analysis of muscle tissue from harp seal

mortalities in Norway, this study aims to determine the origin of this

population. The mtDNA sequences gathered from Norway will be compared

with previous data gathered from ancient and modern seal populations.

Material and Methods

Material The tissue samples of 30 harp seals from Norway were provided by Dr Anne

Kirstine Frie; they come from the collection of the Marine Research Institute in

Tromso, Norway.

Laboratory Procedures

The samples were processed in three steps: DNA extraction, PCR

amplification of a mtDNA fragment (the control region) and purification of PCR

products. After each stage the samples were ran through a gel

electrophoresis in order to check for the presence of DNA bands and confirm

the absence of contamination in negative controls.

DNA extraction – High salt method A small amount of tissue was taken from each sample and cut finely. This was

then placed into a 1.5ml microcentrifuge tube and labelled with the sample ID.

In each batch one tube was always used as a control without any tissue

added. 500µl of TNE buffer, 25µl of SDS and 10µl of proteinase K were added

to each tube and mixed by vortexing. The TNE buffer was a mixture of TRIS,

NaCl and EDTA. The samples were sealed with a parafilm to prevent the lids

from opening and incubated overnight at 55°C. After this 250µl of 6 M NaCl

was added and shaken by hand. The samples were then microcentrifuged at

12-14000 rpm for 10 minutes. The supernatant was removed and transferred

to new labelled tubes, leaving a pellet of cell debris at the bottom of the tube.

An equal volume of 100% ethanol (~750µl) was added to the supernatant and

gently mixed by inverting the tube, then placed at -20°C for 30 minutes. The

sample was then centrifuged again at the same speed for 20 minutes. The

supernatant was removed and disposed of leaving a pellet of DNA. 1000µl of

ice-cold 70% ethanol was added and mixed by inverting. The sample was

centrifuged at the same speed for 5 minutes. The supernatant was again

removed without dislodging the DNA pellet. In order to remove all of the

ethanol, the samples were centrifuged again briefly to bring all remaining

ethanol to the bottom so it can be removed. The sample was left to air dry,

and when dry was suspended in 100µl of elution buffer (Tris-EDTA buffer).

Samples were frozen for storage. A negative control was added to each set of

samples that underwent the extraction procedure.

Gel electrophoresis After DNA extraction 10µl of each sample was ran through a gel

electrophoresis. The gel was first made by dissolving 0.8g of agarose in 80ml

of 0.5M TBE (Tris, boric acid and EDTA) buffer and heated until the agarose

had dissolved. 1.2µl of ethidium bromide was added and mixed. Once it was

cool enough to hold, the solution was poured into an empty mould with

enough wells to accommodate the samples, and left to set for 30 minutes.

10µl of each DNA sample had 2µl of loading buffer added to it. When the gel

had set the well dividers were removed and the gel, still in the mould, was

placed into the electrophoresis tank. The tank was filled with enough TBE

buffer to cover the gel. 10µl of a 50bp ladder was used for comparison with

the samples and added to the first well. The DNA samples and the negative

control, were each pipetted into individual wells. The electrophoresis machine

was switched on at 100V for 20 minutes. After this time the gel was removed

and placed onto a UV transilluminator, showing which wells contain DNA and

which do not. Samples that appeared to carry no DNA were no further

processed.

PCR – amplification of mtDNA control region The extracted DNA samples were first vortexed and centrifuged for one

minute once brought back to room temperature. Each sample was transferred

to a 0.2ml PCR tube that was labelled with the sample identification number.

The tubes were put on ice to prevent any reaction prematurely taking place.

1.8µl of DNA extract was added to each tube, and then a reaction mixture

consisting of 8µl of PCR Master Mix (Thermo Scientific), 0.2µl of BSA, 2µl of

primers and 4µl of water was added to the DNA extract. The tubes are put on

ice to prevent any reaction prematurely taking place. They were then placed

in a thermal cycler, PCR, conditions: 3 minutes of DNA denaturation at 95°C,

followed by 36 cycles: 30 seconds of DNA denaturation at 95°C, 1 Minute of

annealing of primers to the DNA template at 55ºC and 1 minute of elongation

of a new DNA strand at 75ºC; and the finally step was 10 minutes of

elongation at 70ºC. A negative control was added to each set of samples for

which the PCR was ran. After the PCR was completed the samples were put

through a gel electrophoresis to check whether the PCR was successful and

the negative control clear.

Purification of Samples

PCR products were purified using GeneJET PCR Purification Kit (Thermo

Scientific). To each PCR product, 14µl of binding buffer was added and the

sample was vortexed. After this 14µl of isopropanol was added and the full

contents of the PCR tubes were transferred to column tubes. The column

tubes were centrifuged for 1.30 minutes, after which 700µl of wash buffer was

added. The samples were centrifuged for a further 1.30 minutes. The column

was then removed from the tube where it was placed and put into a new

Eppendorf tube with the lid removed. These

were centrifuged for 3.30 minutes. The column was added to a new

Eppendorf tube with the lid attached and 15µl of elution buffer was added and

left for 2 minutes. After this time the samples were centrifuged for 1.30

minutes. 2 µl of the purified PCR products were ran through a gel

electrophoresis to see their DNA content.

Successfully amplified PCR products (with a visible DNA band on an agarose

gel) were sent for sequencing to an external service. Overall I estimate that I

spent around 110 hours in the laboratory collecting my data.

Using gene analyzing software (Mega), the sequenced samples were edited

using this software so that any mistakes during sequencing could be

corrected. After this the final sequences could be compared to other

sequences gathered of the harp seal control region of mtDNA. This allowed

for statistical analysis to be performed on samples from different populations.

Results

Out of the 30 samples initially extracted, the mtDNA control region sequences

(519 bp) were obtained for 21 samples. These sequences were compared

with the analogous, but shorter, sequences (335 bp) of the modern harp seals

from the White Sea (N= 12) and subfossil harp seals from the Baltic Sea (N=

21) provided by the supervisor, as well as one harp seal sequence from

Greenland downloaded from GenBank.

Among the Norwegian individuals analyses, there were 21 different

haplotypes, i.e. each individual had a different haplotype. Among the White

Sea samples there were 12 different haplotypes, and among the Baltic Sea

samples 20 different haplotypes. Figure 3 shows that between all three

populations three samples share the same haplotype (A124 from the Baltic

Sea, MP56/11 from Norway and MO58 from the White Sea) and two samples

in the White Sea and Norway share the same haplotype (MP37/11 from

Noway and MO56 from the White Sea).

The nucleotide diversity for the 519 bp sequences obtained for the Norwegian

harp seals in this study was 0.025 with a standard error of 0.004. When

aligned with previous data from the White and Baltic Sea, for which shorter

sequences (335 bp) were obtained, the nucleotide diversity for Norwegian

harp seals was 0.028 with a standard error of 0.005, and the same estimate

was obtained for the Baltic Sea. The White Sea subpopulation had a

nucleotide diversity of 0.032 with a standard error of 0.006 (Table 1).

The effective population size (NE) was estimated from the equation

NE = π / (µxg), where π - the nucleotide diversity of the population, µ -

mutation rate and g - generation time, was used to calculate the effective

breeding population size for each population. The mutation rate used was 3 x

10-7, and the generation time 11 years. The effective population size was

estimated at 8485 for Norway and the ancient Baltic Sea population and 9697

for the White Sea population (Table 1).

Pairwise genetic distances between the three populations considered here

were similar for each pair and had values between 0.029 and 0.030 (Table 2).

Table 1. Genetic variability and effective population size in the harp seal

populations.

Population Sample

size

N

haplotypes

Haplotype

diversity

Nucleotide

diversity

NE

Norway 21 21 1 0.028 (SE 0.005) 8485

White Sea 12 12 1 0.032 (SE 0.006) 9697

Baltic Sea 21 20 0.986 0.028 (SE 0.005) 8485

Table 2. Pairwise genetic distances between harp seal populations

Baltic Sea White Sea

Norway 0.029 (SE 0.005) 0.030 (SE 0.005)

White Sea 0.030 (SE 0.005) -

Figure. 3 – Phylogenetic tree

of mtDNA haplotypes of

three populations: the Baltic

Sea, White Sea and Norway.

The tree was constructed

using the neighbour joining

method. The individuals

A124 from the Baltic Sea,

MP56/11 ALL from Norway

and MO58 from the White

Sea all appear to be the

same as well as the

haplotypes, MP37/11 ALL

and MO56 White Sea.

Legend: White Sea

Norway

Baltic Sea

 

Discussion

One hypothesis as to where the Norwegian harp seals came from is the White

Sea breeding colony, which is spatially closest to the Norwegian coast.

Analysis of data collected from this study and comparisons made with data

from previous studies indicated that it is likely that most or even all individuals

from Norway came from the White Sea. The phylogenetic tree of harp seal

mtDNA haplotypes supports this. The tree shows how the Norwegian and

White Sea individuals do not form distinct clades and are in fact intermixed,

with many haplotypes from Norway being more similar to that of the White

Sea than to other haplotypes from Norway. Also two haplotypes are shared

between these populations, even though both populations have high

haplotype diversity. Despite these results reinforcing the hypothesis that

Norwegian harp seals may originate from the White Sea, it cannot be

excluded that other populations, particularly the second-closest Greenland

population may also contribute migratory individuals to Norway. To test this,

the control region sequences of mtDNA of all harp seal populations should be

included in the analysis. Unfortunately this data is not currently available.

Figure. 3 also shows that the individuals from Norway are not related as they

have no common female ancestor. This could imply that large numbers of

individuals migrate along the Norwegian coast, and that they do not consist of

groups of related individuals.

The results of this study also shed some light on the origin of the ancient, now

extinct harp seal colony from the Baltic Sea. Surprisingly, the pair-wise

genetic differentiation between the ancient Baltic population and the

contemporary populations from the White Sea and Norway was comparable

to the differentiation between these two populations, as well as to the diversity

within each of the three populations. Moreover, one mtDNA haplotype is

shared between all the three populations. This may indicate that the ancient

Baltic Sea population and the modern Norway population both originated from

the southwards migration of individuals from the White Sea, although the

sample size isn’t currently large enough to exclude alternative scenarios.

As expected it was found that the White Sea population of harp seals had the

largest nucleotide diversity of the three populations (0.032), whereas the

Baltic Sea and Norway populations had exactly the same nucleotide diversity

(0.028). It could be expected that the group diversity for the Baltic and Norway

populations is smaller because they likely originated from a fraction of the

White Sea population, and therefore would have a lower diversity. With both

the Baltic and Norway populations having the same diversity, the migrating

seals may have taken the same route from the White Sea, which goes on past

the Norwegian coast. The ancient population migrating to the Baltic may have

continued further south, past the Baltic straits and colonized the Baltic, which

at that time may have held a more similar environment to that of Norway now

due to the decreased temperature of the period. The Baltic Sea still freezes

during winter (Haapala & Lepparanta, 1995) meaning that in a colder climate

it would have been habitable for harp seals. Now perhaps, with the climate

being warmer, harp seals are taking the same migration route but stay further

north, rather than continuing south into warmer waters. Harp seals have a

seasonal southward migration in autumn prior to the advance of ice in the

winter. This migration is probably food related (Haug et al. 1994) and may be

similar to the previously mentioned longer migration to Norway and the Baltic

Sea. Nilssen et al. (1995), suggests that the collapse in fish stocks such as

the herring (Clupea harengus) and capelin (Mallotus villosus) in the Barents

Sea are responsible for the migration onto the Norwegian coast.

However, harp seals from the Norwegian coast are in a worse condition than

that of the Barents Sea (Nilssen et al. 1995). As mentioned it is possible that

the harp seals southward migration is in search of food, but the food that they

may have found may have been of a poor quality or in not enough

abundance. Nilssen et al. (1995) have found that the main prey species of

harp seals off the coast of Norway were gadoids (Gadidae). This fish family is

more resistant to stomach acid than the usual Barents Sea prey of harp seals.

This could account for the poorer condition of harp seals found of the

Norwegian coast. Perhaps the ancient harp seal population of the Baltic Sea

suffered similar problems. It could have been that the food resources of the

Baltic harp seal was very poor. This saw their condition deteriorate, and

resulted in eventually their condition deteriorating so much that they became

extinct.

A common mtDNA haplotype was found in samples from the Baltic Sea,

Norway and the White Sea. This suggests that in this case the same

haplotype has migrated from the White Sea to the Baltic and Norway during

independent migration events separated by several thousand years. It could

be possible for the same haplotype to remain in the White Sea population until

an opportunity arose once more for individuals to migrate southwards.

Another haploype match between Norway and the White Sea can be seen

adding further evidence for the hypothesis that the Norway population

originated in the White Sea. Figure 3 shows a very diverse range of

haplotypes as there are very few clusters of individuals of the same

geographic origin, suggesting that ultimately they all could have came from

one population, which is most likely the White Sea.

Also present on the phylogenic tree is a group of five individuals that are all

from the Baltic Sea. This may be due to the fact that the Baltic population, that

may have only been started by a small group of migrating individuals, had

been occupying the Baltic Sea around for at least 2,000 years (Stora &

Ericson, 2006). This meant that with a small starting population interbreeding

may have been present, resulting in some seals having similar mtDNA.

The even spread of haplotypes from different geographic locations, seen in

Figure 3 is concordant with what was found by Carr et al. (2008) in a larger

investigation on harp seals from the Greenland Sea, White Sea and

Newfoundland Sea Front and Gulf populations. That study looked at the

cytochrome b region of mtDNA, and the phylogenetic tree based on these

sequences showed a fairly even spread of haplotypes from different

geographic locations (Carr et al. 2008). This could imply that there has been

some migration between populations similar to what has been found in the

present study. If it is possible for populations as geographically distant as up

to 5000 km to be mixed, then the potential of the populations in this study to

be mixed and interbreed, is certainly possible.

Analysis of the effective female population size showed that the population in

Norway and the Baltic Sea had the smallest effective size after the White Sea.

This followed what was expected from the hypothesis that the Baltic and

Norway populations originated from the White Sea. This is because the White

Sea had the largest diversity and therefore it would be expected that it would

have a larger population. This estimate takes into account the breeding

females, because only females will pass on their mtDNA, which makes it more

relevant to the study. Perry et al. (2000) calculated the effective population

size of harp seal populations by using the nucletide diversity index. They

estimated that the effective population size of the Greenland Sea was

137,000 whereas the Northwest Atlantic had an effective population size of

around 37,000. The White Sea harp seal stock was estimated at around

600,000 (Nilssen et al. 1995). The differences between the estimates in the

study by Nilssen et al. (1995) and the present study results from differences in

the mutation rates assumed. The census size of the Norwegian population

was estimated at 3238 (Nilssen et al. 1995), which is more consistent with the

estimates of the effective population size in this paper.

Through the laboratory process a total of seven samples were lost due to

them showing up as negative for DNA after a PCR. This was followed by two

more samples being lost after sequencing due to the poor nature of the

mtDNA sequenced. The quantity and quality of the DNA extract may have

been low due to some samples being highly degraded, which lead to PCR

failure. As this was the first experience of the author with the molecular

genetic laboratory work, human error also cannot be excluded. Time and

financial constraints were also an issue in the loss of samples as samples for

which the DNA extraction failed were not re-analysed. Otherwise, it might

have been possible to obtain the mtDNA sequences for all 30 samples. There

is little that can be done for the samples lost in processing, although one idea

would be to process each sample using two independent DNA extractions to

minimise the failure due to human error. However, this was impossible due to

financial constraints. For future studies it would be interesting to continue

collecting DNA samples from the harp seals form the Norwegian coast to build

up and add to the data that already exists. It is also important to collect the

data from other harp seal populations, and especially from Greenland. With

more data the origin of the harp seals migrating to Norway can be established

with a high confidence. More data could also help answer the question of

where the ancient Baltic population originated from.

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