1. 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

2. 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

3. 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.

4. 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.

5. 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.

6. 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

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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

13. 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) -

14. 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

15. 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

16. 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.

17. 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

18. 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

19. 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.

20. References
Benjaminsen, T. (1979) “Pup production and sustainable yield of White Sea
harp seals.” Fiskeridirektoratet, Institute of Marine Research, Directorate of
Fisheries, Bergen.
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Brown, Terry. (2010). Gene cloning and DNA analysis: an introduction. John
Wiley & Sons. 334-335.
Carr, S. M., Marshall, H. D., Duggan, A. T., Flynn, S., Johnstone, K. A., Pope,
A. M., & Wilkerson, C. D. (2008). Phylogeographic genomics of mitochondrial
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Forsten, A., & Alhonen, P. (1975). The subfossil seals of Finland and their
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