The handle http://hdl.handle.net/1887/33217 holds various files of this Leiden University dissertation.
Author: Osinga, Nynke
Title: Comparative biology of common and grey seals along the Dutch coast : stranding, disease, rehabilitation and conservation
Issue Date: 2015-06-09
Section
Genetic variation
Studies of the of genetic variation in common seals of the Wadden Sea conducted in the early 1990’s concluded that the Wadden Sea common seal population is depauperate of genetic variation. However, the Wadden Sea common seal population has shown a recovery in recent decades. The genetic status of the recolonised grey seals had not been surveyed yet.
Because of the recent population recoveries, there was a need to re-assess the current level of genetic variation for both seal species (Chapter 4). Small populations may have a high or low frequency of particular alleles. This can become visible as a releatively high occurrence of pigment disorders, i.e. albinistic or melanistic animals. The strong fluctuations in population size of common and grey seals in Dutch waters, may also have led to an increased occurrence of such pigment disorders. To test this hypothesis, stranding data of live and dead stranded seals were analysed for the occurrence of colour aberrations (Chapter 5).
B
Chapter
An update on the genetic status of common seals and grey seals of the Wadden Sea
Nynke Osinga1,2, Anique L. Kappe3, Louis van de Zande4, Helias A. Udo de Haes1, Paul M. Brakefield1,5
1. Institute of Environmental Sciences (CML) and Institute of Biology Leiden (IBL), Leiden University, The Netherlands, 2. Seal Rehabilitation and Research Centre (SRRC), Pieterburen, The Netherlands, 3. Gendika, Veendam, The Netherlands, 4. Center for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands, and 5. Department of Zoology, University of Cambridge, United Kingdom.
Submitted for publication
4
4
Abstract
In the 1970s, the Wadden Sea common seal (Phoca vitulina) population reached a historical low point after centuries of intensive hunting followed by exposure to pollution.
Grey seals (Halichoerus grypus) were once abundant in Dutch waters, but disappeared entirely in preceding centuries due to intensive hunting. A heightened conservation concern together with a strong appreciation of the Dutch for their largest wildlife species, have now made these seals key species for nature conservation in the Netherlands. In recent decades, the Wadden Sea common seal population has grown and grey seals have recolonised Dutch waters. The goal of this study was to re-assess the genetic variation now that the seal populations are recovering. To achieve this goal, we studied the current genetic status of both species of seal using ten microsatellite markers. For both seal species, we found a level of genetic variation that was amongst the lowest recorded for pinnipeds. However, the levels were more robust than reported for endangered or inbred mammal species. We conclude that although human activities may have contributed to the low diversity recorded, they do not appear to have unduly impacted the level of genetic variation of Wadden Sea common and grey seals.
111 The genetic status of common and grey seals
4
Introduction
The two species of seals occurring in Dutch waters are the common seal (Phoca vitulina vitulina) and the grey seal (Halichoerus grypus). Common seals in Dutch waters are part of a Wadden Sea population unit, ranging from the Dutch Wadden Sea to the Danish Wadden Sea (Goodman 1998). Grey seals in Dutch waters are part of the Northeast Atlantic grey seal population. This population is centred around the British Isles, ranging from Iceland, eastward along the coast of France, and north to Norway and the Kola Peninsula (Bonner 1981; Haug et al. 1994; Boskovic et al. 1996).
The structure and distribution of genetic variation of populations can be strongly influenced by historical events. Several studies have linked the lower genetic variation of European common seals, compared to subspecies in North America, to a severe bottleneck during the last ice age or even recolonisation from ice age refugia (Stanley et al. 1996; Kappe et al. 1997). Furthermore, the Wadden Sea common seal population reached a historical low point in the 1970s due to centuries of intensive hunting, followed by exposure to pollution (Koeman et al. 1973; Van Haaften 1974, 1978; Reijnders 1980; ‘t Hart 2007). In 1977, there were only 430 common seals in the Dutch Wadden Sea (Van Haaften 1978). Although, the population size in previous centuries is unknown, common seals must have been very abundant considering that they sustained a high hunting pressure over many centuries (‘t Hart 2007).
The remains of grey seals have been found in archaeological studies of dwelling mounds distributed along the coast of the Wadden Sea (Clason 1988) and it is therefore assumed that grey seals were once abundant in Dutch waters but disappeared due to intensive hunting (Van Bree et al. 1992). Grey seals began to settle again in the Dutch Wadden Sea from the 1980s onwards (‘t Hart et al. 1988; Reijnders et al. 1995). They originated from the coasts of England and Scotland, most likely from the Farne islands where seals were culled and harassed in the 1960s, 1970s and early 1980s (Hickling 1962; Van Haaften 1974;
Thompson & Duck 2010).
Currently, the numbers of both common seals and grey seals are increasing in the Wadden Sea (TSEG 2013). There are, however, remarkable differences in disease rate between the two seal species. Analyses of stranding data have revealed that common seals suffer more frequently from disease than grey seals (Chapter 2). The principle causes of this high frequency of diseases are phocine distemper and parasitic bronchopneumonia.
Outbreaks of the phocine distemper virus (PDV) resulted in the mortality of half of the number of common seals in Dutch waters in 1988 and again in 2002 (Osterhaus & Vedder 1988; Rijks et al. 2005). Parasitic bronchopneumonia in seals is caused by the nematodes Parafilaroides gymnurus and Otostrongylus circumlitus. Its occurrence has increased sharply since the late 1990s and it is currently the primary cause of disease in juvenile common seals (Chapter 1). Phocine distemper virus is more pathogenic for common seals than for grey seals; indeed, PDV was not identified as a cause of death in grey seals during the investigations of the 2002 epidemic (Rijks et al. 2005). Also, grey seals rarely die of parasitic bronchopneumonia (Chapter 2).
4
Previous studies of genetic variation in seals of the Wadden Sea used various molecular techniques, including allozyme electrophoresis (Swart et al. 1996), the random amplified polymorphic DNA (RAPD) technique (Kappe et al. 1995) and the multilocus DNA fingerprinting technique (Kappe et al. 1995; Kappe et al. 1997). These studies concluded that the Wadden Sea common seal population is depauperate of genetic variation. Higher levels of genetic variation were found for other P. vitulina subspecies as well as for grey seals (Kappe et al. 1995; Kappe et al. 1997). Nowadays, markers for microsatellite DNA are often used for surveys of genetics among populations (Hoelzel 1998). Microsatellite markers consist of short tandem repeat DNA sequences and are considered to be selectively neutral. They typically show very high levels of polymorphism with many alleles per locus, and can be sensitive markers even in populations with overall low levels of genetic variation. A previous study by Goodman (1998) employing microsatellites revealed a high level of genetic variation in the common seals of the Wadden Sea but this was not observed in another survey performed by Kappe (1998).
Since the genetic studies in the 1990s, there has been an increase in the Wadden Sea common seal population, with more than 7,000 animals counted during aerial surveys of the Dutch Wadden Sea in 2013 (TSEG 2013). In addition to population growth, migration between the common seal population units and subsequent gene flow (Goodman 1998) may have increased the level of genetic variation. On the other hand, the genetic variation may have been reduced due to an outbreak of PDV in 2002 that resulted in the mortality of half of the Wadden Sea common seals population (Rijks et al. 2005). The number of grey seals in the Wadden Sea has continuously increased since the 1980s, with more than 2,000 individuals counted during aerial surveys of the Dutch Wadden Sea in 2013 (TSEG 2013). The genetic status of the recolonised grey seals has not been surveyed before the present study.
We re-assessed the genetic variation now that the common seals are recovering in the Dutch Wadden Sea and the grey seals successfully recolonised these waters. We studied the current level of genetic variation for both seal species using ten microsatellite markers.
The results are discussed in relation to a review of studies in other pinnipeds (cf Curtis et al. 2011) and mammalian species that have examined microsatellite variation.
Materials and methods
We investigated hair samples of 24 common seals and 24 grey seals which were admitted to the Seal Rehabilitation and Research Centre (SRRC) in Pieterburen, the Netherlands.
The common seals were orphaned seals (lengths: 62-88 cm) that stranded in the Wadden Sea in the months of June, July and August in 2007 and 2008. The grey seals were orphaned seals (lengths: 77-113 cm) that stranded in the Wadden Sea in the months of December and January in 2007 and 2008.
The hair samples from each seal were incubated for 1 h at 56 °C in 200 µl lysis buffer containing 10 mM Tris-HCL, pH 8.0, 10 mM EDTA, pH 8.0, 100 mM NaCl, 40 mM DTT
113 The genetic status of common and grey seals
4
amd 1mg/ml proteinase K. Genomic DNA was isolated from the extract using a Qiagen QIAamp DNA Mini Kit following the manufacturer’s instructions.
We used the following ten published pinniped loci: 1. 4A3 (Kappe 1998), 2. SGPV3 (Goodman 1995), 3. SGPV10 (Goodman 1995), 4. SGPV11 (Goodman 1995), 5. HG6.1 (Allen 1995), 6. HG8.9 (Allen 1995), 7. HG8.10 (Allen 1995), 8. HGDII (Allen 1995), 9.
71HDZ15 (Huebinger et al. 2007), and 10. 71HDZ301 (Huebinger et al. 2007). The first four primer sets had originally been designed for the common seal (Goodman 1995;
Kappe 1998), the next four for the grey seal (Allen 1995) and the final two for the Steller sea lion (Eumetopias jubatus) (Huebinger et al. 2007).
PCR reactions were carried out in 10 µl reaction volumes containing 0.25 U of Taq polymerase (5 PRIME), 1 x manufacturer-supplied buffer (5 PRIME), 200 µM of each dNTP, 10 pmol of each primer and c. 10 ng of genomic DNA. Loci were amplified using the following PCR profile: an initial denaturing step of 94°C for 5 minutes, followed by 10 cycles of 30 s at 94°C, 1 min at 50°C and 1 min at 72ºC, followed by 30 cycles of 30 s at 94 ºC, 30 s at 52 ºC and 1 min at 72 ºC and finally 10 min at 72 ºC. Forward primers were 5’end labelled with 6-FAM, HEX or NED fluorescent dyes. Amplified fragments were sized on an ABI Prism 3130 Genetic Analyzer using GeneScan 500 ROX size standard (Applied Biosystems, Bleiswijk, the Netherlands). Genotypes were scored by two observers (NO and AK). Results were entered manually into a Microsoft Excel spreadsheet. Allelic diversity and observed and expected heterozygosities were calculated using Arlequin version 3.5.1.3 (Excoffier et al. 2005). Hardy-Weinberg equilibrium was tested for all loci with Arlequin’s exact test using a Markov Chain. Bootstrapping for allelic diversity calculations was done using FStat version 2.9.3.2 (Lausanne, Switzerland) (Goudet 1995).
Results
For some samples and/or loci, PCR tests failed repeatedly before a genotype could be scored. We therefore analyzed the dataset with the following adjustments:1) all tests carried out five times or more were replaced by a missing value; 2) three loci (SGPV3, HG6.1 and 71HDZ301) had more than 5 missing values (for a total of 48 seals) and these three loci were removed from the dataset; and 3) for those seals that still had two or more missing values (0 common seals, 5 grey seals) all data were removed from the dataset.
Comparison of the results from the raw data (included as supplementary materials) with those from the revised data indicated that genotyping errors likely occurred in the poor quality samples. For this reason, we use the revised data for the analysis of our results.
The average observed heterozygosity for common seals was 0.41 with an expected heterozygysity of 0.41 (Table 1). For grey seals, both values were higher, with an average observed heterozygosity of 0.50 and an average expected heterozygosity of 0.56.
The average allelic diversity of common seals was 3.1, with the number of alleles ranging between 2 and 5 (Table 1). The average allelic diversity of grey seals was higher, with a value of 5.4, and the number of alleles ranging between 2 and 11. The revised dataset
4
has a larger sample size for common seals than for grey seals, therefore bootstrapping was applied using the program FStat. Allelic diversity for a sample size of 18 seals resulted in an average allelic diversity of 2.9 for common seals and 5.4 for grey seals.
Tests for Hardy-Weinberg equilibrium resulted in significant heterozygote deficits for four loci (4A3 and HG8.10 for common seals and grey seals, and HG8.9 for grey seals) and one significant heterozygote excess (71HDZ15 for common seals) (Table 1). Overall there was no significant departure from Hardy-Weinberg equilibrium for either species.
Table 1. The number of seals tested (N), allelic diversity (A), observed heterozygoity (Ho) and expected heterozygosity (He), and Hardy-Weinberg equilibrium P-value (P). Significance level indicated as *p<0.05,
** p<0.01, *** p<0.001.
Common seal
Locus N A Ho He P
4A3 24 3 0.29 0.51 0.0011 **
SGPV10 24 2 0.042 0.042 1.00
SGPV11 23 3 0.56 0.55 0.69
HG8.9 24 2 0.42 0.34 0.54
HG8.10 24 5 0.42 0.57 0.0027 **
HGDII 24 2 0.58 0.42 0.13
71HDZ15 24 5 0.54 0.45 0.038 *
Mean 3.11 0.41 0.41
s.d. 1.3 0.19 0.18
Grey seal
Locus N A Ho He P
4A3 19 3 0.26 0.24 1.0
SGPV10 19 2 0.21 0.27 0.37
SGPV11 19 7 0.74 0.75 0.098
HG8.9 18 11 0.67 0.83 0.0062 **
HG8.10 19 7 0.58 0.79 0.018 *
HGDII 18 5 0.78 0.71 0.73
71HDZ15 19 3 0.26 0.32 0.49
Mean 5.41 0.50 0.56
s.d. 3.2 0.25 0.27
1. Allelic diversity after bootstrapping to a sample size of 18 seals is 2.9 for common seals and 5.4 for grey seals.
115 The genetic status of common and grey seals
4
Table 2. Published studies of genetic variation in common seals, grey seals and other pinniped species, including the number of loci (K), the number of seals tested (N), the allelic diversity (A) and the observed heterozygosity (Ho).
Species Type of marker K N A Ho Reference
Common seals Phoca vitulina vitulina
Wadden Sea Microsatellite 7 24 3.1 0.41 Current
study Common seals Phoca vitulina vitulina
European range (six populations) Microsatellite 7 1029 9.7 0.50 Goodman 1998 Common seals Phoca vitulina Richardii
British Columbia and Alaska (3 populations) Microsatellite 5 222 4-12 0.66 Burg et al.
1999
Grey seals Halichoerus grypus Wadden Sea
(NE Atlantic population) Microsatellite 7 19 5.4 0.50 Current
study Grey seals Halichoerus grypus Scotland
(NE Atlantic population) Microsatellite 9 1340 8.0 0.74 Allen 1995
Grey seals Halichoerus grypus Baltic Sea
(Baltic Sea population) Microsatellite 8 131 6.8-7.6 0.67-0.76 Graves et al. 2009
Lake Saimaa ringed seals Pusa hispida
saimensis Microsatellite 8 58-70 2.0 0.25 Palo et al.
2003 Mediterranean monk seals Monachus
monachus monachus Western Sahara Microsatellite 13 43-95 3.0 0.35 Pastor et al. 2007 Mediterranean monk seals Monachus
monachus monachus Eastern Mediterranean Microsatellite 13 7-11 2.6 0.23 Pastor et al. 2007 Hawaiian monk seals Monachus schauslandi Microsatellite 8 2409 3.5 0.48 Schultz et
al. 2009 Northern Sea elephant Mirounga
angustirostris Microsatellite 4 100 2.5 0.45 Hoelzel et
al. 2002 The other 18 pinniped species for which microsatellite genetic variation was studied (see overview published by Curtis et al. (2011)) were reported to have an Ho>0.50
Discussion
The detected level of genetic variation in common seals was higher than previously found with other methods (Swart et al. 1996; Kappe et al. 1995; Kappe et al. 1997). The genetic variation of of Wadden Sea grey seals was found to be higher than that of Wadden Sea common seals.
Common seals
The current level of genetic diversity in Wadden Sea common seals is similar to that found for this population in a microsatellite study conducted in the 1990s (Ho=0.43, n=140;
Goodman 1998). The other microsatellite study conducted in the 1990s (Kappe 1998) revealed a lower level of genetic variation (Ho=0.13, N=27). This may be due to the use of
4
microsatellite loci with relatively low levels of polymorphy. Indeed, locus 4A3 that was also used in this study showed a relatively low level of variation in P. vitulina compared to most other loci (Table 1).
The level of heterozygosity observed for Wadden Sea common seals in this study (Ho=0.41, N=24) was lower than the average level for different common seal populations throughout its European range (Ho=0.50, n=1,029; Goodman 1998; Table 2). Furthermore, our findings support the conclusion of Kappe (1998) that Phoco vitulina vitulina has substantial lower levels of genetic variation than the subspecies Phoca vitulina richardii.
The low diversity recorded for common seal in this study suggests that there has been a demographic contraction in the past. Such a contraction could have occurred at different periods in the species’ history. It is often suggested that European common seals have been absent and recolonised since the last ice age (Stanley et al. 1996; Kappe et al. 1997).
In this regard, it is interesting to note that grey seal remains are found in dwelling mounds (called ‘terpen’) in the north of the Netherlands (dating from the period of 600 B.C. to 1000 A.D.) (Clason 1988), whereas no common seals have been discovered. Thus, common seals may have recolonised the Wadden Sea region in relatively recent times. Additionally, the population crash in the mid-20th century may have lowered the genetic variation of the Wadden Sea common seal. Future studies of genetic variation in museum specimens could provide more information on the levels of genetic variation in previous centuries (Wandeler et al. 2007). Amos and Harwood (1998) in a review of genetic variability studies, state that in many cases the species in question have not spent long enough at sufficiently low numbers to attribute genetic impoverishment to a population bottleneck.
In this regard, the lower level of genetic variation in Wadden Sea common seals appears not to be caused by a true bottleneck, but rather is a result of recolonisation since the last ice age and/or demographic contractions caused by hunting over the past centuries.
Grey seals
The level of genetic variation of Wadden Sea grey seals (Ho=0.50, n=18/19) was lower than that of this species from Scotland (Ho=0.74, n=1340; Allen 1995). These two groups of seals are part of the same Northeast Atlantic population. We postulate that the recolonisation may be reflected in the lower level of genetic variation in the grey seals of the Dutch Wadden Sea. The gene pool of grey seals in Dutch waters may be smaller because of the founding. Philopatry may also explain a more limited gene flow than expected based on dispersal rates. It is interesting to note that Pomeroy et al. (2000) provided evidence for fine scale philopatry in grey seals of the UK. Our results support this finding.
Comparison with other species
Compared to other pinnipeds, the level of genetic variation of Wadden Sea common seals is amongst the lowest (Curtis et al. 2011; Table 2). Pinniped species with a lower heterozygosity levels than that of common seals are species with great conservation concern. For example, Lake Saimaa ringed seals (Pusa hispida saimensis) are a small
117 The genetic status of common and grey seals
4
landlocked subspecies of ringed seals and both subspecies of monk seals are listed as critically endangered (Mediterranean monk seals Monachus monachus monachus and Hawaiian monk seals Monachus schauslandi; The IUCN Red List of Threatened Species).
Northern elephant seals (Mirounga angustirostris), which have been through a severe bottleneck in the 19th century, have a level of genetic variation comparable to Wadden Sea common seals.
Frankham et al. (2002) stated that endangered species usually have about half the genetic diversity of related, non-endangered species. For example, a very low level was found for the Florida panther (Puma concolor coryi) (Ho=0.16, n=10; Driscoll et al. 2002), a species known to suffer from severe inbreeding as manifested by spermatozoal defects, cryptorchidism, cardiac abnormalities and a relative high pathogen-parasite load (Roelke et al. 1993). Based on above studies of Frankham and Roelke, it appears that the current level of genetic variation of both common and grey seals are much more robust than found for species that are classified as inbred or endangered.
Disease susceptibility
Phocine distemper and parasitic bronchopneumonia are diseases that cause high levels of morbidity and mortality in common seals, but not in grey seals (Chapter 2). The relationship between genetic variation and susceptibility to phocine distemper has been investigated by McCarthy et al. (2011). They could not relate variation at immune response genes to the susceptibility to phocine distemper virus in European common seals. We therefore consider it unlikely that the genetic status, as found in the current study using neutral markers, is an explanation for the high pathogenity of phocine distemper to common seals.
As with common seals, parasitic pneumonia is a frequent disease in northern elephant seals (Gulland et al. 1997). Low levels of genetic variation were hypothesized as a potential factor for the increased susceptibility of this species to the lungworm Otostrongylus circumlitus (Elson-Riggins 2001). In contrast, Weber et al. (2000) could not associate the loss of mtDNA genotypes in northern elephant seals with a lowered mean fitness of individuals today. Similarly, for cheetahs, one of the classical examples in conservation genetics, the relationship between low levels of diversity and disease vulnerability was questioned by Castro-Prieto et al. (2011). There is much scientific debate on whether low levels of genetic variation result in a loss of fitness. Another aspect of the debate is that although reduced levels of genetic variation may not have direct apparent fitness consequences, they may lower a population’s ability to react to novel challenges such as emerging diseases (Amos & Balmford 2001). It is therefore difficult to draw conclusions on whether the genetic status of Wadden Sea common seals could be a factor in the vulnerability of this population to disease. Its level of genetic variation is not dramatically low and severe fitness costs such as those found for the Florida panther as a result of founder events and inbreeding are not expected. Future studies of immune response genes, such as those of the major histocompatibility complex (MHC) are needed. Their
4
overall variation or specific MHC alleles may provide more insight in to the common seals vulnerability to parasite infections. Furthermore, other factors that may play a role in the high prevalence of parasitic bronchopneumonia in common seals compared to grey seals need to be studied, such as diet, foraging strategy, and levels of immunosuppressive toxins.
Conclusion
For both seal species, we found a level of genetic variation that was amongst the lowest recorded for pinnipeds. However, the levels were more robust than reported for endangered or inbred mammal species. We conclude that although human activities may have contributed to the low diversity recorded, they do not appear to have unduly impacted the level of genetic variation of Wadden Sea common and grey seals.
Acknowledgements
We are grateful to the colleagues of Gendika for their support with the laboratory work.
We would also like to thank Dr. Jocelyn Elson-Riggins for her helpful comments on the manuscript.
References
Allen P.J. (1995) Molecular analysis of grey sal (Halichoerus grypus) breeding behaviour and social organisation, pp. 199. Darwin College, University Cambridge, Cambridge, United Kingdom.
Amos W. & Harwood J. (1998) Factors affecting levels of genetic diversity in natural populations. Philosophical Transactions of the Royal Society London B 353, 177-86.
Amos W. & Balmford A. (2001) When does conservation genetics matter? Heredity 87, 257-65.
Bodewes R., Morick D., van de Bildt M.W.G., Osinga N., García A.R., Contreras G.J.S., Smits S.L., Reperant L.A.P., Kuiken T. &
Osterhaus A.D.M.E. (2013) Prevalence of phocine distemper virus specific antibodies:
bracing for the next seal epizootic in north- western Europe. Emerging Microbes &
Infections, doi:10.1038/emi.2013.2.
Bonner W.N. (1981) Grey seal Halichoerus grypus Fabricius, 1791. In: Handbook of marine mammals (ed. by Ridgway S.H. &
Harrison R.J.), pp. 111-44. Academic press, London.
Boskovic R., Kovacs K.M. & Hammill M.O.
(1996) Geographic distribution of mitochondrial DNA haplotypes in grey seals (Halichoerus grypus). Canadian Journal of Zoology 74, 1787-96.
Bree P.J.H. van, Vedder E.J. & Hart L. ‘t (1992) Terug van weggeweest: de grijze zeehond in Nederland. Zoogdier 3, 11-5.
Castro-Prieto A., Wachter B. & Sommer S. (2011) Cheetah paradigm revisited: MHC diversity in the world’s largest free-ranging population.
Molecular Biology and Evolution 28, 1455-68.
Clason A.T. (1988) De grijze zeehond Halichoerus grypus (Fabricius, 1791). In:
Terpen en wierden in het Fries-Groningse kustgebied (ed. by Bierma M., Clason, A.T., Kramer, E. & Langen, G.J. de), pp. 234-40.
Wolters-Noordhoff/Forsten, Groningen.
Curtis C., Stewart B. & Karl S. (2011) Genetically effective population sizes of Antarctic seals estimated from nuclear genes. Conservation genetics 12, 1435-46.
Driscoll C., Menotti Raymond M., Nelson G., Goldstein D. & O’Brien S. (2002) Genomic microsatellites
119 The genetic status of common and grey seals
4
as evolutionary chronometers: a test in wild cats.
Genome research 12, 414-23.
Elson-Riggins J.G., Al-Banna L., Platzer E.G., Kaloshian I. (2001) Characterization of Otostrongylus circumlitus from Pacific harbor and Northern elephant seals. Journal of Parasitology 87, 73-8.
Excoffier L., Laval G. & S. S. (2005) Arlequin ver. 3.0: An integrated software package for population genetics data analysis.
Evolutionary Bioinformatics Online 1, 47-50.
Frankham R., Ballou J.D. & Briscoe D.A. (2002) Introduction to conservation genetics, pp.617.
Cambridge University Press, Cambridge.
Goodman S.J. (1995) Molecular population genetics of the European harbour seals (Phoca vitulina) with reference to the 1988 Phocine Distemper Virus epizootic, pp. 195. Darwin college, University of Cambridge, Cambridge.
Goodman S.J. (1998) Patterns of extensive genetic differentation and variation among European harbour seals (Phoca Vitulina vitulina) revealed using microsatellite DNA polymorphisms. Molecular Biology and Evolution 15, 104-18.
Goudet J. (1995) FSTAT, a computer program to calculate F-statistics. Journal of Heredity 86, 485-6.
Gulland F.M.D., Beckmen K.B., Burek K., Lowenstine L.J., Werner I., Spraker T., Dailey M.D. & Harris E. (1997) Nematode (Otostrongylus circumlitus) infestation of Northern Elephant seals (Mirounga angustirostris) stranded along the central Californian coast. Marine Mammal Science 13, 446-59.
Haaften J.L. van (1974) Zeehonden langs de Nederlandse kust. Wetenschappelijke Mededelingen K.N.N.V. 101, 1-35.
Haaften J.L. van (1978) De Waddenzee- zeehonden in 1977. Waddenbulletin 13, 98- 499.
Hart L. ‘t, Moesker A., Vedder L. & Bree P.J.H.
van (1988) On the pupping period of grey seals, Halichoerus grypus (Fabricius, 1791), reproducing on a shoal near the island of Terschelling, the Netherlands. Zeitschrift für Säugetierkunde 53, 59-60.
Hart P. ‘t (2007) Zeehondenjacht in Nederland 1591-1962, pp. 351. Vrije universiteit Amsterdam, Amsterdam.
Haug T., Henriksen G., Kondakov A., Mishin V., Tormod Nilssen K. & Røv N. (1994) The status of grey seals Halichoerus grypus in North Norway and on the Murman Coast, Russia. Biological Conservation 70, 59-67.
Hickling G. (1962) Grey seals & the Farne islands, pp.180. Routledge and Kegan Paul, London.
Hoelzel A.R. (1998) Molecular genetic analysis of populations, pp.445. Oxford University Press, Oxford
Huebinger R.M., Louis E.E., Thomas Gelatt J.R., L.D. R. & Bickham J.W. (2007) Characterization of eight microsatellite loci in Steller sea lions (Eumetopias jubatus).
Molecular Ecology Notes 7, 1097-9.
Kappe A.L., Zande L. van de, Vedder E.J., Bijlsma R. & Delden W. van (1995) Genetic variation in Phoca vitulina (the harbour seal) revealed by DNA fingerprinting and RAPDs. Heredity 74, 647-53.
Kappe A.L., Bijlsma A., Osterhaus A.D.M.E., Delden W. van & Zande L. van de (1997) Structure and amount of genetic variation at minisatellite loci within the subspecies complex of Phoca vituilna (the harbour seal). Heredity 78, 457-63.
Kappe A.L. (1998) Detecting genetic variation, application of molecular techniques in conservation biology, pp. 114. Department of genetics, University of Groningen, Groningen.
Koeman J.H., Peeters W.H.M., Koudstaal-Hol C.H.M., Tjioe P.S. & Goeij J.J.M. de (1973) Mercury-selenium correlations in marine mammals. Nature 245, 385-6.
McCarthy A., Shaw M.-A., Jepson P., Brasseur S.M.J.M., Reijnders P.J.H. & Goodman S.
(2011) Variation in European harbour seal immune response genes and susceptibility to phocine distemper virus (PDV).
Infection, genetics and evolution 11, 1616- 23.
Osterhaus A.D.M.E. & Vedder E.J. (1988) Identification of virus causing recent seal deaths. Nature 335, 20.
4
Pomeroy P.P., Twiss S.D. & Redman P. (2000) Philopatry, site fidelity and local kin associations within grey seal breeding colonies. Ethology 106, 899-919.
Reijnders P.J.H. (1980) Organochlorine and heavy metal residues in harbour seals from the Wadden Sea and their possible effects on reproduction. Netherlands Journal of Sea Research 14, 30-65.
Reijnders P.J.H., Dijk J. van & Kuiper D. (1995) Recolonization of the Dutch Wadden Sea by the grey seal Halichoerus grypus. Biological Conservation 71, 231-5.
Rijks J.M., Bildt M.W.G. van de, Jensen T., Philippa J.D.W., Osterhaus A.D.M.E.
& Kuiken T. (2005) Phocine distemper outbreak, the Netherlands, 2002. Emerging Infectious Diseases 11, 1945-8.
Roelke M.E., Obrien S.J., Martenson J.S. &
O’Brien S.J. (1993) The consequences of demographic reduction and genetic depletion in the endangered Florida panther. Current Biology 3, 340-50.
Stanley H.F., Casey S., Carnahan J.M., Goodman S., Harwoord J. & Wayne R.K. (1996) Worldwide patterns of mitochondrial DNA differentiation in the harbor seals (Phoca
vitulina). Molecular Biology and Evolution 13, 368-82.
Swart J.A.A., Reijnders P.J.H. & Delden W.
van (1996) Absence of genetic variation in harbor seals (Phoca vitulina) in the Dutch Wadden Sea and the British wash.
Conservation Biology 10, 289-93.
Thompson D. & Duck C. (2010) Berwickshire and North Northumberland Coast European Marine Site: grey seal population status.
Report to Natural England: 20100902-RFQ, pp. 37. Sea Mammal Research Unit, Scottish Oceans Institute, University of St Andrews, St Andrews.
TSEG (2013) Harbour seal counts 2013. URL:
http://www.waddensea-secretariat.org/
monitoring-tmap/topics/marine-mammals.
Wandeler P., Hoeck P.E.A. & Keller L. (2007) Back to the future: museum specimens in population genetics. Trends in Ecology &
Evolution 22, 634-42.
Weber D.S., Stewart B.S., Garza J.C. & Lehman N. (2000) An empirical genetic assessment of the severity of the northern elephant seal population bottleneck. Current Biology 10, 1287-90.
