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  • Absence of Genetic Variation in Harbor Seals (Phoca vitulina) in the Dutch Wadden Sea and the British Wash

    JACOBUS A. A. SWART,*:~ PETER J. H. REIJNDERS,t AND WILKE VAN DELDEN:~ *Section of Science & Society, Biological Center, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands t DLO-Institute for Forestry and Nature Research, P.O. Box 167, 1790 AD den Burg, The Netherlands ~/Department of Genetics, Biological Center, University of Groningen, P.O. Box 14, 9750 ~ Haren, The Netherlands

    Introduction

    Populations of the harbor seal, subspecies Phoca vitu- lina vitulina, are found at the northeast Atlantic coasts and Iceland. The population in the Dutch part of the Wadden Sea has undergone a strong decline since the beginning of the twentieth century. The population de- creased from approximately 7000 to only around 500 animals in the late 1970s, first because of overhunting and then because of the effects of environmental pollu- tion ~ei jnders 1976~ 1978, 1980). The population started to recover in the early 1980s but an epidemic viral infec- tion, which started in 1988, resulted again in a large re- duction in population (Reijnders 1989).

    One common consequence of a decline in popt~t ion size is decreased genetic variation. In populations of oth- erwise genetically variable species, the reduction in ge- netic variation following bottlenecks in population size often leads to severe inbreeding and is generally accom- panied by loss of individual fitness. This latter phenome- non, known as inbreeding depression, may increase the chance of extinction of already vulnerable , populations (Van Delden 1992; Brakefield & Saccheri I994; Bijlsma et al. 1994). Low genetic variability reduces adaptive po- tential in variable environments, which is a considerable disadvantage when a species must live in a new environ- ment (Levins 1968; Bryant 1974). Genetic diversity ap- pears to be positively correlated with broader geo- graphic, climatic, and habitat spectra (Nevo et al. 1984; Karron 1987, 1991). Human impacts on the environ- ment create new and often less favorable environments.

    Paper submitted July 11, 1994; revised manuscript accepted Febru- ary 16, 1995.

    Thus, conservationists should be alert to the level of ge- netic variation and should consider action when low ge- netic variability is found in endangered populations.

    Accordingly, it is relevant to determine the genetic variation of the reduced population of harbor seals in the Dutch Wadden Sea. A straightforward method to in- vestigate the level of genetic variation is the electro- phoretic assay of proteins (Lewontin & Hubby 1966; Hubby & Lewontin 1966; Harris 1966; Nevo et al. 1984; Hamrick & Godt 1990). We describe the results of the electrophoretic assessment of allozyme variation in two populations of the harbor seal (Phoca vttulina vitu- lina), one originating from the Dutch Wadden Sea and one from the British Wash. The latter population has not experienced as severe a reduction in population size as has occurred in the Wadden Sea population.

    Materials and Methods

    Blood samples were taken in 1982 (analyzed within three months after sampling) from 38 female seals kept in captivity in the Institute for Forestry and Nature Re- search (IBN) in Texel, The Netherlands. Twenty-one of the animals were offspring of individual females caught in - the Dutch Wadden Sea, and 17 were captured in the Wash in England. Kinship among the seals was not known. Blood sampres (about 10 mL each) were col- lected from the hindflipper vein and immediately centri- fuged'(3000 rpm, 15 minutes, r = 14 cm). The pellets were frozen at -25Q(~ in sealed tubes. Because freezing may damage the blood cells, samples were not washed to re- move possible disturbing ions. To hydrolyze the red blood cells and to stabilize the protein molecules, we added 4 ml of an EDTA/NADP buffer (0.2 M Na2HPO4.H20,

    289

    Conservation Biology, Pages 289-293 Volume 10, No. 1, February 1996

  • 290 Genetic Variation in Harbor Seals Swart et al.

    0.2 M NaH2PO4.H20, 1 mM Na2EDTA.H20, 17 mg NADP/1, 0.02 ml f3-mercaptoethanol/L at 4C), and we shook the samples vigorously for 1 O-15 minutes at room temperature. Subsequently, we shook the samples vigor- ously for 1-2 minutes with one-third volume CCI 4 in or- der to remove fat molecules from the sample. The sam- ples were centr i fuged again (3000 rpm) for 20-30 minutes. The resulting bright red supernatant - -d i rect ly usable for e lec t rophores is - -was divided into aliquots and stored at -70C. Electrophoret ic runs were carried out at 4C and 15 V/cm in horizontal 6% polyacrylamide slabs wi th a part icular e lectrophoret ic buffer (Table 1). Subsequently, the gels were stained in accordance wi th the references in Table 1. The samples were Screened for variation in 21 enzyme and non-enzyme systems. These 21 prote in systems were selected from a total of 37; the other 16 were rejected because no or nonrepro- ducible staining activity was found. The number of stain- ing bands are presented in Table 1.

    Results and Discussion

    None of the 21 prote in systems in the samples from both populat ions showed any variation. This result indi- cates no al lozyme diversity among harbor seals in either

    the British or the Dutch populat ion. This result is in ac- cordance with reports on the absence or low levels of al lozyme variation in other seals such as the northern e lephant seal (M i rounga angust i ros t r i s ; Bonnell & Se- lander 1974), the southern e lephant seal (M i rounga le- on ina , McDermid et al. 1972), the r inged seal (Pusa h i sp ida) , the harp seal (Phoca groen land ica) , and the hooded seal (Cystophora cr is tata , Simonsen et al. 1982a, 1982b). Low levels of variation (in heamopex in and adenylate kinase systems) were found in 25 adult gray seals (Ha l i choerus grypus) and 19 harbor seal pups, the latter originating from the Wash (McDermid & Bonner 1975). Although studies on al lozyme variation have shown that many animal and plant species are ge- netical ly highly variable (Nevo et al. 1984; Hamrick & Godt 1990), low levels of variation are frequently found among carnivorous mammals (Al lendorf et al. 1979; Si- monsen 1982; Simonsen et al. 1982a, 1982b; O'Brien et al. 1983, 1985, 1987; Menotti-Raymond & O'Brien 1993). Our results on al lozyme variation are conf i rmed by a re- cent study by Kappe et al. (1995) employing more pow- erful tools for the est imation of the level of genetic varia- tion. They examined two populat ions of the harbor seal, one from the Dutch Wadden Sea and one from the east- ern coast of Scotland by means of mult i locus DNA fin-

    Table 1. Protein systems investigated and electrophoretic methods and results.

    Protein systems Buffer Reference sta in ing Name Symbol system a technique ~

    Methods Results Number o f bands in red blood cells

    Enzyme proteins Glucose phosphate isomerase GPI A * Isocitrate dehydrogenase IDH B * Hexokinase HK A * Glucose-6-phosphate dehydrogenase G-6-PD A * 6-Phosphogluco.nate dehydrogenase 6-PGD A * Phosphoglucomutase PGM A * Lactate dehydrogenase LDH B * Pyrophosphate phosphohydrolase PP B Harris & Hopkins 1976 cx-Glycerophosphate dehydrogenase ot-GPDH B * Glyceraldehyde-3-phosphate dehydrogenase GA-3-PD C * Glutamate oxaloacetate transaminase GOT A * Xanthine dehydrogenase XDH A * Adenylate Kinase AK A Shaw & Prasad 1970 Malate dehydrogenase MDH B * D(-)-3-Hydroxybutyrate dehydrogenase HBDH B Shaw & Prasad 1970 Esterase EST B * Tetrazolium oxidase TO F * Amylase AMY E *

    Non-enzyme proteins Haemoglobin HB D Benzidine staining BZ D Amidoblack staining AB D

    Brewer 1970 3 Kimura 1976 1 Brewer 1970 1

    aThe buffers were developed in the Department of Genetics, University of Groningen, except buffer system B. A: 0.05 tris citrate, pH 7.0, diluted 1/2; B: 0.1 M tris maleic anhydride, 0.05 M MgCla 0.05 M EDTA, pH 7.0 (Harris & Hopkinson 1976); C: 0.05 M Na2PO 4 citrate, pH 4.5; D: 0.5 M tris boric acid, 6 g EDTA/L; E: 0.05 M tris citrate, pH 8.0; 17." 0.1M boric acid, NaOH to pH 9.0. b Staining techniques were developed in the Department of GeneticS, University of Groningen and are available on request.

    Conservation Biology Volume 10, No. 1, February 1996

  • Swart et al. Genetic Variation in Harbor Seals 291

    gerprinting. Average band-sharing coefficients, S (Wet- ton et al. 1987), were 0.85 and 0.81, respectively, for within-population comparisons and 0.81 for between- population comparisons. These figures were much higher than S -- 0.42 found for a sample (n = 14) of a re- lated species, the gray seal, also caught in the Dutch Wadden Sea. Kappe et al. (1995) calculated that the av- erage heterozygosity in gray seals was about twice that in harbor seals. The low level of genetic variation in the harbor seal was further demonstrated by means of the random amplified polymorphic DNA (RAPD) method ap- plied to the two populations screened by Kappe et al. (1995). Out of 50 randomly chosen primers, 25 gener- ated amplified fragments. All individuals of both popula- tions revealed an identical monomorphic pattern with 23 primers, although only two primers gave polymor- phic bands. Thus, both allozyme and DNA assays indi- cate low levels of genetic variation in the harbor seal. Further studies should include the screening of other subspecies of harbor seals, originating from either the Atlantic or the Pacific Ocean, to access genetic differ- ences among these subspecigs.

    Harbor seals are restricted to coastal areas. The great genetic similarity between the Wadden Sea and British populations may be explained by occasional migrations. Low levels of migration, as low as one animal a year, may prevent genet

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