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1997 Blackwell Science Ltd, Molecular Ecology, 6, 661–666. 662. N. J. GEMMELL ET AL. Table 1 Pinniped species examined in this study, classified as.
Molecular Ecology 1997, 6, 661–666 SHORT COMMUNICATION

Interspecific microsatellite markers for the study of pinniped populations N. J. GEMMELL, P. J. ALLEN, S. J. GOODMAN,* J. Z. REED Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK, *Institute of Cell, Animal and Population Biology, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK

Abstract Microsatellites have rapidly become the marker of choice for a wide variety of population genetic studies. Here we describe 20 pinniped microsatellite markers which have been tested across 18 pinniped species. The majority of these markers have broad utility in all pinnipeds and provide a strong base for detailed population genetic studies in the Pinnipedia. Keywords: microsatellite, pinniped, population genetics Received 3 September 1996; revision accepted 12 February 1997

Introduction Despite intensive study the population structure and reproductive strategies of many pinniped species remain unresolved. Behavioural observations are often difficult given the sheer density and number of animals involved, while mark–release–recapture and radio telemetry studies require large amounts of time and effort to return useful data. An alternative approach is to investigate relationships within and between pinniped populations using molecular genetic technologies. Microsatellites are widely regarded as the most versatile genetic markers yet discovered and their applicability for identifying relationships in natural populations is well documented (Bruford & Wayne 1993; Queller et al. 1993). They are highly polymorphic, predominantly selectively neutral, 2–5-bp repeat sequences which occur scattered abundantly throughout the genomes of all higher organisms (Tautz & Renz 1984; Tautz 1989). Microsatellite length variation is quantified using the polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis, enabling small amounts of poor-quality DNA to be typed to single nucleotide resolution, simplifying comparison between experiments and laboratories. Microsatellites are frequently considered to be highly species-specific. As a consequence, the beginning of any Correspondence: Neil J. Gemmell, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UK. Tel: +44(0)1223-336677; Fax: +44-(0)1223-336676. E-mail: [email protected]

© 1997 Blackwell Science Ltd

microsatellite research on novel species entails the costly and time-consuming process of isolating and developing new markers. However, recent evidence suggests that many microsatellite markers will work within the same genus, and often further (Moore et al. 1991; FitzSimmons et al. 1995; Fredholm & Winterø 1995; Stallings 1995; Coltman et al. 1996; Rico et al. 1996; Valsecchi & Amos 1996). Such markers are highly desirable as they greatly reduce the requirement to develop microsatellites de novo and enable direct comparisons between species. In this paper we report the results of an intensive screen of 18 of the 33 extant pinniped species with a panel of 20 polymorphic microsatellite markers. The majority of these markers have broad utility in all pinnipeds and provide a strong base for detailed population genetic studies in the Pinnipedia.

Materials and methods Pinniped samples A total of 18 species, representing all of the living families constituting the Order Pinnipedia, were used in this study (Table 1). Of these, 12 species are true or phocid seals, while the remaining species include five eared seals and a walrus. Species sample sizes ranged from one to 48 individuals, the largest sample sizes being associated with several concurrent population genetic studies. Samples were received either as tissue preserved in 20% dimethylsulphoxide saturated with NaCl (Amos & Hoelzel 1991),

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Table 1 Pinniped species examined in this study, classified as reported in Riedman (1990). The numbers of species screened from each group are given in parentheses Suborder Pinnipedia Superfamily Phocoidea Family Phocidae Subfamily Monachinae (4 of 8 species) Hawaiian monk seal (Monachus schauinslandi) Mediterranean monk seal (Monachus monachus) Southern elephant seal (Mirounga leonina) Leopard seal (Hydruga leptonyx) Subfamily Phocinae (8 of 10 species) Hooded seal (Cystophora cristata) Bearded seal (Erignathus barbatus) Grey seal (Halichoerus grypus) Harp seal (Phoca groenlandica) Largha seal (Phoca largha) Caspian seal (Phoca caspica) Ringed seal (Phoca hispida) Harbour seal (Phoca vitulina) Superfamily Otarioidea Family Otariidae Subfamily Otariinae (2 of 5 species) Stellar sea lion (Eumetopias jubatus) Southern sea lion (Otaria byronia) Subfamily Arctocephalinae (3 of 9 species) South American fur seal (Arctocephalus australis) New Zealand fur seal (Arctocephalus forsteri) Antarctic fur seal (Arctocephalus gazella) Family Odobenidae (1 of 1 species) Walrus (Odobenus rosmarus)

or as purified DNA in TE. DNA was extracted from tissue samples as described in Gemmell & Akiyama (1996).

Microsatellite analysis Microsatellite markers were developed from three species of pinniped, the grey seal, the harbour seal and the South American fur seal, using the methods described in Rassmann et al. (1991). Thirty-two primer sets were developed of which 18 amplified polymorphic loci in more than one of our three primary species. Only these 18 primer sets, and two other pre-existing primer pairs isolated from the southern elephant seal, were included in the current study. The primer sequences of the microsatellite loci used are shown in Table 2. PCR amplifications were initially carried out in 10-µL reaction volumes using the reaction conditions of Allen et al. (1995). However, in order to improve the specificity of the PCR reaction, at the necessarily low annealing temperatures required for cross-species microsatellite amplification, 60mM tetramethylammonium chloride (TMAC) and 2.5% formamide were added to the reaction mix (Gemmell 1997). The modified reaction mix was as

follows: ≈ 20 ng of genomic DNA, 1 × PARR excellence PCR buffer (Cambio, Cambridge, UK), 60 mM TMAC, 2.5% formamide, 0.1 mM of dGTP, dATP, dTTP, 0.01 mM dCTP, 4 pmol of each primer, 0.25 units of Taq DNA polymerase and ≈ 1 µCi α-32P-dCTP. Reactions were overlaid with mineral oil and thermal cycling carried out as follows: 2 min at 94 °C, 8 cycles of 94 °C for 30 s, 48 °C for 30 s, 72 °C for 40 s and 25 cycles of 94 °C for 15 s, 52 °C for 15 s, 72 °C for 40 s. Microsatellites were resolved on 6% polyacrylamide denaturing gels with a nonrecombinant M13 sequencing reaction and five grey seals of known genotype as size standards. In species where only one or two samples were available, amplification of a microsatellite was considered successful if either one (homozygote) or two (heterozygote) products showed characteristic microsatellite stutter bands (Hauge & Litt 1993; Valsecchi & Amos 1996). An amplification was considered unsuccessful, as opposed to general PCR failure, only if the majority of other samples in the experiment amplified successfully. If in doubt the samples were reamplified and rerun.

Results Twenty primer sets were tested for amplification on 18 pinniped species. The results of these amplifications are presented in Table 3. Of 360 locus/species combinations, 237 (65.8%) amplified polymorphic products. However, this is likely to be a significant underestimate because in 16.6% of all locus/species combinations only one specimen was tested. In some of these cases it is likely that amplification/ polymorphism will have been undetected, due to the sample being homozygous or possessing null alleles (Koorey et al. 1993). In order to test the extent to which this is so, our results were classified into three categories: single-specimen combinations, two-specimen combinations and all others (Table 4, after Valsecchi & Amos 1996). We then examined whether the proportions of loci that were polymorphic, monomorphic or showed no amplification were significantly different from expectation for each of our groups. As expected, the proportion of monomorphic loci and null amplification is much higher when only one specimen was tested (χ21 = 10.924, P 600 000 animals) now, but that this population has been large for some time. At the other end of the spectrum, extremely low levels of polymorphism were observed in the Hawaiian monk seal Monachus schauinslandi. In the five individuals examined, each of which represented one of the five major breeding sites, only three of 20 markers were polymorphic, and these were all dimorphic. While this is a species known to have very low levels of genetic variation between individuals (Kretzmann et al., in press), the levels of variation are markedly less than that observed in four samples from a small, isolated, West African population of its sister species the Mediterranean monk seal Monachus monachus – a species also known to be genetically impoverished, but which is at least threefold less numerous (≈ 400 vs. 1300 animals).

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 661–666

56 19 5 80

157 56 8 221

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237 103 20 360

Taxon-specific markers Several primer sets showed specificity for particular groups, with the most striking differences being observed at the family level. Locus Hgdii is diagnostic for the families Phocidae and Odobenidae. In all species tested, polymorphic microsatellites were only amplified in the Phocidae and Odobenidae, whereas no amplification product was observed in the Otariidae. Other loci including Hg3.7, Hg8.9 and BG, also exhibit family specific amplification patterns, however, in the case of these loci the distinction is based on the size of the amplified product. In otariids and the walrus the Hg3.7 primers amplify polymorphic microsatellites, which are twice as large as those observed in phocids. Similarly, Hg8.9 is large and monomorphic in the Otarioidea, and small and polymorphic in the Phocidae. The reverse is true for BG. Further, more detailed analyses, should enable the identification of markers capable of resolving species and population identity on the basis of allele frequency distribution differences (Paetkau et al. 1995; Reed et al.1997).

Conclusion The large number of primer sets that amplify polymorphic loci successfully in diverse pinniped species augers well for future population studies. Allen et al. (1995) have already illustrated the efficacy of microsatellite methodologies for investigating differentiation and substructuring in pinniped populations. Regrettably, the prerequisite need to clone novel microsatellite markers has limited the application of this technology. However, having now identified more than 10 microsatellite loci with broad cross-species reactivity studies of this type are now conceivable in all pinniped species. Indeed, for many species, we are now in a position where very fine-scale investigations of reproductive strategies are possible.

Acknowledgements We are grateful to the following people for supplying samples and primers: Tom Arnbom, Ian Boyd, Charles Chambers, Claudio Campaigna, Luis Cappozzo, Stephen Davenport, Callan Duck, Ailsa Hall, Rus Hoelzel, Maria Kratzmann, Patricia Majluf, David Paetkau, Teresa Pastor, Patrick Pomeroy, Rob Slade, Paul

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Thompson, Sean Twiss. N.J.G is funded by the Leverhulme Trust and J.Z.R is funded by the National Environment Research Council. All work was conducted in the laboratory of Dr Bill Amos and we gratefully acknowledge his financial and academic support.

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© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 661–666