Heredity 55 (1985) 199-207 The Genetical Society of Great Britain Received 6 February 1985 Lack of biochemical polymorphism in British fallow deer J. M. Pemberton* Wildlife Management Group, Department of Pure and Applied Zoology, University of Reading, Whiteknights, and R. H. Smith P.O. Box 228, Reading RG6 2AJ Seven-hundred and ninety-four samples of fallow deer (Dama dama L.) blood or tissue were collected from 37 sites in England and Wales. A selection of these samples was screened for electrophoretic variation at each of 30 loci (minimum of 88 samples per locus). No genetic variation was found. Possible explanations for the lack of polymorphism are discussed. It is suggested that European fallow deer experienced a genetic bottleneck during a period of captivity in Mesolithic or Neolithic times. INTRODUCTION Electrophoresis has been used to generate genetic Most of the applications mentioned above data about a number of deer species. To date at imply the presence of considerable intraspecific least 70 papers and reports have been written in polymorphism in deer, which is certainly true for this field. The information obtained has been used the four species on which studies have so far con- to study genetic differentiation between species centrated. Most work on the Old World reindeer (Baccus et a!., 1983), subspecies (Gyllensten et a!., (Rangifer tarandus tarandus) has been limited to 1983; Dratch and Gyllensten, in press) and popula- a single locus, transferrin, at which major surveys tions (Soldal and Staaland, 1980; Ryman et a!., have each revealed between 7 and 12 alleles 1980; Gyllensten et a!., 1983; Dratch and (Braend, 1964; Turubanov and Shubin, 1971; Gyllensten, in press) and to examine population Zhurkevich and Fomicheva, 1976; Soldal and subdivision, givinginsightsintodispersal Staaland, 1980; Røed, in press). The remaining behaviour (Manlove et aL, 1976; Ramsey et aL, three species have been surveyed extensively both 1979; Chesser et a!., 1982). Other authors have geographically and in terms of the number of loci sought relationships between performance and screened, and table 1 summarises the principal genotype (Johns et a!., 1977; Smith et a!., 1982; studies. Points to notice are that none of the species Cothran et a!., 1983) or changes in genotype completely lacks detectable variation and that the frequencies over time (Baccus eta!., 1977; Chesser white-tailed deer shows an apparently higher level et a!., 1982), suggesting the action of natural selec- of genetic variation than moose, red deer and tion. Among practical uses, the discrimination of wapiti. Indeed, all the white-tailed deer popula- species from blood spots or tissue scraps is of value tions and some of the moose and wapiti popula- in law enforcement (Oates et aL, 1979; Lawton tions studied have average heterozygosities (H) and Sutton, 1981; McClymont et a!., 1982) and greater than the H for all mammals (H = genetic information is of obvious interest to con- 0.033-0.039)derived by various authors (Powell, servationists, for example in the planning of 1975; Nevo, 1978; Avise and Aquadro, 1982; introductions (Smith eta!., 1976; Ryder eta!., 1981; Baccus et a!., 1983) which argues further against Gyllensten et a!., 1983). A number of uses for the generalisation that large mammals are electrophoresis studies can also be envisaged in monomorphic (see Ryman et a!., 1980). the growing deer farming industry, ranging from The present study was undertaken as a first the assessment of genetic differences between lines step towards using electrophoretically-detectable to paternity testing. variation to investigate the occurrence of *Presentaddress: Department of Zoology, University College inbreeding in fallow deer (Dama dama L.) London, Gower Street, London WC1E6BT. populations (Smith, 1979). Previously published 200 J. M. PEMBERTON AND R. ft SMITH Table I Summary of the mostextensive genetic surveys ofdeer speciesto date Number (and location) of Number Range of average populations of loci heterozygosity Species surveyed screened (H) found Reference white-tailed deer 8 (SE. USA) 35 0049—0092 Smith et a!., in press (Odocoileus virginianus) moose 18 (Scandinavia) 23 0006-0047 Ryman ci a!., 1980 (Alces alces) red deer and wapiti 22 (NW. Europe) 34 0000-0031 Gyllensten ci al., 1983 (Cervus elaphus) 11 (NW. USA) 28 0004-0060 Dratch and Gyllensten, in press electrophoresis and isoelectric focusing studies of blood sampled) while samples of muscle only were fallow deer include a survey of serum proteins, obtained from 51 carcasses in a butcher's cold especially transferrin, by McDougall and Lowe store. (1968), haemoglobin studies by Maughan and Fig. I also gives a rough indication of the Williams (1967), Butcher and Hawkey (1977) and number of animals sampled per site which ranged Lawton and Sutton (1981) and the screening of from one (four sites in Berkshire and Hampshire) LDH by Munday et a!. (1974). Only the Butcher to 126 (Margam Park, South Wales). The "sites" and Hawkey (1977) study suggested a polymorph- themselves consisted of 14 more-or-less enclosed ism in fallow deer. However, besides the small parks and 23 areas holding wild fallow deer. number of loci screened, all the studies mentioned In addition to fallow deer samples, 133 blood involved limited sampling of individuals and/or samples and 14 post mortem tissue sets were collec- populations. Since initial small surveys of the ted from red deer from two sites and blood samples species shown in table I also sometimes failed to were taken from six muntjac (Munliacus reevesi) reveal variation (e.g., transferrin: Braend, 1962; from one site, for comparative purposes. Johnson, 1968; Seal and Erickson, 1969) the pre- Blood was collected into heparinised tubes vious fallow deer studies do not necessarily indi- (Vacutainer and Sterilin). Blood samples from live cate the outcome of a more thorough study. deer and from some freshly shot deer were brought Indeed, given the results of existing large surveys to Reading in a vacuum flask containing melting of deer species (see above), the widespread, geneti- ice and were processed within 48 hours of collec- cally controlled coat colour polymorphism of Brit- tion. Blood samples collected by stalkers were ish fallow deer (Chapman and Chapman, 1975, posted to Reading and processed on arrival. After pp. 23-3 1) and a suggestion of genetically deter- centrifugation (3000 rpm for 15 minutes) the mined skeletal variation in this species (Chapman plasma was drawn off, separated into aliquots and and Chapman, 1969), the present survey was fully frozen. All samples were stored at —30°C. Approxi- expected to reveal genetic variation. mately 2 ml of the remaining red cells were washed and centrifuged two to three times in 8 ml 0.85 per cent NaC1 solution. When a clear supernatant was MATERIALSAND METHODS produced, the red cells were lysed with 1 ml dis- tilled water, divided into aliquots and frozen. Bloodand post mortem tissues were collected from Post mortem tissues collected by stalkers were fallow deer from the sites in England and Wales stored in domestic freezers until retrieved. The shown in fig. 1. In total 794 individual deer from samples were brought to Reading in a vacuum flask 37 sites were sampled in some way, but we empha- packed with solid CO2 to prevent thawing. The size the heterogeneity of the samples. Blood was tissues were kept frozen until immediately before obtained by bleeding live deer (389 animals), by electrophoresis when sections weighing approxi- post mortem sampling (280 animals) and by taking mately 600mg were homogenised in an equal aliquots of samples collected by other researchers volume of distilled water. All homogenising was (43 animals). Post mortem tissue sets consisting of carried out by hand in a ground glass homogeniser muscle, liver and kidney were collected by stalkers (Gallenkamp) or with a pestle and mortar, when from 179 animals (including 148 which were also 200 mg sterilised sand was added to aid grinding. LACK OF POLYMORPHISM 201 • >30 • 11—30 . •<11 ••.I ••.' • S. Figure 1Sites in England and Wales from which fallow deer samples were obtained; size of circle indicates number of animals sampled. Homogenates were centrifuged (3000 rpm for 15 mammals, because they stain up at the same time minutes) and the supernatant was then used in as the "target" loci, or because they are cheap to electrophoresis. stain or easy to resolve. Electrophoresis was carried out using a mixture In a project with a fixed time limit, the number of Shandon and home made equipment. General of loci which can be screened is inversely related procedure for horizontal starch gel electrophoresis to the number of individuals screened at each did not differ importantly from that described by locus. In the present study, after the first few loci Manlove et a!. (1975). Buffer systems and stain had been screened, calculations were made to help recipes were taken from the literature or followed decide how many samples from the "sample bank" those in use in the laboratory of Professor R. J. needed to be examined at a locus and found to be Berry, University College London. In several cases indistinguishable for that locus to be regarded as more than one buffer system was tried. Preferred monomorphic. Taking a locus in which the rarer buffer systems, tissues used and references to allele is at frequency 001 as a minimum criterion detailed methods are given in table 2. Full details of polymorphism and assuming Hardy-Weinberg are given in Pemberton (1983). equilibrium, each individual examined gives a Haemoglobin was focused in 03 mm thick 7 probability of 098 of not detecting such a poly- per cent acrylamide gels containing pH 6-8 morphism. The probability of detecting poly- Ampholines (LKB) at a concentration of 02 per morphism is then (1_0.981)=0.02 after one cent. Seventeen chroma Whatman paper was sample, (1_0.98b0)=0.18 after 10 samples, etc.