Biological Conservation 1992, 61, 133-144

Applications of molecular genetic techniques to the conservation of small populations

Bill Amos*," & A. Rus Hoelzel*,~, b
a Department of Genetics, University of Cambridge, Downing Street, Cambridge, UK, CB2 3EH b NERC, Centre for Population Biology, Imperial College at Silwood Park, Ascot, Berks, UK, SL5 7P Y

Over the last three decades, techniques have become available to screen large numbers of individual animals for genetic variability at the molecular level. Earlier work employed gel electrophoresis to resolve slight changes in the amino acid sequence of proteins. More recently, polymorphism at the level of the D N A itself has become amenable to screening through the discovery of a class of enzymes, restriction endonucleases, which cleave DNA at specific sites. By focusing on specific classes of D N A sequence, each with its own characteristic rate and mode of evolution, different degrees of genetic resolution may be obtained. At the fastest end of the spectrum, the extremely high rates of gain and loss of repeats from minisatellite arrays forms the basis of the technique of DNA fingerprinting, capable of uniquely identifying individuals and assignment of parentage. At the population level, internal mechanisms of molecular turnover result in the homogenisation of repeated sequences and can result in population-characteristic markers. The small extranuclear mitochondrial genome is inherited only through the maternal line and thus provides an informative tool for investigating haplotype lineages. All these techniques, and others, are discussed in relation to how they may be used to determine the basic population parameters which may be important in a critical assessment of an endangered species: the effective population size, population substructure and various aspects of the generation and effects of having low genetic variability. In some instances several techniques may be equally suitable, in others only one will do. We therefore discuss other factors which may influence the choice of methodology, such as approaches for tissue sampling, sample preservation and cost. Amongst these, the technique of PCR (Polymerase chain reaction), a means by which minute quantities of a specific DNA sequence may be enzymatically amplified, can be singled out as one which will influence profoundly the scope and power of future molecular genetic analysis.

INTRODUCTION

The problem of maintaining genetic variation in small populations can be divided into two general concerns: the effect of lost variation on the evolutionary stability of populations; and the effect of inbreeding, outbreeding and heterosis on individual fitness. Both are affected by the 'effective population size' (Ne: the average number of individuals with equal reproductive contribution to the succeeding generation) and are therefore dependent on behavioural characteristics such as
*Order of authorship is arbitrary Biological Conservation 0006-3207/92/$05.00 1992 Elsevier ,Science Publishers Ltd, England. Printed in Great Britain 133

dispersal rate, range and breeding system. In the following review, we will describe how modern molecular genetics can improve the resolution of, and increase the scope for, analyses of behaviour and population parameters related to these conservation issues. Beginning in the mid-1960s, with the development of a method for routine screening of genetic variants by gel electrophoresis (Harris, 1966; Lewontin & Hubby, 1966), a vast body of data on protein variation in natural populations began to amass. It became apparent that small differences between molecules of the same protein were abundant and that these differences were heritable, reflecting minor changes at the level of the DNA itself (see review by Nevo et aL, 1983). Visualisation

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of this variability could be achieved by comparing the mobilities of each form, or isozyme, through starch or polyacrylamide gels under the influence of an electric field. For the first time a wealth of quantifiable genetic variability, comparable between both populations and species, became accessible. This opened a window into the fundamental processes of evolution. The technique was quickly and widely adopted, establishing a vast body of isozyme-based data. In similar fashion, a far-reaching development was achieved with the discovery of a class of enzymes, called restriction endonucleases, which cleave DNA in a predictable way (Lederberg & Meselson, 1964; Meselson & Yuan, 1968). These enzymes recognise short, very precise motifs, generally of between four and six nucleotides. There are now well over 100 known restriction enzymes and the list is continually growing. Most common forms of mutation can be monitored using these enzymes, by examining differences in the length of fragments produced when DNA is cleaved. For instance, if one nucleotide gets replaced by another the resulting sequence change may either delete an old restriction site or generate a new one. In either case one or more DNA fragments will change in length, and this can be detected by gel electrophoresis. Similarly, deletions and nsertions will also result in length changes that may be accurately quantified. This general methodology (restriction fragment length polymorphism = RFLP) has allowed the development of the new methods for genetic analysis described below. The chemical composition of DNA is extremely simple, comprised of only four different nucleotides (the basic building units) arranged in one long linear message. Despite this apparent simplicity it is structurally highly complex. Perhaps only 5% of the genome is represented by regions traditionally thought of as genes (the sequences that encode proteins). The rest ranges from very short motifs tandemly repeated thousands of times, through gene families (genes present as multiple copies in the genome, see Long & Dawid, 1980), to tracts of apparently functionless sequences in which nucleotides seem to be randomly arranged (Dover & Flavell, 1982). Given this diversity it is not surprising to find differences in mutation rates between genomic regions. Furthermore, different forms of mutational event are more prevalent in some classes of DNA than in others.

It is clear that the examination of non-coding regions of the genome can greatly expand our ability to resolve genetic structure at the population level. Some DNA regions vary at a rate that is sufficiently high to mark individuals uniquely, test paternity and trace genealogies (e.g. the minisatellite sequences described by Jeffreys and co-workers 1985a, b). Other regions are conserved within reproductive populations more than would be expected by chance, thereby identifying patterns of reproductive continuity (gene families, e.g. the rDNA gene family, see Dover, 1982; Coen & Dover, 1983). The mitochondrial genome (an independently evolving, extranuclear molecule showing strict maternal inheritance) can be used to examine matrilineal history. It is also sufficiently variable to serve as a potent population marker (Wilson et al., 1985).

EVOLUTIONARY STABILITY OF POPULATIONS

Effective population size

It is a fundamental axiom of evolutionary theory that natural selection acts on a store of genetic variation phenotypically represented in the diversity of individuals. As environments change, whether by catastrophy (e.g. floods, resource failure, etc.) or gradual transition, it is critical that species possess sufficient variation to respond. The most important factor influencing the level of genetic variation, in the absence of strong selection, is the effective population size. This is because genes tend to become fixed into the homozygous condition by the random gain and loss of gametes or individuals in small populations. This effect occurs independently of change due to selection, mutation or immigration and is referred to as genetic drift (see Wright, 1978). One method for the estimation of N e depends on the assumption that mutations in the mitochondrial DNA (mtDNA) accumulate at an approximately constant rate over time. The time since two randomly picked individuals shared a common mother would then be given by the simple relationship: t (in million years) = 0-5 d, where d is the mean pairwise divergence between mitochondrial genomes (see Wilson et al., 1985). The effective population size could then be estimated by dividing t by the mean number of years per generation (Wilson et al., 1985). This estimate,

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however, provides only an average, long-term approximation to N e. An estimate of current Ne can be made by measuring recent temporal fluctuations in allele frequencies between generations (Waples, 1989), or levels of gametic disequilibrium at mul-tiple loci (Hill, 1981). These indirect methods depend on assumptions of selective neutrality, random mating and no migration between genetically differentiated populations. The temporal allele model takes advantage of the prediction that random drift and inbreeding will increase fluctuations between generations. This method generally requires comparisons over a large number of genera-tions, though in some cases comparing cohorts will suffice (Waples, 1990, 1991). The gametic disequilibrium model is based on the fact that drift increases linkage disequilibrium. Variance will be very high by this method unless a large number of loci are investigated. In both cases, molecular methods that measure highly variable regions that are selectively neutral may increase the accuracy of these estimates. Non-genetic estimates can be achieved through the application of mathematical population models (see review in Lande and Barrowclough, 1987), which use data on the number of males and females, fluctuations in population size, and variance in male and female reproductive success. However, acquiring data on male reproductive variance is rarely trivial. One method is to compare the genetic variance of males of reproductive age in a population with the variance of paternally derived alleles in the offspring generation (McCracken & Bradbury, 1977, 1981). The most direct method is to extend observed maternal genealogies through the genetic determination of paternity. In the past, paternity testing was conducted on an exclusion principle. It was possible to rule out individuals as potential fathers if their isozyme profile was inconsistent with allelic variants in the mother and offspring (e.g. McCracken & Bradbury, 1977). However, if enzyme variability is low (as is often the case for large mammals, see Simonsen et al., 1982), then paternal exclusion will not be possible for most males tested. A more powerful form of exclusion based on highly variable RFLPs has recently been used on a natural population of snow geese Anser caerulescens (Quinn et al., 1987). However, another technique, popularly referred to as DNA fingerprinting (Jeffreys et al., 1985a; Smouse & Chakraborty, 1986; Whetton et al., 1987), allows the direct determination of paternity.

DNA 'fingerprints' are based on regions of the genome known as minisatellites (Jeffreys et al., 1985b). These are short, tandemly repeated sequences that occur clustered at as many as one hundred sites (loci) throughout the genome. These arrays are subject to extremely high levels of genetic crossing, tending to produce similarly high variability in repeat copy number. Measured levels of allelic diversity may reach as much as 98% in humans (Wong et aL, 1986), far exceeding those observed for isozyme loci. Visualisation of alleles is achieved using simple electrophoresis and, since minisatellites share a conserved 'core' sequence, 50 or more loci may be surveyed simultaneously. The resulting banding patterns contain enough information to assure confident positive identification of individuals for most species examined to date (Gill et al., 1985; Jeffreys & Morton, 1987; Amos & Hoelzel, 1990). Extensive studies have now been carried out on known human (Jeffreys et al., 1986) and mouse (Jeffreys et al., 1987) genealogies to investigate inheritance. It has been shown that individual bands are inherited in strict Mendelian fashion with low levels of linkage. This is important since it allows the exceptional variability of these sequences to be harnessed to the previously intractable problem of positive paternity analysis. Each parent will donate, on average, half its bands to any given offspring. If the mother is known, then bands attributable to her may be subtracted from the offspring's fingerprint, leaving a subset of bands which must belong to the father. By comparing this subset of bands with the banding patterns of putative fathers it is possible to identify the father with great confidence. The power of this method has been recognised and used in legal test cases involving rape and murder. It is important to note that theoretical considerations indicate that, despite its proven capacity for determining paternity, for genetic relationships more distant than full-sib or parent-offspring, the resolving power of DNA fingerprinting falls off dramatically (Lynch, 1988). Minisatellite sequences have been found to occur in an ever-increasing range of animals. The list includes several insects and fish (Amos, unpublished data), mice (Jeffreys et al., 1987), dogs (Morton et al., 1987), cats (Jeffreys & Morton, 1987), ungulates (Vassart et al., 1987), whales (Hoelzel & Amos, 1988; Amos & Hoelzet, 1990), seals (Amos & Anderson, in prep.) and birds (Burke & Bruford, 1987), for a review see Burke

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(1989). Levels of variability revealed by these studies are not always as high as those originally found in humans. Nonetheless, paternity has been positively assigned in natural populations of birds (Whetton et al., 1987) and seals (Amos & Anderson, in prep.). Furthermore, optimisation of protocols and cloning of species-specific probes could greatly improve non-human applications. Since the discovery of minisatellite-based DNA fingerprinting, a number of other hyper-variable systems have been developed. Two important methodologies depend on the high mutation rates observed amongst simple 'microsatellite' sequences. These tandemly repeated di-, tri- or tetranucleotide repeats, such as (CAC)n or (GATA)n, occur abundantly in eukaryotic genomes. Since each repeat unit within an array is identical, the two DNA strands of a molecule can readily align out of register, a process termed slippage. Resolution of slippage products by DNA repair or other mechanisms can, in turn, lead to gain or loss of repeat units and hence to changes in the length of the array. Simple sequence variability can be visualised in two ways. In the simplest, longer arrays (1-15 kbt) can be analysed in a precisely analagous method to that used for multilocus minisatellite fingerprints (see, for example, Schafer et al., 1988; Weising et al., 1990). Genomic DNA is cut with a 4-base cutter enzyme, the fragments separated on an agarose gel and simple sequences detected with synthetic oligonucleotide probes. This method has been used successfully on a number of species. The second method was developed simultaneously by Tautz (1989), Litt and Luty (1989) and Weber and May (1989) and focuses on short stretches of dinucleotide repeats, perhaps 20-60 bp~/in length. This approach harnesses the power of the polymerase chain reaction (PCR--see below for a description) to amplify specific simple sequence loci enzymatically. PCR primers are constructed for each side of the dinucleotide repeats, in the unique flanking sequences of cloned, sequenced microsatellite loci. The DNA amplified will be determined by the position of the primers and is typically 50-300 bp in length. Such fragments are poorly resolved on agarose gels. However, by employing polyacrylamide 'sequencing' gels, single nucleotide differences may be recorded and simple sequence alleles differing by multiples t kb, kilobasepair. :~bp, basepair.

of two nucleotides are resolved efficiently. Furthermore, since careful construction of the primers allows one to determine precisely the length of the PCR product, and alleles tend to differ by only a few basepairs, it is possible to screen several loci simultaneously. Although this technique is still in its infancy, preliminary results suggest that a typical locus might have six to eight alleles of which three to four account for 90% of alleles scored (Schloetterer, Tautz and Amos, in prep.). This level of variability is considerably lower than has been observed for some minisatellite loci, but is nonetheless greater than that observed for most isozyme loci and RFLPs. However; the microsatellite approach does have a number of advantages when compared with DNA fingerprints. First, it allows efficient screening; it is certainly feasible to screen populations for 10 or more loci, sufficient for powerful exclusion paternity analysis. Secondly, the information yielded is very high quality since each allele is unambiguously described by its precise length. Thirdly, by effectively dissecting out individual loci from a fingerprint, it becomes possible to score both homozygotes and heterozygotes.

Loss of diversity
If a population has been substantially reduced in size, it is important to estimate the expected loss of genetic variance over time and its subsequent rate of recovery. For this purpose it is necessary to distinguish between variation in single locus systems and in quantitative characters. A quantitative character is one that shows continuous variation (e.g. height) and is controlled by the interaction of several genes. Franklin (1980) determined that, for a population at equilibrium and in the absence of selection, an N e of approximately 500 will be sufficient to maintain 'normal' levels of genetic variation for quantitative characters. This is based on the assumption that the additive genetic variance is roughly 10-3 the environmental variance expected of a genetically homogeneous line (Lande, 1976; Hill, 1982), and that variance is lost at the same rate as heterozygosity ( 1 / 2 N e per generation). By contrast, for selectively neutral alleles in a single locus system, an N e several orders of magnitude higher would be required to maintain high levels of heterozygosity (Dobzhansky,1970; Lande & Barrowclough, 1987). This is based on the relationship between the expected equilibrium

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level of mean heterozygosity and the mutation rate as described by Kimura and Crow (1964). Heterosis effects will reduce this requirement (see discussion in Hartl, 1980). Similarly, there is a considerable difference in expected recovery rates. Once above the critical N e, variation in quantitative characters could return to levels typical of large outbreeding populations within 100s or 1000s of generations (Lande, 1977, 1980), compared to 107 generations, assuming neutral mutations (Nei et al., 1975). Although the effect of a brief 'bottleneck' in population size will have a disproportionately large impact on the long-term value of N e (see examples in Lande and Barrowclough,1987), an extreme reduction in heterozygosity only results from extended periods of inbreeding or repeated crashes in a short period (Lewontin, 1974; Lande, 1980). Heterozygosity can be assessed directly from study populations and monitored over time to determine qualitatively if inbreeding is a problem. Unfortunately, there is no standard, acceptable level of heterozygosity. In addition, heterozygosity levels vary greatly between taxa, and in some cases may reflect the demographic structure of the population some time in the past. As we have mentioned above, diversity can take a very long time to recover after a population has been through a severe or extended bottleneck. For some of the single locus genes which code for metabolic enzymes, heterozygosity levels are readily determined by gel electrophoresis for enzyme polymorphisms (Brewer, 1970; Harris & Hopkinson, 1978). Nei (1978) has described a useful set of statistical formulae for the interpretation of this type of genetic variance. If possible, a database on heterozygosity in a non-threatened population of the same species should be acquired as a standard for comparison. The advantages of using enzyme electrophoresis, aside from practical considerations, are twofold. First, there is a vast literature of published levels of enzyme polymorphism from most taxa (see review by Nevo, 1978) to serve as a basis for comparison. Second, the study of loci that are expressed phenotypically allows an investigation into the possible role of natural selection on allele frequency at those loci. An alternative to using isozymes may be microsatellite analysis. Although there is currently no body of data with which to compare results, it might be argued that microsatellite heterozygosity should be roughly constant across broad taxo-

nomic divides. This is because microsatellites are extremely simple, both in their molecular sequence and in the dominant mutational forces which affect them (but see below). A further advantage is that these loci show higher levels of variability than equivalent isozyme loci and hence will have a greater chance of being affected by inbreeding. The probability of a bottleneck having affected the population can be determined by investigating variation at enzyme loci and in mitochondrial DNA (mtDNA). The mitochondrial genome is a double-stranded molecule ranging in size from 15.7 to 19.5kb in multicellular animals (see Fauron & Wolstenholme, 1976; Brown, 1983). It is inherited matrilineally and effectively haploid (Dawid & Blacker, 1972; Avise et al., 1979; Giles et al., 1980). This means that it will show the impact of a population bottleneck more than nuclear DNA. For example, if a diploid organism is reduced to one breeding pair, the population will still have four copies of the nuclear genome, but only one transmissable copy of the mitochondrial genome. Since regions in mtDNA evolve faster than most comparable nuclear loci (see Cann & Wilson, 1983), less variation in the mitochondrial genome will imply a population bottleneck (Ferris et al., 1982, 1983). Given a mean rate of divergence for mtDNA of 2% per million years (e.g. Brown & Simpson, 1982), it is possible to estimate the time since a bottleneck event (Wilson et al., 1985). Monitoring genetic variation of quantitative characters is best done by heritability studies (Lande & Barrowclough, 1987). The level of quantitative genetic variation for a trait can be estimated by measuring the narrow-sense heritability (h2, the ratio of the additive genetic variance to the total phenotypic variance, Hartl, 1981). The narrow-sense heritability of a trait can be estimated as the slope of the regression of the offspring value (some quantifiable measure of the trait) on the value for the mid-parent (the mean value for the two parents). As these estimates are subject to large standard errors, large sample sizes should be used. Full pedigrees for animals in wild populations or in game reserves can be difficult to determine from observational data. However, application of DNA fingerprinting techniques (as described above) can allow genealogies to be traced for up to several generations (limited by the age of extant ancestors) if one parent in each generation can be identified (Jeffreys et al., 1986).

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Population structure
In subdivided populations, the genetic diversity for the species is discontinuous. Regional variants will have adapted to local conditions, as well as having diverged due to stochastic effects. A subpopulation can become genetically isolated by genetic drift alone if there is fewer than one reproductive migrant entering the population per generation (Crow & Kimura, 1970). The representation of homozygotes in the species as a whole will increase, even if local populations are in HardyWeinburg equilibrium (Wahlund, 1928). However, there could be allelic variants specific to local populations. Subdivision may give the species greater flexibility to respond to changes in the environment over evolutionary time. In the event of a catastrophe, subdivision increases the likelihood that at least one population will survive. For this reason it is desirable to be able to recognise and maintain diversity at the subpopulation level. Genetic distance is a measure of gene diversity expressed as a function of genotype frequency (Nei, 1972). It is related to the pairwise comparison of allelic diversity over a number of different loci. To achieve high levels of statistical accuracy, it is useful to measure variation at as many loci as possible. For comparisons of conspecific subpopulations, the resolution of the analytical technique must be high so that relatively short periods of reproductive isolation can be detected. For this reason, the higher mutation rate observed in mtDNA and some well-understood non-coding genomic regions should be investigated. However, there is some danger in measuring genetic distance using non-coding regions. Measures of genetic distance assume that the dominant force generating sequence change is the gradual and constant accumulation of neutral mutations. Non-coding regions, while incorporating neutral mutations, are more susceptible to a variety of non-Mendelian processes than regions under functional constraints (Strachan et al., 1982; Tautz et al., 1986; Hancock & Dover, 1988). These processes have been collectively referred to as genomic turnover mechanisms and act independently of genetic drift (Dover, 1982). Although assuming neutrality produces believable results when coding segments of nuclear and mitochondrial genes are analysed, it may be incorrect by orders of magnitude if variation has been affected by turnover mechanisms (Dover, 1982; Coen & Dover, 1983). In general, unless the evolutionary

force generating diversity is fairly well-understood, it is not possible to interpret the observed pattern of genetic variation correctly at the population or specific level. For this reason we recommend using highly variable protein systems such as transferrin and albumin (Gahne et al, 1977), and mtDNA (see Lansman et al., 1981; Wilson et al., 1985; Moritz et al., 1987). In the case of the former, care should be taken to confirm that these loci are not under significant selection, or closely linked to other loci that are. Another possibility is to use the high levels of variability found at microsatellite loci (Tautz, 1989), since there are some grounds for expecting many of these to be selectively neutral and to mutate at a relatively constant rate. However, this may not be the case. Some simple sequences occur in and around coding sequences, and hence will be constrained by selective forces. Furthermore, although it is currently assumed that simple sequence loci are dominated by the action of slippage (Tautz, 1989), perhaps leading to rather uniform mutation rates, at present we have very few data to confirm this in natural populations. Thus, until these sequences are understood more fully, microsatellite data should be interpreted in the light of data from other genetic markers. Although genomic turnover mechanisms interfere with the measurement of genetic distance, they can facilitate the identification of reproductively separate populations. The process of 'homogenisation' causes sequences that are tandemly repeated in 'gene-families' to evolve cohesively (Ohta & Dover, 1984). The precise mechanism for homogenisation is not well-understood, but the underlying principle is relatively simple (Dover, 1982). During a cell's lifetime, its DNA is constantly being read and repaired. Repeated sequences are particularly susceptible to change caused by this internal activity. Repeat units may be duplicated, lost or 'corrected' with respect to one another. The latter process, where differences between two similar sequences in chance alignment are eliminated by the cell's repair mechanism, can spread mutations throughout the genome. The former process of chance fixation or loss can spread the mutation laterally through the population. Therefore, repeated sequences tend to mutate as single units within reproductive populations. For conservation biology, this phenomenon has the important property of allowing reproductive populations to be identified by characteristic sequences of repetitive DNA.

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One particularly suitable multigene family is that coding for the ribosomal RNAs (the rDNA region, see Coen et al., 1982a). These genes have been characterised in many animals and the structure of the basic repeat unit is well-known (Coen et al., 1982b; Flavell et al., 1986; Tautz et al., 1987). It is comprised of three coding regions separated by longer, fast-evolving spacer regions. The spacer regions provide the greatest resolution for comparing populations. Within each spacer there are a series of subrepeats that are highly complex and often very divergent between species. Studies conducted on Drosophila species have shown that there can be nested subrepeats, each subject to homogenisation at different rates (Tautz et al., 1987). Spacer sequences are thus a potentially rich source of information on the degree of reproductive isolation shown by two populations.

GENETIC VARIANCE AND INDIVIDUAL FITNESS There are at least two ways in which genetic homogeneity can affect the fitness of individuals: through the expression of deleterious homozygous recessive traits and through the loss of heterosis (heterozygote advantage). It is well-established that inbreeding can reduce the fertility of adults and increase juvenile mortality in the short term through the expression of homozygous recessives (Wright, 1977; Rails et al., 1979; Rails & Ballou, 1983). The effect of heterosis is more controversial. Much of the support for heterozygote advantage has come from studies of single locus systems in invertebrates. However, the textbook example is a human study demonstrating that the recessive gene for sickle-cell anaemia is maintained in some populations through malarial resistance in heterozygotes (Allison, 1955; Cavali-Sforza & Bodmer, 1971; Wills, 1981). Although there has been general agreement among population geneticists that heterosis is not a widespread phenomenon (Lewontin, 1974; Eberhart, 1977; Falconer, 1977; Nei, 1983), a considerable variety of fitness characteristics have been associated with heterosis effects. These include survival rates, disease resistance, growth and development rates, and developmental stability (see review by Allendorf & Leary, 1986). Most of the survivorship studies have compared heterozygosity at a particular locus between cohorts (e.g. Koehn et al., 1973; Koehn et al., 1976; Singh, 1982; Singh

& Green, 1984; Foltz & Zouros, 1984). In each of these studies a larger percentage of the older cohorts were heterozygous, implying differential survival and heterozygote advantage. The converse problem, the effect of parentoffspring and sib matings on the viability of their offspring (inbreeding depression), has been consistently demonstrated for captive animals of various species (see Rails & Ballou, 1983). As would be expected, inbreeding is rare in natural populations. Rails et al. (1986) reviewed the literature for 13 bird and 15 mammal species and found a range of 0-19% inbreeding with a mean of 2-4% and a standard deviation of 3.9%. Quite a different problem is sometimes encountered when animals are introduced to a managed population from a distant or captive stock. The general phenomenon is referred to as outbreeding depression (see review by Templeton, 1986). An extreme example was described by Greig (1979) for the Tatra Mountain ibex Capra ibex ibex. A population of this species had been established in Czechoslovakia from an Austrian stock after the local ibex had been hunted to extinction. Several years later two different subspecies from Turkey and Sinai were added to the herd. This cross produced fertile hybrids, but they rutted in early fall and calved in February instead of spring (as the local ibex had). The kids born in February were unable to withstand the cold weather and the entire population went extinct. Two primary mechanisms have been proposed to explain outbreeding depression: local adaptation (illustrated in the above example); and intrinsic co-adaptation. Local adaptation is the least controversial of the two, simply proposing that geographically isolated populations adapt to their local environments. When transferred to a new habitat or climate it is possible that their specific adaptations are illsuited to their new surroundings. Intrinsic coadaptation is said to occur when genetic or karyotypic complexes evolve under the influence of other genes or chromosomes (Templeton et al., 1986). An example is the occurrence of chromosomal races (De Boer, 1982) and their partial incompatibility when individuals from different races are paired (Cicmanec & Campbell, 1977). The existence of chromosomal races can be determined by karyotyping. To some extent the problems described above could be avoided for introductions to managed populations if the genetic characteristics of individual animals could be effectively compared with

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the proposed host population. This may be possible through the analysis of genetic components that are minimally constrained by selection. Highly variable regions such as the minisatellite sequences described above will allow the identification of closely related individuals in many cases. This may well help in avoiding inbreeding depression for controlled matings. Before this is attempted, however, the technique should ideally be calibrated on known genealogies such that average levels of bandsharing are determined and inheritance patterns confirmed for the subject species. The potential problem of outbreeding depression may be detectable using genetic distance measures in genomic components comparable at the population level (e.g. isozyme polymorphism, mtDNA, or multigene family data--though the latter should be handled with caution). The measure which is used must be calibrated, however, since the degree to which populations would have to diverge before outbreeding depression becomes important is not known and may vary between species. To estimate the level of inbreeding in a local population, a simple and often sufficient method is to compare enzyme polymorphisms at numerous (30 +) randomly chosen loci with a larger population of the same species. As in other cases mentioned above, microsatellite loci (about 10) could be substituted for the proteins. An alternative with higher resolution would be to use mtDNA variation. DNA fingerprinting could be used after the level of variation in a non-threatened population of the same species and the segregation behaviour of apparent alleles were determined.

PRACTICAL CONSIDERATIONS The choice of analytical technique will relate, of necessity, to both scientific and practical considerations. The following discussion is intended as an outline guide to the limitations and potential provided by the various techniques described above. Protein electrophoresis is cheap, fast and relatively easy to perform. However, there are problems with comparability. Different tissues often give different banding patterns and samples stored for different lengths of time or under different conditions may be subject to modification. Further, samples taken from different seasons may show differential expression of certain isozymes. Careful collection and storage procedures can control for most of these problems (see Harris &

Hopkinson, 1978). The greatest limiting factor is the relatively low level of variability detected. This reduces its potential resolution, especially at the intra-population level. The direct analysis of DNA holds several advantages. It is comparable between tissues, free from possible seasonal variability, and stable (in some tissues for periods of days, weeks or even months after death). Analysis may be performed on very small samples (e.g. 200-300 mg of mammalian skin or 2 ml of blood, Gill et al., 1985). As described above, these techniques offer greater resolution and flexibility than enzyme electrophoresis, allowing the use of several different approaches simultaneously. The main drawback is the time and expense involved in labwork. Recently, a further, extremely powerful argument in favour of DNA analysis has been added. The polymerase chain reaction (PCR) is a method for enzymatically amplifying specific pieces of DNA, and is capable of producing microgram quantities of DNA from a single target molecule. The principle of the technique is relatively simple. Short pieces of DNA (primers) are synthesised to be complimentary to sequences on either side of the target sequence. When the sample DNA is melted, primer molecules become free to pair with their target sites and a thermo-stable polymerase can then copy the target sequence in both directions, starting with the two primers. Repeated cycles of melting, primer-annealing and polymerase extension result in an exponential synthesis of the target sequence. At its most basic, the technique allows the analysis of minute quantities of DNA. To date, amplification has been achieved from blood stains, single hairs, single cells and even single molecules of DNA. For the conservation biologist this allows many non-intrusive sampling regimes to be employed. For example, whales slough small pieces of skin into the water; this may be collected and analysed (Amos et al., 1992); gorillas leave hair in their nests and this too may be used. On a technical level, PCR also permits novel approaches to be used. Since primer sites can be conserved over considerable evolutionary time periods, a cloned gene, for example the rDNA genes, from one species may be used as a basis for the amplification and cloning of the same gene from another species, bypassing the need for the time-consuming construction of genomic libraries. Furthermore, technology is now available such that the PCR product may be sequenced directly.

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With respect to mitochondrial DNA analysis, specific PCR amplification negates the need for purifying mtDNA away from contaminating nuclear DNA. Coupled with direct sequencing this will generate the highest possible level of genetic resolutions. If non-conserved regions such as spacer sequences are to be investigated, it will often be necessary to clone a species-specific probe. This requires a larger amount of high-quality material for the extraction of high molecular weight DNA and the construction of a genomic 'library' (Glover, 1985). Such material can be obtained from freshly dead animals, or from blood in the case of larger mammals, birds or reptiles. Tissue, particularly that which is less metabolically active such as skin, can remain useful for periods of several days when the ambient temperature is not excessive (Amos & Hoelzel, 1991). For very fresh animals the best sources of DNA, such as the liver, spleen, heart or kidney, can be used. Samples should be frozen to -20C or below. Liquid nitrogen may be preferable in some cases. Where refrigeration is not available, samples should be stored in 20% DMSO saturated with table salt and maintained as cool as possible (Amos & Hoelzel, 1991). If blood is collected, at least 10 ml should be taken, separated by centrifugation, and the white cells frozen to at least -20C. For the purpose of population screening, much less of the same types of material is needed. There are two common methods for analysing the mitochondrial genome. The simpler is to extract whole cell DNA by methodologies which enrich the mtDNA fraction. Samples may be as small as 200-300 mg of tissue or 2 ml of blood, though a larger quantity is preferable. They should be stored frozen. DNA is extracted, cut and probed without further purification as for any other RFLP study, using probes cloned from a related taxon (mouse mtDNA works well for a variety of mammals). The resolution of this method is limited by the lower size of the fragments detectable by Southern blotting (Southern, 1975) and radiolabelled probing. The alternative approach resolves larger amounts of variability by visualising a greater number of smaller fragments. However, this necessitates the purification of mtDNA away from contaminating nuclear DNA, since detection methods (silver staining or radioactive fragment end-labelling) are no longer sequence-specific. To achieve this it is important to avoid any breakdown of the mitochondria or their

DNA. This requires more material and greater stringency during transport and storage. Blood can be used for mtDNA purification when collected in EDTA tubes, separated for white cells, and stored on liquid nitrogen in a salt/DMSO buffer (Hoelzel & Dover, 1989). If PCR is to be used both the quality and quantity of material required is reduced, although, wherever possible, sufficient sample should be taken to allow several repeats. Tissue samples collected for RFLP analysis (including the special cases of DNA fingerprinting and rDNA spacer variants) can be stored frozen or in a 20% DMSO solution saturated with salt for up to several months at room temperature (Amos & Hoelzel, 1991). The system for blood has yet to be rigorously quantified. Preliminary results indicate that it can be stored for long periods frozen, or in a salt/EDTA preservative with mild refrigeration. Radioactively labelled probes are made from cloned sequences, such as the human polycore construct used for DNA fingerprinting. Many useful clones are widely available. Alternatively, probes will have to be cloned de novo, as in the case of studies looking at divergent sequences like the rDNA spacers. As more studies are conducted, the bank of clones available increases and the need for specific cloning is reduced. It is already unnecessary for the majority of studies. The greatest resolution of genetic variation is attained by DNA sequencing. New techniques for direct sequencing PCR products are beginning to make this technique practical for population screening (Casanova et al., 1990; Green et al., 1990; Hoelzel & Green, 1992). However, wherever sample sizes are large, the high cost of both PCR and DNA sequencing may become an important factor. On a more basic level, a frequently used approach is non-specific RFLP analysis. However, this kind of RFLP analysis typically offers little more genetic resolution than protein electrophoresis, without the convenience. Its advantages are those inherent in DNA work, such as requiring very small quantities of tissue, tissue comparability, and less stringent storage conditions.

CONCLUSION As advances in molecular biology continue to improve our understanding of genetic mechanisms and structure, new tools will develop for the

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analysis o f p o p u l a t i o n s . A t p r e s e n t we c a n a l r e a d y go b e y o n d the t r a d i t i o n a l m e t h o d s o f p r o t e i n elect r o p h o r e s i s a n d visualise v a r i a t i o n in the D N A molecule directly. F u r t h e r m o r e , the p o w e r o f P C R opens u p a whole r a n g e o f p r e v i o u s l y u n t h i n k a b l e n o n - i n t r u s i v e sampling o p p o r t u n i t i e s such as shed hair. F o r some applications, such as the c o m p a r i son o f h e t e r o z y g o s i t y levels b e t w e e n p o p u l a t i o n s o r testing the possible selective a d v a n t a g e o f a n isozyme, p r o t e i n electrophoresis is still the m o s t practical technique. H o w e v e r , several p r e v i o u s l y i n t r a c t a b l e p r o b l e m s in c o n s e r v a t i o n biology, such as the d e t e r m i n a t i o n o f N e, d i s c r i m i n a t i o n between r e p r o d u c t i v e l y isolated p o p u l a t i o n s , a n d pedigree a n d kinship assessment, will be greatly facilitated b y the strength a n d flexibility o f molecular techniques.

ACKNOWLEDGEMENTS W e w o u l d like to express o u r g r a t i t u d e to J o h n H a r w o o d for his help a n d advice d u r i n g the p r e p a r a t i o n o f this manuscript. F u r t h e r useful c o m m e n t s were supplied b y Nigel Leader-Williams.

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