Zoo Biology 5:Ql-99 (1986)

Protein Variation, Fitness, and Captive Propagation Philip W. Hedrick, Peter F. Brussard, Fred W. Allendorf, John A. Beardmore, and Steven Orzack

Division of Biological Sciences, University of Kansas, Lawrence (P. W. H.); Department of Biology, Montana State University, Bozeman (P. F. 8.); Department of Zoology, University of Montana, Missoula (F. WA.); Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts (S. 0.); Department of Genetics, University College of Swansea, Swansea, Wales, United Kingdom (J.A.B.)

We examine the possibility of utilizing protein variation (allozymes) as a tool in the preservation and breeding of endangered and captive species. We believe that allozymes provide estimates of the relative amounts of genetic variation within and among populations. However, because of the difficulties encountered in evaluating both heterozygosity and fitness, we conclude that estimates of individ- ual heterozygosity derived from allozyme studies are poor criteria for selecting individuals for breeding in captive propagation schemes. Furthermore, breeding plans designed to maintain rare allozymes in captive populations represent an unwise strategy for the propagation of most rare and endangered animal species. We believe that successful reintroduction is most likely when genetic variation is preserved as found in natural populations.

Key words: allozymes, heterozygosity, management, rare alleles

INTRODUCTION

Since the late 1 9 6 0 ’ ~ ~ biochemical techniques such as protein electrophoresis have provided new insight into the extent and pattern of genetic variation in natural populations. Most species appear to have extensive variation at soluble protein loci [eg, Nevo, 1978; Nevo et al, 19841, with exceptions such as the northern elephant seal [Bonnell and Selander, 19741 and the cheetah [O’Brien et al, 19831. In general, large vertebrates have the least protein variation, small vertebrates have somewhat more, and invertebrates have the most [Nevo, 1978; Nevo et al, 19841.

Our purpose is to examine the possibility of utilizing information about protein variation as a management tool in the preservation and breeding of endangered and captive species. We address the following questions: (1) Is the extent of protein variation, as determined by electrophoretic techniques, useful in evaluating the rela- tive fitness of individuals or assessing the long-term fitness of a species? (2) Should individuals with high heterozygosity be given preference in breeding? (3) Should

Received for publication September 17, 1985; accepted October 22, 1985.

Address reprint requests to Dr. Peter F. Brussard, Department of Biology, Montana State University, Bozeman. MT 59717.

0 1986 Alan R. Liss, Inc.

92 Hedrick et al

breeding programs be designed to maintain rare alleles because these alleles might increase the long-term fitness of the species? Before answering these questions, we will first briefly discuss measures of protein variation, measures of fitness, and several other related topics.

MEASURES OF PROTEIN VARIATION

The extent and pattern of electrophoretically detected genetic variation in pro- teins (allozymes) has been widely documented [eg, Lewontin, 1974; Nevo et al, 19841. Although new techniques now allow characterization of DNA sequences, gel electrophoresis still remains the major tool used to document biochemical variation because of its relatively low cost and since a large number of individuals can be analyzed in a short time period. The genetic variation of individuals, populations, or species can be compared by using a number of standard measures (see Brown and Weir [1983] for a discussion of the statistical properties of these measures). Below we will introduce as a general background several of them: number of alleles, proportion of polymorphic loci, heterozygosity, and the information index. In the following discussion we will emphasize heterozygosity as the measure of choice.

Number of Alleles The simplest measure of genetic variation at a locus is the number of alleles (n)

observed in a sample. The average nunmber of alleles per locus (a) in a sample of m loci is then

The main advantage of this measure is that it has no upper bound, ie, it does not begin to asymptote with only a few alleles. However, n is very sensitive to sample size (rare alleles are often not included in small samples), and it gives equal weight to all alleles regardless of their frequency.

Proportion of Polymorphic Loci Another simple measure of genetic variation is the proportion of protein loci

that are polymorphic in a sample (P) , ie, have two or more alleles in appreciable frequency. A practical approach to defining polymorphism is to arbitrarily decide on a limit for the frequency of the most common allele. In general, polymorphic loci are arbitrarily defined as those for which the frequency of the most common allele is smaller than 0.99 or 0.95. Based on such a definition, then

X p = - m

where x is the number of polymorphic loci. An advantage of this measure is that it separates loci into two categories, monomorphic and polymorphic, giving a crude measure of the distribution of variation over loci. Its main disadvantages are that it is sensitive to sample size (in small samples, some polymorphic loci may be categorized as monomorphic) and that it gives equal weight to all polymorphic loci.

Protein Variation and Fitness 93

Heterozygosity The most widely used measure of genetic variation is the extent of heterozygos-

ity, sometimes called gene diversity. Because individuals in diploid species are either heterozygous or homozygous at a given locus, this measure represents a biologically useful quantity. Assume that the heterozygosity of individual i for a given locus j is Hij , being equal to unity for heterozygotes and zero for homozygotes. Therefore, the observed heterozygosity in a population at locus j is

in a given sample of N individuals. Heterozygosity, unlike the first two measures, can be used to measure the extent of variation in an individual. For example, the observed heterozygosity in individual i is

m 1

Hi, = - c HU m j = l

(4)

in a sample of m loci. In general, the variance of H j is much greater than Hi, because of intrinsic differences in heterozygosity among loci [Nei and Roychoudhury, 1974; Hedrick, 19831. The overall heterozygosity in a sample of N individuals at m loci is then

In most outbreeding animal populations, genotypes at protein loci are near Hardy-Weinberg proportions. In this case heterozygosity at locusj with n alleles is

n

H j = 1 - C p i , k = 1

where Pk is the estimated frequency of allele k . The Hardy-Weinberg heterozygosity in a sample of m loci is then

m

R = c H j . i= 1

(7)

The advantages of heterozygosity as a measure of genetic variation are that it is biologically meaningful and that it is relatively insensitive to sample size. On the other hand, it has several disadvantages. It is highly dependent on the frequencies of the two most common alleles (rare alleles contribute very little), has an upper bound of unity (does not reflect variation after the first few alleles), and is insensitive to differences in allelic frequencies when alleles have intermediate frequencies [Brown

94 Hedrick et al

and Weir, 19831.

Information Index

given locus, is Another measure of genetic variation is the information index, which, for a

Over a sample of m loci, it becomes m

1 7 = - cz; m i = l

(9)

The main advantages of this measure are that it combines the aspects of heterozygosity (allelic evenness) and the number of alleles (allelic richness) and is not bounded by unity. However, an important disadvantage is that it is not readily interpretable in genetic terms.

MEASURES OF FITNESS

The measurement of traits that contribute to the fitness of a genotype or a population has been a major focus of evolutionary research. Even in Drosophilu, an organism that has many advantages in experimental studies, measurement of fitness is still extremely difficult [eg, Hedrick and Murray, 19831. Furthermore, estimation of fitness in captive animals may be misleading when applied to natural populations. We will discuss briefly measures of genotypic and population fitness and some problems related to their measurement.

Genotypic Fitness

The relative fitness of a genotype is its ability to pass on alleles to the next or future generations, relative to that of other genotypes. The relative fitness of a genotype can be divided into four components: those affecting sexual, gametic, fecundity, and zygotic selection [Bundgaard and Christiansen, 19721. Often the zy- gotic level of selection, ie, survival in different life stages or age classes along with the rate of development, is emphasized. However, it is likely that fecundity selection owing to differences in egg or sperm production or fecundity schedules and sexual selection because of nonrandom mating, male vigor, and female receptivity are important in many species. Obviously, there is no a priori reason to assume that an advantage in one fitness component, say survival, would be mirrored in other components. Furthermore, the importance to overall fitness of morphological or physiological traits that are correlated with a component of fitness seems quite conjectural (see below). Prout [ 1965, 19691 , Christiansen et a1 [ 19771, and Hedrick and Murray [1983] have discussed at length a number of other problems associated with fitness estimation.

Protein Variation and Fitness 95

Population Fitness The fitness of a population is both difficult to measure as well as an elusive

concept in itself. One approach to defining and measuring population fitness is to evaluate the adaptedness of a population, ie, the ability of a population to survive and reproduce in a particular environment. Ecological measures such as the intrinsic rate of increase, carrying capacity, biomass, or productivity are often used to indicate the adaptedness of a population. However, such measures may not reflect the ability of a population to survive and reproduce in another environment or the probability that the population will continue to exist for a long period of time. In fact, it is possible that traits such as high productivity in a captive environment, where there is either intentional or unintentional selection for domestication, may actually lower the adapt- edness of a species reintroduced into a natural habitat.

PROTEIN VARIATION AND MANAGEMENT DECISIONS

Allozyme Heterozygosity as an Estimator of Population or Species Heterozygosity and Fitness

The processes of evolution and natural selection depend upon the existence of genetic variation. Although the fitness of a population does appear to be correlated with genetic variation for quantitative traits [eg, Lerner, 1954; Beardmore, 19831, it is unknown whether the extent of variation available for adaptive response in a population is reflected by the extent of variation at protein-coding loci. In some plant species, there appears to be a general association of the extent of protein variation within a population and the extent of morphological variation within a species [Hamrick, 19831. However, in wild barley the distribution of protein and morpholog- ical variation within and between populations is not concordant [Brown et al, 19781.

There are also examples of species with large amounts of genetically determined morphological variation but little allozymic variation [eg, Simonsen, 1976; Jain et al, 19801. This difference may indicate that the extent of variation in regulatory loci [eg, Yamazaki and Yoshinori, 19841 is not necessarily reflected by variability in structural genes. It may also reflect past events in a species’ history, such as a dramatic reduction in population size. The restoration by mutation of genetic variation affecting quanti- tative traits occurs much more quickly than it does for protein-coding loci [Lande, 19761, because mutations that affect quantitative traits can occur at many loci. Perhaps the only general conclusion is that a population or species with high levels of allozyme variation can be expected to have high levels of other types of genetic variation as well, but the absence of protein variation does not necessarily imply the absence of genetic variation for adaptively important characters.

Allozyme Heterozygosity as an Estimator of Individual Genomic Heterozygosity or Fitness

Assume that individuals are classified as heterozygous or homozygous at a number of protein-coding loci. How indicative of the heterozygosity in the whole genome is a ranking based on such a sample of loci? Chakraborty [I9811 examined this question by calculating the expected correlation between the individual heterozy-

96 Hedrick et al

gosity for a given number of loci and the overall genomic heterozygosity in these individuals. Assuming that the expectation of heterozygosity is the same at all loci and that the heterozygosities at different loci are independent, then the expected correlation is the square root of the proportion of Ioci sampled. When the number of genes sampled is a small proportion of of the total genomic number, this correlation is quite low. For example, if 20 loci are examined (an average number in routine electrophoretic surveys) from a pool of 1,OOO loci, the correlation is only 0.141. This suggests that heterozygosity at a few protein-coding loci is not a good estimator of the overall genomic heterozygosity of an individual. Note, however, that the above correlation is probably a lower bound because if the heterozygosities at different loci are not independent, the correlation would be increased.

Some studies have found a correlation between protein variation, either of particular alleles, genotypes, or heterozygosity, and traits related to individual fitness (reviewed by Mitton and Grant [1984]). These corelations suggest that some protein variants may have different intrinsic fitness values or that they are marking selectively important regions of the genome. We should note that in careful experiments in which the genetic background of a protein-coding locus was made isogenic [eg, Dykhuizen and H a d , 19801 or randomized [eg, Yamazaki et al, 19831, there appeared to be few detectable selective differences among individuals carrying different alleles at the locus in question (see Turelli and Ginzburg [1983] for a brief review of the empirical literature and a theoretical study).

Several factors such as genetic drift, selection, or gene flow can increase the statistical association of alleles at different loci (gametic disequilibrium [Hedrick, 19831). Accordingly, protein variants m y sometimes serve as markers of heterozy- gosity over several map units. Likewise, if an individual is homozygous at a protein locus owing to inbreeding, adjacent regions of the genome will have an increased probability of homozygosity as well. As a result, when inbreeding is present, then homozygotes at allozyme loci may have a lower fitness than allozyme heterozygotes because they are more likely to be homozygous for linked detrimental alleles. However, because of the uncertainties involved, using allozyme heterozygosity as a general indicator of overall genomic heterozygosity or fitness is not recommended.

Breeding to Maintain Rare Alleles in a Captive Population

Many samples from natural populations contain certain allozymes in low fre- quency, ie, less than 5 % . Although rare alleles can be useful in determining paternity in managed populations, there are many reasons why selective breeding to maintain these variants is unwise. First, sampling theory predicts that most rare alleles present in natural populations will not be represented in small samples. In addition, most electrophoretic variants themselves are in all probability heterogeneous classes of DNA sequences [eg, Kreitman, 19831. Thus, trying to maintain all the rare alleles present in a species would be a nearly impossible task.

Furthermore, models of selective neutrality predict that populations will have, at low frequency, alleles that are adaptively equivalent to more common ones. In addition, detrimental alleles maintained in a population by a balance between purify- ing selection and mutation will also be present in low frequency. Accordingly, there is no obvious way to differentiate rare alleles that are advantageous, neutral, or disadvantageous, or even to know which category is the most common.

Nevertheless, some conservation programs for plants [eg, Marshall and Brown, 19751 have placed some emphasis on conserving rare variants on the assumption that

Protein Variation and Fitness 97

they may mark genomes of potential adaptive significance, particularly those associ- ated with pest or disease resistance. This strategy may have some merit for conserving genetic variation in organisms that achieve local adaptation by maintaining highly coadapted gene complexes with low levels of gene flow and high levels of self- fertilization. However, in most vertebrates, there is little evidence that local adapta- tion is mediated by selectively favored supergenes (a possible exception is the histocompatibility system [eg, Thomson, 1981; Thomson and Klitz, 1985; Templeton et al, 19861).

We note two practical points. First, the population size necessary to retain a given proportion of rare alleles is much greater than the population size necessary to retain the same proportion of heterozygosity . For example, a one-generation bottle- neck of ten individuals would reduce heterozygosity by only 5 % , but reduce the number of alleles by approximately 69% [Nei et al, 1975; Allendorf, 1986; Fuerst, 19861. While this may not be a serious problem for plant conservation because thousands of seeds can be stored, it is clearly impossible to maintain most animal populations at a size that will retain a high proportion of rare alleles. More impor- tantly, increasing the contribution of individuals with a rare allele at the expense of contributions from other individuals may increase the overall loss of genetic variation in the population. Hence, maintaining a rare allele, which represents only a small portion of the genome, could result in an increase in homozygosity in the rest of the genome. Overall, a breeding scheme that is designed to preserve as much heterozy- gosity as possible appears to be the most prudent course of action.

Genetic Variation, Natural Population Structure, and Eventual Reintroduction

Successful reintroduction is most likely when genetic variation in a captive population is maintained in the form found in natural populations. Thus, having reliable information about natural population structure is an important prerequisite for developing a biologically sound breeding plan. If it is not possible to assay natural populations directly, then tentative inferences about a species’ population structure can be made from natural history information when it is available. When evaluating population structure it is also important to remember that a formerly widespread species may have only recently been divided into small, isolated populations as a result of human activities.

It is important to determine whether most genetic variation occurs within or between populations. If, for example, a high proportion of genetic variation is contained within populations, then an appropriate conservation strategy would be to maintain high heterozygosity within each captive population. Alternatively, if most genetic variation is distributed between populations, then an appropriate policy would be to mimic such a population structure although the heterozygosity maintained in each population may be relatively low. In this regard, maintenance of breeding and social structures found in nature may be more important in maintaining a species than a captive breeding program designed to retain maximum heterozygosity . CONCLUSIONS Is Individual Fitness a Function of Protein Heterozygosity?

Although there may be a relationship between fitness and electrophoretically assayed heterozygosity, it appears to be weak, and the data is equivocal. This is due in part to the difficulties encountered in evaluating both heterozygosity and fitness.

98 Hedrick et al

Accordingly, we feel it unwise to use an estimate of individual heterozygosity derived from electrophoretically detected protein polymorphisms as a criterion for selecting breeding individuals in captive propagation plans. Nevertheless, biochemical methods are useful in assessing the relative amounts of genetic variation between and within populations and in monitoring levels of heterozygosity in captive populations.

Is Future Population Fitness a Function of the Number of Allozymes Preserved?

The evolutionary potential of species depends on the number of alleles present because natural selection requires allelic variation to bring about adaptation to envi- ronmental change. However, natural selection works on additive genetic variance in fitness that is related to heterozygosity, not the number or alleles. Furthermore, a strategy of maintaining all protein variants, particularly rare ones, is operationally impossible. Preserving particular allozymes that have no known fitness advantage can be detrimental because such a strategy can actually result in increasing the overall homozygosity in other parts of the genome.

ACKNOWLEDGMENTS

We appreciate the comments of A.H.D. Brown, R. Chakraborty, C.J. Gorman, M. Soul6, M. Turelli, and J. Wright on earlier drafts of this manuscript.

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