Developmental Genetics 4:379-391 (1984)

Molecular Polymorphism: How Much Is

There and Why Is There So Much?*
Francisco J. Ayala
Department of Genetics, University of California, Davis

The evidence for genetic variation can be traced to Mendel’s experiments: The discovery
of the laws of heredity was made possible by the expression of segregating alleles. Since
that time, the study of genetic variation in natural populations has been characterized by a
gradual discovery of ever-increasing amounts of genetic variation. In the early decades of
this century geneticists thought that an individual is homozygous at most gene loci and that
individuals of the same species are genetically almost identical. Recent discoveries suggest
that, at least in outcrossing organisms, the DNA sequences inherited one from each parent
are likely to be different for nearly every gene locus in every individual: ie, that every
individual may be heterozygous at most, if not all, gene loci. But the efforts to obtain
precise estimates of genetic variation have been thwarted for various reasons.

Key words: genetic variation, molecular evolution, natural selection, DNA polymorphism

INTRODUCTION
This paper consists of two parts. First, I review the question of how much
molecular genetic variation exists in natural populations. Second, I present some
general considerations concerning the processes that contribute to maintain that
variation.
PROTEIN POLYMORPHISMS
Genetic variation is an attribute that cannot be exhaustively measured. It is not
possible, even if we wanted it, to examine every gene in every individual of a given
species, so as to obtain a complete enumeration of the genetic variation in the species.
The well-known solution in such a situation is to measure a sample from the group to
be evaluated. Two conditions need to be met for a valid extension of the results from
a sample to the whole set. First, the sample must be representative or unbiased;
second, the sample must be accurately measured. In the case at hand, the requirement
that the sample be unbiased applies to two levels: (1) the individual organisms sampled
must be, on the average, neither more nor less genetically variable than the population
as a whole; (2) the genes sampled must be neither more nor less polymorphic, on the
average, than the whole genome. And the condition of accuracy requires that genes
that are different be identified as such; ie, it requires that every allelic variant be
recognizable.

*Based on a lecture delivered in September, 1983 at the International Conference in Biochemical and
Developmental Genetics, Kos, Greece.
Received for publication October 23, 1983; accepted December 26, 1983.
Address reprint requests to Francisco J. Ayala, Dept. of Genetics, University of California, Davis, CA
95616.

0 1984 Alan R. Liss, Inc.

380 Ayah
Neither one of these two necessary conditions for valid sampling has been met
in the study of genetic variation. There is no serious difficulty in sampling individuals
that are, on the average, as genetically variable as the population as a whole. An
important consideration is that the individuals sampled not be particularly either
inbred or interrelated; but this is not difficult to satisfy. The difficulty lies in choosing
the genes to be sampled. With the methods of Mendelian genetics, the existence of a
gene is ascertained by examining the progenies of crosses between individuals show-
ing different forms of a given character; from the proportion of individuals in the
various classes, we infer whether one or more genes are involved. By such methods
the only genes known to exist are those that are variable. There is no way of obtaining
an unbiased sample of the genome, because invariant genes cannot be included in the
sample of genes to be examined.
A way out of this problem became possible with the advent of molecular
genetics. The genetic information encoded in the coding sequence of the DNA of a
structural gene is translated into the sequence of amino acids making up a polypeptide.
One can select for study a series of proteins without previously knowing whether or
not they are variable in a population-a series of proteins that, with respect to
variation, are an unbiased sample of all the structural genes in the organism. If a
protein is found to be invariant among individuals, it is inferred that the gene coding
for the protein is also invariant; if the protein is variable, the gene is inferred also to
be variable and one can measure how variable it is, ie, how many variant forms of
the protein exist, and in what frequencies.
Gel electrophoresis is a fairly simple technique that makes possible the study of
protein variation with only a moderate investment of time and money. Since the
1960s, genetic variation has been studied in a large variety of organisms by gel
electrophoresis. It was clear from the beginning of these studies that not all allelic
variants are detected by electrophoresis, and hence that the condition of accuracy is
not satisfied. But because genes for electrophoretic studies can be chosen without
regard to how variable the genes are, many investigators thought that electrophoresis
would provide estimates of variation in structural genes that would be accurate to a
first approximation.
Electrophoretic data give the frequency of electromorphs (proteins that differ in
electrophoretic mobility). Proteins encoded by different alleles may yield indistin-
guishable electromorphs, but as a first approximation it is assumed that each electro-
morph corresponds to only one allele. A variety of statistics can be used to summarize
the amount of genetic variation in a population. The most extensively used measures
are the polymorphism (P) and the heterozygosity (H). P is simply the proportion of
loci found to be polymorphic in the sample. Usually, a locus is considered poly-
morphic when the frequency of the most common allele (electromorph) is no greater
than a certain value, such as 0.99 or 0.95. In outcrossing organisms, H estimates the
average frequency of heterozygous loci per individual or, what is equivalent, the
average frequency of heterozygous individuals per locus. In naturally inbred orga-
nisms, H is a good measure of genetic variation in a population only if it is calculated
from the allelic frequencies as the “expected” frequency of heterozygous individuals
on the assumption of Hardy-Weinberg equilibrium. H is a better measure of genetic
variation than P for most purposes, because it is more precise [ 11. A related measure
also used by population geneticists is the effective number of alleles, n,, which is the
reciprocal of the average frequency of homozygous individuals, ie, 1 / ( 1 - 4 .
 Molecular Polymorphisms-How and Why 381
Electrophoretic studies have established that natural populations of most orga-
nisms possess large stores of genetic variation, even though not all variants are
detected. Table 1 shows that the average heterozygosity is about 6.0%for vertebrates
and about 13.4 % for invertebrates, although considerable heterogeneity exists within
each of these groups. Plants, even those reproducing by self-fertilization, also have
considerable genetic variation. The average proportion of polymorphic loci in a
population lies between 20 and 50% for most animal or plant species.
How accurate are electrophoretic estimates? That is, what proportion of the
total variation is detected by electrophoretic techniques? Electrophoresis cannot, of
course, detect nucleotide substitutions that do not change the encoded amino acids.
The question is what proportion of amino acid substitutions are detected. Some
biologists have argued that electrophoresis detects only substitutions that change the
net electric charge of the encoded proteins and have calculated that about 67% of all
amino acid substitutions are electrophoretically cryptic [2]. It is now known, however,
that electrically neutral charges can, at least in some cases, be detected [3].
The question raised could ultimately be resolved by obtaining the amino acid
sequence of a sufficiently large number of electromorphs with identical electropho-
retic mobility. This is clearly not feasible at present because of the enormous time
and cost required. A variety of other, less satisfactory, methods have manifested the
existence of electrophoretically cryptic variation. The methods used include sequential
electrophoresis, heat denaturation, urea denaturation, and peptide mapping.
Sequential electrophoresis consists of performing electrophoresis of the same
samples under diverse conditions; eg, using different buffers or different gel concen-
trations. If tissue samples or enzymes are exposed to high temparature or some other
denaturing agent such as urea, two proteins with identical electrophoretic mobility
may become distinguishable because one but not the other is denatured by the
treatment. Peptide mapping, or “fingerprinting,” is practiced by digesting the proteins
with trypsin or some other enzyme that hydrolizes the polypeptides into a number of
small peptides; these are then subjected to two-dimensional chromatography or to
chromatography in one dimension and electrophoresis in the other.

TABLE 1. Genic Variation in Natural Populations*

Effective
Average number Average Average number
Number of of loci polymorphism heterozygosity of alleles
Organisms species per species (0 (H) (4)
Animals
Invertebrates 57 22 0.469 0.134 1.15
Vertebrates 68 24 0.247 0.060 I .06
Plants
Self-pollinating 33 14 0.179 0.058 1.06
Outcrossine 36 11 0.511 0.185 1.23
*After Ayala and Kiger [29].

Table 2 summarizes results obtained by sequential electrophoresis and two

denaturation methods in three species of Drosophila-the only organisms in which
several loci have been studied by these methods in a given species. The average
382 Ayala

increase in heterozygosity is 0.04 by sequential electrophoresis and about 0.08 by the

denaturation methods, or an increase in the amount of variation (n:ln, between 12
and 25 % . The methods used tend to uncover more cryptic variation when the loci
sampled are more heterozygous to start with. But the average H of the loci sampled
is 0.181 to 0.410, substantially greater than the average of 0.150 observed in Droso-
phila populations when random samples of loci are assayed. Hence, the increase in
variation detected by these methods on a random sample of loci might be somewhat
smaller than the values shown in Table 2.
The amount of cryptic variation detected at the Adh locus of Drosophila melan-
ogaster by three different methods is displayed in Table 3. As might be expected,
peptide mapping detects more cryptic variation than any of the two other techniques.
Yet the increase in variation, 20%, is not very large. If we assume that this value, as
an average, is an approximate estimate of the amount of cryptic protein variation, we
can calculate the “corrected” amount of genetically determined protein variation in
natural populations (Table 4). With standard electrophoresis, n, = 1.15 for inverte-
brates and, therefore, n: = 1.20 X 1.15 = 1.39, which gives H’ = 0.28. For
vertebrates, n: = 1.28 and H’ = 0.22 and for plants n: = 1.37 and H’ = 0.27. The
average heterozygosity becomes approximately double for invertebrates and plants,
and more than triple for vertebrates.

DNA-SEQUENCE POLYMORPHISM
It has been known for more than a decade that only a small fraction, perhaps
less than 10% of the nuclear DNA of eukaryotes is translated into protein. The
recently developed techniques of DNA cloning and sequencing have shown that genes
are separated from each other by long DNA sequences that do not become transcribed
into RNA. The genes themselves have a complex organization. At both ends they
have relatively short sequences that are present in the mature mRNA transcript, but
do not code for amino acids. Most genes contain, in addition, intervening sequences
(introns), which separate from each other the segments that code for the amino acids
(exons). The introns are transcribed in the nucleus, together with the rest of the gene,
but they are spliced out before the mRNA migrates to the cytoplasm.

TABLE 2. Increase in Genetic Variation Detected in Three Species of Drosophila by Various

Methods*
Standard Additional Increase in
Number electrophoresis method variation
Species Method ofloci H n, H’ n: H’-H nfh,
D pseudoobscura Sequential I3 0.181 1.38 0.221 1.65 0.040 1.12
electrophoresis
D menalogaster Heat 4 0.410 1.73 0.485 2.06 0.075 1.18
denaturation
D subobscura Urea 8 0.379 1.83 0.456 2.42 0.077 1.25
denaturation
*After Ayala [30].
 Molecular Polymorphisms-How and Why 383

The question of how much genetic variation exists in the DNA of an organism
can, thus, be formulated in various ways. One may ask the question about the whole
genome or about particular components such as, for example, the coding segments.
A number of genes have been sequenced in two or more related species, and it has
become apparent that different segments evolve at different rates. This suggests that
different kinds of segments may have different levels of polymorphism, a hypothesis
recently corroborated by direct evidence.
Slightom et al [4] have sequenced two alleles of the *Y gene, which codes for
one of the polypeptides of fetal hemoglobin (Fig. 1). The two alleles are from a single
individual, one allele from the paternal and the other from the maternal chromosome.
The results are summarized in Figure 2. There are 13 substitutions of one nucleotide
by another and three segments deleted in one of the alleles (or inserted in the other).
None of the substitutions occurs in the exons; most (nine) are concentrated in the 5'
half of the long intron. Two deletions are each 4 np long (positions 741-744 and 791-
794 of the sequence); the third consists of 18 contiguous np (starting at position 1080).
If the *y gene is a typical example, it seems likely that at the level of the DNA
sequence every outcrossed individual will be heterozygous at nearly all, if not all,
loci-that is, if the noncoding sequences are taken into account. The question of
heterozygosity needs to be reformulated in terms of the proportion of nucleotide
differences, which may be called nucleotide heterozygosity or nucleotide diversity.
Trying to measure nucleotide heterozygosity, one encounters some ambiguity.
If only substitutions are considered, the nucleotide heterozygosity of *y is 13/1647 =
0.008. If the deletions are also taken into account, the question arises of how they are
to be counted. If each deleted segment is counted as one difference independently of
its length, then there are three additional differences between the two alleles and the
heterozygosity is 16/1647 = 0.010; if each deleted nucleotide is counted as one
difference, then the heterozygosity is 3911647 = 0.024.

TABLE 3. Increase in Genetic Variation Detected by Three Different Methods at the Adh Locus of
Drosophila melanogaster*
Method H'-H n;/n,
Sequential electrophoresis 0.00 1 .00
Heat denaturation 0.02 1.03
Peptide mapping 0.10 1.20
*From Ayala [30].

TABLE 4. Increase in Genetic Variation for Three Groups of Organisms when Electrophoretically
Cryptic Protein Variation Is Taken Into Account*
Electrophoretic
variation Total variation
Organisms H ne H' n;
Invertebrates 0.134 1.155 0.278 1.386
Vertebrates 0.060 1.064 0.217 1.277
Plants 0.124 1.142 0.270 1.370
*It is assumed that the average increase in variation is 20%; ie, ndln, = 1.20. Average values from
Table 1.
384 Ayala

codons 30 31 104 105 146

1 1 1 1 1 1 1 1 1 ~ 1 1 ~ 1 1 ~ 1
bp 0 300 600 900 I200 I500

Fig. 1. Organization of the A-y globin gene. This gene is part of the P-globin cluster located in
chromosome 11. The functional gene consists of three exons (black) separated by two introns (white).
One intron separates the triplets coding for amino acids 30 and 31; the other is between the triplets for
amino acids 104 and 105. The A-y gene consists of about 1,600 base-pairs (bp), 438 of which code for
the 146 amino acids of the polypeptide.

r Substitutions
4-
3-
m
w 2-
0
=W I -
fx
W
LL
k I -
Q
2 -
W
3-
U
m 4-
T

I7 - De/etions/Insertions
18 -
I 1 I I
0 500 1000 I500
SEQUENCE POSIT 10N
Fig. 2. Local distribution of nucleotide differences between two allelic A~ genes. Nucleotide substitu-
tions are shown on top; deletions/insertions on bottom. A diagram of the gene is shown in the middle;
black regions are the exons, white regions are the introns, hatched regions are flanking sequences. Data
from Slightom et a1 [4].

The nucleotide heterozygosity in other genes for which two independent alleles
have been sequenced is given in Table 5. Three genes (Adh in Drosophila, C, in rats,
and *y in humans) have substitution heterozygosities between 1 and 2%. The DNA
sequenced for Adh and C, includes only coding regions and thus no deletions were
observed. For the insulin genes the substitution heterozygosity is only 0.003, but the
 Molecular Polymorphisms-How and Why 385
TABLE 5. Heterozygosity at the Single Nucleotide Level
Length of Heterozygosity
DNA sequence Substitutions
Organism Gene region (base pairs) Substitutions and deletions References
Drosophila Adha 765 0.009 0.009 ~311
melanogaster
Mouse IgG2a 1,108 0.100 0.100 [321
Rat Immunoglobulin C,“ 1,172 0.018 0.018 [331
Man Globin A~ 1,647 0.008 0.024 [41
Man Insulin 2,721 0.003 0.175 [341
aOnly coding sequences are included in these comparisons.

5‘ flanking region contains a deletion/insertion of 467 contiguous np, which are

within a highly repetititve sequence.
The constant region of the heavy chain of mouse immunoglobulin consists of
eight proteins. One of these, y2a, is known to differ extensively from one inbred
mouse strain to another. The gene, ZgG2a, coding for this protein has been sequenced
in two strains. Of the 1,108 bases sequenced, 111 (10%) are different. Only 18
(16.2 %) of these nucleotide substitutions are silent; the others yield different amino
acids in 15% of the sites. There are reasons to presume that the variation observed in
the mouse ZgG2u gene may not be typical of structural loci. Immunoglobulin genes
are very polymorphic; the two alleles sequenced come from two inbred strains, rather
than from outbred individuals; the two proteins were known to be very different
before the DNA was sequenced. Indeed, the frequency of amino acid differences
between the two allele products is one order of magnitude greater than the average
observed in other kinds of protein.
If we exclude from consideration the ZgG2a and insulin genes as being atypical,
it would appear that nucleotide heterozygosity may be around 1 or 2 % . This must be
taken only as a very tentative estimate because of the paucity of the data.
Estimates of nucleotide heterozygosity have been obtained in four species of sea
urchins by DNA denaturation followed by competitive reassociation (“hybridiza-
tion”). This technique is inexact but has the advantage that it assays the complete
genome of an organism. The results for the single-copy DNA are summarized in
Table 6. The estimated frequency of nucleotide substitutions ranges from 2 to 4%.
This is not very different from the I-2% estimate of nucleotide heterozygosity derived
from the sequence data. Thus, although quantitative estimates of the amount of DNA-
sequence variation cannot be provided with confidence for organisms in general,
there can be no doubt that the variation is extensive. If the noncoding regions of genes
are included, it seems likely that most, if not all, genes are heterozygous in every
outbred individual.

SELECTION VERSUS DRIFT

What is the evolutionary significance of this wealth of protein and DNA
variation? Is it adaptive, the stuff from which are built the multifarious adaptations of
organisms to their environments? Or is it for the most part evolutionary noise,
variations that are tolerated by natural selection because they do not modify any
significant function of the organisms?
386 Ayala

TABLE 6. Single Nucleotide Heterozygosity Estimated by Competitive Reassociation

(“hybridization”) of Single Copy DNA in Four Species of Sea Urchins*
Organism Heterozygosity
Strongylocentrotuspurpuratus 0.040
S franciscanus 0.032
S intetmedius 0.030
S drobachiensis 0.020

*From Grula et a1 [35].

All genetic variations arise first by the mutation process, broadly understood so
as to include not only the substitution of one nucleotide by another but also deletions,
duplications, and reorganizations of the DNA. If the mutants modify the adaptations
of organisms, they will increase or decrease in frequency as a result of natural
selection. If they have no effect on adaptation, mutants will drift in frequency as a
consequence of random sampling from generation to generation. The hypothesis that
considers a mutation or a polymorphism as adaptively neutral is the starting null
hypothesis of the population geneticist. In recent years, however, Kimura and others
[5-71 have argued that, with respect to DNA and protein evolution, adaptive neutrality
is no longer just a null hypothesis, but a notion positively supported by evidence.
Two approaches may be followed to test the hypothesis of neutrality versus
natural selection. One consists of testing each particular polymorphism to ascertain
whether natural selection is implicated [see 8-12]. The other approach is global: It
uses theoretical reasoning or empirical evidence to argue for or against the role of
natural selection with respect to a general kind of variation, molecular variation in
the case at hand [eg, 13,141.
I want to examine here two general arguments-one positive, the other nega-
tive-that have been advanced to support the adaptive neutrality of protein variation.
The positive argument relies on the apparent existence of a molecular evolutionary
clock. When the rate of evolution is examined in a protein such as cytochrome c, it is
observed that amino acid substitutions have occurred in different branches of the
phylogeny at different times and at approximately constant rates. What is meant by
the phrase “approximately constant rates” is that the substitutions occur with a
constant probability, but stochastic variation is expected.
Langley and Fitch [ 151 have tested statistically the evolution of seven proteins
in 17 mammals and found that the variance in the rate of amino acid substitutions is
much too large-inconsistent with the hypothesis that the rate was stochastically
constant as predicted by the neutrality theory (Table 7). It is possible, however, to
maintain that the rate is stochastically constant but that it has a variance greater than
expected from a Poisson distribution [16]. One additional problem with this sort of
evidence in support of the neutrality hypothesis is that stochastically constant rates of
molecular evolution are also predicted by models of natural selection [ 171. Therefore,
the existence of a molecular evolutionary clock cannot be used in support of either
the neutrality or the adaptive hypothesis.

HETEROSIS AND GENETIC LOAD

The negative argument offered in support of the neutrality theory is based on
the concept of genetic load. The argument is that if some alleles are less adaptive than
 Molecular Polymorphisms-How and Why 387
TABLE 7. Statistical Test of the Constancy of Evolutionary Rates of Seven Proteins in 17 Mammal
Species*
Degrees
Chi-square of freedom Probability
Overall rates (Comparisons among 82.4 31 4 x 10-6
branches over all seven proteins)
Relative rates (comparisons among 166.3 123 6 X lop2
proteins within branches)
Total 248.7 154 6 x
*After Langley and Fitch [ 151.

others, then a number of individuals would have less than optimal genotypes at each
polymorphic locus subject to natural selection. If the number of such loci is very
large, a population might be unable to withstand the burden of so many poorly fit
individuals. This argument deserves to be examined in detail because the neutrality
hypothesis was largely proposed as the only alternative left for those who rejected
natural selection because of the enormous genetic load that would be created by
ubiquitous protein polymorphisms .
The genetic load argument is strongest in the case of heterosis; ie, when a
polymorphism is maintained owing to the adaptive superiority of the heterozygotes.
Sved et a1 [18], King [19], and others have suggested that an efficient method for
testing whether heterosis plays a major role in natural populations is to compare the
fitness of ordinary outbred individuals with the fitness of individuals homozygous for
a larger-than-average proportion of loci. This method permits one to ascertain whether
heterozygotes are at an overall advantage over homozygotes.
Numerous experiments, particularly in Drosophila, have shown that an increase
in homozygosity results in a decrease in fitness. The experiments published before
1970 were, in general, carried out by measuring particular components of fitness,
mostly viability [20] and fertility [21,22], and were not, in any case, performed under
population conditions [23]. Sved and Ayala [24] devised a method by which fitness
as a whole can be measured in Drosophila flies made homozygous for full chromo-
somes, under conditions of equilibrium population density and a stable age distribu-
tion. This method has now been used in a number of experiments that yield consistent
results in that the fitness of homozygotes for one full chromosome is invariably very
low, in the sublethal range.
The method of Sved and Ayala [24] is as follows. Flies homozygous and
heterozygous for whole chromosomes sampled from a natural population are obtained
using the method shown in Figure 3. The flies recovered in the F3 are used to
establish experimental populations, where the course of natural selection can be
studied over many generations. Since the balancer chromosome inhibits recombina-
tion, only two kinds of viable zygotes can exist at any time-those homozygous for
the wild chromosome and those heterozygous for the wild and the balancer chromo-
some; all zygotes homozygous for the balancer chromosome die before completing
development. If the homozygotes for the wild chromosome have lower fitness than
the balancer heterozygotes, a stable equilibrium will eventually be established be-
tween the two types of flies. The relative fitness of the homozygotes can be directly
calculated from the zygotic equilibrium frequencies. If the balancer heterozygotes
have lower fitness than the chromosomal homozygotes, the balancer chromosome
388 Ayala

Fig. 3. Crosses used to obtain large numbers of Drosophila flies homozygous for a chromosome
sampled from a natural population. A. P generation: A wild male is crossed to females of a balanced
marker stock. The balancer stock contains a chromosome with multiple inversions (to inhibit recombi-
nation) and two mutant markers, one dominant and the other recessive; the other chromosome contains
the recessive mutant marker. F, generation: A single F I male heterozygous for one wild chromosome
and the marker chromosome is crossed to females of the balanced marker stock. F2 generation: Males
and females heterozygous for the same wild chromosome and the balanced marker chromosome are
intercrossed. Three kinds of progeny are expected in the F3 generation: One fourth should be homozy-
gous for the wild chromosome, one half should be heterozygous for the wild and the balanced marker
chromosome, one fourth should be homozygous for the balanced marker chromosome, but this carries
also a recessive lethal gene and these flies die. B. Control crosses are made by intercrossing F2 flies
heterozygous for different wild chromosomes. The wild flies in the F3 generation of these crosses are
heterozygous for different wild chromosomes, and thus are genetically similar to wild flies.

will gradually decrease in frequency; the relative fitness of the two kinds of flies can
then be estimated from the rate of elimination. Control experimental populations are
set up with flies heterozygous for different wild chromosomes, and for these and the
balancer chromosome. The heterozygotes for different wild chromosomes have ge-
netic constitutions comparable to flies in a natural population. The control populations
permit an estimation of the fitness of balancer heterozygotes relative to wild hetero-
zygotes. This estimate of fitness can be used to estimate the fitness of chromosomal
homozygotes relative to flies heterozygous for random combinations of wild
chromosomes.
With this method, overall fitness rather than a specific fitness component is
measured under population conditions. The experiments show that under these con-
ditions all chromosomes become either lethal or semilethal. Figure 4 shows the fitness
distribution of 23 second chromosomes sampled from a natural population of Droso-
phila melanogaster [25]. Although the method is extremely laborious, studies
have been conducted in three species of Drosophila. The results are summarized in
TabIe 8.
In order to estimate the number of loci that can be maintained by natural
selection in view of the fitness experiments, the assumption is made that selective
interactions between loci are multiplicative and that there is no linkage disequilibrium
[26]. If at each locus maintained by heterosis the heterozygote has a 0.01 selective
 Molecular Polymorphisms-How and Why 389

0 .I0 .20 .30 .40 .50 .60

M e a n F i t n e s s of Homozygous Files
Fig. 4. Fitness of Drosophila melanogaster flies homozygous for second chromosomes sampled from
a natural population, The fitness of the homozygous flies is measured in experimental population cages
relative to flies heterozygous for wild chromosomes. When all fitness components are taken into
consideration, virtually all chromosomes have lethal or semilethal effects in homozygous flies.

TABLE 8. Fitness of Homozygotes for Whole Chromosomes (Lethal chromosomes excluded)

Under Population Conditions, in Several Species of Drosophila
Average fitness Reference
Species Chromosome Sample size of homozygotes
D pseudoobscura I1 16 0.37 ~ 4 1
D willistoni I1 15 0.34 [361
D melanogaster X 34 0.60 [371
I1 24 0.15 [381
I1 23 0.19 [251
I1 24 0.08 ~ 7 1
111 14 0.32 [251
I11 14 0.10 I391
I11 24 0.08 ~ 7 1

advantage over either homozygote, then the fitness of a homozygous individual

relative to an individual heterozygous at 200 loci would be (0.99)200= 0.13. This is
approximately the mean fitness of individuals homozygous for a complete second or
a third chromosome in D melanogaster (see Table 8). The second or the third
chromosomes of D melanogaster are estimated to contain each about 40% of the
genome. Therefore, the number of heterozygous loci that could be maintained by
heterosis in the whole genome under the assumptions made could be, approximately,
200/0.40 = 500. This is 10% of the 5,000 loci estimated to be present in D
melanogaster. The heterozygosity (rr) as estimated by electrophoretic methods in D
melanogaster is about 0.10; hence, the evidence indicates that all the polymorphisms
observed by electrophoresis could be maintained by heterotic natural selection. There-
fore, it would seem that arguments of genetic load cannot be used against the
hypothesis that many natural polymorphisms are maintained by natural selection.
390 Ayala
The calculations just made rely on a number of assumptions. A particularly
relevant one is that fitness interactions among loci are multiplicative. Seager et a1 [27]
have performed an experiment to test this hypothesis. The experiment consists of
measuring the fitness of flies homozygous (1) for only the second chromosome, (2)
for only the third chromosome, and (3) for both the second and the third chromosome.
The results are striking. The fitness of D melanogaster homozygous for both the
second and the third chromosomes (0.079 k 0.024) is not significantly different from
the fitness of flies homozygous for only the second (0.081 0.014)or only the third
chromosome (0.080 *0.017). If we assume that fitnesses are multiplicative, the
average expected fitness of the double-chromosome homozygotes is 0.0066 & 0.002;
the observed value is more than ten times greater.
The experiments of Seager et al [27] manifest, therefore, large negative syner-
gistic interactions, If we assume that similar synergistic interactions occur when there
is homozygosis for one full chromosome or less, then the fitness depression observed
in the homozygotes for only one chromosome could account for a number of heterotic
loci much greater than calculated above. And it should be noted in addition that other
forms of natural selection, such as frequency dependence and the associated phenom-
enon of overcompensation [28] cause a lesser genetic load than heterosis and may in
fact increase the ability of a population to exploit the environmental resources.

REFERENCES
1 . Dobzhansky Th, Ayala FJ, Stebbins GL, Valentine JW: “Evolution.” San Francisco, CA: Freeman,
1977.
2. Marshall DR, Brown AHD: The charge-stage model of protein polymorphism in natural popula-
tions. J Mol Evol6:149-163, 1975.
3. Ramshaw JAM, Coyne JA, Lewontin RC: The sensitivity of gel electrophoresis as a detector of
genetic variation. Genetics 93: 1019-1037, 1979.
4. Slightom JL, Blechi AE, Smithies 0: Human fetal ‘7- and Ay-globin genes: Complete nucleotide
sequences suggest that DNA can be exchanged between these duplicated genes. Cell 21:626-638,
1980.
5 . Kimura M: Evolutionary rate at the molecular level. Nature 217:624-626, 1968.
6 . King JL, Jukes TH: Non-Darwinian evolution. Science 164:788-798, 1969.
7. Kimura M, Ohta T: Protein polymorphism as a phase of molecular evolution. Nature 229:467-469,
1971.
8. Marinkovic D, Ayala FJ: Fitness of allozyme variants in Drosophila pseudoobscura. I. Selection at
the Pgm-1 and M e 2 loci. Genetics 79:85-95, 1975a.
9 Marinkovic D, Ayala FJ: Fitness of allozyme variants in Drosophila pseudoobscura. 11. Selection at
the Est-5, Odh, and Mdh-2 loci. Genetical Research 24: 137-149, 1975b.
10. Snyder TP, Ayala FJ: Temperature and density effects on fitness at the Mdh-2 and Pgm-1 loci of
Drosophila pseudoobscura. Genetica 51 59-67, 1979a.
11. Snyder TP, Ayala FJ: Frequencydependent selection at the Pgm-I locus of Drosophila pseudoob-
scura. Genetics 92:995- 1003, 1979b.
12. Tosic M, Ayala FJ: Density- and frequency-dependent selection at the Mdh-2 locus in Drosophila
pseudoobscura. Genetics 97:679-701, 1981.
13. Ayala FJ, Tracey ML, Barr LG, McDonaId JF, Perez-Salas S: Genetic variation in natural popula-
tions of five Drosophila species and the hypothesis of selective neutrality of protein polymorphisms.
Genetics 77:343-384, 1974.
14. Ayala FJ: Protein evolution in related species: Adaptive foci. Johns Hopkins Med J 138:262-278,
1976.
15. Langley CH, Fitch WM: An examination of the constancy of the rate of molecular evolution. J Mol
EVOI3: 161-177, 1974.
 Molecular Polymorphism-How and Why 391
16. Gillespie JH, Langley CH: Are evolutionary rates really variable? J Mol Evol 13:24-34, 1979.
17. Gillespie JH: Polymorphism and molecular evolution in a random environment. Genetics 93:737-
754, 1979.
18. Sved JA, Reed TE, Bodmer WF: The number of balanced polymorphisms that can be maintained in
a natural population. Genetics 55:469-481, 1967.
19. King JL: The gene interaction component of the genetic load. Genetics 53:403-413, 1966.
20. Dobzhansky T, Spassky B: Genetics of natural populations. XXXIV. Adaptive norm, genetic load
and genetic elite in D. pseudoobscura. Genetics 48: 1467-1485, 1963.
21. Gowen JW (ed): “Heterosis.” Ames, 10:Iowa State College Press, 1952.
22. Marinkovic D: Genetic loads affecting fertility in natural populations of Drosophila pseudoobscura.
Genetics 57:701-709, 1967.
23. Latter BDH, Robertson A: The effects of inbreeding and artificial selection on reproductive fitness.
Genet Res 3:llO-138, 1962.
24. Sved JA, Ayala FJ: A population cage test for heterosis in Drosophila pseudoobscura. Genetics
66:97-113, 1970.
25. Tracey ML, Ayala FJ: Genetic load in natural populations: Is it compatible with the hypothesis that
many polymorphisms are maintained by natural selection? Genetics 77:569-589, 1974.
26. Lewontin RC, Hubby JL: A molecular approach to the study of genic heterozygosity in natural
populations. 11. Amount of variation and degree of heterozygosity in natural populations of Droso-
phila pseudoobscura. Genetics 54595-609, 1966.
27. Seager RD, Ayala FJ, Marks RW: Chromosome interactions in Drosophila melanogaster. 11. Total
fitness. Genetics 102:485-502, 1982.
28. Tosic M, Ayala FJ: “Overcompensation” at an enzyme locus in Drosophila pseudoobscura. Genet
Res Camb 3657-67, 1980.
29. Ayala FJ, Kiger JA Jr: “Modern Genetics, 2nd ed.” Menlo Park, CA: BenjaminlCummings, 1984.
30. Ayala FJ: Genetic variation in natural populations: Problem of electrophoretically cryptic alleles.
Proc Natl Acad Sci USA 79:550-554, 1982.
31. Benyajati C, Place AR, Powers DA, Sofer W: Alcohol dehydrogenase gene of Drosophila melano-
gaster: Relationship of intervening sequences to functional domains in the protein. Proc Natl Acad
Sci USA 78:2717-2721, 1981.
32. Schreier PH, Bothwell ALM, Mueller-Hill B, Baltimore D: Multiple differences between the nucleic
acid sequences of the IgG2aa and IgG2ab alleles in the mouse. Proc Natl Acad Sci USA 784495-
4499, 1981.
33. Sheppard HW, Gutman GA: Allelic forms of rat K chain genes: Evidence for strong selection at the
level of nucleotide sequence. Proc Natl Acad Sci USA 78:7064-7058, 1981.
34. Ullrich A, Dull TJ, Gray A, Brosius J, Sures I: Genetic variation in the human insulin gene. Science
209~612-615,1980.
35. Grula JW, Hall TJ, Hunt JA, Guigni TD, Davidson EH, Britten RJ: Sea urchin DNA sequence
polymorphism and reduced interspecies differences of the less polymorphic DNA sequences.
Evolution 36:60-676, 1982.
36. Mourao CA, Ayala FJ, Anderson WW: Darwinian fitness and adaptedness in experimental popula-
tions of Drosophila willistoni. Genetica 43:552-574, 1972.
37. Wilton AN, Sved JA: X-chromosomal heterosis in Drosophila melanogaster. Genet Res Camb
34:303-315, 1979.
38. Sved JA: An estimate of heterosis in Drosophila melanogaster. Genet Res 18:97-105, 1971b.
39. Sved JA: Fitness of third chromosome homozygotes in Drosophila melanogaster. Genet Res 25: 197-
200. 1975.