Procedings of the National Academy of Sciences

Vol. 67, No. 3, pp. 1264-1267, November 1970

Evidence of Heterosis Associated with an Enzyme

Locus in a Natural Population of Drosophila*
Rollin C. Richmondt and Jeffrey R. Powell
THE ROCKEFELLER UNIVERSITY, NEW YORK, N.Y. 10021
Communicated by Theodosius Dobzhansky, August 7, 1970
Abstract. A wild population of Drosophila paulistorum (Andean semispecies)
was analyzed by starch gel electrophoresis for a sex-linked enzyme locus, Tetra-
zolium oxidase. The results of the analysis of F1 progenies from 106 single female
lines from Sao Jose do Rio Preto, Brazil, has allowed us to determine the genotype
of 106 wild-collected females and 104 wild-collected males. The population is
polymorphic for two alleles with frequencies of 0.56 and 0.44. The observed
frequencies among the females of the two homozygotes were 0.23 and 0.13 and of
the heterozygote 0.64. The deviation from the frequencies expected according
to the Hardy-Weinberg formula is statistically significant (P < 0.005). There
is a substantial excess of heterozygotes over the expected number. This observa-
tion is interpreted to mean that this polymorphism is maintained by heterosis.
This is probably the first reported case of heterosis associated with an enzyme
locus in a natural population of Drosophila.

It has now been unequivocally shown that natural populations of organisms

as diverse as man and several species of Drosophila have many enzyme poly-
morphisms.1-3 How these polymorphisms are maintained is an open question.
Lewontin and Hubby have pointed out that the current mathematical formula-
tions of selectional processes in natural populations cannot account for the ob-
served abundance of enzyme polymorphisms in Drosophila populations.4 Sev-
eral authors have suggested more realistic models of selectional processes which
would allow populations to contain many polymorphic loci.5-7 Kimura and
Crow propose that many polymorphisms are present in natural populations
because of the selective neutrality of the alleles involved.8'
The evidence presented below indicates that natural selection can and does
alter the frequencies of some allozyme2 alleles in natural populations. The
polymorphism we have studied appears to be maintained in the population by
selection in favor of individuals heterozygous for two alleles.
Materials and Methods. Single Drosophila paulistorum females inseminated in
the wild were used to start 106 strains. All females were collected at a single locality,
Mirassol, near Sao Jose do Rio Preto, Brazil. As D. paulistorum is a complex of six
partly reproductively isolated semispecies, the strains were crossed to standard tester
stocks to determine to which semispecies they belong.10 All strains from Rio Preto used
in this study were of the Andean semispecies. No other semispecies has been found in
this locality.
One F1 male and six F1 female progenies from each strain were analyzed for the allo-
zymes of Tetrazolium oxidase (To), which is (R. C. Richmond, unpublished data) a sex-
1264
VOL. 67, 1970 HETEROSIS IN DROSOPHILA 1265

linked locus in D. paulistorum. From the genotypes of the progenies, the genotype of
the parents can be determined with high probability. The probability of not detecting
one of the two alleles of the female parent, so that the female would be erroneously
scored as a homozygote is only (0.5)6.
Starch gel electrophoresis was used to detect the various forms of the enzyme. The
gel was prepared from electrophoresis starch (Sigma) in 87 mM Tris-boric acid buffer
(pH 9.0) containing 1 mM EDTA and 0.1 mM 3-NAD+. Each fly was homogenized in
buffer of this composition; the homogenate was absorbed by a small piece of filter paper
and placed in a slot in the gel. A voltage gradient of 20-25 V/cm was applied for 6 hr.
The gel was sliced and stained in 100 ml of 50 mM Tris-HCl buffer (pH 8.5) contain-
ing 20 mg of Nitro Blue Tetrazolium salt (Sigma), 25 mg of O-NAD +, and 5 mg of phen-
azine methosulfate (Sigma). The gel in the stain was exposed to light for 1 hr, the stain
was then removed, and the gel was fixed in acetic acid-methanol-water 1:5:5. Light
catalyzes reduction of the tetrazolium salt to a blue precipitate except where there
is an enzyme (To) capable of oxidizing the tetrazolium salt." The in vivo function of
tetrazolium oxidase is not known and this name is given only to describe its in vitro action.
These bands do not stain with any other common stain and variants behave as single
sex-linked Mendelian factors.
Results. Fig. 1 shows a zymogram of females homozygous and heterozygous

FIG. 1. Tetrazolium oxidase

zymogram of Drosophila females
homozygous and heterozygous
for two allelic variants. The
arrow indicates direction of Ad G
migration. Genotype F
F
F/
/S S

for the two To alleles present in the Mirassol population. The two variants are
designated "F" (fast migrating) and "S" (slow migrating). From the pattern of
the heterozygote, it is obvious that this enzyme is composed of at least two sub-
units.
Table 1 gives the genotype frequencies of the 106 females in our sample as
TABLE 1. Genotype frequencies of wild females of D. paulistorum at the Tetrazolium oxidase
locus. Expected values are calculated for a Hardy-Weinberg equilibrium with
frequencies of the S allele being 0.554 and the F being 0.446. These frequencies
were determined from the sample of 106 mothers and 104 fathers.
F/F F/S S/S Total
Observed 14 68 24 106
Expected 21.1 52.4 32.5
XI = 9.26 P < 0.005
inferred from the genotypes of their progenies. As this locus is sex-linked, no
similar data can be given for males. The expected genotype frequencies were
calculated using the gene frequencies in the natural population (from the sample
of 106 females and 104 males as there was no difference between sexes) and as-
suming a Hardy-Weinberg equilibrium.
Laboratory crosses did not show any segregation distortion associated with this
locus. In three crosses of F/S females and males, 27 F and 28 S males were
scored. Among the female progenies a slight excess of heterozygotes was de-
tected.
1266 GENETICS: RICHMOND AND POWELL PROC. N. A. S.

Five sets of reciprocal crosses between strains homozygous for the two alleles
were made to provide heterozygous females. Salivary gland smears were pre-
pared from five larvae from each cross and examined for the presence of hetero-
zygous inversions in either arm of the X chromosome. Although the X chromo-
some did contain several inversions, no consistent association between any in-
version and the To alleles could be established.
Discussion. Some possible explanations for the excess of females heterozygous
for the To locus are the following.
First, the gene frequencies in the population may not be at equilibrium, that is,
one allele may be in the process of replacing the other and therefore our sample
may be at an intermediate stage in this directional selection process.'2 This
seems unlikely because of the magnitude of the heterozygote excess. Also,
Richmond has noted (unpublished observations) that several widely-distributed
populations of the AndeAn semispecies are segregating for these two alleles. It
seems unlikely that a directional selection process would be occurring simulta-
neously over so wide an area.
Second, it has been shown by Dobzhansky and Pavlovsky'3 that flies hetero-
zygous for a chromosomal inversion are sometimes present in excess of Hardy-
Weinberg equilibrium frequencies. If the To locus were associated with such a
supergene, the heterozygous excess would not necessarily be due to the locus
per se, but rather to its location within an inversion. As mentioned above, no
systematic relationship between heterozygosity for the To locus and heterozy-
gosity for an X chromosome inversion was detected in the material studied.
Third, since the genotypes of the wild-collected females were inferred from
their F, progenies, any systematic bias in the segregation ratio of the To alleles
might produce the results observed. However, laboratory crosses showed no
significant segregation distortion, although a slight heterozygote excess was noted
for certain crosses.
Fourth, heterosis may be acting at this locus. There is evidence that "hetero-
zygous enzymes" may have an advantage over "homozygous enzymes."'4 Such
an advantage may result from association of different subunits giving rise to
an enzyme with different, favorable properties.
Our observations are most consistent with heterotic balance maintaining the
To polymorphism in the population. To our knowledge this is the best evidence
to date for this type of mechanism acting at an enzyme locus in a natural pop-
ulation. However, the possibility that heterosis is acting not at the To locus but
rather at some tightly linked locus or loci cannot be ruled out.
The authors thank Olga Pavlovsky for preparing chromosome slides, Boris Spassky for
classifying the original wild collection, and Dr. A. J. Gallo of the FFCL of Sio Jos6 do Rio
Preto, S.P. who kindly collected the original material. We also thank Professor Th. Dobz-
hansky and Dr. F. J. Ayala for their helpful discussion and criticism of the manuscript.
*
Supported by NSF grant GB-12562 (International Biological Program), AEC contract
AT-(30-1), NIH predoctoral fellowship, 1-Fl-GM33, and an NSF Graduate Fellowship.
t Present address: Department of Zoology, Indiana University, Bloomington, Ind. 47401.
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