The evolution of a small population of

Drosophila melanogaster over three

generations

Wendy V. Tifft

Lab partners:
Hiwot Zewdie
Erica Bahnweg
Danielle Goldberg

BSC2011L section 16

November 11, 2016

Abstract
The Hardy-Weinberg equilibrium theory outlines the conditions that
must be met for a population to be in a state of no evolution. These
criteria are rarely met in the natural world. In the lab this equilibrium is
easy to test in an isolated population of Drosophila melanogaster fruit
flies with known allele frequencies. Even with all other criteria met, it is
hypothesized that lack of a sufficiently large population will result in
evolution as a result of genetic drift in as short as three generations.
Additionally the criteria of no sexual selection may not be controllable
when all mating will occur in the light. A population of 16 flies of known
genotypes was isolated and observed for three generations of
offspring. The flies of each new generation were sorted and counted
based on phenotype. A chi-squared test was used to determine if the
observed allele frequencies differed significantly enough from the
expected allele frequency to suggest evolution of the population.
Although the results of the three-generation experiment are clear, it is
advised that future experiments should be conducted for additional
generations to provide more complete data. In the end the data did not
support the hypothesis and no evolution was observed.

Introduction

Over time the allele frequencies of genes change in a population as

evolution occurs. Evolution is driven by sexual selection, gene flow
between populations, random mutations and natural selection for
advantageous over disadvantageous alleles. Evolution also occurs by
genetic drift where the ratios of one allele to another change over time
due to chance, not advantage. If all drivers of evolution are removed
from a population the ratios of alleles should remain constant. This
situation is called Hardy-Weinberg equilibrium. There are five
conditions that must be observed in order for this equilibrium to be
possible. The first is that the population is sufficiently large so that a
natural drift in alleles will not sway the overall ratios. Second is that
mating must be random, no sexual selection. Third, the population
must be isolated and thus not influenced by migration. Fourth, there
must be no natural selection. And fifth, there must not be any new
mutations in the population being studied. In any population the
likelihood of these criteria being met is unlikely.

In an attempt to observe this equilibrium an experiment was created in

which the phenotypes of a species were observed for several
generations and compared to the expected ratios if Hardy-Weinberg
equilibrium were occurring. A model organism was needed, one that is
diploid and can be expected to predictably demonstrate the known
phenotypes for a locus with exactly two possible alleles. The fruit fly

Drosophila melanogaster was chosen for its short reproductive cycle,

ease of sex determination and observable phenotypic trait of color.
Although some studies revealed that the ratio of body color
phenotypes may change in response to seasonal weather changes
(Chahal et al. 2011), that was not a factor in this experiment because
the fly containers were kept at a constant temperature and humidity
level for the duration.

In the case of the fruit flies there are two alleles for body color; wild
type and ebony, with wild type being the dominant trait. A typical
Punnet square revels that if a homozygous wild type mates with a
homozygous ebony the most likely result will be 25% homozygous
ebony offspring, 25% homozygous wild type offspring and 50%
heterozygous offspring. Phenotypically this would be 25% ebony and
75% wild type. If the population is in Hardy-Weinberg equilibrium this
ratio will remain constant generation after generation.

The experiment was limited to 16 flies to start with; four homozygote

wild type males, four homozygote wild type females, four homozygote
ebony males and four homozygote ebony females. Due to the small
population size it was expected that Hardy-Weinberg equilibrium would
not be present by the end of the experiment. Any drift in gene
frequency would have a significant impact on the population and

render it out of equilibrium and thus evolving. Additionally, a prior

study on the reproductive fitness of ebony Drosophila suggest that
their mating frequency is lower than that of wild type males when
breeding occurs in the light (Burnet et al. 1978) as it did in this
experiment. This demonstration of sexual selection will also play a role
in causing the population to evolve resulting in a higher than 75%
occurrence of phenotypically wild type flies.

Methods
A vial was prepared to store and isolate a breeding fly population. The
vial contained fly food, 10 beads of yeast, and a section of plastic fly
culture netting. The netting gives the flies something on which to land
and and climb. A vial of homozygous wild type flies and a vial of
homozygous ebony flies were retrieved and anesthetized by inserting a
wand soaked in Flynap. Once all flies were asleep a few were removed
from each vial and sorted based on color and sex using the dissecting
microscope. Four wild type males, four wild type females, four ebony
males and four ebony females were put in the newly created vial and
the end was plugged. The vial was kept horizontal to prevent
anesthetized flies from falling in the fly food. The vial was then stored
for 1 week in the light in an incubator at 25C.

At the beginning of weeks two, four and six the adult flies were
anesthetized in the vial, removed and disposed of. The larvae were
observed and then the vial stored for one more week. At the beginning
of weeks three and five a new vial was prepared with food, yeast and
plastic mesh. The flies in the original vial were anesthetized and
removed. They were sorted by sex and color and the data recorded.
The live flies were then placed in the new vial and stored for another
week.

At the beginning of week 7 the flies in the original vial were

anesthetized and removed. They were sorted by sex and color and the
data recorded.

A chi-squared calculation was done to determine if the observed

changes in allele frequency from week to week were significant enough
to suggest the population was evolving. The x2 value was then
compared to the chi-squared table. In this experiment two allele
possibilities were observed making the degrees of freedom 1. The
chart revealed the probability of the observed result and if the
population was indeed in Hardy-Weinberg equilibrium. If the probability
was greater than .05 then the population was considered at
equilibrium.

Results
Week one started with equal numbers of wildtype homozygous flies
and ebony homozygous flies for and allele frequency of 50:50. At the
beginning of week three, the first generation of offspring from the
original flies were sorted and counted. There were a total of 74 flies, 51
being of the wild type phenotype and 23 of the ebony phenotype. This
resulted in an allele ratio of .44 wild type to .56 ebony (Table 1). At the
beginning of week five, the second generation of offspring from the
original flies were sorted and counted. There were a total of 42 flies, 31
being of the wild type phenotype and 11 of the ebony phenotype. This
resulted in an allele ratio of .49 wild type to .51 ebony (Table 1). At the
beginning of week seven, the third generation of offspring from the
original flies were sorted and counted. There were a total of 62 flies, 44
being of the wild type phenotype and 18 of the ebony phenotype. This
resulted in an allele ratio of .46 wild type to .54 ebony (Table 1). The
largest gap in allele frequency occurred in week three with the first
generation of offspring, and then in week five the second generation of
offspring returned back to nearly the same frequency as the initial
population only to diverge again slightly for the third generation (Figure
1).

Table 1: Frequency of wildtype (+) and ebony (e) alleles.

Figure 1: Frequency of wildtype (+) and ebony (e) alleles over three
generations of flies.

Using the observed phenotype data and the formula

p2+ 2 pq+ q2 =1

the genotype frequency was estimated for each generation of flies. For
week one the genotype frequencies were known to be .50 for wild type
homozygous and .50 for ebony homozygous. For week three, the
frequency of genotypes was estimated to be .194 homozygous wild
type, .493 heterozygous, and .314 homozygous ebony. For week five,

the frequency of genotypes was estimated to be .24 homozygous wild

type, .5 heterozygous, and .26 homozygous ebony. For week seven, the
frequency of genotypes was estimated to be .212 homozygous wild
type, .497 heterozygous, and .291 homozygous ebony (Table 2). Figure
2 illustrates the estimated changes in genotype frequency over the
three generations.

Table 2: Estimated frequency of genotypes homozygous wildtype (++),

heterozygous (+/e) and homozygous (ee).

Figure 2: Estimated frequency of genotypes homozygous wildtype (++),

heterozygous (+/e) and homozygous (ee) over three generations of flies.

A chi-squared value was calculated for the fly population with every
new generation and the probability of the value looked up in the chisquared table. X2 for the first new generation in week three was 1.46
indicating a probability greater than .20. For the second generation in
week five the x2 was .032 indicating a probability greater than .80. For
the third generation in week seven the x2 was .537 indicating a
probability greater than .30 (Table 3).

Table 3: Chi-squared calculations for three generations of flies.

Discussion

Allele frequencies over the three generations of offspring studied

remained fairly constant. The probability of the observed frequencies
being what they were due to chance were all well above the .05
probability threshold for Hardy-Weinberg. The null hypothesis of HardyWeinberg equilibrium is, in this case, true and the differences between
what was expected and what was observed did not differ significantly
suggesting no evolution of the population. The initial hypothesis that
evolution would indeed occur was proven false. Ultimately the small
population size did not lend too much weight to genetic drift as was
originally hypothesized. Additionally the lighted breeding environment
did not lead to an increased selection for wild type alleles as suggested
in previously documented studies (Burnet et al. 1978). It is important
to recognize the short duration of this study when compared to other
studies of Drosophila. Previous studies demonstrate that it often takes
more than three generations for significant changes in alleles to occur
(Connolly 1966) but they do occur in less than 10 generations time. A
2005 study mimics the results of this study in its findings that loses in
genetic variation in small populations happened much slower than
predicted in neutral theory (Gilligan 2005). Neutral theory suggests
genetic drift as a main driver of evolution as opposed to natural
selection. If this experiment were to be repeated, a longer time frame
would be advised. Observing more successive generations would be an

improvement and offer more conclusive data regarding the presence of

genetic drift in a small population of flies.
The occurrence of some flies getting stuck in the food and perishing
was constant from generation to generation. There is no evidence to
support that any particular genotype was more likely to be the victim
of such fate and thus this circumstance did not alter the overall results.
Likewise, each time the flies were sorted by hand there were one to
two casualties in the handling of them. Again this was not more likely
to occur with one genotype over another and is not considered
significant to the results. The data resulting from this study is an
accurate reflection of the experiment proposed and completed to test
the likelihood of Hardy-Weinberg equilibrium for three generations of
offspring from a small population Drosophila melanogaster of known
alleles.

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