Mattia Avery 12 April, 2016 Lauren Falkenberg

Evaluating the Effects of Natural Selection in Drosophila melanogaster

Abstract

The purpose of this experiment was to complete an experiment about population genetics using living organisms (Drosophila melanogaster) over the course of several months. Skill sets, such as sexing and tracking phenotypes within a fly population across five generations, allowed evidence of evolution, by either natural selection or genetic drift, to be compounded. By observing the changes in phenotypes among the sample population, and later, the changes in allele frequencies through further data analysis, an illustration of the principles of X-linked inheritance, an example of evolutionary forces as an observable phenomenon, and a demonstration of evolutionary factors contributing to the change in allele frequencies within a population were completed during the experiment. The experiment was accomplished by taking sample populations from the population cage every second week, for twelve weeks. The sample of flies was then sexed and sorted depending on the eye color phenotype. The population was randomly sampled, and a count of the phenotypes for four succeeding generations was completed with accompanying calculations of allelic frequencies from the results. It was hypothesized that natural selection would act on the fly population over the course of an entire semester. This was determined by observing the X-linked trait for eye color and by collecting data from a sexed sample of the population for each generation. After the completion of the experiment, the experiment showed evidence of evolution occurring within the population after testing the results using the principles of Hardy-Weinberg Equilibrium. The data shows that evolution was happening within the population of tested Drosophila, since the data was not in accordance to the expectations of the Hardy-Weinberg Equilibrium, which served as the null hypothesis for the experiment, which states that allele frequencies will remain constant so long as evolutionary influences occur within the population. The calculations recorded in Tables 3-6 found the χ2 and P for all generations were significant, with large χ2 and small P values being the basis for rejecting the HWE. The reason that evolution can be accounted for through the reject of the HWE is attributed to natural selection against white-eyed allele within the population. The overall changes in allele frequencies and selection coefficient against the white-eye allele was proof of evolution. Therefore, it was discovered that the white-eyed color allele (w) was naturally selected against in the breeding population, showing that natural selection was occurring within the evolving population. Due to the small population of flies, and the even smaller sample populations taken from the population cage, though, variances in the trend of data were also attributed to genetic drift. Regardless, evolution was occurring in the Drosophila population over the twelve-week time frame. Introduction

The purpose of this experiment is to perform and evaluate a long-term experiment to illustrate evolution using living organisms. By tracking the phenotypes (allele frequencies) of the population under study and looking evidence of evolution in the form of natural selection or genetic drift by the changes in allele frequencies and further data analysis, a demonstration of evolutionary forces as an observable phenomenon, an illustration of the principles of X-linked inheritance, and a demonstration of factors, other than natural selection, contributing to the change in allele frequencies in the population in

question will be accomplished during the experiment. This experiment falls under the category population genetics, which is the study of the distribution and change in frequency of alleles within populations, and as such, it is instrumental to the field of evolutionary biology (Goldman). Natural selection and genetic drift are both important processes of evolution. In order for natural selection to take place, there must be variation among individuals within a population that is heritable and this variation must allow for certain individuals to be more successful at surviving, mating, or reproducing than others (Goldman). While genetic drift is random, natural selection tends to produce a directional change in allele frequencies that is noticeable from generation to generation. For example, if a specific allele consistently experiences greater success within the population, then, overtime, that allele will become more prevalent in the population (Goldman). The results of natural selection are consistent from population to population, while drift can behave differently in each population. For example, in Geer and Green (1962), the study observed evidence of natural selection in colonies of Drosophila melanogaster. Their study concluded that mate selection was visually dependent. They also found that when wild type, red-eyed male flies competed for mates with white-eyed males, they had a greater mating success (Geer & Green). Genetic drift differs from natural selection in that it is non-directional. It is the result of the random sampling of alleles at each generation. Its affects are usually seen in small populations or populations that are “below an effective population size” (Rich). For example, in Buri (1956) an experiment using small populations of Drosophila melanogaster having equal frequencies of two alleles, bw and bw75, at the brown locus, the data found that, in many of the populations, the alleles became randomly fixed for one or the other allele, which is consistent with the expectations for genetic drift (Buri). Drosophila melanogaster, more commonly known as the fruit fly, is easily cultured and its generation time comprises of two weeks at 21-23 C. Since it occupies little space, yet is large enough to observed phenotypic traits, such as eye-color, with the naked eye, it was the species of choice for the experiment. A fruit fly has four distinct life stages: egg, larva, pupa, and adult (Greer & Green). A fresh culture of D. melanogaster can produce new adults in two weeks, with the time span being 2-3 days for the embryo stage, 6 days in the three larval stages, and six days in the pupal stage. After 12 hours, the adult fruit fly is fertile, but it usually takes between 3 to 7 days for the fly to reach maximum fertility. An adult fruit fly may live for several weeks while remaining fertile during that time (Weigmann et al).

Figure 1. Drosophila melanogaster Life Cycle (K. Weigmann et al, 2009)

At the beginning of the experiment, the original stocks of flies (G0) were homozygous for either the red-eye (wild-type) or white-eye (mutant) phenotype, with red eye type being dominant to white eye type. The gene that codes for eye color is located on the X chromosome in Drosophila. The two alleles are:

w+ = wild type eye color (brick red) w = white-eye allele Females have two X-chromosome and, therefore, have two copies of the gene for

eye color. Therefore, red-eyed females have the genotype w+w+ while white-eyed females have the genotype ww.

Meanwhile, male Drosophila have only one X chromosome, with their other sex determining chromosome being a Y chromosome; red-eyed males have the genotype w+ Y and white-eyed males have the genotype wY. Consequently, when looking at a male fly with the naked eye, the eye color allele that the male fly possesses can easily be identified. By using two alleles for an X-linked gene controlling eye color, it is much simpler to distinguish wild-type males from white-eyed males. Since the gene is X-linked, the allele frequencies for both alleles can be directly calculated for males, while a red-eyed female can have the genotype w+w+ or w+w (Weigmann). Since approximately ½ of females will have a white allele by the end of the experiment, the allele frequency can be indirectly estimated in females by assuming the Hardy-Weinberg equilibrium is present. The Hardy–Weinberg equilibrium states that both genotype and allele frequencies within a breeding population will remain constant from generation to generation only when there is an absence of evolutionary influences, such as gene flow, mate choice, mutation, genetic drift, and meiotic drive (Dawson).

Calculations for the frequency of the white-eye allele in males can be found by taking the number of white-eyed males divided by the total number of males, while the frequency of the red-eyed allele is one minus the frequency of the white-eyed alleles since allele frequencies sum to 1.

To determine the sex of each fly, the fly can either be examined beneath a dissecting microscope or by the naked eye. The best method is to look at the fly’s abdomen; in males, the end of the abdomen is more rounded and is much darker in color than a female’s abdomen. Meanwhile, females will have pointed, light colored abdomens.

The males are also slightly smaller than the females. Every second week for twelve weeks, the flies are to be maintained by

replenishing their food supply in a population cage, which is comprised of four bottles of fly food connected to a plastic container. The flies are also kept in a dark cabinet. During this time, a chosen sample of flies is taken from the entire population. They are then sexed and sorted depending on eye color. By randomly sampling the population, a count of the phenotypes and a calculation allele frequencies could be made and recorded on the data sheets found in the results section.

The purpose of this experiment is to study population genetics by observing five generations of fruit flies. It is hypothesized that the flies would undergo natural selection over the course of an entire semester, which is determined by observing the X-linked trait for eye color and through collecting data by sexing the flies from a sample of the population.

Materials and Methods The culture vials were created by adding commercially prepared fly flakes combined with fly water. This culture medium provided the nutrients for normal larval development, but it also allowed yeast to grow, so the culture vials had to be changed out regularly. Initially, four culture vials were made. About 30 mL of media flakes were added to approximately 30 mL of water in the large bottles.

Once finished, stock bottles containing true breeding male and female flies were then handed out. After sexing the flies under a dissecting microscope, the flies were then sorted based on eye color. After anesthetizing the flies with carbon monoxide gas, the flies were transferred to a plastic culture box, which was corked with foam. The bottles of food attached to the box via adhesive. They were held horizontally to keep the unconscious flies from falling and sticking in the culture medium. Once the flies rose, the box was then labeled. Within the box there were 10 wild-type females, 10 white-eye females,10 wild-type males, and 10 white-eye males. The box was kept in a dark cabinet. The parents were eventually removed so only offspring would be present in the next weeks progeny. After two weeks, the old food bottles were rotated out. These bottles contained larvae and pupae. They were replaced with new food bottles. The old food bottles were sealed with a foam plug and saved for the next week, which was when a full count of the G1 generation was made. After another two weeks, the G1 flies in the food bottles were anesthetized and transferred to a fly pad. The sample population was sexed sorted by the eye color phenotype. Data was then recorded. The numbers allowed for the estimation of allele frequencies within the first generation. New food bottles were placed in the population cage and population cage counts were conducted every two weeks, totaling to five counts through the semester. Once all the data was compounded, results were studied in the section below. Sample calculations to find the frequency of allele types within the population are shown below. To find the frequency of the wild-type allele in males: Example 1, a G1 generation has three red-eyed males out of a sample of four: Frequency of the wild-type allele in males is pm =3/4 = 0.75. To find the frequency of the white-type allele in females:

Example 2, a G1 generation has two red-eyed females out of a sample total of four: Frequency of white-eye females is q2 = 2/4 = 0.5 Frequency of white-eye allele in females is q = = 0.71 Frequency of wild-type allele in females is pf = 1 - 0.71 = 0.29 Frequency of wild-type allele in the whole population pall = (2/3) pf + (1/3) pm pall = [2(0.29) + 1(0.75)] / 3 = 0.44 Calculating the estimation of the selection coefficient against the w allele in males: Equation 1: sm = (qf – q’ m)/1− (qm x qf ) qf = frequency of the white allele in females in the previous generation (using HWE) q'm = frequency of the white allele in males in the current generation qm = frequency of the white allele in males in the previous generation Example 3: In one generation, 60 red-eyed males, 40 white-eyed males, 80 red-eyed females, and 20 white-eyed females are obtained. qf = 0.5 (frequency from G0) q'm = 40/(60+40) = 0.4 qm = 0.5 (1/2 of the males in G0 were white-eyed) sm = (0.5 – 0.4)/[1 – (0.5 x 0.5)] = 0.133 (the selection coefficient against white from generation 0 to 1; s > 0 means selection favors red) Results Table 1: Total Count of Red-Eyed and White-Eyed Sexed Fruit Flies for Five Generations Phenotype Observations Gender Eye Color G0 G1 G2 G3 G4 Male red 10 98 97 83 64 white 10 52 38 9 35 Female red 10 126 125 98 104 white 10 30 54 10 23

In the beginning, 10 wild-type females, 10 white-eye females,10 wild-type males,

and 10 white-eye males were placed into a population cage and allowed to mate over the course of twelve weeks. Every two weeks, a sample of flies were anesthetized and sorted into piles based on sex and eye color. The results for five generations, starting from G0, were recorded in the table above. Eventually, dramatic changes occurred over the course of five generations were noticed, but one trend existed: that the number of red-eyed flies outnumbered the number of white-eyed flies in each sample population over the preceding four generations.

Table 2: Recorded calculations of changes in w and w+ allele frequency over five generations Allele Frequencies G0 G1 G2 G3 G4 Males pm 0.5 0.653 0.719 0.902 0.646 qm 0.5 0.347 0.281 0.098 0.354 Female q^2 N/A 0.192 0.302 0.093 0.181 qf 0.5 0.439 0.549 0.304 0.426 pf = (1 - q) 0.5 0.561 0.451 0.696 0.574

The calculations displayed in the table above were made to find the changes in

red-eyed and white- eyed allele frequencies over the course of five generations. For every generation, recorded values, both of males and females, were taken from Table 1 and calculated to find the changes in allelic frequencies over the course of the twelve weeks. Each value represents the allelic frequencies for both red-eyed flies (p) and white eyed-flies within the population and all data shows a general trend in the four generations white white-eyed allelic frequencies are lower than red-eyed allelic frequencies. This data was later used in the following chi-squared tests below. Table 3: Chi-Square Tests: Testing For Significant Change in Allele Frequencies Across Generations

χ2 test G1 Population

Red-eyed Males

White-eyed males

Red-eyed Females

White-eyed Females

Observed 98 52 126 30 χ2 = 14.106

Expected 75 75 78 78 P= .000173

(Obs-Exp)^2/Exp 7.053 7.053 29.578 29.578

χ2 = 59.076

P= <.00001

Table 3 displays a Chi-Squared Test under the assumption of the Hardy-Weinberg Principle. The calculations above show the number of observed red-eyed verse white-eyed flies, sorted by sex, in the population, compared to the number of flies expected based on the allelic frequencies calculated above, were used to find the χ2 and associated P value for each generation. For each generation, the allelic frequencies from the previous generation were used as the expected allele frequencies for the preceding population. The χ2 and associated P value for each generation was highlighted in blue (males) and red (females). After completing the chi squared tests, it was discovered that in the G1 population, the null hypothesis, the HWE, could not be accepted, since both values for male and females showed a big χ2 and a small P value, indicating evolution was taking place within the sample population.

Table 4: Chi-Square Tests: Testing For Significant Change in Allele Frequencies Across Generations

χ2 test G2 Population

Red-eyed Males

White-eyed males

Red-eyed Females

White-eyed Females

Observed 97 38 125 54 χ2 = .2.55

Expected 88.155 46.845 100.419 77.264 P= .109737

(Obs-Exp)^2/Exp .887 1.670 6.017 7.005

χ2 = 13.022

P= .000308

The same layout in Table 3 is found in Table 4. It was discovered after the χ2 and

P values were calculated for the G2 population that both results were significant and therefore, the Hard-Weinberg Equilibrium was rejected. Table 5: Chi-Square Tests: Testing For Significant Change in Allele Frequencies Across Generations

χ2 test G3 Population

Red-eyed Males

White-eyed males

Red-eyed Females

White-eyed Females

Observed 83 9 98 10 χ2 = 15.278

Expected 66.148 25.852 48.708 59.292 P= 9.3 x 10^-5

(Obs-Exp)^2/Exp 4.293 10.985 49.883 40.979

χ2 = 90.862

P= <.0001

The same layout in Table 4 is found in Table 5. In calculations found in the table above, the χ2 value was calculated based on the HWE, which states the evolution is not happening within the selected populations. Because both values for χ2 and P are considered significant, the HWE had to be rejected.

Table 6:

χ2 test G4 Population

Red-eyed Males

White-eyed males

Red-eyed Females

White-eyed Females

Observed 64 35 104 23 χ2 = 73.132

Expected 89.298 9.702 98.392 38.608 P= <.00001

(Obs-Exp)^2/Exp 7.167 65.965 .3196 8.215

χ2 = 8.8346

P= .002947

The same layout in Table 5 is found in Table 6. After observing the results found

in the G4 population, it was decided that the HWE could not be accepted, as both values for male and females showed a big χ2 and a small P value, indicating evolution was taking place within the sample population. Table 7: Numerical values of the selection coefficient against the w allele in males Generation sm G1 0.204 G2 0.186 G3 0.754 G4 0.052 Average 0.271

The table above displays the calculated numerical values of selection coefficient

in males against the white-eye color allele over the four preceding generations from the initial population of flies. It also takes into account the average over all the generations. In the data above, all numerical values were greater than 0 (s<0), indicating that selection favored the red eye color allele over the white eye color allele.

Figure 1: Plot of change in allele frequencies in males over time

The data displayed in the figure above was based from the data collected in Table 2. The line graph plots two respective lines on a graph the correlates the individual generation of the sample population of male Drosophila to allele frequencies. The red line represents the change in allelic frequency for red-eyed male flies over five generations, whereas the green line represents the change in allelic frequency for white-eyed male flies over five generations. Discussion

It was hypothesized that differential reproductive success of genotypes, by means of natural selection or genetic drift, would occur in the Drosophila melanogaster population, contained in the population cage, over the course of twelve weeks. It was also predicted that the relative change in the proportions of alleles in a population from one generation to the next would indicate evolutionary change.

It was determined by the calculations above that evolution was happening within the population of tested Drosophila. The data found was not in accordance to the expectations of the Hardy-Weinberg Equilibrium, which stated that allele frequencies within a breeding population remain constant from generation to generation only when there is an absence of evolutionary influences (Goldman). The Hardy-Weinberg Equilibrium served as the null hypothesis. Had there been no change in allelic frequencies, no evolutionary influence influences would have occurred, meaning no evolution would have been observed from generation to generation. Because the data found within Tables 3-6 found the χ2 and P for all generations were significant, with large χ2 and small P values being the basis for rejecting the HWE, it was proven evolution was occurring within the populations, allowing for the Hardy-Weinberg Equilibrium to be rejected. In Table 3, G1 in males had a χ2 of 14.106 and a P value of .000173. For females, the values in G1 were χ2= 59.076 while P= <.00001. In Table 4,

0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1  

1   2   3   4   5  

Allele  Frequencies    

Generation  

Change  in  Allelic  Frequencies  in  Males  Over  5  Generations  

Pm  (W)  

Qm  (W+)  

the G2 found the χ2 value in males to be 2.55 while the P value was found to be .109. In females the χ2 was equivalent to 13.022 and the P value was equivalent to .00308. In Table 5, G3 found the χ2 value in males to be 15.278 while the P value was equivalent to 0.3 x10^-5. For females, the χ2 was equivalent to 90.862 and the P value was equivalent to <.0001. And in Table 6, the χ2 value in G4 males was found to be 73.132 and P value was calculated to be <.00001. Meanwhile, the χ2 value in G4 females was found to be 8.8346 and P value was calculated to be <.002947. Since the deviation is significant (with small P values and large χ2 values), then the gene frequencies are changing and thus, evolution is occurring. As can been seen in the data, in the Tables, which have a trend towards significance, and a positive trend in selection coefficient, it is apparent that natural selection is the directionally driving force in the evolution of the Drosophila population. The reason this is can be attributed to natural selection against white-eyed allele taking place during the experiment, which will be further explained below.

During the experiment, according to Figure 1, there was a drastic change in frequencies near the end of the experiment. This can be attributed more to sampling error than genetic drift. Ideally, the frequencies should have changed most rapidly during the first few generations, since the population sizes began small (with only 40 flies) which then reproduced rapidly, but instead, most of the rapid changes began near the end of the experiment. This is likely due to the fact the sample sizes had been much smaller in the later generations than in the earlier generations. As noticed within the tables above, there is a fluctuation within the significance of each generation, likely due to sampling error. The trend of allelic frequencies found more red-eyed alleles than white-eyed alleles within the population, which differed from G0 in that G0 had an equal number of red-eyed alleles (.5) and white-eyed alleles (.5). Had no evolution occurred, the allelic frequencies would not have changed, and the number of red-eyed and white-eyed flies would have remained equal, as there would have been no evolutionary pressures enacting selection on the population. This suggests that red-eyed alleles were being selected for rather than against. This trend was overall noticed in Figure 1 and Table 2, but there are two questionable spikes, one at Generation 3, the other at Generation 4. This is supposed to be because of sampling error. Both the sample count for male flies was low in Generation 3 (92) and Generation 4 (99) in comparison to Generation 1 (150) and Generation 2 (135). This smaller sample size resulted in less obvious signs of natural selection and caused a rapid fluctuation in allelic frequencies. Therefore, the rapid change in allelic frequencies was most likely to be cause by sampling error than genetic drift and the frequencies still have an overall positive trend, suggesting directional change, which is observed in natural selection.

The selection coefficient was mostly consistent throughout the experiment and indicated that the red-eyed male flies were naturally selected for in comparison to white eyed male flies. This suggests the reason why Hardy-Weinberg Equilibrium was proven false. Hardy-Weinberg conditions were used as the null hypothesis, stating that the allele frequencies would remain constant from generation to generation only when there is an absence of evolutionary influences. In G1, the selection coefficient for males was .204, in G2, .186, in G3, .754 and in G4, .0271. These sm values are all greater than 0. Since s was greater than 0, showing that selection favored red-eyed flies, and drove the population in the direction of a higher frequency of red-eyed flies. In order for the population to be driven by selection, a type of evolutionary influence, such as mate

choice, must have occurred within the tested population. The statistically significant deviations from the equilibrium showed that evolution was occurring by selection driving the population towards the red-eyed phenotype. At the end, the selection coefficient was not consistent. It assumed this is not because of genetic drift, but rather sampling error. Less flies were chosen for generations 3 and 4 due to the escape and death of many of the flies from both of these generations. A smaller sampling size most likely affected the selection coefficient recorded in Table 7. Overall, natural selection was proven to occur by a drive in the population towards red eye color alleles over white eye color alleles.

Genetic drift was most likely not as significant as sampling error. As the population sizes within the population cages grew, due to experimental error, many of the flies died when being transferred to a fly pad while many other flies had been dead for so long that by the last two generations, there were hardly enough flies to properly sex. During the fourth generation, many of the flies had yet to hatch, and hatched within the next few hours, after the sample population had already been counted. Therefore, the only flies that had been rounded up had been either dead for a while or few in numbers. The allele frequencies were seen to be less significant in later samples, with smaller χ2 values and larger P values. Though neither of these values in the later samples accepted the HWE, they were ironically much less significant than the earlier samples, despite the earlier samples having less flies to begin with (G1 came from a population of 40 flies). Since genetic drift ordinarily affects small populations or populations that are “below an effective population size”, the effects should have been seen within the first few generations (they should have been more non-directional), but in actuality, the allelic frequencies become more non-directional when smaller sample sizes had been taken, not because the sample population was smaller. The smaller samples sizes gave results that differed more from the previous trend of selective coefficient in earlier generations, which showed white-eyed flies were selected against (as seen in Generation 4, Table 7), and therefore, had allelic frequencies that were less exemplar of natural selection for the wild-type allele. But this was not observed in the early generations with smaller populations. Therefore, it is more likely the sampling error caused fluctuations in the changes in alleles, rather than genetic drift (Rich). Citations Buri, P. 1956. In Hall. B. and B. Hallgrimsson. 2008. Strickberger’s Evolution. Jones and

Bartlett. Sudbury, MA. 759 pages. Dawson, P. S. 1970. Linkage and the Elimination of deleterious mutant genes from

experimental populations. In: Freeman, S, and Heron, J. 2007. Evolutionary Analy-sis. Pearson Education Inc. Upper Saddle River, 196 pages.

Geer, B. and M. Green. 1962. Genotype, Phenotype and Mating Behavior of Drosophila

melanogaster. The American Naturalist. 96: (888): 175-181. Goldman, C. 1991-1992. Natural Selection and Genetic Drift in Drosophila

melanogaster. BIO 150, Biology Department, University of Toronto.

Rich, S.S., A. E. Bell, and S. P. Wilson. 1979. Genetic Drift in Small Populations of Tribolium. Evolution, 33: (2): 579-584.

Weigmann, K., R. Klapper, T. Strasser, C. Rickert, G. Technau, H. Jäckle, W. Janning and C. Klämbt: FlyMove-a new way to look at development of Drosophila. http://flymove.uni-muenster.de/Homepage.html?http&&&flymove.uni-muenster.de/hometxt.html.