Determining the Evolution of Alleles Present in Populations of Drosophila melanogaster by Altering the Conditions of the Hardy-Weinberg Theorem.

April 21, 2010 The Pennsylvania State University Brooke Gushen In Collaboration With: Cara Pinto, Jessica Mitchell, Ellen Broz, and Section 11 Arunima Sen Biology 220W, Section 11

INTRODUCTION: Evolution is essential to life and development of all living organisms. It occurs through numerous processes, such as adaption, genetic drift, gene flow, mutation, and natural selection, to create genetic diversity in a population ("Evolution," 2008). Genetic drift is one of the processes measured in this experiment, and research explains that this process often leads to population differentiation specific to location due to isolated gene pools yielding these specific allele frequencies (Seongho, Dipak, & Holsinger, 2006). All of these evolutionary processes generate changes in species genotypes, however, in the case of Hardy-Weinberg equilibrium, species are maintained in a non-evolving motion. To meet this condition several requirements must be met. In order to resist evolution, the population must be large in size, mate randomly, and experience no mutation, migration, or natural selection (Hass, Burpee & Meisel, 2006). In this state, allele frequencies should remain constant over time, and it should be seen that there is no difference between the observed and expected allele frequencies. In terms of statistics, this superficial population represents the null hypothesis, which states that if there is a statistically significant relationship (p < 0.05), we will see no difference between the observed and expected allele frequencies (Hass, Burpee & Meisel, 2006). Every species has a gene pool that holds a complete set of unique alleles, which serves as a pool for members of the next generation to obtain their genes, giving each a distinctive genetic makeup (Campbell, Mitchell, & Reece, 1994). Drosophila melanogaster is one species that is particularly favored in research due to the simplicity of determining genotypes from phenotypes. This is important because knowing the genotype allows easy conversion to allele counts and frequencies. Differing alleles are attributed to traits such as curly wings, wrinkled wings, sparkly eyes, reduced eyes (eyeless), and also, most commonly, wild-type traits (Hass, Burpee & Meisel, 2006). We will investigate determining how these fruit flies develop wild-type wings or curly wings, and how these traits reflect in alleles in the gene pool of the population. In this experiment, several populations of D. melanogaster will experience different alterations to impact the evolution of the subsequent generations. One population will observe the eyes of the individuals to determine how the alleles change over time in different size populations. We expect that the larger population will be closer

to Hardy-Weinberg Equilibrium, due to the fact that there is more resistance to the changing alleles. In another population, the wings will be observed as either curly or wild-type. It is expected that the allele that yields the most favorable trait will become more dominant in the population. Since curly wings hinder flight, the allele attributed to this trait should lessen in frequency in this population. The final population to be tested is made up of D. melanogaster with differing degrees of wrinkled wings. Wrinkled wings greatly decrease the fitness of the insect, therefore reducing its offspring. In an event where a population with a majority of wrinkled-wing individuals experiences a small migration with wild-type individuals, it is predicted that because the wild-type alleles are significantly more favorable, the population will favor that allele greatly and lead to an increase in frequency after several generations. MATERIALS AND METHODS: Experimental procedures were derived from the following laboratory manual: (Hass, Burpee & Meisel, 2006) In this experiment, three different populations were manipulated and observed to determine patterns in allele frequencies over the course of five weeks. For Population A, the observed individuals had either reduced eyes (ey/ey), sparkly eyes (sp/sp), or wild-type eyes (ey/sp). These traits are not strongly related to natural selection, therefore allowing the difference in two population sizes to be the influencing variable. For this particular population, we did not record the observations, but rather obtained the data from the teaching assistant. From this data, expected allele frequencies were calculated and compared to observed allele frequencies to find a chi-square statistic. This statistic determines if the null hypothesis can be rejected and whether Hardy-Weinberg equilibrium is reached. For Population B, the individuals had either curly wings (Cy/Cy+) or wild type (Cy+/Cy+) wings. A random mixture of the two groups was obtained. Over the course of the five weeks, the ratio of curly to wild-type individuals was found by counting sedated populations of D. melanogaster. They were returned to containers with food after being counted and stored until the next counting session. Once all the data was collected, the allele frequencies were calculated based on the counts of the genotypes. They were then graphed to determine the effects of natural selection on this population. 3

Population C went through the same care and counting procedures as Population B, however this population was impacted by migration in the first week. A population of wrinkly wing D. melanogaster experienced a very small migration of wild-type individuals. Over the course of the five weeks, the counts of the offspring were recorded. With the collected data, allele frequencies were calculated and compared over time. This population shows how natural selection and migration affect a populations allele frequencies. Expected allele and genotype frequencies were calculated by using the Hardy-Weinberg Equilibrium equations: p2 + 2pq + q2 = 1 and p + q = 1. Also, all the calculated values and graphs from the last two populations were finally compared to outputs from Populus to compare to a theoretical model. RESULTS: Population A Effects of Small vs. Large Population Week 1 Week 3 Week 5 Allele Frequency ey (p) sp (q) ey (p) sp (q) ey (p) sp (q) Small 0.480 0.520 0.380 0.620 0.366 0.634 Large 0.356 0.644 0.368 0.632 0.330 0.670 TABLE 1: Allele Frequencies of Population A- Population A is made up of eyeless (ey/ey), wild-type (ey/sp), and
sparkly-eyed (sp/sp) individuals. Allele frequencies calculated from raw data of genotype counts.

Table 1 shows the relatively close ey and sp allele frequencies in the initial small population. Over the course of the five weeks, the ey allele frequency gets steadily smaller as the sp allele frequency gets steadily larger. In the large population, the allele frequencies do not steadily move in one direction, rather both allele frequencies oscillate over the five weeks. Number of ey/ey 79 80.202 0.018 Number of ey/sp 280 277.595 0.021 Number of sp/sp 239 240.202 0.006 X21df = P-value = TABLE 2: Chi-Square Analysis of Genotype Frequencies in Small Population in Week 5 Total 598 598 0.045 0.045 0.832

Observed (O) Expected (E) (O-E)2/E

Observed (O) Expected (E) (O-E)2/E

Number of ey/ey 48 64.11584327 4.051

Number of ey/sp 292 259.7683135 3.999

Number of sp/sp 247 263.1158433 0.987 X21df = P-value =

Total 587 587 9.037 9.037 0.003

TABLE 3: Chi-Square Analysis of Genotype Frequencies in Large Population in Week 5- Observed raw counts of
genotypes are compared to expected counts of genotypes. Expected genotype counts are calculated by determining the theoretical proportions of each genotype based on the Hardy-Weinberg equation. The chi-square can be calculated from the observed and expected values and converted to find the p-value.

Tables 2 and 3 show the counts on the different genotypes in the small and large population. Differences between the observed and expected values can be seen in all the genotypes, however the significance of these differences is dependent on the entire populations size. After calculation, the chi-square statistic in Table 2 gives a p-value that is greater than 0.05, and Table 3 shows a p-value that is less than 0.05. Number of ey 438 574.08 32.256 Number of sp 758 621.92 29.775 X21df = P-value = TABLE 4: Chi-Square Analysis of Allele Frequencies in Small Population in Week 5 Observed (O) Expected (E) (O-E)2/E Number of ey 388 418.109 2.168 Number of sp 786 755.891 1.199 X21df = P-value = Total 1174 1174 3.367 3.367 0.066 Total 1196 1196 62.032 62.032 0.000

Observed (O) Expected (E) (O-E)2/E

TABLE 5: Chi-Square Analysis of Allele Frequencies in Large Population in Week 5- From the raw observed counts
of genotypes, counts of ey and sp alleles can be made. The expected allele counts are calculated by determining the theoretical values from the initial allele frequencies from Week 1. The chi-square can be calculated from the observed and expected values and calculated to the p-value.

Tables 4 and 5 examines the allele counts of the population rather than the genotype counts. Table 4 shows that there are large changes in the allele counts over the five weeks for the small population, and Table 5 shows more minor differences in the allele counts of the large population. The calculated chi-square statistics show that the p-value is virtually nothing for the small population and is greater than 0.05 for the large population.

FIGURE 1: Comparison of Allele Frequencies of Large and Small Populations- The sp and ey allele frequencies are
compared in different population sizes.

Figure 1 shows how in the small population the ey and sp allele frequencies are growing apart consistently over the five weeks. As the sp allele frequency approaches 1.0, it is becoming more prominent in the population. The allele frequencies for the large population, however, stay relatively constant over the five weeks only shifting up or down slightly. This shows little change in allele frequency. Population B Effects of Natural Selection on a Population P Generation F1 Generation F2 Generation + + Allele Frequency Cy (p) Cy (q) Cy (p) Cy (q) Cy (p) Cy+ (q) Group BJC 0.325 0.675 0.185 0.815 0.136 0.864 Section 11 0.306 0.694 0.217 0.783 0.189 0.811 + + + TABLE 6: Allele Frequencies of Population B- Population of curly-winged (Cy/Cy ) and wild-type (Cy /Cy ) individuals.
Cy/Cy individuals immediately die in population. Allele frequencies calculated from raw data of genotype counts. Genotypes converted into allele counts.

Table 6 shows a complilation of data calculated from the recorded genotype counts. From the table, it can be seen that the Cy allele frequencies consistently decrease for both the group and the section data while the Cy+ allele frequencies grow higher.

FIGURE 2: Comparison of Allele Frequencies After Proceeding Generations in Population B- Over the time of
generations, Cy and Cy+ allele frequencies are plotted from both group and section data.

In Figure 2, the data collected by both the group and the section shows that the Cy+ alleles are slowly increasing in the populations gene pool. Also, the Cy alleles are decreasing at the same rate. The Cy+ allele is approaching a frequency of 1.0, driving the Cy allele further to extinction with each successive generation.

FIGURES 3 & 4: Populus Model of Cy+ Allele After Three Generation Based on Group and Section Data - Populus
predicts how the Cy+ allele frequency should progress with the three generations. As the allele frequency approaches 1.0, the allele is becoming more dominant.

Figures 3 and 4, show a slow but steady increase in the Cy+(p) allele over the three generations. This predicted model matches the results gathered and displayed in Figure 2. Population C Effects of Small Wild-Type Migration on Wrinkly-Winged Population P Generation F1 Generation F2 Generation + + Allele Frequency W (p) W (q) W (p) W (q) W (p) W+ (q) Group BJC 0.714 0.286 0.280 0.720 0.000 1.000 Section 11 0.714 0.286 0.090 0.910 0.007 0.993 TABLE 7: Allele Frequencies of Population C- Population C is made up of individuals with severely wrinkled (W/W),
moderately wrinkled (W/W+), and wild-type (W+/W+) wings. Allele frequencies calculated from raw data of genotype counts. Genotypes converted into allele counts.

In Table 7, the populations begin with the same allele frequency for both the group and section. In the 7

beginning, the W allele attributed to wrinkly wings is most prominent. With each successive generation, however, the W allele frequency decreases significantly while the W+ allele frequency sharply jumps up. In the final generation, the W allele is virtually nonexistent, and the W+ is significantly present.

FIGURE 5: Comparison of Allele Frequencies After Proceeding Generations in Population C - Over the time of
generations, W and W+ allele frequencies are plotted from both group and section data.

In Figure 5, the allele frequencies of W+ increase dramatically and quickly reach the 1.0 frequency by the third generation. The W allele is basically extinct by the third generation. The greatest difference between the group and section data is that the group data changes a little less quickly in the second generation but does catch up by the third. Population C shows a much more steep change in the wild-type allele frequency than in Population B.

FIGURES 6 & 7: Populus Model of W+ Allele After Three Generation Based on Group and Section Data - Populus predicts how the W+ allele frequency should progress
dramatically with the three generations.

Figures 6 and 7, show a dramatic and quick increase in the W+(p) allele over the three generations. This predicted model matches the results gathered and displayed in Figure 5, illustrating the slightly slower change in the group population than in the section population.

DISCUSSION Population A Effects of Small vs. Large Population Population A only alters the population size condition in the Hardy-Weinberg Theorem. The mating is random, and there is no known migration, natural selection, or mutation occurring. The results from the chi-square statistic state that in the small population there is a difference in the expected and observed allele frequencies because the p-value was less than 0.05 (Table 3), and a significant conclusion cannot be made about the genotype frequencies because the p-value was greater than 0.05 (Table 2). Due to the populations small size, it is likely that the alleles are subject to natural processes, such as genetic drift or disease, which can lead to deterioration of one allele in the case of one of these events. This is why the HardyWeinberg equilibrium cannot be achieved in small populations. Also, the chi-square statistic concluded that in the large population there is a significant difference in the expected and observed genotype frequencies because the p-value was less than 0.05 (Table 4), yet no significant difference can be drawn about the allele frequencies because the p-value was greater than 0.05 (Table 5). In larger populations, there is more resistance to natural events, therefore it is more difficult to cause a significant drop in one particular allele. Allele frequencies may vary throughout the population, however the vast number acts as a buffer to extreme changes in the gene pool. The large population appears to be close to Hardy-Weinberg equilibrium. Population B Effects of Natural Selection on a Population Population B only alters the natural selection condition in the Hardy-Weinberg Theorem. The mating is random, the populations are large, and there is no known migration or mutation occurring. In Figure 2, it is evident that the allele frequencies are growing apart at a steady rate. Since the wild-type allele is the one increasing, it is more favored by natural selection. This can be explained just by the shape of the different wings. The wild-type wings are flat and can easily be used for flying, while the curly wings are more cumbersome and put these flies at a greater disadvantage. This will therefore effect their ability to mate and produce offspring, lessening their fitness. The curly wing flies, however, are not completely unfit, which explains why there is a slow 9

change in the allele frequency. Natural selection is an effective method for shifting allele frequencies, however, it does take time. The Populus models (Figures 3 & 4) show a reinforcement of Figure 2, therefore it can be concluded that the individuals with the wild-type alleles are more favored in this population, and the allele frequency will continually shift further in that direction. Population C Effects of Small Wild-Type Migration on Wrinkly-Winged Population Population C alters both the migration and the natural selection conditions in the Hardy-Weinberg Theorem. The mating is random, the populations are large, and there is no known mutation occurring. In Figure 5, the allele frequency of W+ is shown to increase drastically over the course of the three generations. This indicates that the allele for wild-type wings is strongly preferred over the allele for wrinkled wings. D. melanogaster with wrinkled wings have extremely low fitness in the sense that their inability to fly prohibits them from effectively mating. This greatly reduces their fitness, and also the migrating wild-type individuals with very high mobility and fitness are able to more efficiently reproduce. Not only are the wrinkle-winged parents experiencing decreased offspring, but the wild-type are experiencing a significant increase in offspring. This leads for a doubled effect on the population. Where Population B was only impacted by one condition, Population C experiences two conditions. Therefore, the unfavored allele is more quickly wiped out of the populations gene pool and dominated by the wild-type allele. The Populus graphs (Figures 6 & 7) also reinforce the data found in Figure 5, establishing that the observed results are accurate. Possible error could be found in the gathering of the weekly populations. It could happen by chance that a vast majority of the wrinkle-wing individuals made it into one population, suggesting that they were actually growing in size. This is highly unlikely but is still a possibility in data collection. Also, the counters could draw incorrect conclusions if a flys wing is broken or flipped up and appearing curly. Recorded results may be baised to the counter. Lastly, incorrect mathematical analysis could have occurred due to the considerable amount of recorded values needed to be sorted through. This study could be continued in future experiments if the conditions were altered in different ways. Perhaps the effect of migration alone could be compared the the effect of natural selection alone to see which is

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more selective towards the wild-type genotype. Another possibility would be to try to create mutations in D. melanogaster and insert these mutants into a population to see at what point they begin to become somewhat favored. Research involving evolution and the Hardy-Weinberg Theorem is crucial to biology because all organisms experience this phenomenon. Luckily, Drosophila melanogaster are easily manipulated insects and have taught the science community a great deal about genetics. This simplified research is the foundation to the understanding we will eventually built up to on our own genes and alleles. REFERENCES: (No Author). 2008. Evolution. Encyclopaedia britannica online. Retrieved (2010, April 20) from http://www.reference.com/browse/evolution Campbell, N.A., Mitchell, L.G., & Reece, J.B. 1994. Biology: concepts & connections. Redwood City, CA: Benjamin/Cummings Pub. Hass, C., D. Burpee and R. Meisel. 2006. Analysis of Evolutionary Forces Acting on Populations of Drosophila melanogaster. Department of Biology, The Pennsylvania State University, University Park, PA. Adapted from Leicht, B. G. 1995. A Laboratory Manual for Biology 220W: Populations and Communities. (Burpee, D and B. G. Leicht, eds.) Department of Biology, The Pennsylvania State University, University Park, PA. Hass, C., D. Burpee and R. Meisel. 2006. Statistical Techniques. Department of Biology, The Pennsylvania State University, University Park, PA. Adapted from Leicht, B. G. 1995. A Laboratory Manual for Biology 220W: Populations and Communities. (Burpee, D. and B. G. Leicht, eds.) Department of Biology, The Pennsylvania State University, University Park, PA. Seongho, S., Dipak, K.D., & Holsinger, K.E. 2006. Differentiation among populations with migration, mutation, and drift: implications for genetic inference. Evolution, 60(1), 1-12.

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