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

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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.

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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 population’s 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 5Allele Frequency ey (p) sp (q) ey (p) sp (q) ey (p) sp (q)

Small 0.480 0.520 0.380 0.620 0.366 0.634Large 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 Number of ey/sp Number of sp/sp TotalObserved (O) 79 280 239 598Expected (E) 80.202 277.595 240.202 598

(O-E)2/E 0.018 0.021 0.006 0.045

X21df = 0.045

P-value = 0.832

TABLE 2: Chi-Square Analysis of Genotype Frequencies in Small Population in Week 5

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Number of ey/ey Number of ey/sp Number of sp/sp TotalObserved (O) 48 292 247 587Expected (E) 64.11584327 259.7683135 263.1158433 587

(O-E)2/E 4.051 3.999 0.987 9.037

X21df = 9.037

P-value = 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 population’s 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 Number of sp TotalObserved (O) 438 758 1196Expected (E) 574.08 621.92 1196

(O-E)2/E 32.256 29.775 62.032

X21df = 62.032

P-value = 0.000

TABLE 4: Chi-Square Analysis of Allele Frequencies in Small Population in Week 5

Number of ey Number of sp TotalObserved (O) 388 786 1174Expected (E) 418.109 755.891 1174

(O-E)2/E 2.168 1.199 3.367

X21df = 3.367

P-value = 0.066

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

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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 GenerationAllele 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 0.864Section 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.

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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 population’s 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 GenerationAllele 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 1.000Section 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.

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

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.

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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 population’s 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

Hardy-Weinberg 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

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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 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

population’s 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 fly’s 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.

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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

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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