Biology

An Experimental Study of Natural Selection and Relative Fitness

Introduction (2/3 or 2/4)

Biological evolution is a fundamental concept in biology that helps us understand the natural world i.e., the history and diversity of life on Earth. At the most basic definition, biological evolution is descent with modification. That is, subsequent generations change over time. Biological evolution can be subdivided into microevolution and macroevolution. Microevolution involves small-scale changes in allele frequencies in a population from one generation to the next. Macroevolution encompasses large-scale changes that produces different species from common ancestors over many generations. Since macroevolution requires an extensive period of time (most are beyond human lifetimes), macroevolutionary studies are largely observational. In other words, we cannot create experiments to test macroevolutionary hypotheses. Instead, we observe patterns and infer the processes from those patterns. Alternatively, microevolution studies require a relatively short period of time such that hypotheses testing can be observational or experimentational (we can create experiments).

Performing microevolution experiments requires an understanding of the Hardy-

Weinberg equilibrium principle. Hardy-Weinberg equilibrium is a simple mathematical model

that assumes a single population’s gene pool does not change in frequency from one generation

to the next. The model is represented by two algebraic equations: the allele frequency equation (p

= 1) and the genotype frequency equation (p2 + 2pq q2 = 1). To illustrate these equations,

let’s consider a simple dominant/recessive relationship of a character (mouse fur color,

represented by the letter “b”) with two traits (brown and white). This means we will have two

alleles and three genotypes. The lower-case b allele represents the white fur trait and the upper-

case B allele represents the brown fur trait, while the white fur phenotype is represented by the

ww genotype and the brown fur phenotype is represented by the WW and Ww genotypes. With

respect to the frequencies, f(w) is represented by and f(W) is represented by p, while ww is 22

represented by , Ww is represented by pq and WW is represented by . This lab consists of using these equations to determine whether microevolution has occurred, so make sure you understand them.

In simpler terms, this means that if 60% of a population of mice have the white fur trait and 40% have the brown fur trait, this proportion will be the same in the next generation regardless of population size. There may be more individuals in the next generation, but the ratio remains the same (three white fur traits to two brown fur traits). As a principle, this expectation makes sense. However, there are mechanisms of evolutionary change that violate this Hardy- Weinberg equilibrium principle that need to be understood.

There are five recognized mechanisms that disrupt the Hardy-Weinberg equilibrium principle: genetic drift, non-random mating, mutation, migration, and natural selection.

• Genetic drift is a completely random process where some individuals in a population die before reproducing, some individuals produce more offspring of a specific genotype, etc. From the example we described above, this would lead to the next generation’s proportion to differ from 60% white fur trait and 40% brown fur trait. Over time, the population could be “fixed.” That is, the population would either be 100% white fur trait or 100% brown fur trait, due to chance alone. It is important to note that genetic drift results in fixation faster in smaller populations than in larger populations. This is because

BIOL251 Spring 2020 Updated 28/01/20 Alejandrino 1

Drosophila Lab: An Experimental Study of Natural Selection and Relative Fitness

the relative contribution to the next generation of each individual in a small population is

larger than each individual in a large population.

· Non-random mating is essentially selective mating. An example could be that the mice

with the white fur would prefer to mate with mice with brown fur. Assuming that brown fur is dominant, the next generation will have more individuals with the brown fur trait, which would lead to a proportion to differ from the previous generation.

· Mutation is another random process. Some mutations can alter a mouse’s fur color, for example, from brown to white. Thus, altering the ratio of 3:2 from one generation to the next. However, it is important to keep in mind that mutations are relatively rare in short timescales, especially for mutations that considerably alter important phenotypes such as fur color.

· Migration is the movement of individuals in or out of a population. If several mice with white fur enter a population of mice with a 3:2 ratio, the ratio will change such that the white fur trait’s proportion increases and the brown fur trait’s proportion decreases. Likewise, if several mice with white fur leave the 3:2 ratio population, the ratio will change such that the white fur trait’s proportion decreases and the brown fur trait’s proportion increases.

· Natural selection is a mechanism of evolution first proposed by Charles Darwin and Alfred Wallace. Three conditions must be met for natural selection to occur: there is variation in a given trait, the trait is heritable, and the survival and reproductive success (fitness) of individuals is variable. In our example population of mice, the trait that varies is fur color (brown and white). We know it is heritable because breeding mice with different fur colors (Mendelian genetics), results in predictable fur color ratios. To the condition of variable reproductive success, not every individual, for one reason or another, will produce the same number of offspring i.e., some individuals are more fit than others. Thus, if these conditions are met, the proportion of the next generation will differ from 3:2.

In this lab, we will experimentally test whether the Hardy-Weinberg equilibrium principle is violated, specifically through the mechanism of natural selection in Drosophila melanogaster fruit flies. D. melanogaster is a model organism that has been used in scientific research for over 100 years. This is mainly due to its short life span, which takes about 10 to 12 days from egg to adult (Figure 1). In addition, many mutant strains have been created that are used to test an infinite number of genetic and microevolution questions.

Figure 1: Above is a diagram of the four-stage life cycle of D. melanogaster, a holometabolous insect, showing the egg, larva, pupa, and adult stages (Salata 2002).

The specific fly strains we will work with are the wild phenotype and the ebony phenotype. The wild phenotype is the normal, unmutated strain of fly that are found in nature and have a grey body color. The ebony phenotype is a mutated strain of fly that have a black body color. These fly strains are easy to identify by the naked eye i.e., without the aid of a microscope. In addition, the allele that is responsible for the wild phenotype is dominant over the ebony phenotype allele. Thus, the genotypes of wild phenotypes are either homozygous dominant or heterozygous, while the genotype of ebony phenotypes is homozygous recessive.

For this experiment, we will set up population cages of D. melanogaster consisting of 40% true-breeding wild phenotypes and 60% true-breeding ebony phenotypes to test whether the Hardy-Weinberg equilibrium principle is violated over a period of one month. If so, we need to determine whether the disruption to the Hardy-Weinberg equilibrium principle is due to natural selection; specifically, is there a difference in fitness between wild phenotypes and ebony phenotypes?

Materials

· lab coats

· gloves

· population cage (check to make sure the centrifuge caps are glued to the bottom)

· centrifuge tubes (x9, make sure these fit into the centrifuge caps of the population cage)

· labeling tape

· marker

· tube holder

· fly media

· distilled water

· vial of true-breeding wild phenotype D. melanogaster

· vial of true-breeding ebony phenotype D. melanogaster

· Fly Nap

· cotton swab

· white paper or paper towel

· paint brush

Methods

1. Form a group of either three or four individuals.

2. Wear lab coats and gloves.

3. Label your population cage with your group name and number each centrifuge tube and

its cap from 1 to 9.

4. Label the tube holder with your group name and place Tubes 7, 8, and 9 into the holder.

5. Attach Tubes 3, 4, 5, and 6 onto the population cage. Keep the caps for the end of the

experiment.

6. Fill Tubes 1 and 2 with a scoopful of fly media, add about 7.5 ml of distilled water to

each tube, and attach them to the population cage.

7. Take your vials of flies to the fume hood and lay them on their sides.

8. Dip one end of the cotton swab into the Fly Nap solution and quickly insert the cotton

swab on the top side of the vial, between the sponge and vial wall. You might want to tap the vial where you will be inserting the cotton swab, so the flies are less likely to escape. Leave the cotton swab in the vial for about 4-5 minutes (until the flies are knocked out). Repeat this process for the other vial with the other end of the cotton swab.

9. Once you are certain all the flies are asleep, pull out the cotton swab and discard it in the white bucket. Bring your vials back to your lab bench and carefully dump the flies out on a white piece of paper or paper towel.

10. Using a paint brush, carefully sort out 20 wild phenotype flies and 30 ebony phenotype flies. Carefully place these individuals into your population cage, making sure they don’t fall into the food or centrifuge tubes (they may get stuck in the food or not get enough air in the centrifuge tubes and may die).

11. Set your population cage (with the centrifuge tubes on the bottom), tube holder, and caps on a bench space along the side or back of the lab. Return the other materials where you got them. Throw your gloves and paper (or paper towel) into the white bucket inside the fume hood. Do not throw anything into the regular trash cans.

12. In two days (outside of scheduled lab times), your group is to fill another centrifuge tube (Tube 3) with food. Be careful in removing and replacing the centrifuge tubes; cover the centrifuge cap on the population cage to prevent the flies from escaping. Continue this procedure every Monday and Wednesday (or Tuesday and Thursday) until all the tubes attached to the population cage are filled with food (Table 1). Be sure you are wearing close-toed shoes, gloves, and a lab coat.

At the end of the third week of the experiment, Tube 1 should be removed from the population cage, capped (but not tight), and placed in the tube holder. Tube 7 should be filled with food and attached to the population cage where Tube 1 was removed (Table

1). 14. Follow this procedure over the next week until Tube 3 has been removed from the

population cage and replaced with Tube 9 (Table 1).

Table 1: Feeding schedule of D. melanogaster populations over a four-week period. Keep in mind that some feedings need to be done outside of scheduled lab times.

Data Collection and Calculations (3/2 or 3/3)

By now, we have been running the experiment for four weeks. Next, we need to collect the data and perform calculations so that we can analyze them in the following week. Before we get started, download the Drosophila Excel file from Moodle. Also, be sure to wear a lab coat and gloves.

15. Use Fly Nap to anesthetize the flies in Tubes 1, 2, and 3, as well as the population cage. Count the number of wild phenotype flies and ebony phenotype flies in each tube and population cage and record these values into the Drosophila Excel spreadsheet (Table 2, Columns 3-5). Tube 1 represents the population after one week, Tube 2 represents the population after two weeks, etc. and the cage represents the population after four weeks.

16. Once you are done gathering the data, return the materials where you got them. Throw your gloves, paper (or paper towel), and cotton swabs into the white bucket inside the fume hood. Do not throw anything into the regular trash cans.

Table 2: Counts of fly phenotypes and calculations of expected phenotypes and relative fitness for the initial week with a hypothetical observed population for Tube 1. Follow the same procedure according to the steps outlined below.

 

17. Remember that we started with true-breeding lines of wild phenotype and ebony phenotype flies. This means that we started with homozygote dominant and homozygote recessive genotypes, but no heterozygote genotypes. This also means that the original genotypic frequency equation (p2 + 2pq q2 = 1) can be reduced to p2 + q2 = 1. This will allow us to calculate the genotype frequencies very easily, by simply dividing the number of one phenotype by the total number of flies (Table 3).

Table 3: Example calculations of genotype and allele frequency for the initial (starting) population. Follow the same procedure to calculate the genotype and allele frequencies

18. Once we have the genotype frequency, we can calculate the allele frequency by square rooting the genotype frequency. Remember that a homozygote has two copies of the same allele (Table 3).

19. Then, using the allelic frequency equation (= 1), we can subtract the frequency of one allele from 1 to get the frequency of the other allele (Table 3). Record these allele frequencies into the Drosophila Excel spreadsheet (Table 2, Columns 6-7).

20. If the population is in Hardy-Weinberg equilibrium, we assume that the original allele frequency will not change, and so the expected number of individuals should always be 60% ebony and 40% wild phenotype. We can calculate the expected number of individuals of ebony and wild phenotype individuals by multiplying the original phenotype frequency by the total number of flies for any given week (Table 2, Columns 8-9).

21. Next, calculate the relative fitness (W) of each phenotype for each week. This is a simple division of the observed number of each phenotype by the observed number of the most numerous phenotype (Table 2, Columns 10-11).

22. Finish calculating the rest of the allelic frequencies, expected phenotypes, and relative fitness for all of the populations and enter them into the Drosophila Excel spreadsheet. Once you are finished, upload the completed file into the Drosophila Data link on Moodle.

Statistical Analyses (3/9 or 3/10)

Thus far, each group has uploaded their calculations of the allele frequencies, the genotype frequencies, and the relative fitness of each type for each week. For statistical analyses, these data should be compiled together and reorganized. I have done the former, but you have to do the latter. The calculations should then be averaged for the whole class, graphed, and statistically evaluated. Chi-squared tests will be used to determine whether the Hardy-Weinberg equilibrium has been violated. In other words, did the population evolve? t-tests will be used to determine whether fitness is different between the two phenotypes, suggesting that natural selection is the mechanism of evolutionary change that occurred. Before you begin, download the Class Drosophila Data file from Moodle.

23. To determine if the allele frequencies are changing over time, you will need to average and for each week and graph the averages. Before you can do this, you will need to rearrange the data so that they are organized by week. What trends do the graph show?

24. To determine if the population is in Hardy-Weinberg equilibrium, you will need to perform chi-squared tests comparing the observed number of individuals to the expected number of individuals. Chi-square tests will need to be done for each week. Are the ratios changing?

25. To determine if there are significant differences in the relative fitness of the two phenotypes, you will need to perform t-tests. There should be four t-tests, one for each week. Which phenotype is more fit?

Specific requirements for the final paper

Introduction: Below is an outline of how the Introduction for this paper should be organized and what information should be included. Be sure to use scientific literature to support your explanations.

· Start with a paragraph that broadly explains what the experiment is about. What is the main idea of the experiment and why is it important to test?

· The next paragraph should be about the Hardy-Weinberg equilibrium principle. Explain what the principle is and how it relates to the main idea of the experiment? Also explain how fitness affects the principle and the main idea of the experiment. The bulk of your references should be in this paragraph.

· The third paragraph should introduce the experimental system. Why is D. melanogaster an ideal organism for the experiment? What are the phenotypes that will be used and why?

· The last paragraph should explain how experimenting on D. melanogaster and the phenotypes will help us understand whether the Hardy-Weinberg equilibrium principle and the main idea (What is your objective?). Don’t forget to include your hypothesis and make sure your references back it up.

Results: Below is a list of specific figures and tables that need to be included in the final paper.

· A figure showing the average allele frequency of both ebony and wild phenotypes with

respect to time.

· A table showing the average expected and observed numbers of individuals with the chi-

squared test results.

· A table showing the average relative fitness over time with the t-test results.

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