AP Investigation #2 Evolution: Hardy Weinberg Teacher s Guide Kit # 36-7402 Call Us at 1.800.962.2660 for Technical Assistance Table of Contents Abstract. 1 General Overview. 2 Recording Data. 3 Materials Checklist. 5 curriculum alignment. 6 Learning Objectives. 7 Time Requirements. 7 Safety Precautions. 8 Pre-Lab Preparation. 9 Student guide contents Introduction and Background. 10 Part 1: build a simple model of allele frequency in a human population using a spreadsheet program (Structured inquiry). 12 Part 2: observations and model (GUIDED inquiry)....15 Part 3: design an experiment (open inquiry). 17 MATERIAL SAFETY DATA SHEETS. 19 **AP and the Advanced Placement Program are registered trademarks of the College Entrance Examination Board. The activity and materials in this kit were developed and prepared by WARD S Natural Science Establishment, which bears sole responsibility for their contents.
abstract The focus of this lab is on developing a mathematical model for biological populations using a spreadsheet program. The lab will start by investigating the prevalence of at least one human phenotype in the classroom population and use this as a basis for model testing. Students will build mathematical models based on the Hardy-Weinberg equilibrium to analyze bottlenecks, genetic drift, and effects of selection in the evolution of populations. Page 1
general Overview The College Board has revised the AP Biology curriculum to begin implementation in the fall of 2012. Advanced Placement (AP) is a registered trademark of the College Entrance Examination Board. The revisions were designed to reduce the range of topics covered, to allow more depth of study and increased conceptual understanding for students. There is a shift in laboratory emphasis from instructor-designed demonstrations to student-designed investigations. While students may be introduced to concepts and methods as before, it is expected that they will develop more independent inquiry skills. Lab investigations now incorporate more student-questioning and experimental design. To accomplish this, the College Board has decreased the minimum number of required labs from 12 to 8 while keeping the same time requirement (25% of instruction time devoted to laboratory study). The College Board has defined seven science practices that students must learn to apply over the course of laboratory study. In brief, students must: 1. Use models 2. Use mathematics (quantitative skills) 3. Formulate questions 4. Plan and execute data collection strategies 5. Analyze and evaluate data 6. Explain results 7. Generalize data across domains The College Board published 13 recommended laboratories in the spring of 2011. They can be found at: http://advancesinap.collegeboard.org/science/biology/lab Many of these laboratories are extensions of those described in the 12 classic labs that the College Board has used in the past. The materials provided in this lab activity have been prepared by Ward s to adapt to the specifications outlined in AP Biology Investigative Labs: An Inquiry-Based Approach (2012, The College Board). Ward s has provided instructions and materials in the AP Biology Investigation series that complement the descriptions in this College Board publication. We recommend that all teachers review the College Board material as well as the instructions here to get the best understanding of what the learning goals are. Ward s has structured each new AP investigation to have at least three parts: Structured, Guided, and Open Inquiry. Depending on a teacher s syllabus, s/he may choose to do all or only parts of the investigations in scheduled lab periods. The College Board requires that a syllabus describe how students communicate their experimental designs and results. It is up to the teacher to define how this requirement will be met. Having students keep a laboratory notebook is one straightforward way to do this. Page 2
Recording Data in a Laboratory Notebook All of the Ward s Investigations assume that students will keep a laboratory notebook for studentdirected investigations. A brief outline of recommended practices to set up a notebook, and one possible format, are provided below. 1. A composition book with bound pages is highly recommended. These can be found in most stationary stores. Ward s offers several options with pre-numbered pages (for instance, item numbers 32-8040 and 15-8332). This prevents pages from being lost or mixed up over the course of an experiment. 2. The title page should contain, at the minimum, the student s name. Pages should be numbered in succession. 3. After the title page, two to six pages should be reserved for a table of contents to be updated as experiments are done so they are easily found. 4. All entries should be made in permanent ink. Mistakes should be crossed out with a single line and should be initialed and dated. This clearly documents the actual sequence of events and methods of calculation. When in doubt, over-explain. In research labs, clear documentation may be required to audit and repeat results or obtain a patent. 5. It is good practice to permanently adhere a laboratory safety contract to the front cover of the notebook as a constant reminder to be safe. 6. It is professional lab practice to sign and date the bottom of every page. The instructor or lab partner can also sign and date as a witness to the veracity of the recording. 7. Any photos, data print-outs, or other type of documentation should be firmly adhered in the notebook. It is professional practice to draw a line from the notebook page over the inserted material to indicate that there has been no tampering with the records. For student-directed investigations, it is expected that the student will provide an experimental plan for the teacher to approve before beginning any experiment. The general plan format follows that of writing a grant to fund a research project. 1. Define the question or testable hypothesis. 2. Describe the background information. Include previous experiments. 3. Describe the experimental design with controls, variables, and observations. 4. Describe the possible results and how they would be interpreted. 5. List the materials and methods to be used. 6. Note potential safety issues. (continued on next page) Page 3
Recording Data in a Laboratory Notebook (continued) After the plan is approved: 7. The step-by-step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc., as well as the weights and volumes used. 8. The results should be recorded (including drawings, photos, data print outs, etc.). 9. The analysis of results should be recorded. 10. Draw conclusions based on how the results compared to the predictions. 11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance, and uncontrolled variables. 12. Further study direction should be considered. The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following questions, which also might arise in peer review: Explain the purpose of a procedural step. Identify the independent variables and the dependent variables in an experiment. What results would you expect to see in the control group? The experimental group? How does XXXX concept account for YYYY findings? Describe a method to determine XXXX. Page 4
Materials checklist Units per kit Description MATERIALS NEEDED BUT NOT PROVIDED Calculators 2 Index Cards, Unlined, 3 x 5 Coins 1 CD ROM, AP Biology Computer to view CD ROM 1 Taste Paper, Control, 100 Spreadsheet program to build model 1 PTC Taste-Test Paper, Pkg./100 1 PTC Taste-Test Paper, Pkg./100 Other materials as determined by students experimental design 1 Instructions (this booklet) Call Us at 1.800.962.2660 for Technical Assistance Or Visit Us on-line at www.wardsci.com for U.S. Customers www.wardsci.ca for Canadian Customers Page 5
This lab activity is aligned with the 2012 AP Biology Curriculum (registered trademark of the College Board). Listed below are the aligned Content Areas (Big Ideas and Enduring Understandings), the Science Practices, and the Learning Objectives of the lab as described in AP Biology Investigative Labs: An Inquiry Approach (2012). This is a publication of the College Board that can be found at http://advancesinap.collegeboard.org/science/biology/lab. Curriculum alignment Big Idea Big Idea 1: The process of evolution drives the diversity and unity of life. Enduring Understandings 1A1: Natural selection is a major mechanism of evolution. 1A2: Natural selection acts on phenotypic variations in populations. 1A3: Evolutionary change is also driven by random processes 1C3: Populations of organisms continue to evolve Science Practices 1.5 The student can re-express key elements of natural phenomena across multiple representations in the domain. 2.2 The student can apply mathematical routines to quantities that describe natural phenomena. 5.3 The student can evaluate the evidence provided by data sets in relation to a particular scientific question. 7.1 The student can connect phenomena and models across spatial and temporal scales. Page 6
Learning objectives The student is able to convert a data set from a table of numbers that reflect a change in the genetic makeup of a population over time and to apply mathematical methods and conceptual understandings to investigate the cause(s) and effect(s) of this change (1A1 & SP 1.5, SP 2.2). The student is able to apply mathematical methods to data from a real or simulated population to predict what will happen to the population in the future (1A1 & SP 2.2). The student is able to evaluate data-based evidence that describes evolutionary changes in the genetic makeup of a population over time (1A1 & SP 5.3). The student is able to use data from mathematical models based on the Hardy-Weinberg equilibrium to analyze genetic drift and effects of selection in the evolution of specific populations (1A3 & SP 1.4, SP 2.1). The student is able to justify data from mathematical models based on the Hardy-Weinberg equilibrium to analyze genetic drift and the effects of selection in the evolution of specific populations (1A3 & SP 2.1). The student is able to describe a model that represents evolution within a population (1C3 & SP 1.2). The student is able to evaluate given data sets that illustrate evolution as an ongoing process (1C3 & SP 5.3). Time Requirements The time constraints on this lab revolve more around students mathematical preparation and competence rather than the activities of the lab. If students have spreadsheet programs available outside of the classroom, this lab can be done as an assignment followed by class discussion to better accommodate variable math preparedness. Page 7
Notes General Safety Precautions The teacher should be familiar with safety practices and regulations in their school (district and state). Know what needs to be treated as hazardous waste and how to properly dispose of nonhazardous chemicals or biological material. Consider establishing a safety contract that students and their parents must read and sign off on. This is a good way to identify students with allergies to things like latex so that you (and they) will be reminded of what particular things may be risks to individuals. A good practice is to include a copy of this contract in the student lab book (glued to the inside cover). Students should know where all emergency equipment (safety shower, eyewash station, fire extinguisher, fire blanket, first aid kit etc.) is located. Make sure students remove all dangling jewelry and tie back long hair before they begin. Remind students to read all instructions, Material Data Safety Sheets (MSDSs) and live care sheets before starting the lab activities and to ask questions about safety and safe laboratory procedures. Appropriate MSDSs and live care sheets can be found on the last pages of this booklet. Additionally, the most updated versions of these resources can be found at www.wardsci.com, under Living Materials http://wardsci.com/article.asp?ai=1346. (Note that in this particular lab, there are no materials that require live care sheets.) In student directed investigations, make sure that collecting safety information (like MSDSs) is part of the experimental proposal. As general laboratory practice, it is recommended that students wear proper protective equipment, such as gloves, safety goggles, and a lab apron. At end of lab: All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Page 8
Notes Pre-Laboratory Preparation Students should review the basics of Mendelian genetics and obtain access to a spreadsheet program prior to the start of this lab. The lab can be done without a spreadsheet program, but it will take considerably longer for students to generate and analyze the data. Page 9
OBJEcTIVES Convert a data set from a table of numbers that reflect a change in the genetic makeup of a population over time and to apply mathematical methods and conceptual understandings to investigate the cause(s) and effect(s) of this change. Apply mathematical methods to data from a real or simulated population to predict what will happen to the population in the future. Evaluate data-based evidence that describes evolutionary changes in the genetic makeup of a population over time. Use data from mathematical models based on the Hardy- Weinberg equilibrium to analyze genetic drift and effects of selection in the evolution of specific populations. Justify data from mathematical models based on the Hardy- Weinberg equilibrium to analyze genetic drift and the effects of selection in the evolution of specific populations. Describe a model that represents evolution within a population. Evaluate given data sets that illustrate evolution as an ongoing process. Introduction In this lab, you will use mathematical probability to examine and predict changes in allele frequencies. By building and refining models, you will learn useful ways to test hypotheses. Background Many scientific problems are so complex, with so many variables that can affect outcomes, that it is often difficult to find a useful place to start. By making the simplest model of the system under study as possible, while knowing that the natural state never presents such simplicity, we can make testable predictions about the validity of the model and refine the model further. For example, the process of natural selection acts on heritable characteristics to change characteristics of populations of organisms over time. Testing all of the variables that could be involved in that process is not the most useful way to determine the validity of the statement. However, the logic underlying the statement can be assessed by making models based on the logic and comparing the predicted outcomes to observations from the natural state. Heritable phenotypic traits of individuals are related to their genotypes. Every diploid individual has two sets of alleles one set from each parent. Mutations, selective pressures, random drift, and population mixing will drive changes in allele frequencies in populations of individuals. In a hypothetical ideal state driven by random mating in a large population that lives in unchanging conditions, the frequency of different alleles in a population will reach a stable state of equilibrium. However, this ideal state is unlikely to be encountered in a natural population since conditions are always changing and will therefore disturb this equilibrium. Different types of changing conditions will change allele frequencies in predictable ways. For example, consider the varied allele frequencies for eye color in humans. This is a polygenic trait that behaves almost like a simple trait, with brown eyes usually appearing to be dominant to paler colors, like blue. Blue eyes are expressed most frequently in northern and central Europe. It is thought that this phenotype conferred a selective advantage by permitting more production of vitamin D in the darker northern latitudes; therefore, the frequency of the allele for blue eyes is higher in northern populations. In the United States, 57% of the population had blue eyes for those born around 1900, while 100 years later this is true for only about 17% of the population. Earlier (continued on next page) Page 10
Notes Background (COntinued) immigration to the United States was mostly from northern European countries causing a relatively high frequency of blue eyes. Blue eye alleles mixed with a brown-eyed population to decrease the frequency of the phenotype and later immigration brought more brown-eyed people into the population. It is expected that the population of blue-eyed individuals will continue to decrease as the frequency of the allele is reduced in the US population through a variety of mechanisms. Materials List MATERIALS NEEDED Description Index Cards, Unlined, 3 x 5 CD ROM, AP Biology Taste Paper, Control, 100 PTC Taste-Test Paper, Pkg./100 Instructions (this booklet) Calculators Coins Computer to view CD ROM Spreadsheet program to build model OPTIONAL MATERIALS ( NOT PROVIDED) Other materials as determined by your experimental design Safety Precautions As general safe laboratory practice, it is recommended that you wear proper protective equipment, such as gloves, safety goggles, and a lab apron. As general lab practice, read the lab through completely before starting, including any Material Safety Data Sheets (MSDSs) and live materials care sheets at the end of this booklet as well as any appropriate MSDSs for any additional substances you would like to test. One of the best sources is the vendor for the material. For example, when purchased at Wards, searching for the chemical on the Ward s website will direct you to a link for the MSDS. (Note: There are no live care sheets included in this particular lab.) At the end of the lab: All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness. Wash your hands thoroughly with soap and water before leaving the laboratory. Page 11
Procedure TipS When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation. Part 1 Structured inquiry/demonstration: Build a simple model of allele frequency in a human population using a spreadsheet program PROCEDURE Structured inquiry 1. Define your assumptions to generate a simplified example of allele inheritance. For example: i. A single trait with 2 alleles, one dominant (D) and one recessive (r). ii. Neither the dominant nor recessive allele confers an advantage. iii. The population under study is large, stable, and randomized. iv. There is no mutation at this locus 2. Assign a relative frequency for both alleles in the population. (We will use frequency and probability interchangeably; for mathematical purposes, express as a decimal.) The frequency should add to 100%. For the sake of this example, we will assume that the frequency of each allele is 50%. Draw a Punnett square with allele frequencies. D 0.5 r 0.5 D 0.5 DD Dr r 0.5 Dr rr 3. Build a model using a spreadsheet program to assign random gamete pairings. Most spreadsheet programs will generate random numbers using the operator RAND. By adding an IF statement, you can use the randomly generated numbers to select an allele at a defined probability. See the figure on the following page. The gametes in columns G and H were generated by typing this formula in the cell: formula = IF(RAND()<E$2, D, r ) The expression can be copied down the column in the spreadsheet to generate as many random zygotes as you would like to analyze. (continued on next page) Page 12
Notes Part 1 Structured inquiry PROCEDURE (Continued) Optional: As an alternative to using a spreadsheet random number generator to get zygote genotypes. Label 10 index cards with the frequency of alleles in the population (if 50%, 5 cards Dominant and 5 cards recessive), turn them over, and repeatedly pull two cards to generate a set of random zygotes (as many as you would like to analyze). A coin flip can also substitute for alleles with 50% frequency. Continue with the same mathematical analyses you would have done on the spreadsheet program. 4. Add up the number of each zygote genotype (DD, Dr, rr) and plot the genotype as a percentage of all zygotes in a graph. TIP: The more zygotes you count, the closer your results will match the chosen probability. Is there a way you can use/set up your spreadsheet program to make this count easier to calculate? In the case of the above starting frequency of alleles, you would expect to see 25% DD, 25% rr and 50% Dr genotypes. What is the relative percentage you would expect to see in phenotypes? 25% recessive phenotype and 75% dominant phenotype. (continued on next page) Page 13
Notes Part 1 Structured inquiry PROCEDURE (Continued) 4. (continued) What is the frequency of alleles in the first generation? The second? Given the initial assumptions, they would not change. 5. Repeat this exercise with different frequencies of alleles in the population and with different numbers of offspring in the populations. What patterns do you see? The smaller the population of offspring or the more extreme difference in frequency, the more likely you will see deviation from prediction. 6. Derive an equation to predict your results given the starting assumptions. TIP: Look at the Punnett square. The frequency of one allele combining with that same allele in the next generation gives a probability of that frequency squared for the next generation. (freq.dom + freq. recessive) 2 =1 or freq.dom. 2 +2 (freqdom freq.recessive) + freq.recessive 2 =1 Page 14
Procedure TipS When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation. Part 2 GUIDED INQUIRY: Observations and model Introduction In this part of the lab you will choose a trait to test and then, based on your data, estimate the frequency of that trait. Procedure 1. Start with a concrete example, like the ability to taste PTC. In this case, the dominant allele (P) permits a person to taste this bitter chemical associated with vegetables such as cabbage. A person with two recessive alleles (p) cannot taste this chemical. Other human traits that can be examined in the classroom include: Sodium benzoate or thiurea can be used as an alternative substance to taste with tasting being dominant to nontasting as with PTC. Attached earlobes is recessive to free earlobes. A widow s peak in the hairline is dominant to straight hairline. Hitchhikers thumb is recessive to a straight thumb where the last joint cannot bend backwards by 45 degrees. The ability to roll your tongue almost into a tube is dominant to the inability to do this. 2. Depending on what trait you chose, sample and/or test your classmates. For instance, if you chose the ability to test PTC, have everyone in your class test whether or not they can taste a difference between the two test papers included in the kit and tabulate the entire class results for those who can taste a difference and those who cannot. 3. Use the equation you derived in Part 1 to estimate the frequency of dominant and recessive alleles for the trait you chose.. Solve for frequency of dominant allele and recessive allele. In the case of PTC, the dominant allele is P and the recessive allele is p. About 75% of Americans can taste PTC. Therefore, the frequency of the recessive allele is the square root of 0.25 or 0.5 or 50% while the frequency of the dominant allele is 1-0.5=50% Page 15
Part 2 assessment questions 1. In the case of selective pressure, what would happen to the frequency of a recessive allele that conferred a reproductive/health advantage when its phenotype was displayed? What if it conferred an advantage only in certain environments, like in cold temperatures, and was a disadvantage in warmer climates? The recessive allele frequency would go up and the dominant allele frequency would go down. In the second case, the frequency of the recessive allele would increase in the cold environment and decrease in the warm environment causing differing phenotypes in populations reproducing in the two environments. 2. What would happen in the case of a recessive allele that conferred a reproductive advantage by being highly attractive to the opposite sex yet conferred a disadvantage for individual health? How would the frequency change? It would depend upon how the health disadvantage affected reproductive fitness. If the health effects only occurred later in life, after reproduction, the frequency would go up despite a shorter lifespan. If the health disadvantage impaired reproductive success to a greater degree than the attractive advantage, then the frequency would go down. 3. If an individual had a new dominant mutation that conferred a reproductive advantage, like increasing the number of surviving offspring by ten-fold, how many generations would it take to reach a 50% frequency? It would mostly depend upon the size of the population. If the population was as few as 10 people, it would only take one generation. 4. How would you modify your model to assess a phenotype that was determined by two genes? The allele frequencies would follow the same rules, but the Punnett square would have to account for more possible combinations: 16 instead of 4. 5. How would you modify your model to assess a phenotype that had more than two allele possibilities in the population (like blood type A, B, O)? While any individual mating constrains a mix to be defined by only two alleles, the population can be imagined like a Punnett square with 3 alleles of given frequency across the top and side. In this case, the frequency of three alleles must add up to 100%. 6. Homo sapiens appears to have become a distinct species about 200,000 years ago. However, the species did not migrate out of Africa until about 50,000 years ago. How do you think the frequencies of alleles in the population may have changed over this period of time and why? A small population could allow genetic drift to have occurred, but more likely, there was a change in selective pressure. The physical environment changed around this time in that there was a short warmer period during the last ice age that may have permitted new migration. Migration probably brought Homo sapiens into contact with Neanderthal which could have resulted in new inter-species competition and selective pressure. Intra-species competition/selection may also play an important role in Homo sapiens evolution to be the highly social animal it is today. Page 16
EXPERIMENT DESIGN Tips The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following questions, which also might arise in peer review: Explain the purpose of a procedural step. Identify the independent variables and the dependent variables in an experiment. What results would you expect to see in the control group? The experimental group? How does XXXX concept account for YYYY findings? Describe a method to determine XXXX. Part 3 Evolution: Hardy Weinberg open inquiry: design an experiment You have built a simple model to look at how allele frequencies are expected to remain the same in stable populations and you have collected data on at least one simple allele frequency in the population of your laboratory. Think about all of the factors that may change allele frequency through evolution, and then choose a hypothetical phenotype controlled by a hypothetical genotype. Modify your model to predict how different types of environmental changes over time would affect the allele and phenotypic frequencies in the population(s). Define your simplifying assumptions and model changes over at least five generations. Before starting your experiment, have your teacher check over your experiment design and initial your design for approval. Once your design is approved, investigate your hypothesis. Be sure to record all observations and data in your laboratory sheet or notebook. Use the following steps when designing your experiment. 1. Define the question or testable hypothesis. 2. Describe the background information. Include previous experiments. 3. Describe the experimental design with controls, variables, and observations. 4. Describe the possible results and how they would be interpreted. 5. List the materials and methods to be used. 6. Note potential safety issues. After the plan is approved by your teacher: 7. The step by step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc. as well as the actual weights and volumes used. Page 17
Notes Part 3: open inquiry (continued) 8. The results should be recorded (including drawings, photos, data print outs). 9. The analysis of results should be recorded. 10. Draw conclusions based on how the results compared to the predictions. 11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance and uncontrolled variables. 12. Further study direction should be considered. Page 18
Material Data Safety Sheet Page 19