Identifying Patterns Over Small Spatial Scales

Labs 3-4 – Patterns of Phenotypic Variation

Identifying Patterns Over Small Spatial Scales

Fall 2015

Abstract: In this two-week study, you will explore the realm of evolutionary and population ecology by examining how traits can phenotypically vary with small-scale environmental change. You will also begin a common garden experiment to test hypotheses about possible causes of phenotypic variation observed in the field. Later in the semester, you will complete the common garden experiment.

Goals (for labs 3-4) are to:

1. measure small-scale phenotypic variation in nature.

2. test hypotheses about the association between phenotypic variation and variation in the physical environment.

3. learn and use some basic statistics and the t test.

4. begin a common garden experiment, an important technique for identifying causes of population variation.

A. Background:

It perhaps does not need to be said that individuals within a species phenotypically differ from each other – but I’ll say it anyway! Individuals within a population and populations within a species phenotypically differ for many traits – though not all. Phenotypic variation makes life interesting. What causes the variation? Some variation is explained by genotypic differences among individuals/populations. Some, however, is explained by variation in individual responses to environmental change. Genetically unique individuals in one environment can look different (or behave or physiologically function differently) than do they do in another environment. For example, students who spend most of the summer at the beach, on average, have darker skin than they do when they are studying indoors in the winter. The world is rampant with variation, but at what spatial scales? Can and do populations show phenotypic variation across small spatial scales, e.g., across a lawn, at different depths in a small lake, at different heights on a wall?

Even at small spatial scales, the environment can show change over space. There can be an environmental gradient. Populations of a single species often grow along such gradients. In such cases, these populations can often be divided into subpopulations, each in its own microenvironment. In this study, we will explore small-scale phenotypic variation within a population found growing along an environmental gradient.

Questions:

a) Can individuals within a single clover population (defined as a group of interbreeding individuals growing in the same location) phenotypically vary across small-scale environmental gradients? If so, what is the pattern of variation? Can a population comprise several morphologically distinct subpopulations?

b) How can the microenvironment change spatially across clover populations?

c) What abiotic and biotic factors contribute to microenvironmental variation?

d) Are subpopulations associated with differences in microenvironment?

Lab 3 – First lab:

B. Hypothesis Development:

To answer the above questions, we will study a population of Trifolium repens, white clover, which belongs to the pea family. This species grows in lawns and is pollinated by bees, which can carry pollen large distances. Therefore, white clover populations can be quite large. Study the diagram below to learn about clover morphology. We will focus on two morphological traits: petiole length, and leaf area.

1. After your instructor divides the class into groups of 3-4 students, each group should get 2 pairs of scissors, 2 lab handouts, pencils, clipboards, and 4 paper towels – 2 labeled site A, and 2 labeled site B, each in a separate baggie. Dampen the paper towels before sealing them in the baggie. Also, get 1 light meter.

2. After your instructor explains how to use the light meter, s/he will take you to a white clover population that extends across two microenvironments.

2. We will measure light intensity in each microenvironment. However, in your group, discuss what other abiotic factors might differ between the microenvironments. Jot down at least 3 ideas.

4. In your group, discuss how petiole length and petiole angle might phenotypically differ in the two microenvironments. Jot down possible differences for:

· Petiole length:

· Petiole angle:

5. Now write down 3 class hypotheses concerning: 1) phenotypic variation in petiole length, b) phenotypic variation in petiole angle, and 2) difference in light intensity between microenvironments. Write the hypotheses below – in the present verb tense.

a) H microenvironmental differences in petiole length:

b) H microenvironmental differences in petiole angle:

c) H microenvironmental differences in abiotic factor:

C. Experimental design:

We cannot collect data for leaf area and petiole length on all plants in the population, or in more than one population. Nor can we measure our environmental variable at every site over the population. So we must take samples for our experiment and test our general hypotheses by looking at differences between samples of data.

D. Methods:

1. Field work:

1) Student groups should spread out across each microenvironment so that each is haphazardly sampled. We should not all collect from the same site within each region. Think about the reason for this!

2) Each group should carefully pull up/break off 2 clover stolons from each region. Each stolon needs to have at least 2 mature leaves and a growing tip (with immature leaves). Pull up only one stolon per plant, not the whole plant, which may have many stolons. Handle the stolons GENTLY.

3) Fold a wet paper towel over each stolon and leaves and return the towels to the baggie.

4) Record the microenvironment from which each sample stolon was taken.

There should be 4 stolons, 2 per microenvironment, for each group of students.

5) Measure the abiotic factor at each collection site, and record these data before returning to the lab. Remember to use the multiplication factor, if relevant, when recording data.

Microenvironment Abiotic Factor (units?)

value

2. Lab work:

1) Give each stolon a unique ID.

2) Each group should measure the petiole lengths of 3 mature leaves per stolon. Keep plants moist while working.

3) For each stolon, measure the petiole angles of 3 mature leaves per stolon.

4) Enter data for petiole length, petiole angle, and light intensity on the class datasheet.

E. Setting up the Common Garden Experiment for Lab 8:

1. Rinse your stolons in water to remove any aphids that might be present.

2. Determine which end of the stolon is the growing tip. Cover the other end into the plant hormone (an auxin) so that powder is on that end and the surrounding stolon.

3. Fill a pot with soil, tamp the pot on a workbench, and add more soil so that the soil comes to the rim of the pot. Stick each clover stolon in a separate pot. Bury 3/4 - 1" of the end that has the auxin. The growing tip should be up.

4. Press the soil down and around the stolon.

5. Label each pot with your names, section number, stolon ID, and microenvironment ID on the white plastic strips provided.

6. You are now ready to place pots on the light racks (manifolds) in the lab. Make sure to place the plants away from the edges of the pans in the light manifold For next week: read “Experimental Design and Really Basic Statistics” and your Statistics Manual to help analyze your data.

Lab 4 – Data Analysis:

Statistics are necessary tools in ecological research. Today you will use statistics to test the class’ hypotheses.

F. Predicted Results: For the two morphological variables and for light intensity, what do the class hypotheses predict will be the results? (State predictions in future tense.)

Prediction Light Intensity:

Prediction Petiole Length:

Prediction Petiole Angle:

G. Data analysis for the field experiment:

1. Make sure that data from all groups are entered on the class datasheet.

2. Using the data, calculate the mean and 2 times the standard error for each variable in each microenvironment. Plot your data for petiole length, petiole angle, and light intensity on three separate graphs.

3. Copy graphs to a single powerpoint page, and add axes labels, legends, etc. for each graph. Check that everything that should be on a graph is there.

4. Print the.ppt page with your graphs.

5. Perform a t-test for each variable to see if any differences between microenvironments are statistically significant. You will first do this entirely manually.

6. After mastering the t-test manually, you will use the Experimental Design and Really Basic Statistics Handout to help you perform the test with the excel statistical add-on.

7. Email your excel and .ppt pages to yourself and/or save on flashdrive. You will need these for Lab 8!

Writing Assignment

Patterns of Phenotypic Variation

1. Which type of experiment did we conduct? (manipulative or descriptive)

2. State the three hypotheses tested. For each, state the alternative hypothesis. Consider all possible alternative patterns, in each alternative hypothesis.

H petiole length:

alternative hypothesis:

H petiole angle:

alternative hypothesis:

H light intensity:

alternative hypothesis:

3. Summarize the methods used to test the hypotheses (7 sentences max.)

4. Leaf Area:

a) Describe the observed results in words (1-2 sentences). Use the past verb tense. Why? In parentheses at the end of your first sentence, cite the relevant graphical figure, for example (Fig. 1).

b) What does the t-test tell you? Finish the sentence: “The t test shows that the differences in (fill in) are (finish)..” (In parentheses at the end of the sentence, state the df, the 2-tailed t value, and the p value.)

c) Do you accept or reject the hypothesis? ______Why? (1-3 sentences). Remember that the t test tells you ONLY if you can reject the NULL = there is NO difference. Also, think about what the data from each sample microenvironment suggest about the whole population.

3. Petiole Length:

a) Describe the observed results in words (1-2 sentences). Use the past verb tense. Why? In parentheses at the end of your first sentence, cite the relevant graphical figure, for example (Fig. 1).

c) Do you accept or reject the hypothesis? ______Why? (1-3 sentences). Think about what the data from each sample microenvironment suggest about the whole population.

4. Light Intensity:

a) Describe the observed results in words (1-2 sentences). Use the past verb tense. Why? In parentheses at the end of your first sentence, cite the relevant graphical figure, for example (Fig. 1).

c) Do you accept or reject the hypothesis? ______Why? (1-3 sentences). Think about what the data from each sample microenvironment suggest about the whole population.

5. Does there appear to be an association between light intensity and leaf area and petiole length? Why might there be an association?