Ecology Laboratory, BIO 320L Morehouse College

Laboratory 2

Causes for Phenotypic Variation

Objectives

1. Describe the natural phenotypic variation that occurs under controlled

environmental conditions in one plant species for traits such as height, internode

length, weight, and photosynthetic and accessory pigment concentrations.

2. Describe how each of three varieties of one plant species responds to changes in

environmental conditions (temperature).

3. Evaluate findings to attribute observed variation to environmental or genotypic

variation or both.

Introduction

Understanding the sources of phenotypic variation in organisms is central to the

understanding of natural variation and the responses of organisms to their environment.

Variation within a species of plant or animal is very common over a geographic range.

Variation in size, shape, coloration, behavior and physiology may be a product of current

environmental differences between sites (phenotypic plasticity), a product of heritable

differences (genotype differences = ecotypes) between the subpopulations at different

sites, or a combination of both. The classical methodology for determining the causes of

variation is reciprocal transplants or transplants to a common environment.

Transplanting individuals possessing different traits to a constant environment or

performing cross transplants between natural sites is a means of evaluating the relative

importance of environmental and genetic variation in producing the observed phenotypic

variation. The finding of persistent differences between subpopulations independent of

environmental conditions suggests that genetic variation underlies observed phenotypic

variation. For example, a species of yarrow, Achillea millefolium, grows at a wide

variety of habitats in California ranging from sea level to more than 3000m elevation.

Plants at a given altitude have different height and biomass compared to plants from other

altitudes even when seeds of plants from different sites are grown under the same

conditions at at sea level (Clausen, Keck, and Hiesey, 1948). This result indicates that

the observed phenotypic variation among the California Achillea is ecotypic, caused by

genotypic differences between populations.

In this experiment, phenotypic variation in a vascular plant species will be

evaluated for both morphological-structural traits (for example, total biomass, internodal

length, and total plant height), and biochemical traits (for example, total chlorophyll and

anthocyanin concentrations). We will work with varieties of rapidly growing mustard

plants, Brassica rapa, (Williams, 1989).

Chlorophylls, of course, are the principal photoreceptor pigments in plants,

located in the chloroplasts. We can quantify the concentration of chlorophylls from

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Laboratory 2

Causes for Phenotypic Variation

Objectives

Describe the natural phenotypic variation that occurs under controlled environmental conditions in one plant species for traits such as height, internode length, weight, and photosynthetic and accessory pigment concentrations.
Describe how each of three varieties of one plant species responds to changes in environmental conditions (temperature).
Evaluate findings to attribute observed variation to environmental or genotypic variation or both. Introduction Understanding the sources of phenotypic variation in organisms is central to the understanding of natural variation and the responses of organisms to their environment. Variation within a species of plant or animal is very common over a geographic range. Variation in size, shape, coloration, behavior and physiology may be a product of current environmental differences between sites (phenotypic plasticity), a product of heritable differences (genotype differences = ecotypes) between the subpopulations at different sites, or a combination of both. The classical methodology for determining the causes of variation is reciprocal transplants or transplants to a common environment. Transplanting individuals possessing different traits to a constant environment or performing cross transplants between natural sites is a means of evaluating the relative importance of environmental and genetic variation in producing the observed phenotypic variation. The finding of persistent differences between subpopulations independent of environmental conditions suggests that genetic variation underlies observed phenotypic variation. For example, a species of yarrow, Achillea millefolium , grows at a wide variety of habitats in California ranging from sea level to more than 3000m elevation. Plants at a given altitude have different height and biomass compared to plants from other altitudes even when seeds of plants from different sites are grown under the same conditions at at sea level (Clausen, Keck, and Hiesey, 1948). This result indicates that the observed phenotypic variation among the California Achillea is ecotypic, caused by genotypic differences between populations. In this experiment, phenotypic variation in a vascular plant species will be evaluated for both morphological-structural traits (for example, total biomass, internodal length, and total plant height), and biochemical traits (for example, total chlorophyll and anthocyanin concentrations). We will work with varieties of rapidly growing mustard plants, Brassica rapa , (Williams, 1989). Chlorophylls, of course, are the principal photoreceptor pigments in plants, located in the chloroplasts. We can quantify the concentration of chlorophylls from

different samples by evaluating the absorption of light at the specific wave lengths at which peak absorption occurs. The absorption of light increases with pigment sample concentration. Anthocyanins are a class of flavonoids, three ring secondary plant compounds, that produce orange to blue colors in leaves, stems, roots, flower petals, and fruits of many plants (Harborne, 1988). There are more than 260 different anthocyanin compounds and these pigments may serve a wide range of functions such as protecting plant cells from ultraviolet light, attracting insect pollinators, and acting as anti-herbivore chemical defenses (Harborne, 1988). Anthocyanin concentrations can be quantified in the same manner as the chlorophylls using a spectrophotometer to evaluate light absorption (Miller, 1973). Hypotheses and Predictions Ho: Variation between the plant varieties results from genetic differences between them. Prediction: A given variety will have a constant phenotype between environments and will remain distinct from other plant varieties. H 1 : Variation between the plant varieties results from environmental differences between the sites where these varieties were growing in nature. Prediction: Phenotypes will change with environmental conditions. H 2 : Variation between the plant varieties results from both genetic differences and environmental variation. Prediction: Intermediate results of the two predictions above. Methods Growing Plants from Seed Seeds from three varieties of the mustard plant, Brassica rapa , will be used in this experiment. Information about the source habitat for each variety is not available but the phenotypes of the source plants are known. Variety TA is from plants that are known to grow tall and contain anthocyanin, variety TC is from plants that grow tall and do not contain anthocyanin, and variety SA is from plants that grow short and have anthocyanin pigments. You will sow seeds two or three weeks prior to the collection of data on the plant phenotypes. Seeds will be planted in plastic quads containing four cells each. You will prepare a total of 6 quads. Place a paper wick in each quad cell (4/quad) and gently pull each wick to ensure that the wick remains sticking out the bottom of the quad when the quad is placed on a flat surface. Fill each quad with prepared soil mixture and wet the soil with water (using spray bottle) until the soil has absorbed water and water drips from the bottom of the quad. Sow two seeds (of a given variety) in each quad cell. You will sow seeds in 6 quads, 2 quads of each of the three varieties of B. rapa (Table 1). Place two seeds on the soil surface in a quad cell, press into the soil with the tip of a pencil to just below the soil surface and cover lightly with soil. One quad sown with seed of each

Working with one quad at a time, cut each plant from the quad at soil level, then measure stem length above soil level, distance from soil to the first node, and internodal distance (distance between the first two nodes) (Figure 2). If a measurement is too small to make, but is not zero, record a value of 0.1mm. Record the individual values for each plant from each quad. Weigh the plants from one quad together. Record the total biomass and the number of plants (normally four) from each quad, so the mean biomass per plant stem can be calculated. A suggested data record format is given below (Table 2a and b). Record data in your laboratory notebook using this format, and later transfer the data to the class computer files. Keep the plants from each quad separate from the plants from other quads. Use labeled plastic weighing boats for the plants from each quad. You and your partner will have one quad of each variety per treatment (two quads of each variety total). You will be informed in class which pigment extraction to perform, half of the class will perform the chlorophyll analysis and the other half will perform the anthocyanin analysis. All four plants from one quad will be used to prepare a single pigment extraction. You and your partner will perform pigment extractions and quantify the pigment concentration from plants of each of the three varieties grown under each of the two environmental conditions (six combinations).

Figure 2. Morphology of 13 day old Brassica rapa (after Williams, 1989). Note that individual plants may vary from this example. Scale units are cm. Table 2a. Laboratory notebook data recording format for morphological data. The Student Group # will be the same for your entire data set, but that information will be required for the class data file. Note that this is identical to the format that will be used for the class data file. An example entry is shown in italics. Treatment Variety Stem Length First Node Internodal Flowering Budding Stem Color Petiole Color thirty TA 73 12 17 no yes purple purple Table 2b. Laboratory notebook data recording format for biomass and pigment extraction absorbance data. You and your partner will perform a pigment extraction and collect absorbance data on either anthocyanin (abs antho 530) or on chlorophyll (abs chloro 415 and abs chloro 662). Note that this is identical to the format that will be used for the class data file. An example entry is shown in italics. Treatment Variety Total mass (g)

of

plants abs antho 530 abs chloro 415 abs chloro 662 thirty TA 0.6 4 0.

Bottle. You will be able to calculate the absorbance values per mg of fresh biomass used in each extraction after you enter your data in the class data file. Chlorophylls: Grind four plants with a glass mortar and pestle containing a pinch of sand and 3ml of acetone. Pipette or pour into two microfuge tubes (make sure the tubes are balanced, contain the same volumes, and place them in opposite positions in the centrifuge rotor), and spin 2 minutes. Fit a 3cc syringe with a filter disk (remove the cap, if present, from the luer-lok end of syringe barrel and twist on the filter disk. Pull the syringe plunger from the barrel and position the end of the filter disk over a small glass test tube. Carefully transfer supernatant (using a clean glass pasteur pipette) to the 3cc syringe fitted with the filter disk and force supernatant through filter into the glass test tube. Adjust the total extract volume in the test tube to 4ml. Stopper the tube, label with tape, mix by shaking, and cover with foil to minimize exposure to light. Rinse the mortar and pestle with water and dry before preparing the next sample of plants. Use the quartz cuvettes with your instructor supervising (handle the quartz cuvettes with care, they are very expensive). Acetone will melt disposable polycarbonate plastic cuvettes. Use acetone as the blank in the spectrophotometer. The spectrophotometer program CHLORO will measure absorbance in the visible range at 415nm and 662nm. Be sure to turn-on the VIS lamp before attempting to read the blank. After you have obtained the absorbance values for your samples, turn-off the VIS lamp. Rinse cuvettes with acetone and pour all acetone waste and acetone extractions in the Acetone Waste Bottle. You will be able to calculate the absorbance values per mg of fresh biomass used in each extraction after you enter your data in the class data file (Table 3). Statistical Data Analysis Enter your data in the Excel spreadsheet that your instructor has created on the computers in the laboratory (Table 3a and b). Compare traits, physical and biochemical, between treatments within each variety, and between varieties within each treatment. Perform chi-square tests on the categorical variables (leaf color, stem color, and flowers), and an analysis of variance (ANOVA) with Scheffe´ comparisons on the continuous variables (see Appendix on Analysis of Data). Your Instructor will explain how these statistical tests are performed. Do all the traits respond to environmental variation in the same way? What is the cause for the differences between the three varieties of plants, genotypic variation, environmental variation or both? Table 3a. Spreadsheet File “Variation-Morpho”. A data spread-sheet form similar to that shown below is already present on the laboratory computers. “Group” is a categorical variable containing the name assigned to identify you and your partner in the data file. “Treatment”, “Variety”, “Flowering”, “Stem Color”, and “Petiole Color” are all categorical variables. Stem “Length”, distance to “First Node”, and “Internodal” distance are all continuous variables measured in millimeters. Group Treatment Variety Length First Node Internodal Flowering Budding Stem Color Petiole Color Two Thirty TA 32 20 13 no yes purple purple Two Thirty TA 25 18 4 no yes purple purple Two Thirty TA 40 24 12 no yes purple purple

Table 3b. Spreadsheet File “Variation-Pigments”. A data spread-sheet form similar to that shown below is already present on the laboratory computers. “Group” is a categorical variable containing the name assigned to identify you and your partner in the data file. “Treatment”, and “Variety” are also categorical variables. “total mass”, “# of plants” per quad, “abs antho 530” (extract absorbance at 530nm), “abs chloro 415”, and “abs chloro 662” are all entered as continuous variables. Group Treatment Variety Total mass (g)

of

plants Abs antho 530 Abs chloro 4 15 Abs chloro 662 Two Fifteen TA 0.129 3 0. Two Thirty TA 0.319 4 0. One Thirty TA 0.620 4 2.31 1. One Fifteen TA 0.558 4 3.70 3. Literature Cited Clausen, J., D.D. Keck, and W.M. Hiesey. 1948. Experimental studies on the nature of species, III: Environmental responses of climatic races of Achillea. Carnegie Institute of Washington Publication, 581: 1-129. Harborne, J.B., 1988. The Flavonoids: Recent Advances. in Plant Pigments , pp. 299-343. Academic Press, NY. Miller, L.P. (ed), 1973. Flavonoids. in Phytochemistry. Volume II, pp. 357-378. Van Nostrand Reinhold, NY. Williams, P.H., 1989. Wisconsin Fast Plants Growing Instructions Carolina Biological Supply Company, Burlington, NC.

Phenotypic Variation in Mustard Plants: A Laboratory Experiment - Prof. Lawrence Blumer, Lab Reports of Ecology and Environment

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