Name: Section: Group Quantifying variation within alfalfa (Medicago sativa) Developed by L. Lucas & Z. Gompert, USU Department of Biology
Objectives: In last week's lab, you quantified variation in two human traits across the few people in your group. In this lab, you will quantify the variation among populations of plants from the same species. • A population is all the organisms of the same species, which live in a particular geographic area, and are capable of interbreeding. • A species is the largest group of organisms in which any two individuals of the appropriate mating types can produce fertile offspring. Many species are made up of multiple populations, but some species only have one. By the end of today's project you will be able to: 1) make measurements across individuals to contribute to a real, long-term dataset (part 1), and 2) quantify and compare variation previously recorded in a trait among populations using a statistical test you have not yet used in this class (part 2).
Background information: Some people call alfalfa (Medicago sativa) the most important invention in 2000 years, because it provided year-long feed supply that enabled a reliable horse and animal culture. Alfalfa originated in the steppes of Asia, and now it is cultivated throughout Asia, Europe, Australia, North Africa, North and South America. There are at least 23 million acres of alfalfa in the US. Alfalfa, and other hay, is the third crop in value in the US, behind only corn and soybeans (Putnam et al., 2001). The primary use of alfalfa worldwide is to feed to dairy production, but it is used to feed horse, sheep, beef cows, and other animals as well. Thus, alfalfa is two steps removed from the dinner plate. However, mention the word “alfalfa”, and most people would associate the word with the sprouts used on their salad (a minor use) or with the Little Rascals. Very few would recognize the important role alfalfa plays in their lives in the form of milk, cheese pizza, ice cream, honey, leather, or wool sweaters. Fewer still would recognize the non-economic roles that alfalfa plays in maintaining a healthy environment (Figure 1).
Figure 1. Alfalfa from Victor, ID, housing several insects called treehoppers.
There is a wide range of alfalfa quality for livestock, from supreme to poor. “Purebred” alfalfa is usually higher quality than weedy (or roadside) alfalfa. It is best to “make hay while the sun shines,” since hay quality decreases when hay is rained on. Alfalfa harvested before bloom produces the highest quality hay which is low in fiber and high in protein. To produce the same amount of protein would require four times the acreage of corn. In addition, plant breeders are developing new alfalfa strains that are even higher yielding and more nutritious for livestock. They are also developing alfalfa plants that tolerate wheel traffic and grazing pressure, plants that tolerate high salinity so that it can be grown on salt-affected soils, and plants that tolerate ammonium-N so that they can endure wastewater or manure applications. Varieties with improved root systems for extracting water and absorbing nutrients or contaminants are being developed.
In terms of alfalfa's effect on the environment, it has deep roots (commonly 9-16 feet) and plentiful above-ground vegetation. Consequently, it protects the soil from becoming airborne and causing dusty conditions, or from being washed into rivers as sediment (millions of tons of soil are lost permanently each year; Putnam et al. 2001). Whereas it is true that alfalfa uses a lot of water per year, it uses water relatively efficiently
Intraspecific variation: alfalfa, pg 1 Name: Section: Group due to its deep roots. In addition to growing long, alfalfa's roots house bacteria (Sinorhizobium meliloti) that fix nitrogen; therefore, no nitrogen fertilizers are needed for alfalfa growth. Moreover, the nitrogen alfalfa's bacteria release into the soil aid the crops farmers grow following the alfalfa.
Alfalfa is the beginning of a food chain for many animals, including arthropods like insects. And alfalfa is a home to many beneficial insects; less than 1% of the alfalfa arthropods inflict damage to alfalfa and cause concern to alfalfa growers. These beneficial insects help control pests on alfalfa and other crops (e.g., Figure 1). In general, alfalfa is genetically resistant to many pests, a result of traditional plant breeding. Consequently, alfalfa does not need a large amount of pesticides each year compared with other crops.
The alfalfa we will study in this lab project specifically are in the new Dr. Gene Miller Life Science Garden Laboratory. You will contribute to the scientific history of these plants. In brief:
• In the summer of 2018: Dr. Zach Gompert and Lauren Lucas, as well as researchers from four other institutions received a five-year federally-funded grant to identify the key determinants of plant-insect- microbe interactions. Filling this gap in our understanding of the diversity of life might help us better predict how global change will affect eco-evolutionary dynamics, patterns of biological diversity, and ecosystem function. We are focused on insects and microbes associated with the human-introduced plant, alfalfa -- where it has escaped cultivation and grows in both disturbed and relatively intact habitat, in particular. This “wild” alfalfa hosts a high diversity of insects and microbes. We are using a combination of observational science, experiments, DNA sequencing, and models to analyze the evolutionary and ecological significance of genetic diversity, phylogenetic diversity of microbiome assemblages, and functional variation. As part of this large project, in summer 2018, we studied genetic differences among wild alfalfa populations and the effects these genetic differences have on plant traits and the herbivores that feed on the plants. The seeds used originally came from the following six alfalfa populations: Alpine, WY (“ALP”), north of Davis, CA (“APLL”), west Reno, NV (“AWFS”), Bonneville Shoreline Trail in Logan, UT (“BST”), Victor, ID (“VIC”), and Verdi, NV (“VUH”) (Figure 2). The plants were grown in a greenhouse. The plants were measured within the first few months of growth, and then were used to feed to caterpillars of three butterfly species, to answer the question: how does genetic variation of alfalfa affect the caterpillars that feed on them? Each caterpillar was fed plants from only one alfalfa population. Caterpillar weight was measured and survival was recorded. So far, we've learned that caterpillars of all three species performed better when reared on alfalfa from two populations in particular, APLL and VIC.
Figure 2. A map of the six populations from which seeds were collected for this project.
• In the fall of 2018: We choose six offspring from each of 30 alfalfa plants from the summer 2018 experiment to transplant into each of six plots (i.e., replicates) in the Science Garden. The BIOL 1615 students quantified variation in three traits across the six alfalfa populations. Then, a small team transplanted these 180 plants into the garden. See Figure 3 for a map of the alfalfa in the garden.
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Figure 3. Science Garden map. The garden is divided into six plots. Plants were randomized within a plot. The top of the map is north (against the LSB building).
• In the summer of 2019: Gompert, Lucas and collaborators continued to study these plants, similar to the summer 2018 research project, as well as other alfalfa in Cache Valley to see if they can predict how herbivores perform across alfalfa genotypes. • And now, in fall 2019, it’s your turn to contribute. Your lab section will measure one of many plant traits to contribute to our growing database, and you will start to explore patterns in the previously collected data. Future students may analyze your data and use it to inform a new experiment.
Intraspecific variation: alfalfa, pg 3 Name: Section: Group Lab safety: Pants and covered shoes are required. No other PPE is required for this project.
Equipment: For leaf area: digital calipers For herbivory: N/A For leaf toughness: penetrometers, rigs, labeled envelopes For insect counts: InsectaZookas, charged batteries, labelled collection cups For seed pods: N/A For seeds within pods: labeled collection tubes For plant height: meter tapes Data recording outside: data sheet on page 5 and clipboard Data analysis: Google sheet shared among group members Interpretation of results: Google doc shared among group members
Methods: 1) Part 1. Contributing to a long-term dataset. Refer to Table 1 for the trait your lab section is assigned.
Table 1. Data to be collected by each lab section in fall 2019. TA Lab sections Trait Savannah Adkins 018, 036, 039 Leaf area Binod Borah 005, 033, 040 Herbivory Rachel Buck 001, 011, 016 Leaf toughness Brenna Decker 010, 034 Insect counts Tim DeLory 007, 024, 028 Seed pods Ellis Juhlin 008, 021, 029 Plant height Guopeng Liang 006, 009, 014 Leaf area Helen Plylar 019, 026, 031 Leaf toughness Jessica Scholz 004, 023 Seeds within pods Zach Schumm 020, 030, 037 Insect counts Megan Sidran 002, 015, 025 Plant height Ellie Smith-Eskridge 012, 022, 032 Seed pods George Waldren 003, 013 Herbivory Sam Willard 017, 027, 035 Seeds within pods
2) During class, your TA will train you on how to measure the particular trait your class was assigned.
3) Then, as a class, you will discuss in detail about how each student will measure the trait consistently across the plants to minimize measurement error and thereby noise in the data set. For example, for leaf area, you would need to decide how each student will choose the leaf they will measure to maximize consistency across plants. What units will you use? Write down your class decisions below:
4) Also, as a class, discuss who will collect data on which plants. For example, Group 1 could collect data on all 30 plants in Plot 1, Group 2 could collect data on all 30 plants in Plot 2, etc.
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5) Use Table 2 to record measurements made while in the field. If a plant cannot be measured for some valid reason, record “NA” for this plant.
Table 2. Trait measurements. Plot # Plant ID Measurer initials Trait value (units: ):
6) Once back in the classroom, add your data to the class’ fall 2019 spreadsheet via your TA’s computer. Return measurement equipment to the cart and clipboards to the classroom counter.
Intraspecific variation: alfalfa, pg 5 Name: Section: Group 7) Part 2. Exploring patterns in previously recorded data. Your TA will talk you through the data that already has been collected on these plants. Your group will choose one previously measured trait to explore (e.g., herbivory measured on May 31, 2019). Specifically, you are exploring differences in this trait among the six populations of alfalfa found in the Science Garden.
8) You will access this dataset via Canvas “Lab Documents”. Make your own copy of his datasheet (File > make a copy; you must be signed into your google account to do this), then work off of your own copy for the data analysis.
9) Start by visualizing the difference among population means with a bar graph for your chosen trait. First you will want to sort the raw data appropriately. Make a new data table that just includes the populations (column 1) and trait means (column 2) and a header in the first row. Use the “average” function to calculate the means. Then, highlight this new data table, and insert chart. Choose the column chart type. Under the customization tab, add a meaningful title, get rid of the useless legend, add x- and y-axis labels, and set the minimum of the y-axis to 0.
10) To test whether there are significant differences between the trait means across the populations (specifically, whether at least one population is significantly different from the rest), you will conduct a statistical test called a Single Factor ANOVA. The p-value you will receive after running the ANOVA is the probability of observing a difference in the means as big as the one you observed by chance. It indicates whether we should view the populations as random samples or whether there are true differences among them. Traditionally, a p-value of 0.05 or lower indicates there are true differences among the populations.
You will need to reformat the raw data, so that there is one column per population, with 30 values per population. You will want to convert any “NA”s to blanks (not 0s). You will need to add the XLMiner Analysis ToolPak first (under Add-ons). In the XLMiner Analysis ToolPak, choose Anova: Single Factor. In “Input Range”, highlight reformatted data table, including the header in the first row. Select “Grouped by Columns”. Select “Labels in First Row”. Leave “Alpha” at 0.05. In “Output Range” click on an empty cell for your results to go. Then choose OK. Record your population means and p-value in Table 3. Does your p-value indicate there are true differences among populations (specifically, between at least of the populations and the others)?
Table 3. Results. Mean trait values (units: ) per population and ANOVA p-value. ALP APLL AWFS BST VIC VUH Trait mean: p-value:
Scientific Reflection (8 pts total): You and your group members will complete the following Scientific Reflection assignment as a group. If a group member leaves before the assignment is submitted, this group member will receive a 0 for the assignment. Complete the final draft of the assignment in a google document shared with all group members. Save the final draft of the document as a pdf. Only one group member needs to upload the pdf to Canvas.
Which trait in the previously collected data did you explore? (1 pt) Were there statistically significant differences among the alfalfa populations? You must refer to the relevant parts of your bar graph (1 pt) and your p-value (1 pt) in your answer to receive full credit. For your bar graph, you will include: an appropriately descriptive title (1 pt), correct axis labels (1 pt), a y-axis that starts at 0 (1 pt), and you will remove the useless legend (1 pt). You must answer these questions in complete sentences in a paragraph to receive full credit (1 pt).
Reference: Putnam, D., Russelle, M., Orloff, S. Kuhn, J., Fitzhugh, L., Godfrey, L., Kiess, A., Long, R. (2001). Alfalfa, wildlife and the environment: the importance and benefits of alfalfa in the 21st century. California Alfalfa and Forage Association: Novato, CA.
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