Name ______ECOLOGY and EVOLUTION Week 1: October 29 – November 2, 2012 Welcome to the third set of Fall semester introductory biology laboratories. The emphasis will be on populations of vertebrate organisms interacting with their environments.
(Kaplan and Phillips 2006)
In lab, you will work with two different species, a frog and a fish. The focus will be on understanding how to work with variation among individuals within populations, which is Darwin’s major insight with respect to articulating the theory of natural selection. Over the course of the next four labs, you will take three approaches to address questions in this field called evolutionary ecology: a) designing and implementing controlled randomized, factorial experiments in the laboratory with the frog species; b) designing and implementing long term comparative experiments in the field with the fish species; and c) learning how computer simulation methodologies allow us to develop biological theory with complex systems without necessarily needing to engage in mathematical analysis. All three of these approaches are used by animal and plant ecologists to study how species evolve to meet the challenges of life in the real world. This week's lab has two parts. In Part 1, you will learn how to design a multi-factor randomized block wet lab experiment with frog larvae (tadpoles) and address the lecture theme of environmental effects influencing development to potentially influence fitness (see Fig.1 above). In Part 2, using both our fish and frog species, you will learn how important it is to get a “feel for the organism” while you learn a new measuring method, and you will become familiar with computer measurement and more advanced statistical analyses.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 1
LAB 1– Part 1. Tadpole morphological plasticity
Before coming to lab, read the lab manual and review the following: • Tadpoles of the United States and Canada: A Tutorial and Key by Ronald Altig, Roy W. McDiarmid, Kimberly A. Nichols and Paul C. Ustach http://www.pwrc.usgs.gov/tadpole/tutorial.htm • What is the function of a tadpole’s spiracle? • Today in lab from your observations of live Bombina tadpoles, how many spiracles do they have, and where are they (or it)? Phenotypic plasticity in tadpoles – Amphibian embryos and larvae are dynamic developmental systems and are used extensively in ecological and evolutionary studies. Today, you will use larvae of the Asian fire-bellied toad, Bombina orientalis, to explore a variety of fundamental issues especially: 1) the importance of single and multiple environmental factors in development and potentially evolution, and 2) multi-factor experimental design. The question - Does a potential environmental stressor (water runoff into breeding pools in an urban setting) vary in effect with a second stressor (the threat of predation)? (Note this sentence could be reversed for emphasis and would then read “Does a predation threat vary in its effect depending on water runoff into amphibian breeding pools?) Inadvertent runoff of common garden plant fertilizers, pesticides, herbicides, and/or motor oils pose potential harm to amphibians and potentially contribute to the amphibian species decline crisis. The Reed Canyon water is a precious resource. Is there reason to be concerned that there may be chemicals getting into it that are harmful to amphibian development? Plus, the myriad of potential artificial chemical stressors on amphibians may synergize with other factors that are also important. In terms of predation, adult male Bombina orientalis have been shown to predate upon Bombina tadpoles, and other studies of predation effects in different species of frog larvae (as seen on video in lecture) have been well documented.
Today, you will begin to look at the effects of our Canyon’s water and variation in exposure to chemical cues that signal potential predation, both separately and in interaction with each other as we learn how to design a symmetrical, randomized, and replicated, two-factor experiment (See Bio Binder Quantitative Experimental Design G- 3+4 and I-11).
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 2 The statistical design of the experiment is formally called a multi-factor, randomized, block design. Specifically, your experiment will be a two-factor (with two levels for one factor and two levels for the other factor), completely crossed, randomized, block design.
Three null hypotheses will be tested simultaneously in this, the simplest and most fundamental of experimental designs that allows for a formal test of factor interaction:
• Ho = There is no significant effect of canyon water presence or absence on size or shape as measured by body length and tail height in Bombina orientalis tadpoles after 2 weeks of exposure. • Ho – There is no significant effect of potential male predator chemical cues presence or absence on size or shape measured by body length and tail height in Bombina orientalis tadpoles. • Ho – There is no significant effect of the interaction of canyon water (+/-) and predator cues (+/-) on the size or shape measured by body length and tail height in Bombina orientalis tadpoles.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 3 Setting Up a Multi-factor Experiment –The physical design (see below) is supported by the statistical framework.
Experimental Design has two components: a statistical design and a physical design. The statistical design ensures that proper thought is given to the data matrix that will ultimately be constructed, AND that JMP statistical analyses can properly test the null hypotheses under examination. The physical design dovetails the actual materials, equipment, space, and time constraints with the statistical design. Both aspects of experimental design must be tended to with careful thought, or the null hypotheses cannot be objectively tested or false conclusions can be made. This happens all the time because it takes training, practice, and (as with all things) using your intuition helps. We will design this experiment for you. It is your job to: 1) understand the design, 2) execute the experiment, 3) perform the statistical analyses of the data, and 4) write a report.
The statistical design - Two factors crossed with two levels of one factor and two levels of the other factor yields four unique treatments. This will be repeated as a whole block during each of the lab sections on each of the days (two rooms x five days = 10 blocks in total). The goal is for each student to monitor two tadpoles through the experiment. There are approximately 20 students in each section. That means in each class we are designing for 40 tadpoles to be divided symmetrically into each of the treatments. Since there are four unique treatments, there will be 10 tadpoles in each treatment in each lab room and day (i.e., a block). The actual sample sizes in the following statistical design chart for your particular section should be filled in by you and modified over the next two weeks. What might cause the sample sizes at the end of the experiment to be different from what we plan? Is this OK? Symmetry is the key element here that protects the fundamental statistical structure. The best we can do, because of space and time constraints is 10 + 10 + 10 + 10 = 40 per block (i.e., 20 students per class and two tadpoles per student) and then take care of our tadpoles as best we can.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 4
Figure 1. Statistical Design. A two by two matrix showing all four possible combinations of the two treatments: Canyon Water vs. Laboratory Water and Predation cues present vs. Predation cues absent. Consider that this is a picture for one laboratory section (i.e. a statistical block). There are 10 sections all together (two on each day for a total of 10 blocks.)
The Physical Design matches the statistical design (see above).
Procedure – Wash your hands with soap and rinse thoroughly to protect the tadpole from any contaminants on your fingers such as hand lotions, antiseptics, perfumes, soaps, nicotine (no time like the present to quit smoking), etc. 1. Obtain TWO HDPE (high density polyethylene) containers with lids. This will be what your tadpoles live in for the next two weeks.
2. Carey or Ned will assign each of you a particular treatment via a randomization process. (Randomization is a technical term here that you should understand.)
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 5 Each of you will get two tadpoles, and one will live in Canyon water (CW) and the other will live in Lab water (LW). You will get one random number defining your treatment of Predation cues added (Pred) or No Predation cues added (NoPred). If you have a Predation cue (Pred) assigned, it is added to both Canyon Water (CW) and Lab Water (LW) containers. If you have a No Predation cue (NoPred) assigned, it is added to both Canyon Water (CW) and Lab Water (LW) containers. Therefore, you will have one tadpole in two of the four possible treatments. Next, you will get another random number for each of the two containers (Canyon and Lab water) locating the position to put your tadpoles on each of the two grids that have been positioned on the lab bench. One tadpole goes into the position on one grid and the other tadpole goes into the same position on the other grid. Depending on the first random number assigned to you both water types get the same predation cue treatment (i.e., Pred vs. NoPred). Then you mark your containers with two of the following colors of tape place on two sides of the container and the lid:
If you get a random number that is: Canyon Water Lab Water 1!12, then Predator cue is added then label the container with Yellow Tape Green Tape 13 ! 24, then No Predator cue is added then label the container with Pink Tape Red Tape
3. In addition to the color of tape for the two containers, the containers should be carefully labeled with a Black Sharpie lab marker on specific color lab tape on two sides AND the lid. A demo will be on display. The label of a container should be exactly in the following form: Day of week, Your Full Name, CW or LW, Pred or NoPred, grid # (assigned in step above)
4. Using a beaker, add 200 ml of specially prepared laboratory water (LW) (from the 50- gallon container in the middle room between the two labs) to the plastic container. This water (i.e. 30% modified Holtfreter's solution) has the following recipe:
NaCl 204.6 g KCl 3.0 g CaCl2 5.9 g MgSO4*7H2O 11.7 g NaHCO3 1.2 g Water added to 50 gallons total volume.
Use 200ml of Canyon water (CW), also in the middle room, for your other tadpole.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 6
5. Use clean forceps and add one dime-sized piece of single-layer boiled spinach to each container (approximately 1.0 cm diameter). Crush the spinach piece as much as possible with the forceps. Do not overfeed, as too much spinach now will require more frequent water changes later.
6. When you are sure that the amount of spinach is right, add one (1) volume of solution, depending on your treatment, using a micropipette (see Bio Binder Micropipettes P-1). Predator Cue Water = 1000!l DI prepared predator cue water or No Predator Cue: = 1000!l DI water
7. Get one clean plastic Petri dish lid and a plastic mm ruler.
8. Get comfortable taking accurate measurements by looking at the ruler and a coin under your dissecting microscope. To the nearest tenth of a mm, how large is your coin?
9. Bring two petri dishes to the middle room where Bob will give you two tadpoles and show you how to handle them. This will be done one student at a time, so be patient.
10. Look at your tadpoles under the scope. Look for the beating heart and blood cells moving. Where is the spiracle? What other parts can you list?
11. Bring your tadpoles to your desk, and gently put the plastic Petri dish and the centered tadpole on top of the mm ruler. a) Let the tadpole calm down by not touching it. b) Also without touching the tadpole, wick off as much excess water as you can from the Petri dish with a KimWipe (in your plastic drawer). c) Gently shake the Petri dish side to side to slide the tadpole into a “lie-flat” body position. d) Move the ruler under the dish to measure while looking through the dissecting microscope. e) Make an estimate to the nearest 0.1 mm (it will be rough) of the body length (line “a”, does not include the tail) and maximum tail height (line “d”) of the tadpole (Fig. 2). A typical tadpole is 8mm body length and 4mm tail height.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 7
Figure 2. Tadpole body length = line “a”, maximum tail height = line “d” Photo from: Van Buskirk and McCollum, 2000
12. Be sure to write these numbers down in your lab notebook along with whether it is the CW tadpole or the LW tadpole and Pred or NoPred cues are added, along with other important details (e.g., time, treatment, grid location - see below).
13. Once you are satisfied with your four measurements (two on each tadpole), gently “pour” the tadpole into the CW or LW container. Place the cover loosely on top without sealing. We want to minimize evaporation, but not seal them in too tightly and risk spills when opening.
14. Using the FireFox web browser, enter your results in the lab data collector found at http://collector.reed.edu. Go to: a) Bob Kaplan b) Find your lab section: Tadpole wk1 Carey or Tadpole wk1 Ned c) Follow the instructions carefully. Abbreviations must match the instructions exactly. d) Make sure you measured in mm (not cm). A typical tadpole is 8 mm body length and 4 mm tail height. If your tadpole is much larger than 10 mm, please check that you are measuring body length and not total length. If your tadpole is smaller than 1, please check that you are measuring in mm, not cm. e) Do not enter units next to your numbers. 15. Get a location for your tadpoles assigned by Carey or Ned. Write the location numbers on the lids and side labels. Put your tadpoles and containers into their proper locations in the grids on the lab bench and record these locations in your notebook.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 8
Figure 3. The 4 x 6 class grids are called randomized blocks. We have randomized both water source treatment and predation cue treatment in each block. We will have 2 randomized blocks in the experiment in each class, at two different locations on the bench. (Note that the spatial blocks within classes become temporal blocks among classes.) Consider that 20 tadpoles per block x 2 blocks per class x 2 classes per day x 5 days = 400 tadpoles. Why is this symmetry important? Why will we not be able to maintain it for very long? (Note there will be 4 extra spots in each block that are unoccupied.)
An important review about randomization and intentional placement: The random positioning of your tadpole in these grids is the second randomization we have used in this experiment. 1) We randomized the treatment that you are responsible for. 2) We randomized the location of your tadpole in the grids. We did each of these randomizations with computer-generated random numbers, There are many ways to generate random numbers. There are also many ways to incorrectly generate what appears to be a random pattern. Ask your instructors for more information about how to randomize.
16. Subsequent care of your tadpoles (5 minutes EVERY DAY for the next two weeks). Make sure to go over this with us before you leave lab today. Bring your id card. The middle door between B5 and B7 has a card reader to let you in.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 9 a) Bring your lab notebook with you when you come to lab for a few minutes each day to check on your tadpole. Record what you did that day: fed, water change, etc. When you go in to check, here is what to check for and what to do: b) Is your tadpole alive? If yes, proceed to step c. If it's not swimming, touch it with a clean disposable blue pipette tip to see if it was just resting. If it's really dead, it will start to decay within a few hours. Pour the contents of the container with the tadpole into the dead tadpole container in the hood. We will take care of it. Record the date and time discovered in your lab notebook. c) Is the water cloudy? (This means bacteria and the oxygen depletion that they cause.) If it needs to be changed, proceed to next step. If not, proceed to step e. d) Water changing: as needed if water is cloudy, too much food was added. i) Get a clean small (40 mm diameter) petri dish and add some Canyon water or Lab water from the middle room (not DI water). ii) Move your tadpole with a plastic spoon into the petri dish. iii) Carefully pour dirty water down the drain. iv) Rinse tadpole container with DI water (left tall tap at sinks). v) Add 200 ml Canyon water or Lab water. Add penny-sized piece of boiled spinach before adding 1000 !l of predator cue water or 1000!l DI water depending on treatment. vi) Move tadpole back into its container. vii) Put the used petri dish in the dishpan by the back sink. e) Does the tadpole have food? If not, add another penny-sized piece of boiled spinach using forceps that are on the spinach box in the refrigerator in your laboratory. Please be sure to put the spinach back in the refrigerator. f) Replace the cover and put the tadpole back in its assigned grid position on the lab bench. Record what happened in your notebook with time and date. g) Come back tomorrow to check both tadpoles again.
During week 3 - Analysis of the class data will be performed in Lab during Week 3. You will be removing your tadpoles, taking the same measurements, entering them into the computer, and analyzing a class dataset. A report will be due the week following Thanksgiving break.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 10 Literature Earl, J.E. & Whiteman, H.H. Evaluation of Phosphate Toxicity in Cope's Gray Treefrog (Hyla chrysoscelis) Tadpoles. Journal of Herpetology 44, 201-208. Kaplan, R. H. and P.C. Phillips. 2006. Ecological and developmental context of natural selection: maternal effects and thermally induced plasticity in the frog Bombina orientalis. Evolution 60:142-156. Relyea, R. A. 2005. The impact of pesticides and herbicides on the biodiversity and productivity of aquatic communities. Ecological Applications 15:618-627. Sih, A., A. M. Bell, and J. L. Kerby. 2004. Two stressors are far deadlier than one. Trends in Ecology and Evolution 19:274-276. Van Buskirk, J. and S. A. McCollum. 2000. Influence of tail shape on tadpole swimming performance. Journal of Experimental Biology 203:2149-2158.
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 11
Lab I Tadpole Plasticity Reed College Bio101/102: Kaplan 12 ECOLOGY and EVOLUTION Week 1: October 29 – November 2, 2012
LAB 1-Part 2. Stickleback fish and Bombina tadpoles Basic computational skills for future work with variation
PLEASE READ the entire lab manual before coming to lab and ask as many questions as you like. Be sure to have your Bio Binder and Lab notebook with you. Today, you will be introduced to two vertebrate species that we will study repeatedly for this part of the semester. You will need to be familiar with three documents in the Bio Binder some of which should be reviewed: Computer Basics E, Measuring with ImageJ F, and JMP Instructions H. Specifically, you will become familiar with: 1) A fish that lives in Reed Canyon, the threespine stickleback, Gasterosteus aculeatus, and the amphibian larvae of Bombina orientalis, that we have studied for years in the field in the Republic of Korea. Bombina are not found in the field in North America. 2) Image analysis methodology via ImageJ.
1. Introduction to the Animals via the Encyclopedia of Life (Complete A and B before coming to lab this week. Your lab notebook is a good place to take notes as you go. Start lab today at Part 2 below.)
(This entire handout, Lab 1 quant skills F2012.pdf is accessible as a document from the Courses Server using AFS [see Bio Binder page E-2]. Clicking on the web links in the document is easier than typing them in.) A. Sticklebacks The threespine stickleback (Gasterosteus aculeatus) is an important model organism for evolutionary, developmental, and genetic studies. This is primarily due to the fact that the fish has long been a well-known study organism from a behavioral and ecological perspective, and its genome is nearly complete in DNA sequence. We started working with stickleback in the Intro. Bio. classes here in the Reed Canyon (http://web.reed.edu/canyon/) in November 2005. There are also two independent student stickleback projects (Nick Manoukis ‘95, Ezra Lencer ’05) and four senior theses (Meng Qi ’05, Max Press ’08, Annika Saltman ’08, and Laila Bryant ’09). This year in lab, you are going to gather and analyze data, and by comparing results, test hypotheses about
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 1 animal dispersion and population size and growth, and add to the long-term database as we track our populations in the canyon. For now, go to the Encyclopedia of Life website (http://www.eol.org/pages/1). This is a new and valuable site, conceived by the respected Dr. E. O Wilson of Harvard and developed through collaborations of the major biodiversity institutions and museums of the world. Enter Gasterosteus aculeatus into the Search EOL… space. Note that there are several different classification systems. For our purposes, find the one that follows the following path: Kingdom Animalia -> Phylum Chordata (chordates) -> Class Actinopterygii (the ray- finned fish group) -> Order Gasterosteiformes (dragonfishes)-> Family Gasterosteidae (sticklebacks and tubesnouts) -> Genus Gasterosteus -> Species Gasterosteus aculeatus (three spine stickleback). You do not have to memorize these, but play with the pictures. In the process, you will see how diverse the fishes are. Notice the tabs at the top of the picture of the stickleback. There is a lot of information there. For more detailed information on sticklebacks you can also check out Susan Foster’s link at http://www.clarku.edu/activelearning/departments/biology/foster/fosterD.cfm.html. We will be referring to this link again later in the semester.
Answer the following questions at home and know the answers for a future exam: Questions you must know how to answer using the Encyclopedia of Life: 1. What are the three most distinctive morphological features of chordates? (Find the answer in the Encyclopedia of Life -under chordates). 2. What is a current estimate of numbers of known and living species of ray-finned fish (as opposed to lobe-finned fishes)? 3. What other kinds of fishes are there besides ray-finned and lobe-finned?
B. Fire-bellied toad - Bombina orientalis– Like stickleback fish, amphibians are vertebrates, and are easy to handle. For an example of the latest research published by the Kaplan Lab, check out this article Kaplan and Phillips, 2006.pdf on the Courses Server. In addition, go to the Reed Library Home Page (http://library.reed.edu/). Search for Bombina in the Reed College Library. On the left, under Format, select “Thesis/dissertation (33)". Note that approximately 33 Senior theses by Reed College students are the first items to appear.
Now go back to the Encyclopedia of Life at http://www.eol.org/pages/1. Enter Bombina orientalis and find the classification system that is similar to the one that follows:
2 Reed College Bio101/102: Kaplan Lab 1Fish and frogs Kingdom Animalia (animals) -> Phylum Chordata (chordates) ->Subphylum Vertebrata - -> Class Amphibia (amphibians) -> Order Anura (frogs and toads) (Amphibia with -> Family Bombinatoridae -> Genus Bombina -> Species Bombina orientalis You do not have to memorize these. Using the AmphibiaWeb website (http://amphibiaweb.org/) browse by family. As with stickleback, the details of morphology, physiology, behavior, and the theory and science behind the experiments we set up today will be discussed in detailed in lecture. The complex life cycle (i.e., metamorphosis) adds an interesting wrinkle. To learn about anuran larvae, link to Tadpoles of the United States and Canada: A Tutorial and Key by Ronald Altig, Roy W. McDiarmid, Kimberly A. Nichols and Paul C. Ustach. http://www.pwrc.usgs.gov/tadpole/tutorial.htm.
Questions you must know how to answer: 1. How many major groups of living amphibians are there worldwide? (from Encyclopedia of Life). 2. How many species of living frogs, salamanders and caecilians are there? (from Amphibia Web; search the database, then about amphibians, then species numbers) 2. Comparative study of shape of two populations of stickleback in the Reed Canyon using a ratio of length to depth - testing univariate differences with Analysis of Variance (ANOVA) Find the two sample sites (Reed Lake and Ritmanis Pond) on the map below (Fig.1). You will be sampling these sites during weeks 2 and 3. You can see live fish in two tanks at the back of the lab today. Hypothesis that you will test today from digitized photos (in question form as opposed to null hypothesis form): Is there a difference in the shape of stickleback in the Reed Lake versus Ritmanis Pond, as determined by a random sample of 5 fish from each location in February 2005? (See Bio Binder Quant. Expt. Design G-2 – What is a Hypothesis?)
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 3
Figure 1. Reed Lake (Left) – Stickleback survey sites – Ritmanis Pond (Right)
4 Reed College Bio101/102: Kaplan Lab 1Fish and frogs 1. Work in PAIRS on the computer. Log in and go to the Courses Server (see Bio Binder Computer Basics E-2). Courses Server/Biology/Bio101 102 Intro Biology/3rd Evolutionary Ecology-Kaplan F12/ Laboratory Materials/Lab 1 Quantitative Skills 2. Drag to your Desktop, two folders called Reed Lake Stickleback and Ritmanis Pond Stickleback. These folders contain images of cleared and stained stickleback studied by Meng Qi ’05 during the course of her Senior thesis (Qi, 2005). She cleared away muscle tissue via trypsin digestion and stained the remaining animal with two different stains, one for bone and one for cartilage.
Figure 2. Stickleback fish stained for bone and cartilage. Photo by Lencer ‘06, 2005. Clearing and Staining by Qi ‘05, 2005.
3. Drag a file from inside one of the fish folders onto the ImageJ icon in the dock. (Go to Bio Binder Measuring with ImageJ F-1+2 and follow directions. Reminder from Bio Binder: before measuring the fish, be sure to pull down Analyze and Set Scale [use 20.0 mm] for each photo individually and measure 20 mm on the ruler in the photo.) After setting the scale and testing quickly with the ruler in the photo, drag the mouse from the nose or snout to the beginning of the tail (as shown in Fig. 2), and you'll see the Standard Length up in the ImageJ tool bar. It is only there until you release the mouse and then you have to measure again to get it back. Do the numbers make sense in relation to the ruler? Most fish are 25-45 mm in standard length.
4. Record in your lab notebook the FishID and location (i.e., whether it is from Reed Lake or Ritmanis Pond). Measure and record Standard Length and the body Depth to the nearest 0.1 mm for 5 fish from Reed Lake and 5 fish from Ritmanis Pond. Be sure to keep the standard length and the body depth for each fish associated with the specific fish ID.
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 5 Analysis 1. Open JMP and within it, open a New Data Table. (See Bio Binder JMP Instructions H-2.)
2. In this Data Table, enter the following 4 column headings: -Fish ID -Location -Standard Length (mm) -Body Depth (mm) Save your JMP file with an informative name to your Desktop. Record the file name in your lab notebook.
3. In your open JMP Data Table, enter: -Fish ID (As you enter a letter, notice that the column icon changes from a blue triangle to a red histogram . This is appropriate for a categorical variable.) -Location: Lake or Pond (This is case-sensitive to register as only two categories.) -Lengths and depths numbers only, mm units go in the column name (Column must be Numeric and Continuous as represented by the blue triangle column icon.) Be sure to Return after your last data entry before analyzing. -Transform length and depth to a shape ratio by adding a new column and naming it. Divide length by depth in column properties via formula. Click on length, then click on divide and then depth. OK, and then OK again. Save your JMP file to the Desktop regularly. Drag from the Desktop to your Home Server at the end of lab. (See Bio Binder E-2.)
4. See Bio Binder JMP Instructions H-7 for Frequency Distribution. Choose shape ratio as your Y (Response) Variable. This will be for both Lake and Pond fish together. Print two copies of the Distribution. Tape into your lab notebook.
5. See Bio Binder JMP Instructions H-10+11 for Analysis of Variance (ANOVA). Choose shape ratio as your Y (Response) Variable. Use Location as Model Effect for ANOVA. Follow instructions on H-10 until you have something like the output example on H-11.
6 Reed College Bio101/102: Kaplan Lab 1Fish and frogs Presentation 1. In JMP, Select and Copy, and then Paste the ANOVA graph and tables into a blank Word document.
2. From the Courses Server, drag a copy of the ANOVA report template.doc file to your Desktop, Open and Save As ... your filename. To help you learn to write figure legends and table captions and to report statistical results properly, this template models them for you. (See Bio Binder Lab Report Instructions S-4.) You can use this file for your report by replacing the existing graphs and tables with your results and modifying them appropriately. The following two pages show the original JMP output and the modified form for turning in as your report. Page 10 points out important differences between the two. Please do not Copy and Paste tables from JMP to the template. Type your JMP numbers onto the template.
3. In your lab notebook, indicate what you think the results show with one simple sentence that answers the following question. Are the fish from the two locations of different shape? (See Bio Binder H-8+9 on P values and reporting statistical results efficiently.) If the ANOVA P-value is <0.05, then you can reject the null hypothesis. If you can reject the null hypothesis, are the fish more sleek or stocky in the lake or the pond? If the ANOVA P-value is >0.05, then you fail to reject the null hypothesis and there is no statistically significant difference in shape between the lake and pond fish that you sampled. (See Bio Binder Statistics Explained I-6-8.) Did other people in the class get the same answer as you?
The null hypothesis you have rejected or not rejected is: There is no statistically significant difference in the ratio of standard length to body depth in a random sampling of stickleback pictures from the lake vs. the pond.
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 7 Original JMP ANOVA output looks like this (your numbers will vary). Note: Your Y axis and response variable will read Shape not Standard length. This ratio is a unitless number.
Extract parts of above JMP output to look like the template on the following page in order to turn in your report.
8 Reed College Bio101/102: Kaplan Lab 1Fish and frogs Names: Ann Jones, Joe Smith Mail Stop #1234 Lab day: Monday Lab instructor: Ned Knight Date: October 29, 2012
Shape comparison of stickleback fish in Reed Lake and Ritmanis Pond (Note: different Y –axis label in your results)
Figure 1. Mean standard length of stickleback fish (Gasterosteus aculeatus) in Reed Lake (N=5) vs. Ritmanis Pond (N=5) with 95% confidence intervals
Table 1. Analysis of Variance for standard length of stickleback fisha in Reed Lake vs. Ritmanis Pond Source DF Sum of Squares Mean Square F Ratio P Value Location 1 703.9 703.9 26.0 0.0009 Error 8 216.6 27.1 Total 9 920.6 a Gasterosteus aculeatus
The mean length for pond stickleback fish (42.6 mm) was statistically significantly greater than the mean length for lake stickleback fish (25.8 mm) (ANOVA, F=26.0, df=1,8, P=0.0009).
Be sure to read page 10 before turning it in.
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 9 Note the differences between the original JMP output and the format to hand in:
-No text goes immediately above the figure.
-Figure legend goes below the figure.
-Table caption goes above the table.
-Numbers in the table have the same number of digits to the right of the decimal place as the original measurements.
-Source of variation has been changed from Model to actual source in this case Location.
-Lettered footnotes go directly below the table. (Note: no footnotes go in figure legends.)
-Description of pattern and testing of null hypothesis goes last.
-If your P value was >0.05, your statement would be more like this:
The mean length for pond stickleback fish (32.2 mm) was not statistically significantly different than the mean length for lake stickleback fish (29.5 mm) (ANOVA, F=0.2, df=1,8, P=0.6797).
-Degrees of freedom (DF) is based on sample size, experimental design, and type of statistical test being done. It is often the sample size -1 or the number of treatments -1.
-For an experiment with this design, Location DF is 2 locations -1 =1 Error DF is Total DF - 1 =8 Total DF is 10 fish -1 =9
-For ANOVA, it is standard to report the treatment DF (Location in this case) and the Error DF.
Please turn in only one copy with both partners' names at the top as in the template.
10 Reed College Bio101/102: Kaplan Lab 1Fish and frogs
3. Allometry (another way to analyze body shape and how it changes over size using bivariate analyses of logarithmic data with two week old (14 dpf) Bombina tadpoles (see BioBinder G-5 on allometric theory)
Below are the two measurements (body length and tail height) that we need to measure using ImageJ (Fig. 3). You can see live tadpoles in tanks at the back of the lab.
Figure 3. Tadpole morphology. From Altig et al., (1999)
The hypothesis that we will test today from digitized photos (in question form as opposed to null hypothesis form): Is there a relationship between the maximum height of a tadpole tail and its body length? 1. From the Courses Server, drag the folder called Bombina Tadpole Pictures 14dpf to the Desktop (photos by Juliana Arrighi ‘07). Use ImageJ to measure Body Length (mm) and Tail Height (mm) for each of 10 tadpole images. Double-check your measurements by looking at the ruler and estimating in mm. Your numbers should be between 2 and 10 mm.
2. Record in your lab notebook, the Tadpole ID and measurements of Body Length (mm) and Tail Height (mm) paired for each tadpole. Note in your lab notebook that these tadpoles are 14 days post fertilization (dpf) at 24 ˚C.
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 11 Analysis 1. Open JMP, and within it, open a New Data Table. 2. In this Data Table, enter the following 5 column headings: -Tadpole ID -Body Length (mm) -Tail Height (mm) -log Body length (Use the Formula -> Transcendental ->Log10 option to transform Body length to logarithms) -log Tail height (Use the Formula option to transform Tail height to logarithms) -Give your file an informative name, and Save it to your Desktop. -Record the file name in your lab notebook. -Enter data, and be sure to Return after your last data entry before analyzing. -Save your JMP file to the Desktop regularly. -Drag from the Desktop to your Home Server at the end of lab.
3. Proceed with Bivariate Regression Analysis. (See Bio Binder JMP Instructions H-13- 14.) Put log Body Length (mm) on the X- axis and log Tail Height (mm) on the Y- Axis. -The equation for the straight line that JMP drew for you will be under the graph.
Presentation 1. Once you have your Bivariate Fit Plot, double click on the X-axis numbers (not on axis label) and select Format: Fixed Dec Width 12 Dec 2. Do this again to the Y-axis. This will present the axis numbers with the same number of significant figures.
2. Select the graph and tables and Copy, and then Paste them into your MS-Word document. -From the Courses Server, drag a copy of the Regression report template.doc file to your Desktop, Open and Save As... your filename. -The following two pages show the original JMP output and the modified form for turning in as your report. Page 16 points out important differences between the two. 3. Indicate what you think the graph shows. -Does body length help predict tail height in your sample of 10 tadpole pictures? -Is the P-value <0.05? -Do the 95% Confidence Intervals include a line with a slope of zero? (See Bio Binder Statistics Explained I-9.)
12 Reed College Bio101/102: Kaplan Lab 1Fish and frogs The null hypothesis you have rejected or not rejected is: There is no statistically significant relationship between log body length and log tail height among tadpoles in pictures sampled.
But a deeper hypothesis and the reason for the logarithmic transformation is about shape change over size (i.e., does the slope of the line differ significantly from 1.0?) m Note: log Body Depth = m * log Body length + b and Body Depth = b*body length If m = 1.0 then the equation is that of a straight line and Body Depth does not change proportionally to body length. If m > 1.0 then the function has an upward curve to it. If m < 1,0 it flattens out.
(Despite whether or not your line was significant in the first place, does the slope that you computed tend towards isometry, positive allometry or negative allometry?)(Bio Binder G-5).
Original JMP regression output looks like that on the following page (numbers will vary and note axes in the example are NOT in logarithmic units but yours should be [log Tail Height mm and log Body Length mm]).
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 13
Extract parts of the preceding JMP output to look like the template on the following page.
14 Reed College Bio101/102: Kaplan Lab 1Fish and frogs Names: Ann Jones, Joe Smith Mail Stop #1234 Lab day: Monday Lab instructor: Ned Knight Date: October 29, 2012
Bombina orientalis tadpole body shape analysis
Figure 2. Relationship between body length and tail height for Bombina orientalis tadpoles (N=10) with 95% confidence bands for the slope
Tail Height (mm) = -0.12 + 0.56*Body Length (mm) R2=0.62
As body length increases, tail height statistically significantly increases in B. orientalis tadpoles (ANOVA, F=12.8, df=1,8, P=0.0072). About 62% of the variation in body length explains the variation in tail height.
Add a sentence indicating whether the data suggest isometry, or positive or negative allometry.
Be sure to read page 16 before turning it in.
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 15 Note differences between the original JMP output and the format to hand in:
-No text goes immediately above the figure. -Figure legend goes below the figure.
-Put linear equation and R2 below the figure legend. -Numbers have same number of digits to the right of the decimal place as original measurements.
-Description of pattern and testing of null hypothesis goes last. -The % of variation explained comes from the R2 value.
-If your P value was >0.05, your statement would be more like this:
There is no statistically significant relationship between body length and tail height in B. orientalis tadpoles (ANOVA, F=0.1835, df=1,8, P=0.6797).
-Save files from the Desktop to your Home Server folder.
Due at end of lab today: -Make sure that both partners' full names are on both reports. -Upload two separate one-page pdf reports (stickleback fish ANOVA and Bombina tadpole Regression) to the Lab Report Drop Box on the Courses Server (files.reed.edu) before you leave lab today. Use the standard format for filenames: M_Able_Bell_fish.pdf and M_Able_Bell_tadpole.pdf References Arrighi, J. 2007. Making sense of unpredictable environments: variable temperature effects on embryonic growth in the Oriental Fire-Bellied toad (Bombina orientalis). Reed College Senior Thesis. Reed College. Portland, OR. Lencer, E. 2005. Morphological variation and demography of stickleback populations in the Reed College Canyon. Diack Summer Field Research. On file in Brehm Biodiversity Center. Reed College. Manoukis, N. 1995. A population study of threespine sticklebacks (Gasterosteus aculeatus) in the Reed College Canyon during the summer of 1995. Diack Summer Field Research. On file in the Brehm Biodiversity Center. Reed College. Qi, M. 2005. Morphological variation in populations of threespine stickleback (Gasterosteus aculeatus) in the Reed College Canyon. Reed College Senior Thesis. Reed College. Portland, OR.
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16 Reed College Bio101/102: Kaplan Lab 1Fish and frogs VOLUNTEERS NEEDED BEFORE LEAVING LAB TODAY
Remember there were two parts to today’s lab. The first was setting up your tadpole plasticity experiment. The second was learning how to measure and analyze morphological data. But, today we also have to prepare for next week’s lab in the field, and we need volunteers before leaving lab today.
Volunteers: Please sign up on the sheet on the instructor's bench in your lab to be among the 20-40 volunteer students who will set fish traps on Sunday November 4th. We will be here to help you from 3-4 pm.
There will be another volunteer opportunity to set traps on Sunday November 11th 3- 4pm.
EVERYONE Practice putting a trap together and have one of us check your work. Two hooks and two eyes match on one side to make a hinge, and two single eyes match on the other side and are secured by a clip attached to a rope.
When you leave lab today, walk North on the basement level, and turn left to exit and to see the Physics loading dock where you will meet to find hipwaders, walking sticks, and traps next week.
Be sure to come to lab lecture on Monday 12-12:50 pm for more details.
On your regular lab day, please come inside to lab to receive instructions. We will go out together to collect the traps. Please dress for the weather.
Lab 1 Fish and frogs Reed College Bio101/102: Kaplan 17