BIOL 217 FIELD TRIP TO COLLECT GOLDENRODS Please carpool to this site to limit cars making same trip. ACRES Mengerson Reserve is about two miles northeast on Stellhorn Blvd, just west of the Target shopping area at Stellhorn and Maplecrest. We will meet and park behind the post office, turning in after the Subway or, perhaps more safely, by entering the shopping center and going around after the Pizza Hut. We will meet HALF AN HOUR later than usual to give you time to get there. Also, this meeting will not take the full lab period.
BIOL 217 GALL COMMUNITIES Week 1 Page 1 of 7 We ve gotta lotta gall(s) Introduction Goldenrods (Solidago) are a very prominent plant in old fields and along roadsides in northern North America. The purpose of this lab is to examine interactions between goldenrods, insects which cause a gall to form on the stem of the plant, and the insects which can parasitize the gall formers. Goldenrods flower in late summer and are very apparent due to their tall structure, abundance and the bright yellow color of their flowers. By the fall, many goldenrod plants have galls (roundish growths) on their stems. These are usually caused by one to three species of insects whose larvae bore into the stem tissue of the plant and subvert the plant s genetic system into producing a gall which aids the larvae in thermoregulation and protection. The most common galls, ball galls (Fig. 1), are caused by one species of fruit fly, Eurosta solidaginis (Tephritidae: Diptera). These flies are not the same as Drosophila, or other small fruit flies with which you may already be familiar. The adult fly emerges in May from the gall and Figure 1. A goldenrod ball gall. Photo by Ken Storey. lays eggs in the terminal buds of Solidago. The hatched larva bores into the meristematic stem tissue and a characteristic round gall forms. The larva is full grown by mid-september and it enters a larval diapause over the winter and pupates in the spring. Elliptically-shaped woody galls (no figure) are caused by the larvae of a small moth called Gnorismoschema gallaesolidaginus (Gelechiidae: Lepidoptera). These galls are usually found lower down the stem of the goldenrod plant than those formed by E. solidaginis. Figure 2. A rosette gall. Photo credit: Hilton Pond Center. A third common gall, the rosette gall (Figure 2) is caused by the midge Rhopalomyia solidaginis. This midge causes a proliferation of leaves at the tip of the growing stem forming a dense rosette of leaves. Inflorescence production on stems with this gall usually results from the production of side branches from the main stem.
Although the galls provide protection for the larvae, they are not 100 percent effective. The fly larvae, for example, suffer considerable mortality from two parasitic wasps. Eurytoma obtusiventris (Eurytomidae: Hymenoptera) causes the fly to pupate in mid- August, consumes the fly and remains inside the puparium until the spring. Eurytoma gigantae consumes the fly larva and also eats some of the gall before pupating and emerging in the spring. A beetle larva, Mordellistena unicolor (Mordellidae: Coleoptera), bores in the wall of the gall and may occasionally eat the fly larva. Small dipteran (fly) larvae of the family Cecidomyidae may be found living in the gall walls as inquilines (i.e., commensals) without harming the larva. Finally, other predators, mainly birds, occasionally eat the larvae. The birds peck a hole in the gall and extract the insects. The above account of the system outlines the types of effects each of the elements has on one another. Our aim is to try to obtain something more than a purely descriptive summary of the system. We will attempt to quantify some of these interactions in order to gain a more precise understanding of the system s dynamics. We will investigate the effects of the galls on the goldenrod by determining if the reproductive potential of a plant is altered by the presence of a gall. This can be done by collecting goldenrods both with and without stem galls. The plans will then be dried, separated into their component organs, and weighed. The reproductive effort (ratio of inflorescence biomass to total biomass), the stem biomass/total biomass ratio, and the leaf biomass/total biomass can then be calculated. We will also estimate the mortality rates of Eurosa due to the parasites and other causes and determine whether the mortality rate is related to gall size. Methods Week 1: Field Collection We will travel to an old field to make our collections. See the associated map. Each group will collect three sets of galls. The first two sets of collections are designed to test the effects of galls on the growth and reproduction of goldenrods. For both ball galls and rosette galls, each group should collect 15 goldenrods with galls and the uninfected, health goldenrod closest to each infected stem. We will not work with elliptical galls in this lab, so do not include them in your sampling. Stems should be harvested at ground level and each pair of stems should be taped together and numbered. This is very important because we are making pair-wise comparisons. The third set of galls will be used to analyze sources of mortality in Eurosta solidaginis, the insect which causes ball galls. Each group should analyze a minimum of 25 ball gall. Since you will have already collected a set of 15 ball galls you will need to collect at least 10 additional ball galls. Pack your goldenrods into your plastic bag and take them to the lab, where we will hold them until the following week. It is okay to fold your goldenrods so that they fit.
Week 2: Laboratory Analyses To quantify the effects of galls on goldenrods, for each of your plants, measure gall type, the pair number (1 15), total height, and then the percentage of total weight in each of the following categories: stems, leaves, and flowers. You will use an electronic balance to measure masses. A table (better a spreadsheet so you can do calculations) with each of these bits of information (plus more below) in each row would be good. Calculate the total weight of each plant as the sum of the three kinds of tissue (stem, leaves, and flowers). This would be another column. To study the sources of mortality in the gall-forming insect and to test for relations between survivorship and gall size, we will dissect ball galls to study. The following information can be added to your table. Measure the diameter of each gall. Carefully dissect each ball gall and record the numbers of parasitoids, predators, inquilines, and mortality using the following characterizations of what you find inside: The pupa of a gall-forming Eurosta solidaginis larva (Figure 3a on next page). The pupa of the wasp Eurytoma obtusiventris, a parasite of the Eurosta larva (Figure 3b). This parasite causes the host to pupate prematurely (about mid- August) and then consumes it. The wasp larva then remains inside the empty puparium throughout the winter. The pupa produced is only ~ 2.2 x 7.4 mm, several times smaller than the pupa of the host. Another parasite, Eurytoma gigantea, this time in larval form (Figure 3c). This species consumes its host by the end of August, then remains in larval form in the central cavity throughout winter. It pupates there in spring. The central cavity is enlarged by E. gigantea and is usually filled with large black frass pellets. A larvae (caterpillar in form) of Mordellistena unicolor (Figure 3d). This is an inquiline (something which takes advantage of the pre-constructed cavity to live in it). IT eats through the epidermis of the gall and consumes the E. solidaginis pupa. Predated. Nothing inside, chiseled hole on outside of gall. This means a bird dug in there and ate the larvae or pupa. Natural mortality. All those larvae that are found for reasons such as improper development of the gall, imprisonment and the death of the larva in resins, and unknown reasons.
Figure 3. Interiors of goldenrod galls. From left to right: a) Eurosta pupa, b) pupa of Eurytoma obtusiventris, c) larva of Eurytoma gigantea, d) larval Mordellistena unicolor. Analysis of Data Calculate the percent of goldenrod galls in each of the classes (a-f) defined earlier. Then calculate the total percent parasitized and the total percent predated. Is there a relation between gall size and mortality rate? Use a Chi Square test to test for statistical significance. How to do this is described via example below. Determine the weight of stem, leaves, and flowers and the total weight for each plant. Calculate the percent of total above-ground biomass in inflorescence, stems, and leaves. Compare the data from the gall-bearing stalks and the normal stalks. Are there any differences in regard to mean or relative amounts of biomass among plant organs? Perform statistical tests (paired t Tests) on the data. Examples of how to do this are described below. You can use any statistical programs that you are familiar with and have available. If you are not familiar with a statistical program, you can use Minitab which is available on the student computer network at IPFW. Lab Reports You will report out from this study with a lab report. It is due from each group two weeks after the second lab of this project. Design the lab format following the format we discussed. Source of this Lab Variations of this lab are very popular with academics. They seem to be derived ultimately from the goldenrod research lab of Warren Abrahamson at Bucknell University. The website for the labs is: http://www.facstaff.bucknell.edu/abrahmsn/.
Using Chi Square (X 2 ) Contingency Test to Evaluate Gall Size vs. Mortality Data We have collected data on the relation between gall size and the survivorship of Eurosta in the ball galls of goldenrod. Consider the hypothesis that there is a relation between the size of the gall and the probability of natural mortality. This hypothesis could be tested in two ways. In the first, a t-test (independent, unpaired) could be run to determine whether the mean size of the galls with the live larvae differs from the mean size of galls with natural mortality. The second possibility is a chi square contingency test. This test is more powerful because it is more sensitive to extreme values. Chi square tests are based on actual counts in various categories. Generally, the grouping of data is arbitrary but categories with only the very low counts (say less than 5 should be combined). Chi square (X 2 ) is calculated by squaring the difference between the observed and expected values and dividing by the expected value. X 2 = (Obs. - Exp.)²/Exp. Where there is more than one cell you add the values together to get your total X 2. Thus: X 2.. The following contingency data is based on collections by two groups of ecology students during October 1986, at the Lahmeyer field (gall diameters in mm). Category <15 15-17 18-20 21-23 >24 Total Dead obs 6 17 5 4 2 34 exp 3.09 12.36 8.83 7.51 2.21 Live obs 1 11 15 13 3 43 exp 3.91 15.64 11.17 9.49 2.79 Totals 7 28 20 17 5 77 The expected value for each cell is calculated by multiplying the column total by the row total and dividing by the grand total. Notice that according to our null hypothesis, the expected proportion for live and dead larvae is the same for each size category. For example, in the <15 mm dead category the expected value is (7x34)/77=3.09. This is less than the number of dead larvae but more than the number of live larvae. For this first comparison, X 2 would be (6 3.09) 2 / 6, or 1.411. You would have to do this eight times and then sum. With the X 2 test, the degrees of freedom is equal to the number of columns (categories) minus one (5 1 = 4, in this case), times the number of columns minus one, which conveniently is one, so df equals 4. The cutoff for alpha < 5% for df = 4 is 9.488, so if your X 2 value is greater than that, the difference is significant. What is your X 2, and is it significantly greater than expected? When evaluating such data, you can also look at where values are higher or lower than expected so as to interpret the pattern. You might also graph your original data to see if it shows you a pattern.
Using a t Test for Paired Samples to Investigate Gall Impacts on Growth To test hypotheses about differences between two means when data are paired due to similarity on some factor or factors being controlled, we usually use the paired t test. In this test, we compute differences between the two members of each pair and examine the null hypothesis that the mean difference is zero. For this lab, we will be testing for differences between plants with and without galls, with pairs matched for local conditions. Compute t values as shown in the example below either manually or using a calculator. If you have access to computer programs that calculate t, you can save time. Sample Paired t Test Plant pair # Galled plant Ungalled plant Dif d² 1 6 3 3 9 2 14 8 6 36 3 8 4 4 16 4 4 6-2 4 5 16 9 7 49 6 7 2 5 25 7 19 12 7 49 d = 30 d² = 188 The format of the paired sample t Test is t equals the mean difference divided by the standard deviation of the differences, or t = d / s d. The mean difference is d = d/n = 4.29. The standard deviation (s d ) is the square root of the variance (s d 2 ) divided by (sample size - 1): s d 2 = (n d 2 - ( d) 2 )/(n*(n 1)) (you can find other algebraic variants of this this one is an equality for the standard version (a cheat )) = (7 (188) 30 2 ) / (7*6) = 9.90 s d = (s d 2 /(n-1)) = (9.90/6) = 1.28 t = 4.29/1.28 = 3.61 df = degrees of freedom = n 1 = 6 With α = 0.05, the minimum significant t = 2.45, so the difference is significant. For this imaginary variable, galled plants had a significantly greater magnitude (of whatever it was we measured).
You can set up data tables like the sample below, remembering to use proportions of biomass as data. The calculations are somewhat tedious, but not very difficult. In the sample calculations, the values of t required for significance were determined from a statistical table. You will need 6 such tables, 3 to make comparisons between the three plant parts for each of the gall types. Or a table with more columns - Sample Table for Calculation of t Pair # Galled Ungalled Difference d 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 = =