Taylor presented a poster of her summer research on fitness of native, non-native, and hybrid Echinacea plants at the Tennessee Louis Stokes Alliance for Minority Participation Conference. The meeting was held February 25-26, 2016 at the University of Tennessee-Knoxville. Taylor was awarded 3rd place in Science Poster Presentation category. Yay, Taylor!
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Now that I’ve finished collecting data from the Echinacea heads collected from Staffanson Prairie Preserve in 2015, I am able to start doing some data analysis. While the ultimate goal is to compare the data from 2015, a non-burn year, to previous burn years, I first want to come to a good understanding of what reproductive success looked like in 2015. For all of the analyses I will be doing, I am using computer software called R. R is a very flexible program that allows you to employ a wide range of graphical and statistical techniques including modeling, running tests, and clustering. Although R does have a somewhat steep learning curve, I have been learning many useful techniques in Stuart’s class on R at Northwestern and am confident in my ability to properly analyze the data I have collected. The histograms below show seedset, which is the proportion of achenes that contain a seed and can range from zero to one, for the entire head, as well as for the top, middle, and bottom sections of each head. For the entire head, the sample has a range from 0 to 0.86, with a mean of 0.48 and a median of 0.57. While the middle and bottom portions of the head had similar seedsets, the top portions of each head appear to have lower seed set on average when compared to the rest of the head. This is consistent with the findings of previous years and suggests that florets that are receptive to pollen later in the season may have diminished reproductive success.
An important variable in Echinacea reproduction, spatial isolation, is modeled in the below plot as a predictor of seedset. This plot highlights the importance of doing a careful visual exploratory data analysis before diving into more complicated statistical analyses. While there does appear to be the expected inverse relationship between seedset and spatial isolation, upon looking at this plot Stuart was immediately able to tell me that the two points on the left of the plot are the result of erroneous data. None of the plants have a nearest neighbor less than 5 cm away, so there must have been an error either in data entry or during the recording of GPS coordinates. Because we have records for the correct GPS coordinates of every plant, this will be a very easy error to fix, but had Stuart not looked closely at this plot, I may have done the entire analysis with incorrect data.
 The functional trait machine used in the Kittelson et al. paper. In 2015, we continued to study the effects of inbreeding on Echinacea angustifolia fitness. This experiment was planted in 2001 where each plant was produced from one of three cross types, depending on the relatedness of the parents: between maternal half siblings; between plants from the same remnant, but not sharing a maternal or paternal parent; and between individuals from different remnants. We continued to measure fitness and flowering phenology in these plants. This year, of the original 557 plants in INB1, 157 were still alive. Of the plants that were alive this year, 23.4% were flowering and 24.9% have never flowered. Among the plants that were flowering, average head counts was 2, with a maximum of six heads. Read previous posts about this experiment. Start year: 2001 Location: Experimental plot 1 Overlaps with: Phenology and fitness in P1 Products:The team collected fitness measurements during our annual assessment of fitness in all plants in P1. The below papers were published in summer 2015: Kittelson, P., S. Wagenius, R. Nielsen, S. Qazi, M. Howe, G. Kiefer, and R. G. Shaw. 2015. Leaf functional traits, herbivory, and genetic diversity in Echinacea: Implications for fragmented populations. Ecology 96:1877–1886. PDF Shaw, R. G., S. Wagenius and C. J. Geyer. 2015. The susceptibility of Echinacea angustifolia to a specialist aphid: eco-evolutionary perspective on genotypic variation and demographic consequences. Journal of Ecology 103:809-818. PDF The oldest Echinacea plants in experimental plot 1 will turn 20 this year. They are part of the 1996 cohort, which was planted in a common garden experiment designed to study differences between remnant populations and assess life history traits as they grew. Stuart sampled about 650 seeds (achenes) from eight remnant populations in and near Solem Township, representing the range of modern prairie habitat from small patches along roadsides to a large nature preserve. In 1996, he transplanted seedlings on a 1m x 1m grid, randomly assigning the location of each individual. Every year, members of Team Echinacea assess survival and measure plant growth and fitness traits including plant status (whether it is flowering or basal), plant height, leaf count, and number of flowering heads. We harvest all flowering heads in the fall and obtain their achene count and seed set in the lab. Of the original 650 individuals, 304 were alive in 2015. This year, 136 individuals from the 1996 cohort were flowering with a total of 303 heads. At present, these heads are in the lab where they await processing to find their achene count and seed set. We used 31 plants (45 flowering heads) from the 1996 cohort as maternal plants in crosses for the most recent heritability of fitness experiment (qGen3). We also used five plants from the 1996 cohort (8 heads total) as part of the pollen exclusion and addition experiment. We covered their heads with pollinator exclusion bags for the duration of the season. Read more posts about this experiment.  Stuart passes out pollen to Gina and Ben for crosses between 1996 cohort plants in the qGen3 experiment Start year: 1996 Location: Experimental plot 1 Overlaps with: phenology in experimental plots, qGen3, pollen addition/exclusion Products:
Much of the work I have been doing up to this point has been to determine a single number for each head—the proportion of all achenes on a given head that contain a fully formed seed, or seed set. This gives a good indication of how successful that plant was in terms of reproduction. The most likely reason that an achene does not contain a seed is that the flower did not receive compatible pollen, either due to a lack of mates or due to a limitation on the part of the pollinators. In order to determine seed set, I need two numbers: the total number of achenes and the number of achenes containing an embryo. While the achenes could be counted by hand, this would be a tedious and error-prone process. Instead, the achenes were placed on a glass tray and scanned into the computer and counted digitally. It is possible to determine whether or not an achene contains a seed by several methods. Germination experiments are useful because every achene that germinated certainly contained a seed, but they can be time-consuming and demand lots of attention and resources. Another possibility is to weigh the achenes. Heavier achenes are much more likely to contain a seed, and lighter achenes are most likely empty. We chose to use x-ray, which allows us to see directly inside of each achene. When achenes are x-rayed, empty achenes are barely visible while seeds show up as opaque. Ideally, all achenes could be easily categorized into “empty” or “full,” but some achenes are partially full, likely meaning they were initially fertilized but full seed growth was not entirely successful. Together, these numbers are very important in allowing us to make inferences about what conditions are best for Echinacea reproduction.  Randomized achenes ready to be x-rayed.
 This is what x-rayed achenes look like. Achenes that contain a seed show up with a white oval in the center.  Full, partial, and empty achenes are counted on the computer and entered into an spreadsheet. Randomization is a critical aspect of any experiment. In almost all cases, the population being studied is much too large to study every individual, so a sample of the population is studied with the assumption that trends and relationships seen in the sample are also present in the population as a whole. In order for this to be a good assumption, the sample must be completely random in order to eliminate any bias towards a specific type of individual. In an ideal world, all samples would be completely random, but this is not logistically possible in many cases. For example, at Staffanson Prairie Preserve, there are thousands of Echinacea that bloom every year. It would be a near impossibility to visit every single plant or even to select a completely random sample of plants within the preserve. For this reason, the Echinacea Project created a 10-meter wide transect through the preserve and studies the plants that fall within this transect. While this is not a truly random sample, it is able to approximate the range of conditions seen throughout the preserve. Another example of randomization is something I’ve been working on in the lab for the last week. Many of the heads contain several hundred achenes, so x-raying all of them to determine whether or not they contain a seed would be extremely time consuming and difficult. In order to simplify the process, I am randomly selecting 1/6th of the achenes from each head in order to estimate seed set for each head. While this will not give me the exact seed set, it will give me a very good approximation that will be sufficient for our analyses. Pictures of the randomization process are shown below—achenes are randomly dispersed on a wheel divided into twelve labeled sections of equal size. Next, two letters are selected from a list of random letters, and the achenes that fall within these sections are selected to be x-rayed.  Achenes on the randomization wheel  Randomized achenes–labeled and ready for x-raying Katherine Muller’s paper “Echinacea angustifolia and its specialist ant-tended aphid: a multi-year study of manipulated and naturally-occurring aphid infestation” was selected by the Editors at Ecological Entomology as the most interesting paper in the current issue (41:1). This means that her paper will be highlighted on the Journal’s website and made Open Access for the next two months, along with a summary of the paper and an image of the ants attending the aphids. Congratulations, Katherine! This paper was based on Katherine’s MS thesis in the Plant Biology and Conservation graduate program at Northwestern University. Katherine is now in a Ph.D. program at the University of Minnesota. Here’s the text that is on the Journal’s main page … Aphid abundance was manipulated on the perennial coneflower Echinacea angustifolia, which hosts a specialist aphid (Aphis echinaceae) tended by ants. Both have undergone extensive habitat loss and fragmentation. Aphids did not harm host performance after two years, though they did accelerate seasonal senescence. This experiment found a negative association between aphids and other herbivore damage, suggesting ant protection. However, observations showed the opposite trend, with larger plants more likely to have aphid infestation and leaf damage. The results suggest plant size drives foliar herbivory more than aphid infestation. The paper was co-authored by Stuart Wagenius, Katherine’s MS adviser.  Here are some of those cute little aphids!  This collection plant had some of the most aphids we’ve seen yet in one place!  Specialist aphids, Aphis echinaceae, on a head of Echinacea angustifolia  Ants tending Aphis echinaceae For the past three weeks, I’ve been hard at work collecting the achenes from Echinacea heads that were collected last summer from Staffanson Prairie Preserve. A little bit of Echinacea anatomy to give you a better idea of what I’m talking about: each head typically consists of 100 to several hundred small flowers or florets. After the head has matured and the florets have finished blooming, every flower produces one fruit, known as an achene, regardless of whether or not it was fertilized. Back at the lab, we clean all the achenes off the heads in order to count them and determine whether or not they contain a viable seed.  An intact head  Achenes In addition to cleaning the heads, I have also been separating the achenes by where on the head they were located. The florets bloom row-by-row starting from the base of the head and working their way up, so we know that the florets at the bottom bloomed first and the ones at the top bloomed last. Looking at reproductive success via seed set on the top, middle, or bottom of each head will thus give an indication of how the mating scene and pollen availability changed over the course of the mating season.  Hard at work cleaning a head Cleaning the heads is the first step in determining seed set, the primary measure of reproductive success I’ll be using for this project. Seed set is defined as the proportion of seeds that were successfully fertilized, and this number can range from zero to nearly 100%. While there are many reasons that fertilization may have failed, the primary reason is most likely a lack of compatible pollen. Now that all the achenes have been removed from the heads, I can determine what proportion of achenes on each head contains an embryo using x-ray. I’ll go into more depth about the x-raying process in another post. In 2015, we continued the study of mating compatibility in the remnants that began in 2014. This experiment is designed to assess population level compatibility and to investigate whether difference in flowering phenology and distance between plants predict whether or not a cross will be compatible. We do this by randomly selecting focal plants from remnant populations and then choosing pollen donors which are representative of the ‘extremes’ of these variables–early flowering, late flowering, nearest to focal plant, and furthest from the focal plant.  Bracts are painted to identify the pollen donor for each style of the focal plant that is being crossed This past summer we conducted this study in six of our largest remnant populations with approximately ten focal plants at each for a total of 228 pairwise crosses. Occasionally we were unable to collect pollen from the most ‘extreme’ individuals because they flowered asynchronously with the focal plant, and in those cases we chose the most ‘extreme’ individual available. Excluding all other pollinators, we performed hand-crosses between the focal plants and their pollen donors and assessed style persistence the following day to evaluate the compatibility of each cross. Read more posts about this experiment here. Start year: 2014 Location: large remnant populations Overlaps with: comprehensive compatibility Products: The 2015 data from this experiment has been combined with the 2014 dataset and awaits analysis. Team members who have worked on this project include: Danny Hanson (2015), Amy Waananen (2015), and Claire Ellwanger (2014). Flog posts authored by these team members may provide additional detail about day-to-day activities associated with this experiment. I’ve been helping out around the lab since the fall, but this is my first post on the Flog, so I’ll go ahead and introduce myself. I’m Gordon, and I’m a senior at Northwestern University studying Environmental Science and Chemistry. I have the awesome opportunity to conduct an independent study here at The Echinacea Project—not only will I learn research techniques and scientific writing skills, but I’m also able to get class credit for my research, allowing me to devote more time to the project.
Me with one of the many heads I’ve dissected The main focus of my research is how fires affect the reproductive success of Echinacea. The existing scientific literature suggests that fires (or a lack thereof) redistribute resource availability, giving a survival advantage to certain species of plants. For example, a fire can burn tall prairie grasses to the ground, allowing shorter plants to access sunlight and contributing to their survival. In areas where prairie fires are suppressed, which includes many locations where prairie remnants exist today, it is thought that plants that benefit from fires will become scarcer. However, there is scant scientific literature regarding how fires influence the reproductive success of prairie plants.
At Staffanson Prairie Preserve, prescribed burns are conducted every five years, providing an ideal setting in which to conduct this observational study. I will be examining at plants that flowered both in 2015 (a non-burn year) and in a previous burn year. By comparing their reproductive success in both the burn year and non-burn year, I hope to gain an understanding of what influence fires may have on Echinacea reproduction. The measures I will use to study reproductive success will include: achene count, which indicates resource availability and reproductive effort; style persistence, which measures the pollen availability and limitation; seed set, which measures the success rate of seed production; and fecundity, which serves as an indication of total reproductive success.
Stay tuned for weekly updates about my procedure and progress over the next several months! |
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