I’ve had an amazing semester here in the Echinacea Lab, and today I presented my final update on my internship project. I was able to receive feedback on how to improve both my project and presentation skills. One of the suggestions I received was to add more background to my presentation, so here is some supplemental information to go along with the PowerPoint (attached below). I was working with the P01-nat batch for two consecutive years, 2021 and 2022. I was looking at the plant health indicators of number of basal leaves and length of the longest basal leaf from 2021 because plants receive energy through photosynthesis. My though process was that leaves that are longer and more abundant would lead to a greater ability of an individual plant to photosynthesize and therefore invest more energy in reproduction. I was looking at the reproductive effort and success in the following year, 2022, since Echinacea angustifolia are long-lived perennials. The individual plants that I was working with were originally planted in 1996, so they were pretty well established in the study sites in Minnesota.
I also received feedback on my experimental design, including changing my experimental design a little bit. My current study is phrased as causally linked factors but is more in line with exploring an association between basal leaves and reproductive effort and success rather than a causation. In order to explore more of a causal relationship, one of the suggested studies was to clip leaves so that there was a randomized manipulation on the plants instead of an observational study. Limiting the basal leaves of random plants could allow for a stronger causal relationship to be established between the two factors. A second suggestion to strengthen my current study was to include data from multiple years, since Echinacea angustifolia are long lived and potentially have certain years where reproductive effort spikes over their life cycle and doesn’t spike again, as is one of the potential implications in Sophia’s pollen limitation study.
My hypotheses were not supported by my data, but they still have implications for the further potential future study I mentioned above. The data I collected did not support my hypothesis because the p-values were too high, meaning the data was not statistically significant. I received a suggestion that I should investigate one plant seen in the total achene count graphs (slide 6 of my presentation) that had no basal leaves but produced 200 achenes in the following year. It is possible that this individual only had cauline leaves in 2021, in which case it wouldn’t be relevant to a correlation between basal leaves and reproductive output. Two directions for additional studies that I suggested were the relationship between plant height and reproductive output and overall reproductive fitness as it relates to the number of heads on a plant. The latter question is one that I was going to explore, but I chose to combine the data for my study so that each data point represented the plant as a whole.
I am really grateful to have had the opportunity to be a part of the Echinacea lab this fall. I learned a lot about working in a lab and data analysis using R. I want to give special thanks to Wyatt, Stuart, and Sophia for helping me with R and my project overall, and to all the volunteers and student workers who helped me count, classify, and randomize the 2022 P01-nat data.
This week I worked on randomizing and counting. Thanks to the help of the student workers and volunteers, there is only about a quarter of the P01-nat batch left to randomize. I am hopeful that I will be able to complete the randomizing by the end of next Tuesday.
Today I spent my day counting achenes from the scans that I completed earlier this month. The counting process takes place on the ACE website. Each individual scan requires three different people to count it, and then the average of those three counts is taken as the most accurate one. I am completing one of the three counts for the entire batch. The ACE website is very user friendly and straight forward. The images are counted in a random order to keep the process as accurate as possible. To begin counting, I click the “count achenes” button on the dashboard (Right of Image 1). The first thing I do for every new scan is to confirm that the file name matches the envelope displayed in the scan (Image 2). After I confirm the file name and letno, I click on each achene and a blue dot is overlayed on the image (On the right of Image 2). The software keeps track of how many achenes I have counted in the image. Once I have double checked that I have accounted for every achene, I click “submit count”. The website goes back to the dashboard, and I begin the process over again. As of the end of the day today, I am 55.68% done with counting P01-nat (On the bottom of Image 1). Many thanks to the volunteers for helping me reach this point!
Image 1. ACE dashboard Image 2. A scan after all achenes have been counted.
This week I worked on scanning, randomizing and counting for P01-nat. The batch is now entirely scanned, and the images are up on the ACE website for counting! The other volunteers and I have completed 26.2% of counting as of the end of the day today. We are half done with the randomizing for the batch, and I am hoping to have that step of the process completed by the end of next week. Today I was also trained in how to prepare sheets for x-ray (end result of that process pictured below in Image 1), and I should be completing that process with P01-nat within the next couple of weeks! I have also included a picture of the end result of the rechecking process, which I detailed in my post last week (Image 2).
Image 1. Informative achenes ready for scanning.Image 2. Labeled envelopes and bags after rechecking is completed.
For my research project, the Echinacea heads in batch P01-nat have to go through the processes of cleaning, rechecking, scanning, counting, randomizing, x-raying and finally classifying before I can analyze the data. In my last blog post I detailed the methods of randomizing, and in this one I will be describing the processes of cleaning, rechecking, and scanning, the latter two steps which I have been working on in the past couple of weeks for P01-nat.
Cleaning and rechecking are very similar processes. During the cleaning process, I gently crush the Echinacea head against a Pyrex dish to separate the achenes from the head, and I use forceps to carefully remove the achenes that don’t fall out on their own. Once all of the achenes are removed from the receptacle, I put the chaff in one envelope and achenes in another and label each envelope with my initials, the date, the twist tie color, and the contents of the envelope. I put the twist tie, empty receptacle, and the two coin envelopes in the small brown paper bag. I initial and date the data sheet next to the proper letno. The rechecking process is done to check for achenes that might have been missed during the initial cleaning. For this step, only the chaff, receptacle, and twist tie are looked at again by a second person. At the end of rechecking, letno stickers are placed on the coin envelopes and the coin envelopes are then placed in numerical order in cereal boxes in preparation for scanning (Fig. 1). The same information that was recorded in the data sheet for the cleaning is recorded again under a new column for rechecking. Stay tuned, photographs of this process will be added next week!
The method for scanning the achenes is straight forward. The scanning setup is pictured below (Fig. 1 and 2). I scan each envelope of achenes one at a time by scattering the achenes evenly on a glass bottomed scanning tray, which is placed directly on the scanner. I place the coin envelope, letno label down, underneath the scanning tray and make sure that no achenes are behind it. Each scan is done at 400dpi, and saved to the server in the P01-nat folder under the same file name as the letno in the format “N-number-letter”. Once all of the achenes in each set are scanned, they are moved to the randomizing tray, which is a process I detailed in my last post. The digital files will be uploaded to the ACE website for counting once all of P01-nat has been scanned, which could be as early as next week!
Fig. 1 – Coin envelopes of achenes scanned and waiting to be scanned. Fig. 2 – Scanning setup.
I start the process of randomizing with the coin envelopes of re-checked achenes. The first part of the process is spreading the achenes out evenly across a circle that is sectioned off into 11 parts, each identified with a letter. Using a random letter generator, I select two sections of achenes. I put the rest of the achenes back into their original coin envelope. I sort and count the achenes that were selected by the random number generator into two groups, informative and uninformative. Achenes are uninformative if they are broken, underdeveloped, predated or ray achenes because these types of achenes are either known to be sterile or the seed could have fallen out during the cleaning process. I then put a label with identification information on both a white coin envelope and a clear plastic baggie. On the white envelope, I write the number of informative and uninformative achenes as well as my initials and the date. I put the uninformative achenes in this envelope and the informative achenes in the clear plastic baggie to be x-rayed. I record the data for the number of uninformative and informative achenes in the 2022 randomizing data sheets and put the coin envelopes back in their original box and the randomized ones in a new box so that they can be prepared for x-raying. This week I switched from the bbMost 2022 batch to a smaller batch that will be more manageable to complete during the remainder of my semester long internship with the Echinacea Project. I will be carrying out the randomization using this method after the rechecking and scanning of the new batch is completed.
This week I started the process of randomizing the echinacea achenes in preparation for being x-rayed. I am working with the 2022 achenes and the 2021 field data for my project to determine if health traits can be predictors of reproductive health in the following year. The purpose of randomizing the achenes is to get a sample of achenes to x-ray that is representative of the total number of achenes on that head. I start the process of randomizing with the already re-checked achene coin envelopes. The first part of the process is spreading the achenes out evenly across a circle that is sectioned off into 11 parts (on the right of the picture below). Once all of the achenes are spread out into sections, I use a random letter generator through the ACE website which gives me the two sections of achenes I am going to be working with. I move these achenes to the counting sheet (on the left of the picture below). I put the rest of the achenes back into their original coin envelope to keep them separate from the ones I am going to be sorting.
I examine each of the achenes on the counting sheet and sort them into the bottom numbered spots if they are uninformative or into the top numbered spots if they are informative. Achenes are uninformative for x-ray if they are broken, underdeveloped, predated or ray achenes. These are deemed uninformative because they are either known to be sterile or the seed could have fallen out if the achene was crushed during the cleaning process. I then label a white coin envelope with my initials, the date, and the number of uninformative and informative achenes in the batch. I put the uninformative achenes in this envelope and use a clear plastic baggie for the informative achenes that are going to be x-rayed. I put a label on both the envelope and clear plastic baggie with the assigned head number, batch number and if they are informative or not. I record the data in the 2022 randomizing data sheets and put the coin envelopes back in their original box and the randomized ones in a new box so that they can be put on x-ray sheets during the next step. So far I have randomized 24 out of approximately 500 heads in the bbMost batch for 2022!
It’s the beginning of my fifth week and so far I have learned the first three steps of the ACE process, cleaning echinacea heads, rechecking, and counting achenes. I have been practicing and refining all of these skills over the past few weeks and last week I participated in the trial rechecking assembly line with several other volunteers. As for counting, my official achene count is up to 14,485!
I am currently working on choosing my research topic for the remainder of my semester. The three ideas that I am choosing between are seed predation in E. angustifolia, climate change and flowering times of echinacea angustifolia, and physical characteristics of echinacea angustifolia that could be predictors of survival of the individual or reproductive fitness. I have chosen to use my time in the echinacea lab to investigate the relationship between the basal and cauline leaf characteristics and the survival of individual E. angustifolias. Pictured below is an example of a cultivar basal leaf rosette from an echinacea at Chicago Botanic Gardens. I am excited to explore this question further throughout the semester.
I am interested in plant conservation and learning how a long-term research project is conducted. I am also interested in seeing what impacts of climate change can be observed in a study like the Echinacea Project.
Statement
I am from New Hampshire and enjoy photography, especially nature and wildlife photography.
