Assessing the genetic diversity of native and non-native Phragmites (common reed) in Wisconsin Rachel Bautzmann, Mentor: Dr. Tippery University of Wisconsin – Whitewater, Department of Biology Introduction Phragmites australis (common reed) is a species of grass that can be found in Wisconsin and much of the northern part of North America There are both native and non-native subspecies While there has been previous research completed on samples from Wisconsin, we aimed to expand the number of locations where P. australis had been collected from Many of these locations included the northern and central regions of Wisconsin We are looking to determine the genetic variation between the two subspecies to help predict the ecological impact the non-native species may pose to Wisconsin We hypothesized that the native species would be found more in northern and western Wisconsin while the non-native species would be found closer to Lake Michigan and the eastern side of the state Results From looking at previous year’s results, as well as looking at the results from the samples tested this year, we still see a wide degree of genetic variation in both native and non-native subspecies Comparing the native and non-native species directly, results have shown that the non-native species holds more genetic variation between samples than the native species does (Figure 1) By analyzing the data using principal components analysis, two distinct groups can be seen and these different groups coincide with either the native or non-native subspecies (Figure 2) Figure 3. Our results from Genious show that the primer pairs were successful in producing DNA fragments and the samples tested in this plate contain similar alleles. The red numbers indicate a ladder where base pairs are located. The peaks in green and blue indicate the alleles present (signified by the number of base pairs long that DNA strand is) that the primer HEX or FAM amplified in a sample of Phragmites. Figure 4. Map of Wisconsin showing the locations and different types of alleles present. The map on the left represents the native species while the map on the right represents the non-native species samples. This specific map shows the results for the primer HEX-14. Insert picture of phragmites Figure 1. Map of Wisconsin showing the locations and different types of alleles present. The map on the left represents the native species while the map on the right represents the non-native species samples. The color of the pie chart indicates which allele (number of base pairs) is present in that sample. Sample numbers (located next to the pie chart) from “040” and above indicate new samples added to the map from our studies completed this summer. This specific map indicates alleles found with the FAM-04 primer set. Discussion We had success amplifying eight primer pairs and obtained results that are reproducible By using the data collected at the molecular level, we are able to make distinctions between the native and non-native subspecies Compiling this data allows us to see where certain alleles are more frequent and which subspecies they belong to As more samples are collected from different locations throughout Wisconsin we can continue to track allele trends in these two subspecies After looking into our results, it looks as though sample “P044” on the australis (non-native) chart has genotypes that are abnormal and might warrant a further analysis Our results also show that there is more genetic variation among populations of the non-native species Continuing to receive samples from throughout the state will allow us to track invasion trends of the non-native species Captions to be set in Times or Times New Roman or equivalent, italic, between 18 and 24 points. Right aligned if it refers to a figure on its right. Caption starts right at the top edge of the picture (graph or photo). Methods Over 100 samples were sent to us from various locations throughout Wisconsin Samples were stored in the freezer to preserve tissue DNA extraction was completed on each of these samples using a CTAB/chloroform method After extraction samples were diluted to 10ng/ul and measured on a Nanodrop to ensure DNA was present in the extractions 8 microsatellite pair primers were used to measure differences in lengths of spacers to determine the population the samples belonged to Microsatellite forward primers had a fluorescent tailed regions Polymerase Chain Reaction (PCR) was completed on each sample with all 8 of the primers To ensure that the PCR reactions were successful, gel electrophoresis was used Once PCR success was determined, samples were sent to a lab to to be analyzed These results were then examined in the program Geneious (Figure 3) Acknowledgements Our research was funded by the University of Wisconsin-Whitewater through the Undergraduate Research Program and we received all of our samples from numerous individuals throughout the state of Wisconsin. Figure 2. The results from principal component analysis from samples with microsatellite genotype data. Those numbers colored blue represent the native species while brown represents the non-native species. References: Phragmites photos taken from: https://www.olanderpark.com/tops-works-to-reduce-invasive-phragmites/ https://www.watershedcouncil.org/phragmites.html