Hybridization of Sclerocactus glaucus and Sclerocactus parviflorus By Natalie Murrow 1 and Erryn Schneider 1 Mentored by Anna Schwabe 2 Frontiers of Science Institute , School of Biological Sciences 2 University of Northern Colorado Abstract There is concern that two species of cactus, Sclerocactus glaucus and Sclerocactus parviflorus, are hybridizing. Sclerocactus glaucus is a small cactus native to Colorado and is listed as threatened under the endangered species act. (U.S fish and Wildlife Service, 2010). Populations of S. glaucus may still be declining although no complete inventory data is available (The Center for Plant Conservation, 2010). Hybridization can have potentially negative effects on less common species such as S. glaucus. This study will look at genetic microsatellites to determine if S. glaucus and S. parviflorus are hybridizing. Genetic data has been gathered from samples of S. glaucus and S. parviflorus from 35 unique populations in the Grand Junction area by the Denver Botanic Gardens. Preliminary studies have been conducted on nine populations of S. glaucus and four populations of S. parviflorus. The results from this study indicate hybridization between the two species although more data is needed. Introduction Sclerocactus glaucus, commonly known as the Colorado hookless cactus, is an endangered species (Recovery Outline, 2010), and hybridization with Sclerocactus parviflorus would contaminate the gene pool, leaving few if any genetically pure S. glaucus in the wild. This would lead to an inability to save it from other threats because the species would have essentially disappeared. The purpose of this study is to determine if the two species are hybridizing and, if they are, whether there are genetically pure populations of S. glaucus so that scientists and conservationists can find and conserve them (Schwabe, Ramp-Neale, & McGlaughlin, 2011). An additional goal of this study is to learn more about the two species, finding out not only if they are hybridizing but where the populations come from and what we can do to stop the hybridization and conserve S. glaucus (Schwabe 2011a). It is hypothesized that S. parviflorus is hybridizing with S. glaucus. This is believed to be the case due to various changes in the genetic code as well as the appearance of populations S. glaucus that make them different from other populations. Because S. parviflorus is both closely related to S. glaucus and lives in the same range, it is very possible that the changes are due to S. parviflorus hybridizing with certain populations of S. glaucus (Recovery Outline, 2010). The genetic changes of S. glaucus reflect the genome of S. parviflorus, supporting the hypothesis further (Schwabe, 2011a). S. glaucus is an important part of Colorado’s biodiversity. But being endangered, there are many threats that may force it from its place in the ecosystem, such as illegal collection of specimens or oil and gas development (Recovery Outline, 2010). One of the most pressing threats is the possible hybridization of S. parviflorus with S. glaucus (2011 Colorado Native, 2011). If S. parviflorus genes are infiltrating populations of S. glaucus, as is believed to be the case, then S. glaucus will effectively go extinct (Schwabe, 2011a). Therefore, it is very important to find out if the two species are, in fact, hybridizing, so that this rare Colorado species can be saved from extinction. Results Because the study was not completed at the time of this poster being written, there are only preliminary results to present. Anna Schwabe (2011a) compared two populations of S. glaucus in a preliminary study, one isolated across a river and the other nearby two populations of S. parviflorus. The population that was not isolated showed more genetic similarity on nine loci to the populations of S. parviflorus than to the isolated S. glaucus. The full study will compare 15 loci on 792 different individuals from 35 distinct populations (Schwabe 2011b) to determine whether they truly are hybridizing. Sclerocactus glaucus being collected References Text: Recovery outline for the Colorado hookless cactus. (2010, April). Retrieved from U.S. Fish and Wildlife Service website: cactus_recovery%20outline_Apr% pdf Schwabe, A. (2011a, June 20). Sclerocactus glaucus hybridization. Reading presented at UNC Plant Population Genetics Lab, Greeley, CO. Schwabe, A. (2011b, July 19). The process of sequencing and comparing DNA. Address presented at UNC Plant Population Genetics Lab, Greely, CO. Schwabe, A., Ramp-Neale, J., & McGlaughlin, M. E. (2011, July). Analysis of nuclear DNA from S. glaucus and S. parviflorus to determine the level of directionality and hybridization between these two species. Poster session presented at Botany 2011, St Lewis, MO. Abstract retrieved from: 2011.botanyconference.org/engine/search/index.php?func=detail&aid=292 Science plan for McInnis Canyons National Conservation Area. (2010, September). Retrieved from: File.dat/McInnis%20Canyons%20Science%20Plan.pdf Sclerocactus parviflorus. (n.d.). Retrieved from Flora of North America website: Colorado Native Plant Society grant recipients. (2011). Retrieved from Colorado Native Plant Society website: Pictures: Erryn (left) and Natalie (right) extracting DNA Grinding samples with liquid nitrogen Conclusion The hypothesis that S. glaucus and S. parviflorus are hybridizing is supported by the preliminary data. Because the population of S. glaucus that was not isolated was more similar to the S. parviflorus than the S. glaucus population that was, it is likely that the population of S. glaucus and the populations of S. parviflorus are hybridizing. This data is all preliminary, however. This correlation by no means conclusive, but it does give incentive to continue with testing. Acknowledgements Thanks so much to our general sponsors, Space Connections Inc. for Natalie and The FSI Alumni of 1979 for Erryn, for giving us the opportunity to attend this wonderful and enriching program. Thank you also to our research sponsors, The Edward Madigan Foundation for Natalie and The Kinder Morgan Foundation for Erryn, for sponsoring our research and allowing us to help with this project. We would also like to thank Lori Ball for organizing FSI and for making this entire program happen. None of this would be here without you. Thank you to our teachers, Nick, Nathan, Abby, and Zabedah, for teaching us so much through out the course of the program and helping our research, writing, and science skills grow. Also, thank you so much to Anna Schwabe, our research mentor, who took us on and taught us a lot about DNA and working in a lab. We loved working with you, and we learned so much! Procedure DNA was extracted using the Qaigen DNeasy DNA extraction kit from samples of both S. glaucus and S. parviflorus and 2 µl of the sample were run on a gel to show the extraction was successful. This was the only portion of the procedure that the two of us participated in. The rest of the process is extensive and will take a long time to fully complete. Following extraction, a microsatellite library was made. A microsatellite is a repeating section of DNA (e.g. ATATATAT), which is highly variable and also quite common, allowing the specific genetic sequence to be compared and relationships to be found. To create the library, the DNA was cut up with restriction enzymes and mixed with chemo luminescent tags of nucleotide repeats that are in a solution. The tags attached to the microsatellite regions, allowing for the microsatellite to be identified and sent out to be sequenced. A primer was then developed from the microsatellite sequences to amplify the microsatellite region. The DNA was then diluted and replicated using the primers in a PCR (polymerase chain reaction). These fragments were then compared to determine relationships between them. Incubating samples during extraction A successful DNA extraction of S. glaucus from the Cactus Park population S. glaucus sample Labeled tubes for extraction Centrifuge with samples Space Connections, Inc. The FSI Alumni of 1979 The Edward Madigan Foundation