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Using ‘DNA Barcoding’ to Determine Host Fish Usage of Freshwater Mussels in the St. Croix River Purpose: To build a library of genetic sequences for all known species of freshwater mussel found in the St. Croix River to act as a molecular key for identification of unknown juvenile mussels. To determine the efficacy of DNA barcoding of the juvenile stage of freshwater mussels for which morphological identification can prove difficult and time consuming. To examine which mussel species utilize which species of fish as hosts in the wild. Alexander A. Howe, Sarah L. Boyer, Mark C. Hove, Daniel J. Hornbach Macalester College – St. Paul, MN An adult Quadrula metanevra taken from the St. Croix River. A small tissue sample from the mantle tissue was taken before returning it to the water. COI Phylogeny Red: Juveniles and their grouping. Green: Adults sequenced in lab Black: Sequences from GenBank Materials and Methods: Available COI and ND1 sequences for native St. Croix mussel species were obtained from GenBank. For species with less than two COI and ND1 sequences available on GenBank, mantle tissue clippings from adult mussels were obtained from the St. Croix River. Juvenile mussels from naturally infested fish were obtained from several fish species caught in the St. Croix River. The fish were brought back to a wet lab, sorted by species, and put into separate tanks. The tanks were periodically siphoned and the siphonate was carefully examined for any juveniles. Juveniles were stored in ethanol until subsequent extraction DNA was extracted from the mantle tissue of the adults and from the entire juvenile with a Qiagen DNeasy Tissue Kit and amplified for COI and ND1 using separate sets of primers and PCR profiles. A QIAquick PCR Purification Kit was used to purify successful PCR products. Florescent tags were added to the amplified sequences through cycle sequencing and cleaned with a Beckman’s magnetic plate and beads. Sequencing was preformed with a Beckman-Coulter CEQ8000 Genetic Analysis System. Sequences were edited using CodonCode Aligner and aligned with MacClade. Phylogenetic trees were created with the program PAUP and posterior probability values were obtained using Bayesian Analysis with the program Mr.Bayes. Results: Introduction: North America has the most diverse assortment of freshwater mussels in the world, yet over 70% are federally listed as endangered, threatened, or of special concern. Mussels are an important part of a healthy aquatic ecosystem, filtering large quantities of organic matter from water. Freshwater mussels have a complex life history. The larvae of most freshwater mussels, called glochidia, are obligate parasites of fish and attach to their fins, gills, or scales for dispersal and metamorphosis into the juvenile stage. Host specificity means that conservation efforts aimed at endangered species of mussels must include the fish host(s) as well. Identification of glochidia in the wild to the species level based on morphology alone can be very difficult and in some cases impossible due to their small size. This makes it difficult to identify what species of mussel are parasitizing what species of fish. DNA Barcoding uses a short, standardized, mtDNA sequence (~650bp) to identify an organism to the species level. Cytochrome c oxidase subunit I (COI) is the generally accepted barcoding gene. NADH dehydrogenase 1 (ND1) is another mitochondrial gene that is commonly used in bivalve studies. Using both COI and ND1 allows for a higher identification success rate than using one gene alone would. mtDNA is used because it has a fast mutation rate allowing for significant differences between species, but little difference within species. Identification to the species level based on genetic data is possible. With a library of known adult mussel sequences it becomes possible to identify the juveniles that fall off of naturally infested fish. 8 of 9 larvae analyzed from spotfin shiner group with Fusconaia flava. 1 groups with Pleurobema sintoxia. All larvae analyzed from walleye group with both Lampsilis higginsii and Actinonaias ligamentina, though 2 larvae appear to be different fromthe rest. Results are consistent for larvae from spotfin shiner for both COI and ND1. Bayesian osterior probability values (shown in red for selected groupings) are very high for both, giving strong support for these groupings. Two views of a juvenile freshwater mussel which fell off of a bluntnose minnow. At this age and size it can be very difficult to identify the species by morphology alone. ND1 Phylogeny Red: Juveniles and their grouping Green: Adults sequenced in lab Black: Sequences from GenBank Conclusion and Future Work: The results shown here indicate the viability of DNA barcoding as a method for species identification of freshwater mussel larvae as an alternative to morphological identification. The resolution of identification that DNA barcoding provides seems to generally be quite strong, however in the case of the larvae from the walleye it is unable to resolve between Lampsilis higginsii and Actinonaias ligamentina. The resolution will likely become enhanced as more adult voucher sequences are added to the dataset. There is still much work to be done. Around 85 more adult voucher specimens will be sequenced to enhance the sequence library and will cover every species of mussel known to the St. Croix. Approximately 65 more unknown mussel larvae coming from 4 fish will be sequenced to examine in greater detail the efficacy of DNA barcoding on freshwater mussels. Acknowledgments: Thanks to Suzy, Emily, Scott, the Hornbach lab, and the Hove lab for all their time and help.