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A DNA BARCODING APPROACH TO IDENTIFYING NEWLY TRANSFORMED JUVENILE MUSSELS RECOVERED FROM NATURALLY INFESTED FISHES
Sarah L. Boyer, Biology Department, Macalester College, Saint Paul MN
DNA barcoding is a method for species identification using a short stretch of mitochondrial DNA. We have developed a DNA barcoding approach to the identification of recently transformed freshwater juvenile mussels, which are in many cases impossible to identify to species using morphology alone. We have built reference sequence datasets for two mitochondrial genes from adult mussels of known identity, and successfully used our method to identify juveniles recovered from multiple species of naturally infested fishes in the St. Croix River.
We sequenced two mitochondrial loci: ND1 (NADH dehydrogenase subunit 1) and COI (cytochrome c oxidase subunit I). For 7 of the 40 unionid mussel species native to the St. Croix River, sequences from both loci were already publicly available on GenBank. For all mussels native to the St. Croix River without at least two sequences from different specimens for both genes, collection of mantle tissue clips from adult mussels was performed in the St. Croix and upper Mississippi River. Using DNA extraction, PCR, and DNA sequencing, we were able to build a reference dataset for 37 of the 40 mussels in the St. Croix River.
Three species of fish were collected from the St. Croix and subsequently held in aquaria: walleye, freshwater drum, and spotfin shiner. To ensure that mussels collected from the parasitized fish represented species that successfully metamorphose on the host species in question, as opposed to species that infest but do not metamorphose on the host, we waited for transformed juveniles to fall from the fish by natural means and collected them by siphoning aquaria, rather than attempting to excise them from the fish. Juvenile mussels were photographed and DNA was extracted from each specimen, and we used PCR and DNA sequencing to generate sequences from our genetic loci of interest.
We successfully used phylogenetic analysis of sequences from adult and juvenile mussels to identify juveniles with a high degree of confidence. From the drum we identified Ellipsaria lineolata, Leptodea fragilis, and Potamilus ohiensis; from the spotfin shiner we identified Fusconaia flava and Pleurobema sintoxia, and from walleye we narrowed down the identification of juveniles to either Lampsilis higginsii or Actinonaias ligamentina.