Development of Molecular and Morphological Resources for Identification and Monitoring of Freshwater Mussel Species in the Genera Fusconaia and Pleurobema in the Green River, Kentucky

Freshwater mussel species in the genera Fusconaia and Pleurobema are particularly challenging to identify in the field. In this study, mussels from these genera were collected from the Green River, Kentucky for genetic and morphological analyses. I used molecular markers to detect any cryptic species within these genera and to test for genetic differentiation between two closely related nominal taxa P. rubrum and P. sintoxia using both mitochondrial (ND1, COI, 16S rRNA) and nuclear (ITS1) DNA sequences. After species identification, I used microsatellite DNA markers to estimate genetic diversity and effective population sizes (Ne) of species of Pleurobema. I used microsatellite primers that were developed for P. clava and P. pyriforme in previous studies, as well as microsatellites that I developed for P. plenum. Finally, I assessed morphological variation in my study species and developed dichotomous keys for the identification of both live mussels and shells. My results suggest that P. rubrum and P. sintoxia are the same species based on the mitochondrial DNA analyses, as there were few genetic differences between them. My results showed phylogenetically distinct lineages for F. flava, F. subrotunda, P. cordatum and P. plenum but no cryptic species were detected in the Green River. Current and contemporary Ne showed that these species have large population sizes that should allow for avoiding inbreeding and maintaining their evolutionary potential. Large genetic diversity as well as long-term effective population size could be the result of these species historically occurring as much larger assemblages that extended into the Ohio River and its numerous tributaries. The last objective was to assess morphometrical differences among these species. Using Canonical Variate Analysis, I found discernable morphological differences between the investigated species of Fusconaia and Pleurobema. The two Fusconaia species were morphologically different from the Pleurobema species. However, the Canonical Variate Analysis did not show differences among the Pleurobema species. I used decision tree analysis to develop a dichotomous tree, and random forest analysis was used to aid in the development of a dichotomous key by finding the most important diagnostic characters to distinguish these mussels. I then used the less subjective and easier to identify characters for the development of my dichotomous keys for live mussels and shells. However, both keys need to be tested in the field to determine their effectiveness. I could not separate P. rubrum and P. sintoxia mussels for morphometric analysis due to the lack of genetic differentiation and the inconsistent identification by the experts. However, I did describe a few individuals that look like P. rubrum and P. sintoxia to the eye of the experts. The description of these individuals matched previous descriptions of these mussels. Future studies need to assess taxonomic relationships among these species using genomics approaches, which might result in better node resolution. High genetic diversity and large effective population numbers for Pleurobema species suggest that these species' populations are genetically healthy. However, these results need to be interpreted carefully, and I therefore recommend additional studies to assess life history, habitat, host-fish availability, and current reproduction of these mussels in the Green River.