Select freshwater mussels in the genera Fusconaia, Pleurobema, and Pleuronaia were collected primarily in the upper Tennessee River basin from 2012 to 2014 for phylogenetic and morphological assessments. Freshwater mussels in these genera are similar in appearance, hence the need for phylogenetic verification and morphological assessment. Phylogenetic analyses of the mitochondrial gene ND1 and the nuclear gene ITS1 revealed three unrecognized, phylogenetically distinct species. These species were separated from their closest congener by 2.85%, 3.17%, and 6.32% based on pairwise genetic distances of ND1. Gaps created from aligning ITS1 sequences were coded as fifth characters, which phylogenetically separated most closely related species. Analyses of ND1 agreed with previous literature on the phylogenetic distinctiveness of Pleuronaia species, with the exception of the DNA sequences of P. gibberum, which grouped outside this genus based on the analyses conducted in this study.

Morphological variation was recorded for eight of the species to include quantitative and qualitative characters as well as geometric morphometric analyses. Three decision trees were created from quantitative and qualitative characters using classification and regression tree analyses. The best-performing tree used quantitative and qualitative characters describing shell-only scenarios and obtained 80.6% correct classification on terminal nodes. Canonical variates analysis on geometric morphometric shell data revealed large morphological overlap between species. Goodall's F-tests between pairs of species revealed significant differences (a=0.05) between all but one species pairs; however, examination of landmarks on shells concluded large overlap of landmarks between species pairs. Lack of morphologically distinct characters to readily identify these phylogenetically distinct species indicates large morphological overlap among these species. Biologists need to be cognizant that morphologically cryptic species may exist in systems often explored.

Three dichotomous keys were created from classification trees to identify select individuals in the genera Fusconaia, Pleurobema, and Pleuronaia; two of these keys, one for shells and one for live mussels were tested by participants with varying mussel identification skills to represent novices and experts. Both keys used continuous (quantitative) and categorical variables to guide participants to identifications. Novices, who had no prior mussel identification experience, correctly identified mussels with a 50% accuracy using the shell key and with a 51% accuracy using the live key. Experts, who had at least three years of experience identifying mussels, correctly identified mussels with a 58% accuracy using the shell key and with a 68% accuracy using the live key; however one expert noted that they did not use the live key to correctly identify one mussel. Morphological overlap of variables between mussels likely resulted in failure to consistently identify mussels correctly.

Important management decisions and project implementations require accurate assessment of species' localities and populations. Incorrect species identification could hinder species' recovery efforts or prevent projects that otherwise could have continued if species are misidentified. If a mussel collection is thought to be a new record or could affect a project, I recommend that molecular genetic identifications be used to verify the species identity.