Phylogenetic assessment of endangered and look-alike Pigtoe species in a freshwater mussel diversity hotspot.

The Green River in Kentucky in the eastern United States is a freshwater mussel biodiversity hotspot, with 71 known species. Among them, the endangered coexists with other morphologically similar species in the genera and , known colloquially as "pigtoes." Identification of species in these genera is challenging even for mussel experts familiar with them. In our study, the correct identification of these species by experts ranged from 57% to 83%. We delineated taxonomic boundaries among seven species and tested for cryptic biodiversity among these look-alike mussels utilizing mitochondrial and nuclear DNA sequence variation. Phylogenetic analysis of combined (1215 bp) mitochondrial DNA cytochrome oxidase I () and NADH dehydrogenase 1 () genes showed five well-diverged groups that included , , , and as distinct clades, with and grouped into a single clade. While our mitochondrial DNA analyses did not distinguish and as phylogenetically distinct species, the typical shell forms of these two nominal taxa are very distinct. Further phylogenetic analysis using nuclear ribosomal transcribed spacer region subunit I () DNA sequences also showed that and were not distinct lineages. No cryptic species were detected in the and samples analyzed from the Green River. The highest haplotype diversity (), average number of nucleotide differences (), and nucleotide diversity () were observed for at both the ( = 0.896,  = 3.805,  = 0.00808) and ( = 0.984,  = 6.595,  0.00886) markers, with similarly high genetic diversity in the other taxa. Our results give managers confidence that cryptic taxa do not occur within or among these morphologically similar species in the Green River, and populations appear genetically diverse, indicative of large and healthy populations.