Genetic and morphological evidence for substantial hidden biodiversity in a freshwater crayfish species complex

North America is a center of biodiversity for freshwater crayfish, with >300 described species, mostly in the family Cambaridae. However, the phylogenetic and phylogeographic relationships among these taxa are poorly understood, despite their ubiquitous occurrence in North American freshwater habitats. Here, we target the geographically widespread Orconectesvirilis and its sister taxa for genetic and morphological investigation. We used sequence data from two mitochondrial genes (16S rRNA and cytochrome oxidase I) and from one nuclear gene (glyceraldehyde-3-phosphate dehydrogenase), as well as morphological data from the male gonopod, to gain insights into phylogenetic relationships among individuals collected in the northeastern United States, where this taxon is considered to be invasive, and from Kansas. Our data reveal evidence for three cryptic species in our collections, in addition to O.virilis sensu stricto; all four clades are separated by both mitochondrial and nuclear sequence data. In addition, two of the newly discovered species, O.sp. nov. A and O.sp. nov. B, are distinguishable from O.virilis by significant differences in gonopod morphology (no data on gonopod morphology were available for O.sp. nov. C). O.sp. nov. A was collected at numerous sites in Massachusetts; additional sampling is required to determine if this species is native to New England. Two other taxa, O.sp. nov. B and O.sp. nov. C, were both collected from sites in eastern Kansas, and historically have probably been considered to be O.nais. In summary, this investigation indicates that this species complex may harbor a great deal of unknown biodiversity over the rest of its described range, and suggests that further attention to the phylogenetics and phylogeography of North American crayfishes may yield interesting insights into the evolution of biodiversity.