Population genomics and mitochondrial DNA reveal cryptic diversity in North American Spring Cavefishes (Amblyopsidae, Forbesichthys)

The North American freshwater genus Forbesichthys is composed of facultative cave-dwelling fishes restricted to springs and caves in southern Illinois, southeastern Missouri, southwestern Kentucky, and central Tennessee. These fishes were previously considered a single species, the Spring Cavefish (F. agassizii), but recent molecular evidence led to the recognition of the Shawnee Hills Cavefish (F. papilliferus). The Shawnee Hills Cavefish is hypothesized to be restricted to Illinois, Missouri, Kentucky and north-central Tennessee, whereas the Spring Cavefish is restricted to the Eastern Highland Rim of central Tennessee. However, the distributions of Forbesichthys are difficult to ascertain due to their intermittent appearance in surface springs, making sampling challenging. We assessed the species status, distribution, connectivity, and population sizes of the Forbesichthys spp. using Restriction-site Associated DNA sequencing (RADseq) and the mitochondrial NADH dehydrogenase 2 locus. Our results corroborate the recognition and hypothesized distributions of the Shawnee Hills Cavefish and Spring Cavefish. Furthermore, we suggest the recognition of three Evolutionary Significant Units (ESUs) and two Management Units (MUs) within the Shawnee Hills Cavefish. Although all populations analyzed appear to have reasonable genetic diversity and population stability over time, this regionalization has implications for both groundwater policy and management. Our study provides important information relevant to understanding potential population distributions and the identification of unique lineages that may deserve additional protection.