Distribution Changes, Genetic Population Structure, and a Novel Environmental DNA (eDNA) Detection Method for Darters (Subgenus Nothonotus) in the Upper Ohio River Watershed

In the upper Ohio River watershed three species of small-bodied benthic fish the Bluebreast Darter, Etheostoma (Nothonotus) camurum (Cope), the Tippecanoe Darter, Etheostoma (Nothonotus) tippecanoe Jordan and Evermann, and the Spotted Darter, Etheostoma (Nothonotus) maculatum Kirtland previously existed in disjunct distributions due to poor water quality and habitat degradation. Signs of recovery indicated that these species were moving from areas of refugia into the deeper mainstem waters of the Allegheny and Ohio rivers and expanding their distributions. To provide information for the proper conservation management of these species this dissertation was divided into three stages: 1) distribution records were updated by performing State-mandated electrified-benthic trawling and compiling as many historic and contemporary records as possible, 2) the genetic diversity and genetic population structure of E. camurum was assessed using six polymorphic microsatellite loci, and 3) environmental DNA (eDNA) methods with species detection from water samples via fragment analysis were developed to assist current survey methods which are costly, time consuming, and may be harmful to the fish. The surveys and compilation of data showed that E. camurum and E. tippecanoe are utilizing deeper habitat (than previously reported) in the tailwaters of the navigational lock and dam system, and have nearly continuous distributions from the upper Allegheny River downstream into the Ohio River. Etheostoma maculatum showed a less robust expansion and a more limited use of the tailwater habitat. The genetic assessment of E. camurum indicated high genetic diversity within their populations with no evident signs of isolation or inbreeding. The genetic population structure of E. camurum was weak indicating that the navigational lock and dam system was not strongly influencing gene flow between the populations. In addition, there were signs of a newly advancing population. With eDNA methodologies, a protocol was developed that successfully detected E. tippecanoe eDNA from water samples taken from the Allegheny and Kiskiminetas rivers and Deer Creek in Harmarville, PA. A second set of PCR primers were developed that have the potential to detect all three focal species using eDNA from water samples