North American freshwater mussel species have experienced substantial range fragmentation and population reductions. These impacts have the potential to reduce genetic connectivity among populations and increase the risk of losing genetic diversity. Thirteen microsatellite loci and an 883 bp fragment of the mitochondrial ND1 gene were used to assess genetic diversity, population structure, contemporary migration rates, and population size changes across the range of the Sheepnose mussel (Plethobasus cyphyus). Population structure analyses reveal five populations, three in the Upper Mississippi River Basin and two in the Ohio River Basin. Sampling locations exhibit a high degree of genetic diversity and contemporary migration estimates indicate that migration between populations within river basins is occurring, although at low rates. but no migration is occurring between the Ohio and Mississippi river basins. No evidence of bottlenecks was detected, and almost all locations exhibited the signature of population expansion. Our results indicate that although anthropogenic activity has altered the landscape across the range of the Sheepnose, these activities have yet to be reflected in losses of genetic diversity. Efforts to conserve Sheepnose populations should focus on maintaining existing habitats and fostering genetic connectivity between extant demes to conserve remaining genetic diversity for future viable Sheepnose populations.

Mussels were collected by snorkeling or SCUBA. Samples for DNA extraction were collected by taking a small (~1mm) biopsy of mantle tissue (Berg et al. 1995) or by using cytology brushes that were swabbed over the mantle tissue of mussels to accumulate mucous and sloughed cells (Henley et al. 2006). Biopsy samples were stored in 95% ethanol and DNA was extracted from mantle tissue samples using the Qiagen DNeasy® Blood and Tissue Kit (Qiagen # 69506) according to the kit instructions. Cytology brush samples were stored in the lysis buffer provided with the Puregene Buccal Cell Core Kit B (Qiagen) and DNA was extracted following the kit instructions. The loci markers were developed by Genetic Identification Services, Chatsworth, CA. Polymerase chain reactions (PCR) were performed using 10 µL reactions (~2 ng of genomic DNA was used in each reaction). The standard M13 protocol (Schuelke 2000) was used with the florescent dye labeled with HEX (Applied Biosystems). Reagents for a 10 µL reaction included: 6.6 µL H₂0, 1 µL Biolase Buffer (10x), 0.3 µL MgCl₂, 0.8 µL dNTP, 0.1 µL forward primer, 0.1 µL reverse primer, 0.05 µL M13 labeled oligo, 0.05 µL Biolase Taq polymerase, and 1 µL template DNA. Reactions were run in Eppendorf Master Cycler thermal cyclers under consistent conditions (95°C/5min; [94°C/30 sec.; 62°C/60 sec.; 72°C/30 sec.] X 10 cycles; [94°C/30 sec.; 55-59.4 °C/60 sec.; 72°C/30 sec.; 72°C/20 min] X 25 cycles; 72°C/4 min).