Since the early Holocene, fish population genetics in the Laurentian Great Lakes have been shaped by the dual influences of habitat structure and post-glacial dispersal. Riverscape genetics theory predicts that longitudinal habitat corridors and unidirectional downstream water-flow drive the downstream accumulation of genetic diversity, whereas post-glacial dispersal theory predicts that fish genetic diversity should decrease with increasing distance from glacial refugia. This study examines populations of seven native fish species codistributed above and below the 58 m-high Niagara Falls – a hypothesized barrier to gene flow in aquatic species. A better understanding of Niagara Falls’ role as a barrier to gene flow and dispersal is needed to identify drivers of Great Lakes genetic diversity and guide strategies to limit exotic species invasions. We used genome-wide SNPs and coalescent models to test whether populations are: (1) genetically distinct, consistent with the Niagara Falls barrier hypothesis; (2) more genetically diverse upstream, consistent with post-glacial expansion theory, or downstream, consistent with the riverscape habitat theory; and, (3) are migrating either upstream or downstream past Niagara Falls. We found that genetic diversity is consistently greater below Niagara Falls and the falls are an effective barrier to downstream migration, but at least some species have likely dispersed upstream past the falls since the time of their formation yet before construction of the Welland Canal. Models restricting migration to after opening of the Welland Canal were generally rejected. These results help explain how river habitat features affect aquatic species’ genetic diversity and highlight the need to better understand post-glacial dispersal pathways.

These are both original data and permutations of data generated via a three-enzyme restriction site associated DNA (3RAD) library preparationa and sequencing pipeline applied to seven native fish species that are codistributed above and below Niagara Falls, in the Niagara River between lakes Erie and Ontario at the Canada – United States border. The seven species examined are: Ambloplites rupestris, Ameiurus nebulosus, Catostomus commersoni, Micropterus salmoides, Moxostoma macrolepidotum, Moxostoma valenciennesi, and Perca flavescens. Raw 3RAD sequence reads were clustered and SNPs were called using the program Pyrad. Pyrad outputs provided here are the unlinked SNP matrices and the VCF files generated for each species. Also provided are the input files for our principle coordinate analyses in .xls format, input files for our diveRsity analyses in Genepop format, and input files for our fastsimcoal2 demographic model testing analyses.

Each data set contains data for each of the seven species *except* the fastsimcoal2 datasets, which exclude Moxostoma valenciennesi, for which there were two few individuals to run demographic analyses.