The use of genetic methods to quantify the effects of anthropogenic habitat fragmentation on population structure has become increasingly common. However, in today's highly fragmented habitats, researchers have sometimes concluded that populations are currently genetically isolated due to habitat fragmentation without testing the possibility that populations were genetically isolated before European settlement. Etheostoma raneyi is a benthic headwater fish restricted to river drainages in northern Mississippi, USA, that has a suite of adaptive traits that correlate with poor dispersal ability. Aquatic habitat within this area has been extensively modified, primarily by flood-control projects, and populations in headwater streams have possibly become genetically isolated from one another. We used microsatellite markers to quantify genetic structure as well as contemporary and historical gene flow across the range of the species. Results indicated that genetically distinct populations exist in each headwater stream analyzed, current gene flow rates are lower than historical rates, most genetic variation is partitioned among populations, and populations in the Yocona River drainage show lower levels of genetic diversity than populations in the Tallahatchie River drainage and other Etheostoma species. All populations have negative F IS scores, of which roughly half are significant relative to Hardy-Weinberg expectations, perhaps due to small population sizes. We conclude that anthropogenic habitat alteration and fragmentation has had a profoundly negative impact on the species by isolating E. raneyi within headwater stream reaches. Further research is needed to inform conservation strategies, but populations in the Yocona River drainage are in dire need of management action. Carefully planned human-mediated dispersal and habitat restoration should be explored as management options across the range of the species.

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The Yazoo Darter, Etheostoma raneyi (Percidae), is an imperiled freshwater fish species endemic to tributaries of the Yocona and Little Tallahatchie rivers of the upper Yazoo River basin, in northern Mississippi, USA. The two populations are allopatric, isolated by unsuitable lowland habitat between the two river drainages. Relevant literature suggests that populations in the Yocona River represent an undescribed species, but a lack of data prevents a thorough evaluation of possible diversity throughout the range of the species. Our goals were to estimate phylogenetic relationships of the Yazoo Darter across its distribution and identify cryptic diversity for conservation management purposes. Maximum likelihood (ML) phylogenetic analyses of the mitochondrial cytochrome b (cytb) gene returned two reciprocally monophyletic clades representing the two river drainages with high support. Bayesian analysis of cytb was consistent with the ML analysis but with low support for the Yocona River clade. Analyses of the nuclear S7 gene yielded unresolved relationships among individuals in the Little Tallahatchie River drainage with mostly low support, but returned a monophyletic clade for individuals from the Yocona River drainage with high support. No haplotypes were shared between the drainages for either gene. Additional cryptic diversity within the two drainages was not indicated. Estimated divergence between Yazoo Darters in the two drainages occurred during the Pleistocene (<1 million years ago) and was likely linked to repeated spatial shifts in suitable habitat and changes in watershed configurations during glacial cycles. Individuals from the Yocona River drainage had lower genetic diversity consistent with the literature. Our results indicate that Yazoo Darters in the Yocona River drainage are genetically distinct and that there is support for recognizing Yazoo Darter populations in the Yocona River drainage as a new species under the unified species concept.

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