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Species endemic to coldwater springs in the southeastern United States are some of the rarest and most imperiled in this region, yet little is known about their genetic composition and conservation needs. Here, microsatellite based levels of genetic diversity and estimates of effective population size (N e ) were compared between a narrow spring endemic fish, Etheostoma nuchale, and its widespread stream-dwelling relative, E. swaini. We applied several analytical methods to assess how demographic history is reflected in contemporary levels of genetic diversity for populations of E. nuchale. Phylogenetic analyses based on mitochondrial and nuclear DNA sequence data revealed a complex history among E. nuchale and E. swaini, but suggested ancient divergence and historic periods of isolation since colonization of spring habitats by E. nuchale. Populations of E. nuchale have levels of genetic diversity approximately one-half that of E. swaini, a result most likely due to founder effects and recent bottlenecks. Statistically significant F st values (0.05-0.27) and STRUCTURE analyses implied high levels of differentiation among E. nuchale populations. Estimates of current N e suggest relatively consistent levels across populations of E. nuchale, but one population may suffer from habitat degradation. We suggest that high levels of population structure and low levels of genetic diversity may be typical in other spring endemics inhabiting this region. Therefore, effective management planning for these unique species will require a detailed knowledge of the genetic and demographic history of each population.

Incongruence between recognized taxonomy and phylogenetic relationships between two species from a diverse clade (Percidae: Etheostomatinae) of stream fishes was found in a mitochondrial (mt) DNA gene tree. Two darters in subgenus Oligocephalus, Etheostoma uniporum current darter and Etheostoma caeruleum rainbow darter were sampled throughout their sympatric distribution in the Ozark Highlands of the central United States. Sequences from cytochrome (cyt) b and the first intron of the nuclear marker S7 were analysed separately using maximum parsimony and Bayesian methods. Cyt b recovered both species as polyphyletic; E. uniporum haplotypes were interspersed within E. caeruleum. However, both species were monophyletic and non-sister taxa based on S7. The cyt b gene tree pattern is caused by introgressive hybridization resulting in the mtDNA replacement of E. uniporum haplotypes by those of E. caeruleum. Some E. uniporum haplotypes are shared with geographically proximate E. caeruleum, and this is consistent with recent hybridization, while other E. uniporum haplotypes indicate historical sorting of introgressed lineages. The mechanism of introgression is likely asymmetric sneaking behaviour by male E. uniporum, a mating tactic observed in related species. MtDNA replacement may have occurred in E. uniporum due to drift fixation in a historically small female effective population. Additional evidence for darter hybridization will likely be discovered in future molecular genetic surveys of the nearly 200 species in eastern North America.

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