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Samples of Luxilus cornutus, Luxilus chrysocephalus, and their hybrids were collected along hypothesized routes of dispersal from Pleistocene refugia to examine the significance of geographic variation in patterns of introgression between these species. Patterns of allozyme and mitochondrial DNA (mtDNA) variation were generally consistent with those from previous studies. Tests of Hardy‐Weinberg equilibrium revealed significant deficiencies of heterozygotes in all samples, indicating some form of reproductive isolation. Mitochondrial DNAs of each species were not equally represented in F1 hybrids; however, this bias was eliminated when the two largest samples were excluded from the analysis. Backcross hybrids exhibited biased mtDNA introgression, as samples from Lake Erie (eastern) and Lake Michigan (western) drainages showed significant excesses of mtDNAs from L. chrysocephalus and L. cornutus, respectively, relative to frequencies of diagnostic allozyme markers. The extent and direction of allozyme and mtDNA introgression was quantified by calculating isolation index values from morphologically “pure” individuals of each species from each locality. Analysis of variance of these measures identified limited introgression of allozyme variants with no geographic pattern, but significant differences in direction of mtDNA introgression between drainages (i.e., postglacial dispersal route). Association between patterns of mtDNA introgression and dispersal route across the latitudinal width of the contact zone is best explained by genetic divergence during past isolation of ancestral populations from these drainages. These results identify a significant role for historical effects in the evolution of reproductive isolation and the process of speciation.
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