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Comparative analyses of populations at different stages of divergence can yield insights into the process of speciation. We assess population structure, gene flow, and maternal ancestry at five locations containing sympatric members of the Simulium arcticum complex at different stages of chromosome divergence. We analyze both nuclear and mitochondrial DNA markers, including 11 microsatellite loci, as well as COI, COII, cytb, and ND4 gene sequences. Simulium negativum, representing the later stages of divergence, shows both nuclear and mitochondrial differentiation when compared with allopatric and sympatric chromosomal forms, as well as both low contemporary and historical gene flow in sympatry. At intermediate stages of chromosome divergence, populations differ at nuclear, but not mitochondrial, loci in allopatry and sympatry. In one comparison of intermediate stage chromosomal forms (S. arcticum sensu stricto and S. apricarium), populations demonstrate low contemporary, but higher historical, gene flow in sympatry. In a second sympatric comparison (S. arcticum s. s. and S. brevicercum), both contemporary and historical gene flow are high. All analyses of sympatric populations at the earliest stages of chromosome divergence demonstrate panmixia; yet, some nuclear differentiation in allopatry is apparent. These findings suggest that molecular divergence is tracking chromosome divergence along a chromosomally-defined continuum of speciation in black flies.

Mitochondrial based phylogenetic reconstructions often show deviations from species-level monophyly. We used the Simulium arcticum species complex (Diptera: Simuliidae) as a model system for interpreting non-monophyly in light of chromosomal data supporting species status of siblings. For cytogenetic identification of morphologically indistinguishable black fly sibling species, larvae must be preserved in Carnoy's solution, a fixative known to degrade DNA. Consequently, we reconstructed phylogenetic relationships based on 12S, COII, cyt b, and ITS-1 gene sequences obtained from larvae sampled from presumed taxon-pure localities. As species composition at 'taxon-pure' sites may have changed at the time of sampling, we performed a second study that aimed to: (1) assess phylogenetic relationships among cytologically verified members of the S. arcticum species complex using COI and COII gene sequences; (2) determine whether useable genetic information could be gleaned from Carnoy's fixed specimens; and (3) determine the extent to which Carnoy's fixative degrades DNA over time. We consistently obtained genetic data from material stored in Carnoy's solution for two to three months. Genetic analysis of samples fixed in Carnoy's solution for up to six years indicates that larvae preserved for a maximum of five years can provide useable information for molecular analysis. Our preliminary and cytologically confirmed phylogenetic analyses demonstrate that mitochondrial DNA fails to resolve species-level monophyly of chromosomally distinct S. arcticum taxa. As results of analyses based on cytologically verified larvae mirror those of our preliminary study, we rule out imperfect taxonomy as the reason for species-level non-monophyly. Although we cannot confidently reject either inadequate phylogenetic information or incomplete lineage sorting as the cause of non-monophyly, the sharing of alleles between sympatric siblings suggests introgressive hybridization between taxa. We conclude that the patterns present in the S. arcticum phylogeny likely represent the initial stages of chromosome based sibling speciation.