Good species behaving badly: Non-monophyly of black fly sibling species in the Simulium arcticum complex (Diptera: Simuliidae).

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.