The complete mitochondrial cytochrome oxidase II gene was sequenced from 17 black flies, representing 13 putative species, and used to infer phylogenetic relationships. A midge (Paratanytarsus sp.) and three mosquitoes (Aedes aegypti, Anopheles quadrimaculatus, and Culex quinquefasciatus) were used as outgroup taxa. All outgroup taxa were highly divergent from black flies. Phylogenetic trees based on weighted parsimony (a priori and a posteriori), maximum likelihood, and neighbor-joining (log-determinant distances) differed topologically, with deeper nodes being the least well-supported. All analyses supported current classification into species groups but relationships among those groups were poorly resolved. The majority of phylogenetic signal came from closely related sister taxa. The CO-II gene may be useful for exploring relationships at or below the subgeneric level, but is of questionable value at higher taxonomic levels. The weighting method employed gave phylogenetic results similar to those reported by other authors for other insect CO-II data sets. A best estimate of phylogenetic relationships based on the CO-II gene is presented and discussed in relation to current black fly classification.

Mitochondrial DNA (mtDNA) was extracted from pooled field-collected samples representing six species of black flies (Cnephia dacotensis, Simulium bivittaum, S. johansenni, S. luggeri, S. piperi, S. vittatum) and compared by restriction fragment length polymorphism (RFLP) analysis. Morphospecies were molecularly distinct, with few shared restriction fragments. Eleven populations of S. vittatum were found that appeared to be homogeneous for a single mitochondrial haplotype. Ten other populations of S. vittatum showed extensive mitochondrial heterogeneity. In part, these samples contained mixtures of two cytologically recognized siblings: IIIL-1 and IS-7. About 70% of the mitochondrial genome of a population pure for sibling IIIL-1 was cloned as five HindIII fragments, which were used as hybridization probes to examine individual black flies. Thirteen mtDNA haplotypes involving permutations of 10 HindIII restriction sites were identified in individual black flies examined from 26 populations. DNA from 168 larvae cut with both EcoR1 and HindIII revealed five additional haplotypes. One HindIII haplotype was present in 84% of 390 larvae examined and predominated in every population examined from New York to California and in both the IIIL-1 and IS-7 siblings, Nebraska populations had individuals with nearly all known haplotypes. The most common haplotype was usually the only form present in warm, silty streams with organic enrichment. Rarer haplotypes were found in cool, spring-fed streams but without clear geographic or phylogenetic components.

Sequences are given for nine complete genes and one partial mitochondrial tRNA gene of the black fly, Simulium vittatum (Zetterstedt). Sequenced tRNA genes were for alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, leucine(CUN), lysine, serine(AGN), and valine. Nucleotides were aligned with the same previously sequenced genes in Aedes albopictus Skuse and Drosophila yakuba Burla. A cluster of six tRNA genes, which differ in arrangement in Ae. albopictus and D. yakuba, was amplified by PCR and found to have the same position and orientation in S. vittatum as in D. yakuba. Overall, similarity with either D. yakuba or Ae. albopictus was 86%. Sequences that were common to the three insects suggest that black flies and mosquitoes are as divergent from each other as either is from Drosophila. Sequences for nine species of black flies were obtained for tRNA leucine(CUN) from DNA amplified with another primer set. Little variation occurred within the tRNA gene but, by including the flanking regions to provide 175 base pairs, a phylogeny of the nine species was obtained that was largely consistent with current classification.