Molecular systematics of the Simulium jenningsi species group (Diptera: Simuliidae), with three new fast-evolving nuclear genes for phylogenetic inference.

A molecular phylogeny was inferred for the 22 nominal species of black flies in the Simulium jenningsi species group, which includes major pests of humans and livestock in North America. Females are structurally monomorphic, presenting a problem for identification of the pests. For each species, we sequenced approximately two kilobases from the mitochondrial genome (ND2, Cox I, proximal one-half of Cox II) and about six kilobases from the nuclear genome (ca. 2 kilobases each from 3 rapidly evolving nuclear genes: big zinc finger [BZF], "5-intron gene" [5intG], and elongation complex protein 1 [ECP1]) and analyzed them phylogenetically using maximum likelihood and Bayesian methods. The three nuclear loci have not previously been used in phylogenetic studies. The mitochondrial region recovered 6 group members as monophyletic. BZF, 5intG, and ECP1 sequences each permitted identification of 13 species and recovered the S. fibrinflatum and S. taxodium subgroups. Simulium aranti Stone and Snoddy and S. luggeri Nicholson and Mickel were consistently recovered at the base of the group. Simulium ozarkense Moulton and Adler, S. dixiense Stone and Snoddy, S. krebsorum Moulton and Adler, and S. haysi Stone and Snoddy branched off before two well-supported sister groups of the remaining species. This remainder consisted of species occupying slow, sandy lowland streams-S. definitum Moulton and Adler, S. jonesi Stone and Snoddy, and the S. taxodium subgroup (S. taxodium Snoddy and Beshear, S. chlorum Moulton and Adler, S. confusum Moulton and Adler, and S. lakei Snoddy)-as sister to two clades of species inhabiting swift, rocky upland streams-the S. fibrinflatum subgroup (S. fibrinflatum Twinn, S. notiale Stone and Snoddy, and S. snowi Stone and Snoddy) and a clade comprised of S. anchistinum Moulton and Adler, S. jenningsi Malloch, and S. nyssa Stone and Snoddy, plus species having cocoons without anterolateral apertures (S. infenestrum Moulton and Adler, S. podostemi Snoddy, S. penobscotense Snoddy and Bauer, and S. remissum Moulton and Adler). Simulium snowi Stone and Snoddy is here considered a synonym of S. notiale Stone and Snoddy. Trees inferred from BZF and 5intG were largely concordant with those from ECP1, but slightly less resolved. Combining mitochondrial and nuclear data sets did not greatly improve the performance of the ECP1 data set alone. We, therefore, propose ECP1 as the gold standard for identification of members of the S. jenningsi group. Maximum likelihood analysis of combined sequences from all three nuclear genes, with three morphological constraints imposed, yielded a tree proposed as the best hypothesis of relationships among group members, based on all available data.