Phylogenetics and molecular identification of the Ochlerotatus communis complex ( Diptera: Culicidae) using DNA barcoding and polymerase chain reaction- restriction fragment length polymorphism

The Ochlerotatus communis (De Geer, 1776) complex consists of four cryptic mosquito species in North America, including: O. communis, Ochlerotatus churchillensis (Ellis and Brust, 1973), Ochlerotatus nevadensis (Chapman and Barr, 1964), and Ochlerotatus tahoensis (Dyar, 1916). Most of the morphological characters used for the identification of these species are quantitative and overlap across species. Here we evaluated the efficacy of DNA barcoding for identification of three members of the communis complex (O. nevadensis is not included in this study) and developed diagnostic restriction fragment length polymorphism (RFLP) patterns for O. communis and O. churchillensis. A phylogeny of 23 Ochlerotatus Lynch Arribalzaga, 1891 species was inferred using mitochondrial cytochrome c oxidase subunit I gene sequences. All species included in our analysis within the O. communis complex were delineated using cytochrome c oxidase subunit I barcodes. However, this complex was recovered as paraphyletic with respect to Ochlerotatus abserratus (Felt and Young, 1904) and Ochlerotatus implicatus (Vockeroth, 1954), indicating the need for increased genetic and taxonomic sampling to infer the phylogenetic relationships of these taxa. The RFLP profile for multiple field specimens of O. communis was distinct from all RFLP patterns for O. churchillensis, and this method can be used as an efficient molecular method for the identification these species.