Abstract
One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome c oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the Simulium (Boreosimulium) annulus species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species Simulium ustulatum n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the S. annulus group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California's Central Valley could make it vulnerable, given climate change and increasing land development.