Abstract
The inability to distinguish between species can be a serious problem in groups responsible for pathogen transmission. Culicoides biting midges transmit many pathogenic agents infecting wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a known vector, but limited species-specific characters have hindered vector surveillance. Here, genomic data were used to investigate population structure and genetic differentiation within this species complex. Single nucleotide polymorphism data were generated for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses suggest the occurrence of two additional cryptic species within this complex. All five species were significantly differentiated in both sympatry and allopatry. Evidence of hybridization was detected in three different species pairings indicating incomplete reproductive isolation. Additionally, COI sequences were used to identify the hybrid parentage of these individuals, which illuminated discordance between the divergence of the mitochondrial and nuclear datasets.