Sample collection and sequencing
Culicoides midges were collected from 17 sites across the United States and Canada (Table 1). Specimens were collected either as pupae and reared to adulthood, or as adults using CDC light traps baited with CO2 and UV light (Bioquip 2836BQ). Individuals morphologically assigned to the C . variipennis complex were sorted out from the by-catch and stored in 95% ethanol at -80 °C. Total DNA was extracted from individuals (females only) using a Puregene extraction protocol (Gentra Systems, Inc., D-5500A) with the addition of glycogen (ThermoFisher, R0561) to increase yields. The DNA quality was checked using gel electrophoresis and DNA concentration was measured using a Qubit 3.0 fluorometer and a Qubit dsDNA HS assay kit (Invitrogen, Q33230). A total of 300-400 ng of DNA per sample was sent to Floragenex, Inc. for library preparation using the protocol from Truong et al. (2012). DNA was digested using the restriction enzymesMseI and PstI . After PCR amplification, the samples in each plate were pooled and sequenced on a lane of single-end 100bp sequencing on a HiSeq4000 at the University of Oregon Genomics Facility, Eugene, OR.