Microsatellite genotyping
Genomic DNA was extracted using the EZNA® Tissue DNA extraction kit (Omega Bio-tek; Norcross, Georgia), following the manufacturer protocols. For adult males, prior to extraction, the wings and genitalia were removed and stored as vouchers. For adults and larvae, the remaining body parts were then homogenized, using 3/16” stainless steel beads (GlenMills Inc.; Clifton, New Jersey) with a FastPrep-24 Sample Homogenizer (MP Biomedicals, Santa Ana, California). After extraction, 24 polymorphic microsatellite loci (Havill et al., 2017) were genotyped at the DNA Analysis Facility on Science Hill at Yale University, using a Thermo Fisher Scientific 3730xl DNA Analyzer. Fragment lengths were determined using the microsatellite plugin in the software program Geneious v. R11 (https://www.geneious.com) in comparison to the GeneScan 500 LIZ size standard (Thermo Fisher Scientific; Waltham, Massachusetts).
Only individuals from which ≥ 20 microsatellite loci were successfully amplified were included in analyses. In addition, since winter moth has been reported to hybridize with Bruce spanworm in all of its invaded regions (Andersen et al., 2019a) the dataset was further filtered to remove hybrids by comparing assignment probabilities based on 12 polymorphic loci that co-amplify in both species (described below). Microsatellite genotypes are provided as a tab-delimited Structure-formatted supplemental file titled “WinterMothOriginsStructure.txt”.