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”.