DNA extraction and microsatellite genotyping
For the Massachusetts transect from 2007-2016, up to 20 moths were selected haphazardly from each trap’s pooled sampling (i.e., from combining the contents of the two collection events). For the Massachusetts transect traps from 2017 and 2018, and from all Connecticut transect traps, 20 moths were selected haphazardly from each collection event (i.e., each weekly or bi-weekly collection event was processed independently). Prior to the isolation of genomic DNA, the wings and uncus were preserved as morphological vouchers as these contain characters that may be useful for species identification (Griffin, Chandler, Andersen, Havill, & Elkinton, 2020; Troubridge and. Fitzpatrick 1993). The remaining body parts were placed in a 2.0 ml microcentrifuge tube (USA Scientific Inc., Ocala, FL) with 100 µl of PBS buffer (Sigma Aldrich, St. Louis, MO) and homogenized with a sterile 3/16” stainless steel bead (GlenMills Inc., Clifton, NJ), using a FastPrep-24 Sample Homogenizer (MP Biomedicals, Santa Ana, CA). DNA was extracted and purified using the EZNA® Tissue DNA extraction kit (Omega Bio-tek; Norcross, GA), following the manufacturer protocols.
From each sample, 11 polymorphic microsatellite loci were amplified following Havill et al. (2017), and run with the GeneScan 500 LIZ size standard (Thermo Fisher Scientific; Waltham, MA), on a Thermo Fisher Scientific 3730xl DNA Analyzer at the DNA Analysis Facility on Science Hill at Yale University. Fragment lengths were scored in the software GENEIOUS v. R11 (https://www.geneious.com), using the microsatellite plugin.