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.