Microsatellite genotyping
To document temporal and spatial nuclear genetic variation in H. horticola wasps, we genotyped 323 wasps (including 195 females) using the 14 microsatellite-loci and genotyping conditions developed by Couchoux et al. (Couchoux, Seppä, & van Nouhuys, 2015b). Couchoux et al. (2015b) demonstrated that there is no linkage disequilibrium between each pair of these microsatellite loci. The forward primers were labelled with either FAM, HEX, or TAMRA fluorescent dye (DNA Technology A/S), and used in multiplex non-overlapping PCR reactions using Qiagen Multiplex PCR kit (Cat. #206143, Qiagen, USA) as described by Couchoux et al. (2015b). Diluted PCR products were genotyped on an automated ABI 3730 DNA Sequencer (Applied BiosystemsTM, USA). The sizes were called using Genescan-500 ROX size standard. We manually curated the genotypes for each sample using the GeneMapper® Software 5 (Applied BiosystemsTM, USA).
For the purpose of the genetic analyses described below, we separated the samples into the five localities they were collected from (North Finström, North Föglö, Seglinge-Kumlinge, Saltvik or Sottunga, Figure 1b), or into 12 spatio-temporal groups according to their geographic origin and their collection time interval (Finström 1992-97, 2003-08, 2009-11, Föglö 2000-09, 2010-13, Seglinge-Kumlinge 2000-09, 2010-11, Saltvik 1999, 2005-09, 2010-13, and Sottunga 2002-04, 2005-09; Figure 3, Table 2). Each of the 12 spatio-temporal groups was designed to include specimens from roughly either the 90’s, the 2000’s, and the 2010’s, from each locality, but due to the smaller sample size, the samples from Sottunga were divided between two temporal groups: the early and the late 2000’s.