Samples preparation
All H. horticola wasps emerging from the field-collected caterpillars were individually preserved in ethanol in the freezer (-20°C) until use. The DNA was extracted from the abdomen of each wasp using a Qiagen DNeasy blood and tissues extraction kit, following the manufacturer’s protocol (Qiagen®, USA). We amplified the mitochondrialCOI gene by PCR using the primer pair LCO/HCO developed by Folmer et al. (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994).
Mitotype and Wolbachia-infection status
Cytoplasmic entities such as the mitochondria and Wolbachiasymbionts are passed on only from mothers. These maternally inherited entities can thus change frequency in a population at different rates than genotypes determined using nuclear microsatellite markers. To evaluate maternal inheritance, we sequenced a mitochondrial gene and screened for Wolbachia in the majority of our samples.
Two common H. horticola mitotypes were previously characterised from the Åland islands (Duplouy et al., 2015). We determined the mitotype of 222 wasps picked at random from the 324 samples genotyped above (37 from Finström, 41 from Föglö, 43 from Seglinge-Kumlinge, 63 from Saltvik and 38 from Sottunga) by direct Sanger sequencing theCOI gene described above using an ABI 3730 DNA Sequencer (Applied BiosystemsTM, USA). These samples included males and females from the five localities and each collection year.
Duplouy et al. (Duplouy et al., 2015) showed earlier that about 50% of the H. horticola population on the Åland islands is infected by the Wolbachia strain w Hho. We screened forw Hho in 296 samples (90 from Finström, 41 from Föglö, 40 from Seglinge-Kumlinge, 86 from Saltvik and 39 from Sottunga), picked at random from the 324 samples genotyped above, using the primer pair 81F/691R to amplify the conserved Wolbachia wsp gene (Zhou, Rousset, & O’Neil, 1998).