Sample 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 for the purpose of another study (Duplouy et al 2015), using a Qiagen DNeasy blood and tissues extraction kit, following the manufacturer’s protocol (Cat. #69506, Qiagen, USA). We amplified the mitochondrialCOI gene by PCR using the primer pair LCO/HCO and PCR conditions developed by Folmer et al. (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994) and including two positive samples from Duplouy et al (2013) and a negative control, to check for the quality of the DNA extracts.
Mitotypes and Wolbachia-infection status
Cytoplasmic entities such as the mitochondria and Wolbachiasymbionts can have profound impacts on their hosts but 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 in the Åland H. horticola population, we look at differences in the prevalence of the mitochondrion and Wolbachia -infection in most of the male and female samples genotyped in this study. For this, we used sequences of the 5’end of the mitochondrial gene COI and screenings of Wolbachia that were previously done by Duplouy et al. (2015). The same authors (Duplouy et al., 2015) characterized two common mitotypes (GenBank #KF722993 & 94, for mitotype-C and –T, respectively) from 222 of the 323 H. horticola wasps used in the present study, including 37 wasps from Finström, 41 from Föglö, 43 from Seglinge-Kumlinge, 63 from Saltvik and 38 from Sottunga. The 5’end of the COI gene was Sanger sequenced with an ABI 3730 DNA Sequencer (Applied BiosystemsTM, USA). Additionally, Duplouy et al. (2015) showed earlier that about 50% of the H. horticolapopulation on the Åland islands is infected by the Wolbachiastrain w Hho (ST435 from the Wolbachia -PubMLST database, (Baldo et al., 2006)). They screened for w Hho in 296 of the 323 wasps used in this study, including 90 from Finström, 41 from Föglö, 40 from Seglinge-Kumlinge, 86 from Saltvik and 39 from Sottunga, using the primer pair 81F/691R to amplify the conserved Wolbachia surface protein (wsp) using PCR conditions described by (Zhou, Rousset, & O’Neil, 1998), with two positive controls from (Duplouy et al., 2013) and a negative control.