Microsatellite genotyping
To document temporal and spatial nuclear genetic variation in H.
horticola wasps, we genotyped a total of 324 wasps (124 males/194
females/6 unknown) using the 14 microsatellite-loci developed by
Couchoux et al. (Couchoux, Seppä, & van Nouhuys, 2015b). 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 as described by Couchoux et al.
(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). Several samples were independently
genotyped multiple times to clarify uncertainties in the genotypes.
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 in an
attempt to include specimens from either the 90’s, the 2000’s, and the
2010’s from each locality. Due to the lack of samples from other
decades, the samples from Sottunga were divided between the early and
the late 2000’s groups.
We analysed the genetic structure of wasps collected from the five
localities and the spatio-temporal groups using two independent spatial
Bayesian clustering analyses, with the ‘clustering of groups of
individuals’ settings implemented in the BAPS software (Corander,
Marttinen, Sirén, & Tang, 2008; Corander, Waldmann, & Sillanpää,
2003). We ran two spatial Bayesian clustering analyses with respective
admixture analyses (Figure 3) to evaluate the degree of admixture at
each locality, and in each locality through time. We used the software
GenoDive 2.0b27 (Meirmans & van Tienderen, 2004) to calculate
population genetics measures using only the samples that were
successfully genotyped for at least seven microsatellite markers
(N=309). Hymenoptera males are haploid while females are diploid. The
analyses described above were thus conducted twice: (1) with both sexes
included, but all males considered as homozygote diploids in order to
fit the input format of the software used to define the different
genetic clusters/genotypes, and (2) with only the females, to avoid
overestimating the impact of the haploid males. We tested Hardy-Weinberg
equilibrium at each locus in each locality and for each of the 12
spatio-temporal groups (Table 3). We also calculated the
FST-values between localities and between the 12
spatio-temporal groups. Finally, using only the female samples, we
calculated the observed (HO ) and expected
heterozygosity (HE ) at each locus, as well as the
inbreeding coefficient (Fis ).