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Fig. S1. A. abietis/viridis (gallicola; Aschering) feeding on the primary host tree spruce. An egg cluster is seen between the wings of the insect. Photograph by Holger Daims.

Fig. S2. Phylogenetic relationships of A. laricis/tardus, A. abietis/viridis and A. cooleyi/coweni with other insects based on a concatenated dataset of the cytochrome c oxidase 1 (coI, 634 bp) and elongation factor 1-alpha (ef1alpha, 616 bp) genes. A maximum likelihood tree (PhyML) is shown with selected members of the Aphididae and the Phylloxeridae as outgroup. Maximum likelihood (1000 replicates) and maximum parsimony (1000 replicates) bootstrap values, TREEPUZZLE support values and Bayesian posterior probabilities are indicated at the inner nodes. The letters (G = 60 ± 11.84, F = 65.05 ± 12.03) indicate the estimated divergence times (millions of years ± standard deviation) of the Adelgidae (Havill et al., 2007). Names of host trees are indicated on the right. GenBank/EMBL/DDBJ accession numbers of coI and ef1alpha genes are given in squared brackets. Bar, 10% estimated evolutionary distance.

Fig. S3. Semi-thin cross-section of the abdomen of A. abietis/viridis (Klausen-Leopoldsdorf) stained with toluidine blue showing the location of bacteriocytes longitudinal to the gut. Bar represents 20 μm. g = gut, b = bacteriocytes containing symbionts, s = storage tissue, m = muscle.

Fig. S4. Intracellular localization of bacteriocyte-associated symbionts of A. cooleyi/coweni (Madison) by fluorescence in situ hybridization. A probe mix labeled with FLUOS (green) and targeting most bacteria, a probe double labeled with Cy3 (red) and specific for the betaproteobacterial symbiont ‘Candidatus Vallotia cooleyia’ (ValCo-458), and a probe labeled in Cy5 (blue) and specific for the gammaproteobacterial symbiont ‘Candidatus Gillettellia cooleyia’ (GilCo-576) were used simultaneously. The combined signal of the bacterial and the symbiont-specific probes appears yellow for ‘Candidatus Vallotia cooleyia’ and blue-green for ‘Candidatus Gillettellia cooleyia’, respectively. Bar represents 10 μm.

Table S1. Primers and probes used in this study. Additional information on oligonucleotide probes is available at probeBase (Loy et al., 2007).

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