PCR products were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced with primers fD1, rP2 and R1087 (5’-CTCGTTGCGGCACTTAACCC-3’), gyrA-5F, and gyrB-F1, respectively. Sequencing was done in the Department of Entomology at the Max Planck Institute for Chemical Ecology (Jena, Germany) or commercially by SEQLAB Sequence Laboratories (Göttingen, Germany).
Bacterial sequences were deposited in the GenBank database under following accession numbers: KM035545 – KM035652 (16S rRNA genes), KM035653 – KM035673 (gyrA genes) and KM035674 – KM035755 (gyrB genes). Diversity of bacterial strains in individual beewolf antennae Bacterial micro-colonies selleck compound were isolated from individual antennae of two different Philanthus multimaculatus and one Philanthus psyche
female with serial dilution in 24-well plates with liquid medium SB203580 mouse as described above. Individual micro-colonies were carefully transferred by pipette into 96-well PCR plates with 100 μl PCR lysis solution A without proteinase K (67 mM Tris–HCl (pH 8.8); 16.6 mM (NH4)2SO4; 6.7 mM MgCl2; 6.7 μM EDTA (pH 8.0); 1.7 μM SDS; 5 mM β-mercaptoethanol) ; samples were heated at 95°C for 5 min to destroy bacterial cells. Afterwards, gyrB gene fragments were amplified, purified and sequenced as described above. Obtained sequences were aligned and manually curated using Geneious software version 6.0.5 (Biomatters Ltd., http://www.geneious.com/). Clomifene Phylogenetic analysis 16S rRNA, gyrA and gyrB gene sequences of isolated symbionts were aligned with those obtained from field-collected beewolves
as well as representative outgroup sequences of free-living Streptomyces and other actinomycete strains (Additional file 4: Table S4). Alignments of individual genes were concatenated for phylogenetic analyses. Approximately-maximum-likelihood trees were reconstructed with FastTree 2.1 using the GTR model, with local support values estimated by the Shimodaira-Hasegawa test based on 1,000 resamplings without re-optimizing the branch lengths for the resampled alignments . Bayesian inferences were run with MrBayes 3.1.2 [42–44], with the different genes defined as separate partitions in the concatenated alignment. The searches were conducted under the GTR + I + G model, with 4,000,000 generations per analysis. Trees were sampled every 1,000 generations. We confirmed that the standard deviation of split frequencies was consistently lower than 0.01, and a “burnin” of 25% was used, i.e. the first 25% of the sampled trees were discarded. We computed a 50% majority rule consensus tree with posterior probability values for every node.