Rickettsia multilocus phylogenetic analysis
To better resolve the phylogenetic relationships between BOLDRickettsia contaminants, a multilocus approach was employed on a subsample of 186 Rickettsia -containing samples. To this end, 2 further housekeeping genes (16S rRNA , gltA ) and the antigenic 17KDa protein gene were amplified from the respective templates.
Overall, 135 extracts successfully amplified and gave a high-quality sequence for at least one gene. No intragenic or intergenic recombination was detected for any of the gene profiles. A phylogram, including 99 multilocus profiles containing at least 3 of the 4Rickettsia genes of interest (including COI ), allocated strains to both Limoniae and Leech subclades of the Torix group (Figure 4) and these subclades were derived from similar hosts. For example, specific families (Hemiptera: Psyliidae and Hymenoptera: Diapriidae) were present in both Leech and Limoniae groups. A full list of multilocus profiles and Rickettsia group designation can be found in Table S7.
The multilocus study also provided evidence of co-infection withRickettsia . During Sanger chromatogram analysis, double peaks were occasionally found at third codon sites from protein coding genes. This pattern was observed in 6/10 Philotarsus californicusindividuals and in one member of each of the Psilidae, Sciaridae , Chironomidae and Diapriidae (Table S7). Where double peaks were observed, this was found consistently across markers within an individual specimen. This pattern indicates co-infectingRickettsia strains in hosts is a widespread phenomenon of the Torix group.