Further phylogenetic analysis
COI sequence alone provides an impression of the frequency with which Rickettsia associates are found in barcoding studies. However, they have limited value in describing the diversity of theRickettsia found. To provide further insight into the diversity of Rickettsia using a multilocus approach, we obtained 186 DNA extracts from the archive at the Centre for Biodiversity Genomics (University of Guelph, Canada) that had provided Rickettsiaamplicons in the previous screen. Templates were chosen based on varied collection site, host order and phylogenetic placement. Multilocus PCR screening and phylogenetic analysis of Rickettsia was then completed, using the methodology in Pilgrim et al. 2017. However, slight variations include the exclusion of the atpA gene due to observed recombination at this locus. Furthermore, the amplification conditions for the 17KDa locus was changed because a Torix Rickettsiareference DNA extract (Host: Simulium aureum ) failed to amplify with the primer set Ri_17KD_F/ Ri_17KD_R from Pilgrim et al. 2017. Subsequently, a 17KDa alignment from genomes spanning the Spotted fever, Typhus, Transitional, Belli, Limoniae groups, and the genusMegaira was generated to design a new set of primers using the online tool PriFi (Fredslund, Schauser, Madsen, Sandal, & Stougaard, 2005).
Once multilocus profiles of the Rickettsia had been established, we tested for recombination within and between loci using RDP v4 (Martin, Murrell, Golden, Khoosal, & Muhire, 2015) using the MaxChi, RDP, Chimaera, Bootscan and GENECONV algorithms with the following criteria to assess a true recombination positive: a p-value of <0.001; sequences were considered linear with 1000 permutations being performed. Samples amplifying at least 3 out of 4 genes (16S rRNA , 17KDa , COI and gltA ) were then concatenated and their relatedness estimated using maximum likelihood as previously described. The selected models used in the concatenated partition scheme (Chernomor, von Haeseler, & Minh, 2016) were as follows: 16S rRNA : TIM3+F+R2; 17KDa : GTR+F+I+G4;COI: TVM+F+I+G4; gltA: TVM+F+I+G4. Accession numbers for all sequences used in phylogenetic analyses can be found in Table S1.