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.