Table 1.
MLST marker genes.
Figure 1.
Bacterial ML phylogeny generated from 16 S ribosomal RNA encoding sequences.
Terminal branches are labelled by genus and species, pathotype, strain and/or original host designations, GenBank accession numbers as well as pairwise nucleotide sequence identity percentages as calculated from a p-distance matrix with respect to the ‘Rickettsiella ixodidis’ GSU sequence. Numbers on internal branches indicate bootstrap support values; branches that do not receive >90% bootstrap support are represented by dashed lines. The phylogram has been rooted using Escherichia coli as outgroup. The size bar corresponds to 5% sequence divergence. To enhance resolution, the upper clade of the phylogram that comprises Rickettsiella-like bacteria, has been extended into a cladogram. GenBank accession numbers AM490937-39 and EU430248-50 designate partial 16 S rRNA gene sequences comprising only 40–70% of the complete 16 S rRNA marker sequence. The tree has been reconstructed from ClustalX aligned sequences; an essentially identical ML tree has been generated from a T-Coffee based nucleotide sequence alignment (not shown).
Figure 2.
Significance testing evaluating the 16 S rRNA gene based taxonomic assignment of ‘Rickettsiella ixodidis’.
Cladogram presenting the backbone tree topology generated by pruning off the terminal ‘Rickettsiella ixodidis’ branch from the 16 S rRNA gene based ML phylogeny of Figure 1. Terminal branches are labelled by genus and species, pathotype and/or original host designations as well as GenBank accession numbers. A set of 51 candidate topologies to be evaluated by the 1sKH test was generated by re-grafting the ‘R. ixodidis’ branch to any of the 51 branches of the backbone topology. Numbers on branches indicate the p-value attributed by the 1sKH test with respect to the 16 S rRNA gene alignment to the candidate topology that carries ‘R. ixodidis’ grafted to the respective branch. Candidate topologies that remain unrejected under application of a 5% significance threshold are indicated by the p-value printed in bold type; “B” designates the most likely tree.
Figure 3.
Bacterial phylogenies generated from MLST marker sequence alignments.
A–D, F: Bacterial ML phylogenies generated from hypervariability filtered ftsY (A), gidA (B), rpsA (C), and sucB (D) gene sequences or a concatenation of these (F). Terminal branches are labelled by genus and species, pathotype and/or strain designations, GenBank accession numbers, and pairwise nucleotide sequence identity percentages respect to the ‘Rickettsiella ixodidis’ GSU sequence as calculated from a p-distance matrix with. Numbers on internal branches indicate bootstrap support values; branches that do not receive >90% bootstrap support are represented by dashed lines. Trees were rooted using Escherichia coli as outgroup. The size bar corresponds to 5% sequence divergence. E: Extended majority rule consensus tree topology generated from 24 phylogenies reconstructed by the ML, ME, or NJ method from hypervariability filtered nucleotide or deduced amino acid sequence alignments of the ftsY, gidA, rpsA, or sucB marker. For compatibility with the other markers, specimens ‘R. agriotidis’, ‘R. pyronotae’ and ‘Diplorickettsia massiliensis’ were removed from the ftsY data sets prior to single phylogeny reconstruction. Terminal branches are labelled by genus and species, pathotype and/or strain designations. Internal branches collapsing under the strict consensus criterion are represented by dashed lines; the frequency of occurrence across the aggregated set of phylogenies is indicated as a percentage value on top of the respective branch.
Figure 4.
Generation of candidate tree topologies for likelihood-based significance testing based on MLST markers.
15 candidate topologies were generated from a four organism basic tree (A) comprising Coxiella burnetii (acronym “Cbu”) as outgroup together with three Rickettsiella strains in positions a–c. The candidate topologies tested (B) were generated from this basic tree by the following steps: 1. Permutative addition of three Rickettsiella strains representing the species Rickettsiella grylli (“Rgr”) and the pathotypes ‘Rickettsiella melolonthae’ (“Rme”) and ‘Rickettsiella tipulae’ (“Rti”) to positions a–c generates a maximum of six tree topologies that are pair-wise identical, i.e. a total of three different topologies. 2. Subsequent addition of the pathotype ‘Rickettsiella ixodidis’ (“Rix”) to any of the five branches indicated by a black dot in any of the previously generated trees.
Table 2.
Significance testing results for 16 S rRNA and MLST markers in a five organism model.