Genomic diversity across the Rickettsia and 'Candidatus Megaira' genera and proposal of genus status for the Torix group

Abstract

Members of the bacterial genus Rickettsia were originally identified as causative agents of vector-borne diseases in mammals. However, many Rickettsia species are arthropod symbionts and close relatives of 'Candidatus Megaira', which are symbiotic associates of microeukaryotes. Here, we clarify the evolutionary relationships between these organisms by assembling 26 genomes of Rickettsia species from understudied groups, including the Torix group, and two genomes of 'Ca. Megaira' from various insects and microeukaryotes. Our analyses of the new genomes, in comparison with previously described ones, indicate that the accessory genome diversity and broad host range of Torix Rickettsia are comparable to those of all other Rickettsia combined. Therefore, the Torix clade may play unrecognized roles in invertebrate biology and physiology. We argue this clade should be given its own genus status, for which we propose the name 'Candidatus Tisiphia'.

Fig. 1. Genome wide phylogeny of Rickettsia and ‘Ca. Megaira’.

Maximum likelihood (ML) phylogeny of Rickettsia and ‘Ca. Megaira’ constructed from 74 core gene clusters extracted from the pangenome. New genomes are indicated by ◄ and bootstrap values based on 1000 replicates are indicated with coloured diamonds (red = 91–100, yellow = 81–90, black < = 80). New complete genomes are: RiCimp, RiClec and MegNEIS296. Asterisks indicate collapsed monophyletic branches and “//” represent breaks in the branch. Accessions used are provided in Supplementary Data 1.

Fig. 2. Genus and species level clustering across Rickettsia and ‘Ca. Megaira’.

Frutcherman Reingold networks of pairwise (a) Average Amino Acid Identity (AAI) with edge weights >65% similarity and (b) Average Nucleotide Identity (ANI) with edge weights >95% similarity across all genomes. AAI and ANI illustrate genus and species boundaries, respectively. The 13 current cluster names are annotated over the 23 species clusters found in the ANI network. New genomes are named and have a green outline. Node fill colours indicate Rickettsia (Dark blue), ‘Ca. Megaira’ (orange), Torix/ ‘Ca. Tisiphia’ (purple), Orientia outgroup (light blue). Source data are provided in Source Data.

Fig. 3. Gene content comparison.

Shared and unique gene clusters across genus putative genus clusters Rickettsia, Rhyzobius, Torix and ‘Ca. Megaira’ as suggested by GTDB-tk. Vertical coloured bars represent the size of intersections (the number of shared gene clusters) between genomes in descending order with known COG functions displayed in coral and unknown in blue. Black dots mean the cluster is present and connected dots represent gene clusters that are present across groups. Numbers in parenthesis represent the number of genomes used in the analysis. Source data are provided in Source Data.

Fig. 4. Gene cluster accumulation analysis.

a Pangenome accumulation curves. b Core genome accumulation curves. c The unique genome of Rickettsia (red) and Torix (turquoise) clades as a function of the number of genomes sequenced. Each point represents the mean value while error bars represent ± standard deviation based on 100 permutations. Source data are provided in Source Data.

Fig. 5. Comparison of metabolic potential across selected Rickettsia and ‘Ca. Megaira’.

Heatmaps of predicted KEGG pathway completion estimated in Anvi’o 7, separated by function, and produced with Pheatmap. High to low completeness is coloured dark to light blue. Species groups are indicated with a unique colour as shown in the legend. Pathways of interest are highlighted in red: a The pentose phosphate pathway only present in Torix and ‘Ca. Megaira’, b The biotin pathway present only in the Rhyzobius Rickettsia Oopac6. c NAD biosynthesis only present in Moomin Rickettsia. d dTDP‐L‐rhamnose biosynthesis pathway in Gdoso1, Choog2, Drufa1, and Blapp1. SFG is Spotted Fever Group. Source data are provided in Source Data.

Fig. 6. Workflow diagram for extraction, assembly and analyses performed in this study.

Workflows for genome assemble are illustrated for (a) long read host insect sequences and (b) short read host insect sequences. Purple highlights Torix Rickettsia and orange highlights ‘Ca. Megaira’ and red highlights Transitional Rickettsia. Sequencing technologies used vary with source and include Illumina short read sequencing, BGI DNBseq, Oxford Nanopore and PacBio.