Lucy A Weinert; John H Werren; Alexandre Aebi; Graham N Stone; Francis M Jiggins

“ For the phylogeny that contains missing data, we used only the Bayesian approach.
Split networks for each of the four genes were constructed using the neighbour-net method in SplitsTree4 [70] , [71] . Networks represent multiple trees simultaneously, and they can therefore identify conflicting signals in the data. These may arise from either genetic exchange between bacterial strains, or from systematic error in the underlying model of evolution. ”

“ For those Rickettsiathat are vertebrate pathogens but vectored by arthropods, the effects of the bacteria on their arthropod hosts are generally poorly understood [57] .Rickettsia prowazekiiis clearly pathogenic to infected lice, and transmission through humans is essential to the maintenance of the bacteria in arthropod populations. In every other case, human infections are accidental, but transmission through other vertebrates may allow the bacteria to persist in populations. Many of the bacteria that can infect vertebrates are also transmitted vertically by the arthropod host [60] . ”

“ We show that Rickettsiaare primarily arthropod-associated bacteria, and identify several novel groups within the genus. Rickettsiado not co-speciate with their hosts but host shifts most often occur between related arthropods. Rickettsiahave evolved adaptations including transmission through vertebrates and killing males in some arthropod hosts. We uncovered one case of horizontal gene transfer among Rickettsia, where a strain is a chimera from two distantly related groups, but multi-gene analysis indicates that different parts of the genome tend to share the same phylogeny. ”

“ We use the recently published Orientia tsutsugamushigenome [19] to design PCR primers allowing amplification of rapidly evolving genes from strains that lie between the genera Rickettsiaand Orientia. To include other hosts, we also searched published metagenomic databases for Rickettsiasequences. With these data, we have been able to produce the first well-resolved phylogeny of the entire genus Rickettsia, showing how the vertebrate Rickettsiarelate to the other taxa. Our phylogeny has allowed us to identify and name additional novel groups. ”

“ By mapping host species onto our phylogeny, we are able to make inferences about patterns of host-switching in the genus. It is clear from Figure 1 that Rickettsiabacteria have an extremely diverse host range, occurring in arthropods, vertebrates, plants, amoebas, ciliates, annelids and hydrozoa, and that there have been numerous shifts between these hosts. The earliest shift splits the genus into two major divisions: the hydra and torix groups and all other arthropod Rickettsia. As mentioned, the hydra group are symbionts of protists and undetermined hosts. ”

“ Rickettsiaare intracellular symbionts of eukaryotes that are best known for infecting and causing serious diseases in humans and other mammals. All known vertebrate-associated Rickettsiaare vectored by arthropods as part of their life-cycle, and many other Rickettsiaare found exclusively in arthropods with no known secondary host. However, little is known about the biology of these latter strains. Here, we have identified 20 new strains of Rickettsiafrom arthropods, and constructed a multi-gene phylogeny of the entire genus which includes these new strains. ”

“ This raises many questions about how these bacteria are maintained and spread throughout populations of arthropods and vertebrates. Rickettsiaare known to distort the sex ratio of their hosts by male-killing and inducing parthenogenesis, and are also horizontally transmitted through vertebrates and plants. However, these phenotypes are probably not manifest in the majority of strains discovered and so there may be other ways in which Rickettsiaare maintained in host populations. ”

“ This has important implications for the evolution of Rickettsia, as genes can sweep through different genetic backgrounds of bacterial strains, thereby potentially increasing the spread of genes altering bacterial pathogenicity. Recombination can also complicate the inference of relationships between strains, as recombination violates the assumption that a strain has one evolutionary history. ”

“ The test statistic for a given gene is generated by comparing the maximised likelihood score for that gene with topology unconstrained, with the likelihood obtained when topology was fixed at the maximum likelihood topology obtained from the concatenated dataset. ”

“ Pelagibacterspecies account for 26% of the bacterial rDNA sequences from sea water [29] and have the smallest genomes of free-living bacteria. About 525–775 million years ago there was a transition to living within cells, followed by a split into endosymbionts of protists (Holospora) [30] , [31] and a clade that primarily infects arthropods. Holosporaspecies infect the nuclei of paramecium and are generally considered pathogenic to their hosts; for example, Holospora undulatacan sterilise their hosts, reduce the rate of asexual division and increase host mortality [32] . ”

“ Each dataset was forced to adopt the topology from the concatenated dataset and the log likelihood of this tree was compared with the log likelihood of the unconstrained tree. The taxa used in this analysis are shown in Figure 1b.Phylogeny of Rickettsia . The name of the host prefixed by (s) is given where the bacterium does not have a species name, as well as names for each Rickettsiagroup, life history and host order. (a) Bayesian phylogeny using concatenated sequences of atpA, coxA, gltA, 16S. Posterior support for each node is shown. ”

“ Even though recombination machinery has been detected in Rickettsiagenomes [47] , this is the first evidence that housekeeping genes recombine, and could have implications for the inference of relationships, especially since housekeeping genes (in particular gltAin Rickettsia) are often used to build phylogenies. Therefore recombination should be investigated more fully, especially when using single genes to build phylogenies. These ancient recombination events involve the adalia group and the rhizobius group, as well as the transitional group. ”

“ Four Rickettsiain this group are indeed known to infect sap sucking arthropods (a whitefly, a leaf hopper, an aphid and a red spider mite) , and three of these group separately from the other members of the bellii group (Figure 1) . However, we have uncovered a large number of predatory insect hosts in this group, and sap sucking insects in other groups (a spittlebug symbiont is in the transitional group) . Therefore, the view that members of the bellii group are mainly associated with herbivorous arthropods is not supported by these new data. ”

“ The Bayesian analysis incorporated four Markov chains (three heated and one cold chain) , consisting of 1,000,000 generations with sampling every 100 generations. Two simultaneous runs with different random start trees were performed, and the first 25% of samples were discarded as burn-in. For the Bayesian analysis including missing data, the data were partitioned for the four different genes and assigned the appropriate evolutionary model (given above) , then unlinked so that the parameters were estimated separately and allowed to have a different evolutionary rate. ”

“ Our data also have implications for transmission. We have shown that R. felis (transitional group) ,R. canadensis (canadensis group) and R. bellii (bellii group) are more closely related to Rickettsiain non-blood feeding hosts than to those found in other blood feeding hosts. Therefore, are these strains even transmitted horizontally? As far as we are aware, even in cases where the bacteria can infect vertebrates (as is the case with R. felis) , there has been no recorded instance of transmission back to arthropods (i.e. ectoparasites can not pick up the infection from vertebrates) . ”

“ There are 11 strains of Rickettsiathat infect ladybird beetles, and nine of these cluster in a single monophyletic group. The ones that cluster elsewhere are probably not male-killers (male ladybird beetles are also infected at high prevalence [26] ) . A third possible source of ecological clustering relates to herbivorous hosts. Such clustering may reflect ecology in two possible ways. ”

“ As well as this, Gillespie et al. [14] found that many of the genes on the R. felisplasmid have a closer relationship to the bellii group. This evidence suggests that conjugation with the bellii group Rickettsiamay have an important role in the evolution of the groups containing vertebrate pathogens.
We also detected recombination within the coxAand gltAgenes. This is particularly surprising given that the individual gene topologies did not seem to conflict in any way (Table 2) . ”

“ Elateridae in the bellii group, while the gltAand 16Ssequences (unshaded) are most similar to (s) Pediobius rotundusin the transitional group. (b) A hybridisation network of the concatenated sequences of atpA, coxA, gltAand 16S. A neighbour-net split network was generated and splits were then filtered by weight to include only the (s) C. rufasplit. A hybridisation network was then performed on the split network to provide an explicit example of descent from the two different groups. We did, however, detect some evidence of recombination events within two of the four genes. ”

“ Maximum likelihood phylogeny based on complete sequences of atpA, coxAand gltA. Bootstrap support is given as a percentage above the node, and posterior support from a Bayesian tree is given as a decimal below the node. aPreviously characterised groups. bOnly circumstantial evidence connects the trait to the strain. It is also important to investigate the influence of missing data on the phylogeny. Therefore, we constructed a second tree that included only taxa with complete sequences for the three genes atpA, coxAand gltA (excluding 16Sdue to its marginally significant SH test) . ”

“ Kim, SY & Darby, AC, “The Orientia tsutsugamushi genome reveals massive proliferation of conjugative type IV secretion system and host-cell interaction genes”, Proceedings of the National Academy of Sciences of the United States of America 104: 7981–7986, pmid:17483455, DOI 10.1073/pnas.0611553104 1 2 Jiggins, FM & Tinsley, MC, “An ancient mitochondrial polymorphism in Adalis bipunctata linked to a sex-ratio-distorting bacterium”, Genetics 171: 1115–1124, pmid:16079227, DOI 10.1534/genetics.105.046342 ↑ Chen, DQ ”

“ Verni, F & Rosati, G, “A bacterium belonging to the Rickettsiaceae family inhabits the cytoplasm of the marine ciliate Diophrys appendiculata (Ciliophora, Hypotrichia) ”, Microbial Ecology 49: 434–442, pmid:16003470, DOI 10.1007/s00248-004-0055-1 ↑ Seshadri, R ”

“ Meng, XY & Fukatsu, T, “Rickettsia symbiont in the pea aphid Acyrthosiphon pisum: Novel cellular tropism, effect on host fitness, and interaction with the essential symbiont Buchnera”, Applied and Environmental Microbiology 71: 4069–4075, pmid:16000822, DOI 10.1128/AEM.71.7.4069-4075.2005 1 2 Azad, AF & Beard, CB, “Rickettsial pathogens and their arthropod vectors”, Emerging Infectious Diseases 4: 179–186, pmid:9621188 ↑ Kontsedalov, S ”