Previous phylogenetic analyses of Wolbachia sequence data focused upon relationships among Wolbachia strains, but relationships with other members of the Rickettsaceae have not been examined in any great detail [6, 7]. Other analyses have focused upon relationships within genus Rickettsia[8] or genus Ehrlichia[9]. In our analysis, we used 16S rDNA sequences from Rickettsia, Wolbachia, and Ehrlichia to specifically and extensively test whether Wolbachia is a sister taxon to genus Ehrlichia and/or Rickettsia. In addition, amino acid sequences from a subset or Rickettsia, Wolbachia, and Ehrlichia species were also analyzed. Our results confirm and augment previously published trees, which indicate that Wolbachia and the genus Ehrlichia together comprise a sister taxon to the genus Rickettsia. This clustering is extremely robust; analyses using different outgroups, and sequences from a variety of Ehrlichia, Rickettsia, and Wolbachia taxa converge upon the same topology, with good resolution of major branches (Figure 1). Different tree building algorithms (maximum likelihood, parsimony) likewise yield similar topologies. A phylogeny derived from amino acid sequences of GroEL proteins is consistent with the phylogeny inferred from rDNA sequences, although the high level of divergence manifest among these genes precludes unambiguous alignment of rickettsial GroEL sequences to homologous sequences from taxa outside the Rickettsiae. Instead, we aligned representative GroEL amino acid sequences from the three rickettsial genera, and constructed an unrooted tree (Figure 2). This tree is congruent with the tree derived from 16S rDNA sequences. Our analyses confirm monophyly for Wolbachia taxa, including representatives of Wolbachia strains infecting filarial nematodes. When Ehrlichia sennetsu is included in the analysis, this genus is shown to be paraphyletic, with Ehrlichia sennetsu basal to a node linking Wolbachia taxa and all other Ehrlichia taxa included in these analyses (Figure 3). These results indicate that Wolbachia and Ehrlichia lineages shared a common ancestor, and that this ancestral lineage diverged from the genus Rickettsia before the Wolbachia-Ehrlichia split. This conclusion has been implicit in previous studies [10–12]; here we make these relationships explicit, and examine the consequences for our understanding of Wolbachia evolution.
These results have important ramifications for future work on members of this clade. As has been previously noted, phylogenetic analyses of diverse Wolbachia strains imply horizontal transfer of Wolbachia between hosts [1, 7, 10] and has been demonstrated under laboratory conditions [13, 14]. Because the infection frequently results in similar manifestations in taxonomically divergent hosts, this implies bacterial interaction with conserved aspects of host cell and reproductive biology [15]. The obligate endocellular existence is common to all three genera, and presumably represents the ancestral condition of all of these genera. However, the most parsimonious explanation for the evolution and maintenance of traits that alter reproduction, a defining characteristic of the Wolbachia lineage, is to assume that this ability appeared as an evolutionary novelty in the direct ancestor of extant Wolbachia strains. The alternative hypothesis requires numerous independent acquisitions by an endosymbiont of the capacity to induce Cl, alter sex ratios, suppress production of males, and/or feminize host embryos. Hence, the most likely explanation is that a Wolbachia ancestor acquired the ability to manipulate arthropod reproduction in a manner that facilitated bacterial transmission into the next generation, and this property persists in surviving lineages.
There are two reported instances of Rickettsia species which affect host reproduction, one which causes female-biased sex ratio in mites, and a second which causes male killing in ladybird beetles [16]. These two species are fairly distantly related within the genus Rickettsia[8], and intervening members of this genus have not to date been shown to influence or affect reproduction. Therefore, given current information, and in contrast with the situation in the Wolbachia clade, we conclude that these instances are most likely independent acquisitions of the capacity to influence reproduction in their respective hosts. Should further investigation of Rickettsia host reproduction provide evidence of extensive manipulation of host reproduction, we would have to revisit our conclusions. However, since Wolbachia would seem to be selected for efficient vertical transmission, while Rickettsia and Ehrlichia need to maximize the efficiency of horizontal transfer, this does not seem especially likely. This raises questions of a possible trade-offs in life history strategies – did Wolbachia specialization on reproductive manipulation in arthropod hosts cause a loss in the ability to infect vertebrate hosts? Or did loss of the ability to infect vertebrate hosts foster specialization in vertical transmission, and hence, reproductive manipulation? Answers to these questions may come from genetic and molecular studies of the underlying mechanisms involved in Wolbachia-mediated alterations in host reproductive biology.
