"Candidatus Midichloriaceae" fam. nov. (Rickettsiales), an ecologically widespread clade of intracellular alphaproteobacteria

Abstract

"Candidatus Midichloria mitochondrii" is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus. Bacteria phylogenetically related to "Ca. Midichloria mitochondrii" (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales. This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family "Candidatus Midichloriaceae." In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

Fig 1

Majority rule consensus cladogram of the four Rickettsiales families plus MALOs based on the four 16S rRNA gene sequence data sets (Ac, Al, Bc, and Bl) implementing BI, ML, and NJ inference methods. Support values inferred from the four aligned data sets are reported on the branches, with the Bayesian posterior probability above and the maximum-likelihood (left) and NJ (right) bootstrap values below. The asterisk represents the sole branch that does not agree with the majority rule consensus cladogram (i.e., MALOs were found to be the sister clade of Rickettsiaceae in the tree inferred with NJ on data set Bc).

Fig 2

Unrooted Bayesian phylogram of the Rickettsiales based on the 16S rRNA gene sequence Al alignment. Bayesian posterior probability and ML bootstrap values are reported for the main lineages. (Phylograms obtained with BI, ML, and NJ on the four alignment data sets are shown in Fig. S1 to S12 in the supplemental material.) The scale bar indicates the distance in substitutions per site.

Fig 3

Bayesian phylogram of MALOs/“Ca. Midichloriaceae” fam. nov., obtained from the 16S rRNA gene sequence Al data set. The values on the nodes are the Bayesian posterior probabilities and maximum-likelihood bootstrap percentages (values below 50% are not reported). The names after the accession numbers are those of the hosts of the included MALO bacteria. Where 16S rRNA gene sequences were obtained from environmental samples, the source is indicated in parentheses. Visual representations of hosts (or sources) of the bacteria are mapped beside the terminal tips. The scale bar indicates the distance in substitutions per site.

Fig 4

Maximum-likelihood ultrametric tree, obtained from the Al data set, depicting the identified ESUs within Rickettsiales plus MALOs/“Ca. Midichloriaceae” fam. nov. (the tree was processed in order to drop all branches with 0 length; see Materials and Methods). Clusters of ESUs are highlighted in red; black terminal tips indicate the identified single entities. The likelihood-through-time plot is mapped on the tree. The vertical blue line T1 shows the maximum-likelihood transition point of the switch in branching rates. T2 shows a second likelihood peak corresponding to the family rank on the tree. The scale bar on the x axis indicates the time unit from the root of the ultrametric tree (time −1.0) to the tips (time 0).

Fig 5

Principal-coordinate analysis based on Euclidean distances of the pairwise nucleotide distance matrix obtained from the 16S rRNA gene sequence Al data set. The explained variances are as follows: 1st axis, 52.6%; 2nd axis, 24.2%; 3rd axis, 12.3%. The four Rickettsiales families plus MALOs are labeled.