ABSTRACTIn the past 10 years, the number of endosymbionts described within the bacterial orderRickettsialeshas constantly grown. Since 2006, 18 novelRickettsialesgenera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts fromParameciumcollected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium “CandidatusBealeia paramacronuclearis” occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, “CandidatusFokinia cryptica,” whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that “CandidatusBealeia paramacronuclearis” clusters with the so-called “basal”Rickettsiales, and “CandidatusFokinia cryptica” belongs to “CandidatusMidichloriaceae.” We obtained tree topologies showing a separation ofRickettsialesinto at least two groups: one represented by the familiesRickettsiaceae,Anaplasmataceae, and “CandidatusMidichloriaceae” (RAM clade), and the other represented by “basalRickettsiales,” including “CandidatusBealeia paramacronuclearis.” Therefore, and in accordance with recent publications, we propose to limit the orderRickettsialesto the RAM clade and to raise “basalRickettsiales” to an independent order,Holosporalesord. nov., insideAlphaproteobacteria, which presently includes four family-level clades. Additionally, we define the family “CandidatusHepatincolaceae” and redefine the familyHolosporaceae.IMPORTANCEIn this paper, we provide the characterization of two novel bacterial symbionts inhabiting the sameParameciumhost (Ciliophora, Alveolata). Both symbionts belong to “traditional”Rickettsiales, one representing a new species of the genus “CandidatusFokinia” (“CandidatusMidichloriaceae”), and the other representing a new genus of a “basal”Rickettsiales. According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of “traditional”Rickettsialesthat we split into two orders:Rickettsiales sensu strictoandHolosporalesord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated.
The phylogenetic position of enteromonads: a challenge for the present models of diplomonad evolution