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Recently, the family  Midichloriaceae  has been described within the bacterial order  Rickettsiales . It includes a variety of bacterial endosymbionts detected in different metazoan host species belonging to Placozoa, Cnidaria, Arthropoda and Vertebrata. Representatives of  Midichloriaceae  are also considered possible etiological agents of certain animal diseases.  Midichloriaceae  have been found also in protists like ciliates and amoebae. The present work describes a new bacterial endosymbiont, “ Candidatus  Fokinia solitaria”, retrieved from three different strains of a novel  Paramecium  species isolated from a wastewater treatment plant in Rio de Janeiro (Brazil). Symbionts were characterized through the full-cycle rRNA approach: SSU rRNA gene sequencing and fluorescence  in situ  hybridization (FISH) with three species-specific oligonucleotide probes. In electron micrographs, the tiny rod-shaped endosymbionts (1.2 x 0.25–0.35 μm in size) were not surrounded by a symbiontophorous vacuole and were located in the peripheral host cytoplasm, stratified in the host cortex in between the trichocysts or just below them. Frequently, they occurred inside autolysosomes. Phylogenetic analyses of  Midichloriaceae  apparently show different evolutionary pathways within the family. Some genera, such as “ Ca . Midichloria” and “ Ca . Lariskella”, have been retrieved frequently and independently in different hosts and environmental surveys. On the contrary, others, such as  Lyticum , “ Ca . Anadelfobacter”, “ Ca . Defluviella” and the presently described “ Ca . Fokinia solitaria”, have been found only occasionally and associated to specific host species. These last are the only representatives in their own branches thus far. Present data do not allow to infer whether these genera, which we named “stand-alone lineages”, are an indication of poorly sampled organisms, thus underrepresented in GenBank, or represent fast evolving, highly adapted evolutionary lineages.

“Candidatus Fokinia solitaria”, a Novel “Stand-Alone” Symbiotic Lineage of Midichloriaceae (Rickettsiales)