Members of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont “Candidatus Megaira”, which is phylogenetically closely related to the pathogen Rickettsia. “Candidatus Megaira” was first described as an intracellular bacterium in
several ciliate species. Since then it has been found in association with diverse evolutionary distantlyrelated hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species “Candidatus
Megaira polyxenophila”, and the multidisciplinary description of a novel species, “Candidatus Megaira venefica”, presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic
analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic
protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different
separate species-level clades of “Candidatus Megaira”, and we designed a set of specific probes allowing
easy recognition of the four major clades of the genus.