ABSTRACT.    Polynucleobacter necessarius (Betaproteobacteria) was first described as endosymbiont of strain E24 of the ciliate Euplotes aediculatus. Further studies showed that the Polynucleobacter-Euplotes association is an obligatory symbiotic system between a monophyletic group of Euplotes species and bacteria belonging to the genus Polynucleobacter. In this system, neither the host nor its symbiont are able to survive independently. Recent studies revealed the existence of free-living populations of Polynucleobacter-like bacteria which are phylogenetically closely related to the endosymbiotic ones, but never share associations with Euplotes in the natural environment. Nevertheless, phylogenetic analyses based on 16S/18S rRNA data suggest that Euplotes species, during their evolution, recruited Polynucleobacter bacteria as symbionts more than once. Moreover, in two cases, we observed different bacteria as obligate symbionts. These bacteria are the first characterized representatives of a phylogenetic lineage branching in a basal position in respect to the genus Polynucleobacter. Likely, they constitute a new genus for which no free-living relatives have been described so far. These data suggest that the original obligate symbionts belonged to this newly discovered genus, and that, only subsequently, in most cases they have been replaced by Polynucleobacter bacteria. This new hypothesis can be tested by increasing the amount of data (i.e. the number of investigated Euplotes species and the number of strains per species) and by learning more about the functional significance of the association. At present, the genomes of two Polynucleobacter strains, one free-living and one obligatory endosymbiotic, are on the point of being completely sequenced. The comparative analysis of these two genomes will provide unique insights in the evolutionary adaptations taking place during the early phase of endosymbiosis. The obtained data will also contribute to shed light on the metabolic interactions between Euplotes and its obligate symbionts.