Harnessing chemosynthetic symbionts is a recurring evolutionary strategy. Eukaryotes from six phyla as well as one archaeon have acquired chemoautotrophic sulfur-oxidizing bacteria. In contrast to this broad host diversity, known bacterial partners apparently belong to two classes of bacteria—the
Gamma
- and
Epsilonproteobacteria
. Here, we characterize the intracellular endosymbionts of the mouthless catenulid flatworm genus
Paracatenula
as chemoautotrophic sulfur-oxidizing
Alphaproteobacteria
. The symbionts of
Paracatenula galateia
are provisionally classified as “
Candidatus
Riegeria galateiae” based on 16S ribosomal RNA sequencing confirmed by fluorescence in situ hybridization together with functional gene and sulfur metabolite evidence. 16S rRNA gene phylogenetic analysis shows that all 16
Paracatenula
species examined harbor host species-specific intracellular
Candidatus
Riegeria bacteria that form a monophyletic group within the order
Rhodospirillales
. Comparing host and symbiont phylogenies reveals strict cocladogenesis and points to vertical transmission of the symbionts. Between 33% and 50% of the body volume of the various worm species is composed of bacterial symbionts, by far the highest proportion among all known endosymbiotic associations between bacteria and metazoans. This symbiosis, which likely originated more than 500 Mya during the early evolution of flatworms, is the oldest known animal–chemoautotrophic bacteria association. The distant phylogenetic position of the symbionts compared with other mutualistic or parasitic
Alphaproteobacteria
promises to illuminate the common genetic predispositions that have allowed several members of this class to successfully colonize eukaryote cells.