Evolutionary assembly of a unique purple-green photosymbiosis revealed by expanded ciliate diversity

Symbioses are widespread in nature and have been the source of much evolutionary innovation. While some types of symbioses evolved multiple times, others are extremely rare. Only two purple photosymbioses between heterotrophic eukaryotes and intracellular purple bacteria have been documented. What factors prevent the more frequent establishment of purple photosymbioses? To shed light on this question, we investigated the evolutionary history of the purple-green ciliate Pseudoblepharisma tenue (Spirostomidae) using a phylogenetic and comparative approach and newly discovered species. We sampled about 30 new isolates of spirostomid ciliates from Germany and South Korea, inferred a comprehensive and robust phylogeny based on >200 proteins, and resolved the sister relationship between Pseudoblepharisma and Spirostomum. Furthermore, we characterized P. tenue's sister species, here renamed Pseudoblepharisma chlorelligerum, and revealed that it constitutes a quadripartite symbiosis between a ciliate, a green alga, and two non-photosynthetic bacteria. This oxygenic photosymbiosis is presumed to be supplemented with amino acids by its bacterial symbionts. In addition, we discovered three colorless, non-photosymbiotic Pseudoblepharisma species, which branch as sister to the photosymbiotic P. tenue and P. chlorelligerum. Our phylogenetic and comparative genomic analyses suggest that the green algal symbionts of P. tenue predated the acquisition of purple bacterial symbionts, and that the ancestor of the extant Pseudoblepharisma species was non-photosymbiotic and facultatively anaerobic. These data allowed us to hypothesize on the evolutionary steps that led to the origin of P. tenue and thus bring us closer to explaining the conditions that led to the evolutionary emergence of a unique purple-green symbiosis.