Codependence of individuals in the Nephromyces species swarm requires heterospecific bacterial endosymbionts

Symbiosis is one of the most important evolutionary processes shaping the biodiversity on Earth. Symbiotic associations often bring together organisms from different domains of life, which can provide an unparalleled route to evolutionary innovation. 1–4 The phylum Apicomplexa encompasses 6,000 ubiquitous animal parasites; however, species in the recently described apicomplexan family, Nephromycidae, are reportedly non-virulent. 5, 6 The members of the genus Nephromyces live within a specialized organ of tunicates, called the renal sac, in which they use concentrated uric acid as a primary nitrogen source. 7, 8 Here, we report genomic and transcriptomic data from the diverse genus Nephromyces, as well as the three bacterial symbionts that live within this species complex. We show that the diversity of Nephromyces is unexpectedly high within each renal sac, with as many as 20 different species inhabiting the renal sacs in wild populations. The many species of Nephromyces can host three different types of bacterial endosymbionts; however, FISH microscopy allowed us to demonstrate that each individual Nephromyces cell hosts only a single bacterial type. Through the reconstruction and analyses of the endosymbiont bacterial genomes, we infer that each bacterial type supplies its host with different metabolites. No individual species of Nephromyces, in combination with its endosymbiont, can produce a complete set of essential amino acids, and culture experiments demonstrate that individual Nephromyces species cannot form a viable infection. Therefore, we hypothesize that Nephromyces spp. depend on co-infection with congeners containing different bacterial symbionts in order to exchange metabolites to meet their needs.