Novel nitrifying symbiont lineages are vertically inherited and widespread in marine sponges

Ammonia-oxidising archaea and nitrite-oxidising bacteria are common members of marine sponge microbiomes. They derive energy for carbon fixation and growth from nitrification - the oxidation of ammonia to nitrite and further to nitrate - and are proposed to play essential roles in the carbon and nitrogen cycling of sponge holobionts. In this study, we characterise two novel nitrifying symbiont lineages, Ca. Nitrosokoinonia and Ca. Nitrosymbion in the marine sponge Coscinoderma matthewsi using a combination of molecular tools, single-cell imaging techniques, and physiological rate measurements. Both represent a new genus in the ammonia-oxidising archaeal class Nitrososphaeria and the nitrite-oxidising bacterial order Nitrospirales, respectively. Furthermore, we show that larvae of this viviparous sponge are densely colonised by representatives of Ca. Nitrosokoinonia and Ca. Nitrosymbion indicating vertical transmission. In adults, the representatives of both symbiont genera are located extracellularly in the mesohyl. Comparative metagenome analyses and physiological data suggest that ammonia-oxidising archaeal symbionts of the genus Ca. Nitrosokoinonia strongly rely on endogenously produced nitrogenous compounds (i.e., ammonium, urea, nitriles/cyanides, and creatinine) rather than on exogenous ammonium sources taken up by the sponge. Additionally, the nitrite-oxidising bacterial symbionts Ca. Nitrosymbion may reciprocally support the ammonia-oxidisers with ammonia via the utilisation of sponge-derived urea and cyanate. Interestingly, comparative analyses of published environmental 16S rRNA amplicon data revealed that Ca. Nitrosokoinonia and Ca. Nitrosymbion are widely distributed and predominantly associated with marine sponges and corals, suggesting a broad relevance of our findings.