In this study we describe the major lithoheterotrophic and autotrophic processes in 21 microbial sponge-associated phyla using novel and existing genomic and transcriptomic datasets. We show that a single gene family, molybdenum-binding subunit of dehydrogenase (coxL), likely evolved to benefit both lithoheterotrophic and organoheterotrophic symbionts, through adaptation to different inorganic and organic substrates. We show the main microbial carbon fixation pathways in sponges are restricted

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... o specialized symbiotic lineages within five phyla. We also propose that sponge symbionts, in particular Acidobacteria, are capable of assimilating carbon through anaplerotic processes. However, the presence of symbionts genomically capable of autotrophy does not inform on their actual contribution to light and dark carbon fixation. Using radioisotope assays we identified variability in the relative contributions of chemosynthesis to total carbon fixation in different sponge species. Furthermore, the symbiosis of sponges with two closely related Cyanobacteria results in outcomes that are not predictable by analysis of -omics data alone: Candidatus Synechococcus spongiarum contributes to the holobiont carbon budget by transfer of photosynthates, while Candidatus Synechococcus feldmannii does not. Our results highlight the importance of combining sequencing data with physiology to gain a broader understanding of carbon metabolism within holobionts characterized by highly diverse microbiomes.