Museomics reveals uncultured symbionts with biosynthetic potential in nudibranchs

BACKGROUND: Museum specimens are widely used for PCR-based pathogen detection, yet their potential for metagenomic discovery of beneficial microbes remains underexplored, largely due to difficulties in distinguishing true symbionts from contaminants. Here, we use metagenomics of museum specimens to uncover symbioses in endangered or difficult-to-collect animals, such as nudibranchs. To date, Doriopsilla is the only nudibranch demonstrated to harbor an uncultured symbiont involved in chemical defense, leaving it unclear whether comparable associations occur in other nudibranchs. We hypothesized that bona fide symbionts should belong to abundant, uncultured lineages consistently present across individuals of the same host taxon collected across space and time. RESULTS: Using ethanol-preserved specimens archived for up to 30 years, we doubled the number of available nudibranch microbiome datasets and found that dominant uncultured symbionts are rare, with most nudibranchs likely relying on alternative chemical defense mechanisms. An exception were Polycera and Felimare that contained two previously unknown symbionts, Candidatus Polyceribacter and Candidatus Felimaribacter, from distinct uncultured orders that are globally rare in marine metagenomes. These symbionts encode diverse biosynthetic gene clusters exhibiting strain- and species-level microdiversity consistent with metabolites previously reported from their hosts. Their restricted host distribution, phylogenetic distinctiveness, and phylogenetic similarity to symbionts of sponges or corals that are not nudibranch prey, support long-term evolutionary specialization and functional convergence. Fine-scale diversification further suggests host-driven microbial adaptation following symbiosis establishment. CONCLUSIONS: Overall, this study establishes museomics as a robust framework for symbiosis research and advances understanding of the evolutionary and chemical ecology of host-microbe interactions in rare marine invertebrates. Video Abstract.