Hydrocarbon seep biofilms share a common functional organization across diverse substrates

Hydrocarbon seeps sustain productive microbial biofilms across diverse natural and anthropogenic surfaces. Yet, it remains unclear whether these taxonomically distinct communities represent substrate-specific assemblages or converge on a common ecological organization. We addressed this question using genome-resolved metagenomics and metatranscriptomics of biofilms colonizing authigenic carbonates, deep-sea shark egg capsules, crustacean carapaces, artificial settlement structures, and plastic debris within the Palmahim Disturbance cold seep in the southeastern Mediterranean Sea. We recovered 565 metagenome-assembled genomes spanning diverse bacterial and archaeal lineages and reconstructed their metabolic potential, interaction traits, ecological roles, and transcriptional activity. Although community composition differed markedly among substrates, all biofilms converged on a conserved ecological organization. Methane- and sulfur-oxidizing microorganisms formed complementary primary-producing guilds that sustained specialized heterotrophic communities partitioning extracellular protein and polysaccharide degradation, nutrient acquisition, fermentation, and cofactor provisioning. Interaction-associated traits, including pili, secretion systems, quorum sensing, and vitamin biosynthesis, were likewise distributed among complementary populations rather than broadly shared across the community. Methanotrophs diversified primarily through lineage radiation while retaining a remarkably conserved interaction repertoire, whereas sulfur-based primary production recruited evolutionarily distant bacterial lineages. Genome-resolved transcriptomics further demonstrated that these complementary guilds were simultaneously active despite contrasting dominant primary producers in collagen-rich shark egg and plastic biofilms. Together, our results show that persistent seep geochemistry organizes biofilms into conserved ecological guilds, while substrate properties primarily determine which microbial lineages occupy those roles. These findings establish a genome-resolved ecological framework for understanding how complex microbial ecosystems assemble and function across heterogeneous methane-fueled surfaces.