Cold seeps are widespread deep-sea ecosystems sustained by methane-rich fluid seepage and host dense chemosynthesis-based biological communities. In 2016, a methane-driven chemosynthetic system was discovered on the Rio Grande Cone, in the Western South Atlantic Ocean, but the structure and drivers of its prokaryotic communities remained poorly understood. Here, we investigated archaeal and bacterial communities associated with deep-sea sediments across three geographic areas (A, C, and E) and a vertical gradient of up to 18 m below the seafloor, encompassing sediment layers within and below the sulfate-methane transition zone (SMTZ). Community composition was assessed using high-throughput sequencing of the 16S rRNA gene (V3-V4 region), processed into amplicon sequence variants (ASVs), and related to local geochemical gradients using multivariate analyses. To disentangle the ecological responses of methane-cycling taxa from the broader microbiome, the prokaryotic community was analyzed by contrasting the ANME-SRB consortium with the remaining archaeal and bacterial taxa. Both groups exhibited significant spatial structuring across areas and sediment layers. Methane concentration and depth were the dominant drivers shaping both ANME-SRB and the remaining prokaryotic community, with conductivity further influencing the latter. Core microbiome analysis revealed a small number of widespread taxa accounting for a large proportion of total community abundance, including an atypical dominance of the archaeal genus Sulfophobococcus. Functional predictions indicated a predominance of sulfur- and nitrogen-related metabolisms, with no clear depth-structured metabolic profiles across the SMTZ. Overall, our results highlight how local geochemical gradients shape both methane-cycling and non-methane-cycling prokaryotic assemblages in a poorly explored South Atlantic cold seep, providing a baseline for future genome-resolved investigations of microbial functioning in this system.
Keywords: chemosynthetic communities; deep‐sea microbiome; metabarcoding; methane seep; sulfate–methane transition zone.
© 2026 The Author(s). Ecology and Evolution published by British Ecological Society and John Wiley & Sons Ltd.