Seepage area of the cold seep exhibits strong homogeneous selection on prokaryotic community assembly and supports high depth variability of both archaeal and bacterial communities.

Haima Cold Seep is an active cold seep system in the South China Sea with a thriving biological community that plays an important role in the global carbon cycle. Despite its ecological importance, we know little about the variability of its microbial communities and depth-related structure. In this study, we comprehensively investigated the microbial depth variability and its underlying assembly process in the seepage and non-seepage areas of Haima Cold Seep. We found significant inter-group differences in archaeal (ACC) and bacterial (BCC) other than eukaryotic (ECC) community compositions, between the seepage and non-seepage areas. The seepage area had significantly more archaeal and bacterial 16S rRNA gene copies than the non-seepage area and showed greater fluctuation with depth. Anaerobic methanotrophic archaea (primarily, ANME-3) and sulfate-reducing bacteria (primarily, SEEP-SRB1) were predominant in the seepage area, suggesting that ANME-3 might work in conjunction with sulfate-reducing microorganisms to support the carbon cycle in the cold seep environment. Moreover, there were more lineages found only in specific depth ranges, supporting higher depth variability of both ACC and BCC in the seepage area than in the non-seepage area. The greater depth variability of ACC and BCC in the seepage area appeared to be primarily driven by stronger homogeneous selection imposed by environmental factors (e.g., ammonium). By contrast, eukaryotic community assembly was influenced by random processes (primarily drift) and exhibited no depth dependence. Our findings may help broaden our understanding of the cold seep ecosystem and thus provide clues for its resource utilization.IMPORTANCEMarine cold seeps are characterized by the discharge of hydrocarbons and reducing fluids. Rising geological fluids in cold seeps may act as physical transport vectors for deep biosphere microorganisms from the subsurface environment to the surface environment, and thus may influence the depth patterns of microbial community assembly. Despite the ecological importance of microbial communities in cold seeps, we have limited knowledge about their responses to environmental changes along sediment depth and the underlying processes driving these responses. Our study showed that compared with non-seepage area, seepage area exhibited stronger homogeneous selection on prokaryotic community assembly, had more depth-related specialized microorganisms, and supported higher depth variability of both archaeal and bacterial communities. Our findings may provide a theoretical basis for protection and resource utilization of the cold seep ecosystem.