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Deep-sea methane seeps host highly diverse microbial communities whose biological diversity is distinct from other marine habitats. Coupled with microbial community analysis, untargeted metabolomics of environmental samples using high resolution tandem mass spectrometry provides unprecedented access to the unique specialized metabolisms of these chemosynthetic microorganisms. In addition, the diverse microbial natural products are of broad interest due to their potential applications for human and environmental health and well-being. In this exploratory study, sediment cores were collected from two methane seeps (-1000 m water depth) with very different gross geomorphologies, as well as a non-seep control site. Cores were subjected to parallel metabolomic and microbial community analyses to assess the feasibility of representative metabolite detection and identify congruent patterns between metabolites and microbes. Metabolomes generated using high resolution liquid chromatography tandem mass spectrometry were annotated with predicted structure classifications of the majority of mass features using SIRIUS and CANOPUS. The microbiome was characterized by analysis of 16S rRNA genes and analyzed both at the whole community level, as well as the small subgroup of Actinobacteria, which are known to produce societally useful compounds. Overall, the younger Dagorlad seep possessed a greater abundance of metabolites while there was more variation in abundance, number, and distribution of metabolites between samples at the older Emyn Muil seep. Lipid and lipid-like molecules displayed the greatest variation between sites and accounted for a larger proportion of metabolites found at the older seep. Overall, significant differences in composition of the microbial community mirrored the patterns of metabolite diversity within the samples; both varied greatly as a function of distance from methane seep, indicating a deterministic role of seepage. Interdisciplinary research to understand microbial and metabolic diversity is essential for understanding the processes and role of ubiquitous methane seeps in global systems and here we increase understanding of these systems by visualizing some of the chemical diversity that seeps add to marine systems. 

Abstract In the past decade, thousands of previously unknown methane seeps have been identified on continental margins around the world. As we have come to appreciate methane seep habitats to be abundant components of marine ecosystems, we have also realized they are highly dynamic in nature. With a focus on discrete depth ranges across the Cascadia Margin, we work to further unravel the drivers of seep‐associated microbial community structure. We found highly heterogenous environments, with depth as a deterministic factor in community structure. This was associated with multiple variables that covaried with depth, including surface production, prevailing oxygen minimum zones (OMZs), and geologic and hydrographic context. Development of megafaunal seep communities appeared limited in shallow depth zones (~ 200 m). However, this effect did not extend to the structure or function of microbial communities. Siboglinid tubeworms were restricted to water depths > 1000 m, and we posit this deep distribution is driven by the prevailing OMZ limiting dispersal. Microbial community composition and distribution covaried most significantly with depth, but variables including oxygen concentration, habitat type, and organic matter, as well as iron and methane concentration, also explained the distribution of the microbial seep taxa. While members of the core seep microbiome were seen across sites, there was a high abundance of microbial taxa not previously considered within the seep microbiome as well. Our work highlights the multifaceted aspects that drive community composition beyond localized methane flux and depth, where environmental diversity adds to margin biodiversity in seep systems.