Award: OCE-1634002

Methane seep and vent ecosystems represent localized areas of novel microbial and animal diversity, enhanced biomass, and productivity fueled by microbial capture and conversion of chemical energy, primarily methane and hydrogen sulfide. The magnitude, temporal, and spatial extent of influence of these chemically supported communities on the surrounding deep-sea environment is not yet understood. This information is of central importance for informing policy relating to ecosystem preservation and sustainability in the context of the growing global interest and technology development for resource extraction in the deep sea. To begin to develop this fundamental ecological framework for deep-sea methane seep ecosystems, we conducted a multi-year research expedition to three distinct seep environments along the Costa Rican Margin- an area known for its pervasive methane seepage and diverse symbiotic and non-symbiotic animal communities residing in both soft bottomed sediments and in association with methane-derived carbonate rocks and pavements. Our multi-disciplinary research incorporated both in situ field seafloor transplant experiments and laboratory-based geochemical, isotopic, molecular, and microscopy analyses, with an emphasis on quantifying the diversity, interactions, and activity of chemosynthetic microorganisms, namely methane-oxidizing archaea and bacteria, which form base of the food web in seep ecosystems. Notable discoveries include the documentation of a new syntrophic microbial partnership between a specific lineage of methane-oxidizing archaea (ANME-2b) with a previously unrecognized sulfate-reducing bacterial partner within the Desulfobacteraceae (called SeepSRB1g) that appears to be capable of fixing nitrogen within this symbiosis- a trait that had previously been ascribed to the ANME archaea. Active nitrogen fixation within methane oxidizing consortia may be spatially controlled, with cells towards the interior of large consortia showing higher levels of N2 incorporation. This work expands our understanding of the specificity of methane-based syntrophy in seeps and the diversity of microorganisms that contribute bioavailable nitrogen within these highly productive deep-sea ecosystems. A detailed molecular and geochemical analysis of the bacterial and archaeal communities associated with sediments and carbonates within zones of active methane seepage and on the perimeter of visible seeps was conducted for Mound 12, one of several prominent seamounts off Costa Rica. Our work documented steep spatial gradients in the sediment-hosted methane-oxidizing community and corresponding methanotrophic activity that positively correlated with active seafloor seepage. This community dynamic contrasted that observed in the coexisting endolithic (rock-hosted) methane-oxidizing community from seep carbonates, where carbonates observed on the periphery outside the area of seepage still retained abundant methane-oxidizing taxa and methanotrophic activity, albeit at lower rates than that observed in the central area of methane-venting. Similarly, reciprocal ecological transplant experiments with carbonates within and outside the seep revealed greater shifts in the microbial community with an increase in methanotrophic taxa when transferred from a low seepage area into an active seep relative to those samples transplanted from active seepage to the periphery over a period of 13 months, suggesting these rock hosted communities are dynamic and respond positively to methane. Collectively, our findings support the inclusion of seafloor carbonates as part of the greater chemosynthetic ecosystem within and beyond the boundaries of visible seepage, serving as a unique habitat for methanotrophic communities and, as discussed below, contributing to habitat expansion of associated chemosymbiotic animals. Working with our collaborators with expertise in animal symbiosis and invertebrate taxonomy, we uncovered a previously unknown novel symbiosis between feather duster worms and methane-oxidizing bacteria. Feather duster worms are common in seep ecosystems, frequently colonizing hard substrates and forming dense colonies on the seep periphery. Historically these worms were presumed to meet their nutritional needs by filter feeding. The observation of unusual morphology of the respiratory plume of these worms shipboard and their anomalously negative stable carbon isotope values prompted a more comprehensive investigation, revealing that these worms have established a remarkable intimate intracellular symbiosis with methanotrophic bacteria in their respiratory plumes and through digestion of these methane eating symbionts, the worms supplement their nutritional demands from methane. This discovery helps to explain the dense colonies of feather duster worms on what was previously considered the periphery of the seep ecosystem. The expansion of the spatial footprint of known symbiotic animals relying on chemical energy from deep sea methane seeps contributes to our main proposal objectives of defining the boundaries and extent methane seepage influences the ecology of life in the deep sea. Last Modified: 11/23/2021 Submitted by: Victoria Orphan