Natural 14C abundances and stable isotopes suggest discrete uptake routes for carbon and nitrogen in cold seep animals

Cold seeps, where geofluids containing methane and other hydrocarbons originating from the subseafloor seeps through the sediment surface, play important roles in the elemental and energy flux between sediment and seawater. These seep sites often harbor communities of endemic animals supported by chemolithoautotrophic bacteria, either through symbiosis or feeding. Despite these animal communities being intensively studied since their discovery in the 1980's, the contribution of carbon from seep fluid to symbiotic microbes and subsequently host animals remains unclear. Here, we used natural-abundance radiocarbon to discern carbon sources: the ambient bottom water or the seeping geofluid. The C-14 concentrations were measured for vesicomyid clams, a parasitic calamyzine polychaete, and a siboglinid tubeworm species from four different cold seep sites around Japan. We found most vesicomyid clams exhibiting C-14 concentrations slightly lower than that of the ambient bottom water, suggesting up to 9% of C for chemolithoautotrophy originates from geofluid DIC. The different extent of fluid contribution across species may be explained by different routes to incorporate DIC and/or different DIC concentrations in the geofluid at each seep site. Stable nitrogen isotopic compositions further suggested N incorporation from geofluids in these clams, where the burrowing depth may be a key factor in determining their delta 15N values. The siboglinid tubeworm showed a clear dependency for geofluid DIC, with a contribution of > 40%. Our results demonstrate the effectiveness of C-14 analyses for elucidating the nutritional ecology of cold seep animals and their symbionts, as was previously shown for hydrothermal vent ecosystems.