Metabolic and ecological controls on the stable carbon isotopic composition of archaeal (isoGDGT and BDGT) and bacterial (brGDGT) lipids in wetlands and lignites

The glycerol dialkyl glycerol tetraether (GDGT) lipids of archaea and bacteria have been extensively studied in both modern and ancient wetlands, but their specific biological sources and biogeochemical significance in such settings remain unclear. The stable carbon isotopic composition of GDGTs, and associated tetraether lipids such as butanetriol - DGTs (BDGTs), can help reveal aspects of the carbon metabolism of their source organism. Here we present an in-depth compound-specific analyses of archaeal (isoGDGTs and BDGT-0) and bacterial (brGDGTs) tetraether lipid δ 13 C values from the Florida Everglades wetland, supplemented by 16S rRNA gene profiling of the archaeal community. We interpret this alongside GDGT δ 13 C analyses from three additional freshwater wetland sediments, and two ancient wetland deposits (lignites) from the Miocene. Our data suggests that isoGDGT-0 δ 13 C values, which range from –22.5‰ to −36.5‰ across our wetland sites, could to some degree reflect the relative importance of the acetotrophic and CO 2 -reducing hydrogenotrophic methanogenic pathways, though definitively delineating between the two is challenging due to (a) potential incorporation of dissolved inorganic carbon (DIC) by CO 2 reducers, and (b) the diluting effect of GDGTs derived from seemingly heterotrophic/fermentative Bathyarchaeia, the most abundant archaeal class in the Everglades. In the Everglades, which harbours abundant Methanomassiliicoccales, BDGT-0 is significantly more 13 C-depleted than all other lipids, with a δ 13 C signature at certain depths (∼−40.0 to −45.0‰) that is consistent with an important source from these (likely) methylotrophic methanogens. This adds to a growing body of evidence on the underappreciated role of methylotrophic methanogenesis, largely driven by Methanomassiliicoccales , in the freshwater methane cycle. However, slightly more 13 C-enriched values (∼−35.0‰) at certain depths in the same site provide evidence for additional, possibly heterotrophic sources under certain conditions, and BDGT-0 δ 13 C values in the Miocene lignite (≈ δ 13 C of TOC) are consistent with a solely heterotrophic source. Where available for measurement, δ 13 C values of isoGDGTs-1 to -3, averaging –32.7 ± 2.3‰, were significantly more 13 C-depleted than isoGDGT-0, suggesting a different mixture of sources, likely including an autotrophic/mixotrophic component derived from archaea belonging to the Crenarchaeota, Nitrososphaeria (formerly Thaumarchaeota) and/or MBG-D/DHVEG-1. Finally, brGDGT δ 13 C values are generally consistent with their proposed bacterial heterotrophic origin and biosynthetic effects, averaging out at around 4‰ depleted relative to total organic carbon (TOC). Importantly, we find no significant variation between the δ 13 C values of different brGDGTs across our wetland types. This highlights that spatial and temporal changes in commonly applied brGDGT-based proxies likely do not co-occur with changes in the heterotrophic lifestyle of their producer(s), discounting a possible confounding factor in brGDGT palaeothermometry. Taken together, our results yield insights into wetland archaeal communities and the lipids they produce, and underline the applicability of tetraether δ 13 C analyses in probing aspects of microbial carbon cycling in modern and ancient wetland sediments.