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Freshwater reservoirs are an important source of the greenhouse gas methane (CH<sub>4</sub>) to the atmosphere, but global emission estimates are poorly constrained (13.3 – 52.5 Tg C yr<sup>-1</sup>), partially due to extreme spatial variability in emission rates within and among reservoirs. Spatial heterogeneity in the availability of organic matter (OM) for biological CH<sub>4</sub> production by methanogenic archaea may be an important contributor to this variation. To investigate this, we measured sediment CH<sub>4</sub> potential production rates, OM source and quantity, and methanogen community composition at fifteen sites within a eutrophic reservoir in Ohio, USA. CH<sub>4</sub> production rates were highest in the shallow riverine inlet zone of the reservoir, even when rates were normalized to OM quantity, indicating that OM was more readily utilized by methanogens in the riverine zone than in the transitional or lacustrine zones. Sediment stable isotopes and C:N indicated a greater proportion of terrestrial OM in the bulk sediment of this zone. Methanogens were present at all sites, but the riverine zone contained a higher relative abundance of methanogens capable of acetoclastic and methylotrophic methanogenesis, likely reflecting differences in decomposition processes or OM quality. While we found that methane production rates were negatively correlated with autochthonous carbon in bulk sediment OM, production rates were positively correlated with indicators of autochthonous carbon in the porewater dissolved OM. It is likely that both dissolved and bulk OM affect CH<sub>4</sub> production rates, and that both terrestrial and aquatic OM sources are important in the riverine methane production hotspot. Instructions to run analysis: 1. Go to "GitHub project" component and download as zip file ( 2. Go to "data" component and download raw data as zip file. 3. Unzip project directory. 4. Move raw data zip file to data/raw folder in project directory and unzip. 5. Run "analysis_sequence.R" script in R.
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