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The Nara, Sioux, and Mackinac ecotones were sampled in summers 2018 and 2019, while the Saganing and Wildfowl ecotones were only sampled in summer 2020. Although we had planned to sample all 5 sites in summer 2020, this was prevented by limitations to field work caused by the COVID-19 pandemic. Each interface was sampled across 1-3 sampling days in each year due to the number of transect points and the duration of incubations. Sampling days in each ecotone were typically sequential, but in some cases, there was a day (and in one case 3 days) between sampling due to inclement weather. On the first day at each ecotone, a transect of 8-15 points was established that encompassed the wetland - stream - lake ecotone. Based on visual classifications of the 83 total transect points among all transects, 37 were wetland, 18 were transition zones from wetland to stream, 15 were stream, 2 were stream to lake transition zones and 11 were lake sites.

We deployed nutrient diffusing substrates (NDS, Tank et al. 2017) in summers 2019 and 2020 at all transect points except those that were too deep, had high wave action, or had no standing water (see Tables S3 and S4 in Supplemental Information). NDS were constructed using 45 mL plastic containers filled with a 2% (by weight) agar solution amended with 0.8 M N added as NaNO3 (N treatment), 0.05 M P added as NaH2PO4 (P treatment), both (N+P treatment), or neither as a control treatment. A 25 mm porous porcelain disc (Leco Corporation, St. Joseph, MI) was placed on top of each hardened NDS for biofilms to grow on. At transect points, a total of 16 NDS were deployed with 4 control, 4 N, 4 P, and 4 N+P replicates. NDS were deployed at transect points two weeks prior to the first sampling day at a site. After these two weeks, the discs were collected, wrapped in aluminum foil and frozen for later analysis of biofilm biomass using chlorophyll-a and ash free dry mass (AFDM), which provides an estimate of the total organic material present in a sample. Laboratory analysis of chlorophyll-a followed standard method using a Thermo Scientific 10s UV-Vis spectrophotometer and ethanol extraction (APHA 2005). All discs were also analyzed for AFDM as the difference between the mass of the oxidized samples and the initial dry samples. AFDM samples were dried at 50 °C, weighed for dry mass and then oxidized in a muffle furnace at 550 °C, rewetted, and dried before a final weighing.

Chamber incubations were used to measure rates of N2 fixation and denitrification in all transect points. The chambers used during these incubations varied by substrate type. 2-L polycarbonate food storage containers were used for larger macrophyte substrate (Gettel et al. 2007, Eberhard et al. 2018). The chamber lids were sealed airtight with a Viton o-ring, and were fit with a 13x20 mm septum for sample collection. For sediment and smaller macrophyte substrate, chambers were made from pint size glass mason jars and lids were similarly fit with an airtight sampling septum. Macrophytes were collected using chamber lids to approximate surface area of macrophyte to sample, then pulling from the root and placing in chambers. Sediment substrate was collected using a 7 cm diameter suction corer to collect ~200-400 mL of sediment that was then placed into chambers. For each transect point, there were 1-4 sample chambers and 1-4 blank chambers, with each sample chamber having a paired blank chamber. The blank chambers were filled with stream water and incubated along-side sample chambers to simulate an environment with minimal N2 fixation or denitrification to control for chamber effects. Blank chambers were confirmed to have minimal to no N2 fixation or denitrification activity. Nitrogen fixation and denitrification rates were measured and calculated as described in Eberhard et al. 2018.

We also measured a variety of environmental covariates. We measured depth at each transect point and measured canopy cover (%) using a spherical densiometer (Lemmon 1956). We also collected ~40 mL water samples from each transect point. The water was filtered using Millipore 0.45 μm nitrocellulose membrane filters into 60 mL bottles. Samples were frozen until laboratory analysis for NO3--N, ammonium (NH4+-N), soluble reactive phosphorus (SRP), total dissolved phosphorus (TDP), dissolved organic carbon (DOC), and total dissolved nitrogen (TDN) following methods described in Eberhard et al. 2018.

Water was also filtered using Millipore 0.45 μm nitrocellulose membrane filters into 60 mL amber bottles with no headspace for DOM analysis. Samples were kept refrigerated until analysis for absorption and fluorescence spectra using a Horbia Aqualog fluorometer (Horiba-Jobin-Yvon Aqualog C; Horiba Co., Edison, New Jersey) in 1 cm quartz cells (Starna Cells, Inc) to determine fluorescence excitation-emission matrices (EEMs). Samples were analyzed and indices were calculated as described in detail in Meingast et al. 2020.