These methods, instrumentation and/or protocols apply to all data in this dataset:| Methods and protocols used in the collection of this data package |
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Stream surveys:
We surveyed 50-meter stream reaches beginning above the gauging weir for numbered watersheds (1, 2, 3, 4, 5, 6, and 9) and quasi-random 200-meter stream reaches in Bear Brook and Paradise Brook. In each stream reach, we conducted longitudinal surveys (along the stream) and lateral surveys (across the stream). For the longitudinal surveys, walking upstream, we recorded stream characteristics at every meter: depth in the center of flow, habitat type (pool, riffle, slide, or cascade), substrate (boulder, bedrock, cobble, pebble, sand, or wood), and any present organic material (e.g., stick, algae, leaf litter, bryophyte). For our lateral surveys, in the 50-meter stream reaches (W1-6, 9), we randomly selected 10 longitudinal meter markers to conduct transects. We divided the 200-meter stream reaches (Bear and Paradise Brook) into four 50-meter segments and randomly selected 10 longitudinal meter markers to conduct lateral transects in each of the segments. At the lateral transect markers, we recorded the width, bryophyte coverage, and leaf litter coverage for both wetted and active channel cross sections. We defined the active channel margins by breaks in slope. We avoided seeps and additional nutrient inputs in our stretches.
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Nutrient incubation:
We collected 18 bryophyte samples from each of the 200 meter reaches in Bear Brook and Paradise Brook, for a total of 36 samples which were used in lab incubations to measure nutrient uptake. All samples were collected using a circular cutter with a 6.8 cm diameter. Using data from our 2022 longitudinal surveys, we used a stratified random selection of sampling sites where we observed bryophytes, with the goal of collecting replicate samples from across the distribution of habitat types, for a total of 6 samples per habitat (cascade, pool, and slide) in each of the two streams.
Once all samples were collected, we prepared our bryophyte samples for nutrient incubation. We drained excess water from bryophyte samples on a sieve and placed them right-side-up in open pint mason jars. These jars were each filled with 400 mL of water from Paradise Brook, removing air bubbles caught in the bryophyte samples. We placed these jars into a tray using randomly generated numbers to assign location in the tray and water sampling order. The tray was pre-filled with enough water to submerge the bottom half of the jars and maintain them at ambient summer water temperatures (12C) in a refrigerator. The jars were then covered by foil and allowed to rest for 24 hours in the dark at 12C to allow suspended sediments to settle.
After this settling period, the overlying solutions were mixed by withdrawing water near the top of the bryophyte clump with a syringe and reintroducing that water near the top of the jar. This was repeated three times. After mixing we removed 120 mL of stream water and measured initial nitrate and phosphate concentrations for these pre-incubation samples. The volume of water removed was replaced by 120 mL of nitrate and phosphate stock (1.06 mg/L NO3-N and 0.012 mg/L PO4-P, respectively, made from KNO3 and KH2PO4). The samples were incubated for 12 hours under a grow-light and 12 hours in the dark at 12oC. We collected 60 mL of water after both 12-hr light and dark incubations were completed, filtering the samples through a Whatman GF/F glass fiber filter (0.7 µm). The filtered water samples were frozen until they could be analyzed on an ion chromatograph (Dionex ICS-2000 with an AS-18 analytical column) for their nitrate and phosphate concentrations. Here, we have only included nitrate data because almost all samples reported phosphate levels that were below detection after incubation.
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Bryophyte masses:
In addition to the 36 bryophyte clumps collected from Bear Brook and Paradise Brook for nutrient incubation, we collected samples from at least 5 lateral transects in each upper watershed (W1-6, 9), for a total of 84 bryophyte samples for nutrient stock analysis. To prepare the sample for desiccation, depending on the size of the sample, we rinsed it in a tub with 2 L of DI water or a specimen cup with 100 mL of DI water. We then poured the sample through stacked sieves (1.19 mm and 125 μm) into a pre-weighed container and recorded the mass of the filtrate. Coarse materials caught in the top sieve (>1.19 mm) were placed in a paper bag, while fine materials caught in the bottom sieve (between 125 μm and 1.19 mm) were placed in pre-weighed aluminum tins. All samples were air dried and weighed for months, which may have introduced variability in mass measurements due to water retention. Once dry mass was recorded for all samples, they were heated to 550C for 2 hours in a muffle furnace to combust all organic material in accordance with methods described by (Benfield et al. 2017). The resulting ash-free dry mass was then weighed.
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