For complete methodology, see Ramus et al., 2017 (doi:10.1073/pnas.1700353114). In summary:
The experiment was carried out on intertidal mud and sandflats located within the Zeke’s Island National Estuarine Research Reserve (33.95 N, 77.94 W), North Carolina, USA. We manipulated six densities (n = 8 per treatment) of the nonnative seaweed Gracilaria vermiculophylla in 48 large 25 square meter plots over a 10-month period. We selected three low-intertidal flats spanning over 1 km in the reserve that differed in area, flow regimes, Gracilaria cover, grain size, and proximity to the Spartina salt marsh. The three flats represented the continuum of estuarine habitats where Gracilaria naturally occurs in this area. We established the 48 plots along the mean low water line at 5-m intervals by adding 3-m steel rebar 1.2 m into the substrate at each plot-corner. Treatments were randomly assigned to the plots to avoid potentially confounding small-scale effects of site (and all plots had only few Diopatra tubes). Gracilaria was fixed in a plot with metal 'u-pegs' (constructed from clothes hangers) by physically staking handful-sized 'clumps' of loose thalli to the sediment surface. Pegs were flushed with the sediment surface to avoid above-surface experimental artifacts. Our six treatments were based on the total number of pegs per 25 square meters (arranged in squared grids) as follows: 0 (0×0), 9 (3×3), 36 (6×6), 100 (10×10), 225 (15×15), and 400 (20×20). Gracilaria was collected from nearby locations and added to plots in the u-peg grids in August 2013. Treatments were maintained and response variables quantified approximately monthly from September 2013 to June 2014 (treatments were maintained and measured at total of 10 times). For each plot visit we quantified the cover of Gracilaria (in 10 randomly placed 0.25 square meter quadrats per plot) and seven ecosystem functions (see next section for detail) before maintaining Gracilaria densities (by replenishing u-pegs devoid of Gracilaria and manually removing Gracilaria from control plots).

To examine the effect of Gracilaria on epifauna, we positioned a 0.25 square meter quadrat in the center of each plot and collected all Gracilaria and its associated epifauna into a ziptop bag. In the laboratory, Gracilaria was rinsed in freshwater and shaken for about 1 min to remove epifauna, which were captured on a 500 micron sieve. Epifauna were identified and enumerated to broad taxonomic groupings (typically family level) under a stereomicroscope (~18x, Nikon SMZ800). For simplicity, all faunal data were standardized to unit area. Taxonomic richness was rescaled to unit area using the species-area relationship and assuming a conservative value of 0.15 for z.

To quantify whether Gracilaria attenuates hydrodynamic forces, we used gypsum dissolution blocks that dissolve at a rate proportional to water velocity and thus represent an integrated proxy for tidal currents and wave exposure. We created gypsum blocks as hemispheres (⌀ = 6.5 cm) from dental plaster (Die Keen, Heraeus Kalzer), covered on the bottom with two layers of polyurethane to ensure that an equal surface area would be subject to dissolution. Gypsum blocks were dried at 60 degrees C for a minimum of 24 h before recording the initial mass and deploying one block flush with the substrate surface in the center of each plot for 4 d. Following retrieval, gypsum blocks were dried and reweighed, and the dissolution rate calculated as grams of gypsum dissolved per day. Because lower dissolution rates indicate greater flow reduction, dissolution rates were reflected using the equation –fi+max(fi), so that greater flow reduction corresponds with a positive contribution to ecosystem functioning.

To examine the effect of Gracilaria on sediment stabilization, we marked all corner poles at 20 cm above the substrate surface in August 2013. The distance between the marking and substrate surface was measured with a ruler to the nearest 0.5 cm at the end of each month. We calculated the monthly (30 d) change in height in cm by subtracting the final from initial distance to the substrate (using the average of the 4 corners per plot) and correcting for the time interval between measurements. Accretion and erosion are represented as positive and negative values, respectively.

To assess the effectiveness of Gracilaria as a nursery habitat for commercially and recreationally important species, we sampled the entire plot using a 1.2-m high × 6.7-m wide nylon seine net (The Fish Net Company, Jonesville, LA; mesh size = 3.175 mm) during a falling tide. Upon completion of a pass, we swiftly pulled the net taught, tilted it into a horizontal position, and lifted it from the water into an adjacent boat (R/V Adelaide) in a single motion. Organisms (greater than 1 cm) retained on the boat were identified to the family-level and enumerated before being returned to the water. Abundances were reported per unit area (dividing by 25 square meters) and richness data were rescaled to unit area using the species–area relationship and assuming a conservative value of 0.15 for z.

To quantify the effect of Gracilaria on decomposition processes, standing dead Spartina stems were collected from adjacent salt marshes, washed, and dried at 60 degrees C for a minimum of 72 h (until no further weight loss). We pooled multiple stems to achieve an initial mass of 7.0 +/- 0.5 g and placed them inside a mesh litter bag, which was closed and deployed on the sediment surface in the center of each plot. Bags were retrieved just prior to the next treatment maintenance. Remaining stem material was washed, dried, and weighed and decomposition rate was reported as the mass lost in grams per month.

To simplify our analyses and remove temporal autocorrelation, we calculated the average response of each function in each plot using the full 10 month data set (48 plots sampled each month). At the end of the experiment we measured four additional functions (months 8 through 10). Because we did not have seasonal data for these responses they were excluded from the main analysis of multifunction effects.

To sample benthic infauna, triplicate core samples (5-cm diameter, 15-cm depth, volume = 294.5 cubic cm) were taken equidistant along a diagonal transect of each plot on June 25, 2014. The three sediment core samples from each plot were pooled into a ziptop bag. Upon return to the laboratory, the content of each bag were drained and rinsed over a 1-mm mesh sieve to remove fine sediments. Infauna retained on the sieve were preserved in 75% ethanol. The 1-mm mesh-size was chosen to concentrate sampling efforts on juvenile and early life stages of crustaceans, molluscs, and larger polychaete taxa. Infauna were identified and enumerated under a stereomicroscope (~18x, Nikon SMZ800) to families, and, in some cases, phyla. Infaunal data were standardized and rescaled to unit volume (using the reciprocal of 0.8836 L and a conservative z of 0.15) following the same methods described previously for epifauna and nursery functions.

To evaluate the effect of Gracilaria on ray foraging activity, we counted the number of ray holes in each plot on 3 to 4 different days in a given month. We here report the average number of ray holes standardized to unit area (by dividing by 25 square meters) during a given low tide on a single day.

To investigate the association of waterfowl with Gracilaria, we delimited the 48 plots into four sites based on spatial proximity (plots 1-12, 13-24, 25-36, and 37-48) and surveyed all waterfowl activity occurring within a site (containing 12 plots) for a 15-min period during low tide. Bird counts were made through binoculars from our research vessel from a distance of about 100 m to avoid disturbances arising from our presence. We tallied the number of birds initially present, and that became present, within the boundaries of each plot during the observation period. After completing the 15-min observation of a site, we moved to a new vantage point for observing the next 12 plots. Hence, by repeating this procedure at all sites, all 48 plots were sampled with equivalent effort in a ~1 h period. Because measurements were made on 1 to 3 different days in a given month, we present the average number of birds tallied per unit area (by dividing by 25 square meters) per unit time (by multiplying by 4; 15 min x 4 = 60 min = 1 h) of low tide.