Electronic Supplementary Material - Appendix 1
Detailed methodology
This study was conducted at Svartberget Experimental Forest (64°14′ N, 19°46′ E) in the central boreal zone, near Vindeln Sweden [1]. The experimental site consists of an approximately 120 year old closed canopy Norway spruce (Piceaabies(L.) Karst.)forest, occurring on a course grained podzol developed in glacial till [2]. The understory layer at the site is dominated by ericaceous shrubs, mainlyVacciniummyrtillusL. and a minor component of V. vitis-idaeaand the graminoid species Deschampsiaflexuosa(L.) Trin. The forest floor at the site also has an extensive feather moss cover, consisting primarily of two species, Hylocomiumsplendens(Hedw.) B.S.G. andPleuroziumschreberi(Bird). These two feather mosses comprise a majority of the bryophyte layer (> ~80% bryophyte cover), and approximately 40% of the understory biomass in the control plots. Background atmospheric Nr deposition levels for the area are estimated to be approximately 2kgNha−1year−1[3].
In 1996, a replicated randomized block design experiment was established at the site consisting of three levels of N addition (0, 12.5, and 50 kg ha-1 yr-1), with treatments applied to 0.1 ha (n=6) plots. The plots have been fertilized every year directly after snow melt (i.e. May) since 1996 by spreading solid granules of NH4NO3by hand throughout each plot. The lowest N fertilization level of 12.5 kg N ha-1 yr-1 was chosen to represent upper level Nr deposition rates in the boreal biome [4]. The higher N fertilization level of 50 kg ha-1 yr-1 was chosen because a variety of other fertilization experiments in boreal forests have applied fertilizers at or above this rate [5]. To our knowledge, this is the longest running experiment in a boreal forest environment where realistic levels of N-deposition are applied to replicated stands at a relatively large scale.
Biomass estimates of the two most abundant feather mosses, P. schreberi and H. splendens, were made by measuring the shoot density per unit area, and the mean shoot mass for each species in each of the 0.1 ha treatment plots (n=6). The moss shoot density for P. schreberi and H. splendens was measured in five 10 cm diameter circles within each 0.1 plot, placed in the center and half way between the center and each corner. The mean shoot mass was estimated from 25 shoot samples collected at each of these five locations within each plot. The biomass of each moss species per hectare was then estimated by scaling up the number of shoots to a per hectare basis, and then multiplying this value by mean shoot mass. Total feather moss biomass was then estimated by summing the individual biomass estimates of P. schreberi and H. splendens. Biomass of Deschampsiaflexulosa, a common understory grass species, was estimated using the point intercept method [6]. This method involves downwardly projecting100 points throughout the plot, which provides an estimate of percent cover. Percent cover is subsequently converted to biomass using allometric equations that were previously established using destructive sampling [6].
Nitrogen-fixation was estimated in these samples using the acetylene reduction method [7], whereby the conversion of acetylene to ethylene by the nitrogenase enzyme is used as a proxy for estimating N2-fixation. For each moss species, ten shoots were collected from each of the 5 sub-sampling location with each plot, at five different sampling periods between May and October. These shoots were divided into separate 22 ml GC vials (10 shoots per vial), resulting in 5 vials per species per plot at each sampling period [8]. The shoots in each vial were misted with de-ionized water and then sealed with a septa cap, after which 10% of the headspace was replaced with acetylene using a syringe [8]. The addition of acetylene to each tube was staggered such that ethylene in each tube could be analyzed on the GC exactly 24 hours later. After acetylene injection, sample tubes were placed in an incubator at 18 °C, and a light intensity of 150 µmol m-2 s-1. Ethylene concentrations in each sample tube were measured on a Perkin Elmer, Clarus 500 GC, with Turbomatrix 40 headspace injector. The samples were then injected isothermically onto a 30 m Perkin Elmer Elite-Alumina capillary column (0.053 mm ID). Ethylene concentrations were also measured in acetylene-only tubes and moss only tubes (i.e. no acetylene injected) to ensure that ethylene measured in sample tubes was derived via the nitrogenase enzyme. Analytical grade ethylene standards were used to estimate the ethylene concentration in each sample tube. The values for the three vials per plot were averaged to provide a single plot-level value. Following analysis, all moss samples were dried at 65°C for 48 h and weighed, which allowed us to calculate N-fixation per unit mass per day (µmol g-1 day-1) using the universal gas law. These values were further converted to µg N-fixed per moss mass using a ratio of 3 mol of ethylene reduced per mol N, which has previously been shown using 15N techniques to be appropriate for both P. schreberiand H. splendens[9, 10].
For data collected from both moss species, we first analyzed the data using a two-factor blocked ANOVA, with moss species (H. spendens and P. schreberi) and N-treatment (0, 12.5, or 50 kg N ha-1) serving as fixed factors, and using an alpha of 0.05. Because block was never a significant factor, analysis was redone without a blocking factor. For any response variables in which N-treatment had a significant effect, as well as variables derived from the sum of the two species (i.e. total biomass and total N-fixation rates) we further analyzed the data using a one-factor ANOVA, followed by S-N-K post-hoc tests when appropriate. All data were transformed (Log(X+1))when necessary prior to analysis, and non-transformed data are graphically depicted. All analysis were conducted using SPSS version 20.0.
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