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 (Picea abies (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, mainly Vaccinium myrtillus L. and a minor component of V. vitis-idaea and
the graminoid species Deschampsia flexuosa (L.) Trin. The forest floor at the site also has an
extensive feather moss cover, consisting primarily of two species, Hylocomium
splendens (Hedw.) B.S.G. and Pleurozium schreberi (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 2 kg N ha−1 year−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 NH4NO3 by 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 Deschampsia flexulosa, a common understory grass species, was
estimated using the point intercept method [6]. This method involves downwardly projecting
100 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
15
N techniques to be appropriate for both P. schreberi and 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 onefactor 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.
1.
Ahti T., Hämet-Ahti L., Jalas J. 1968 Vegetation zones and their sections in Northwestern Europe.
Annales Botanici Fennici 5, 169-211.
2.
Driessen P., Deckers J., Spaargaren O., Nachtergaele F. 2000 Lecture notes on the major soils of
the world. Diagnostic horizons, properties and materials. In World Reference Base for Soil Resources
(WRB) (ed. Driessen P.M.), p. vi + 334 pp. Rome, Food and Agriculture Organization (FAO).
3.
Phil-Karlsson G., Akselsson C., Hellsten S., Karlsson P.E., Malm G. 2009 Övervakning av
luftföroreningar i norra Sverige - mätningar och modellering. (Svenska Miljöinstitutet.
4.
Gundale M.J., Deluca T.H., Nordin A. 2011 Bryophytes attenuate anthropogenic nitrogen inputs
in boreal forests. Glob Change Biol 17(8), 2743-2753. (doi:10.1111/j.1365-2486.2011.02407.x).
5.
de Vries W., Solberg S., Dobbertin M., Sterba H., Laubhann D., van Oijen M., Evans C., Gundersen
P., Kros J., Wamelink G.W.W., et al. 2009 The impact of nitrogen deposition on carbon sequestration by
European forests and heathlands. For Ecol Manage 258(8), 1814-1823.
(doi:10.1016/j.foreco.2009.02.034).
6.
Nordin A., Nasholm T., Ericson L. 1998 Effects of simulated N deposition on understorey
vegetation of a boreal coniferous forest. Funct Ecol 12(4), 691-699.
7.
Schöllhorn R., Burris R.H. 1967 Acetylene as a competitive inhibitor of nitrogen fixation.
Proceedings of the National Academy of Science USA 58, 213-218.
8.
Gundale M.J., Wardle D.A., Nilsson M.C. 2010 Vascular plant removal effects on biological N
fixation vary across a boreal forest island gradient. Ecology 91(6), 1704-1714.
9.
DeLuca T.H., Zackrisson O., Nilsson M.-C., Sellstedt A. 2002 Quantifying nitrogen-fixation in
feather moss carpets of boreal forests. Nature 419, 917-920.
10.
Lagerström A., Nilsson M.-C., Zackrisson O., Wardle D.A. 2007 Ecosystem input of nitrogen
through biological fixation in feathermosses during ecosystem retrogression. Funct Ecol 21, 1027-1033.