Botany
Responses of mosses Sphagnum capillifolium and Polytrichum strictum to nitrogen deposition in a bog: height growth, ground cover, and CO2 exchange
Journal: Botany
Manuscript ID cjb-2015-0183.R1
Manuscript Type: Article
Date Submitted by the Author: 13-Nov-2015
Complete List of Authors: Juutinen, Sari; University of Helsinki, Department of Environmental Sciences Draft Moore, Tim; McGill University Laine, Anna Maria; Department of Forest Sciences, University of Helsinki Bubier, Jill; Mount Holyoke College Tuittila, Eeva-Stiina; University of East Finland De Young, Allison; McGill University Chong, Mandy; McGill University
Keyword: moss, peatland, photosynthesis, chlorophyll fluorescense, respiration
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Responses of mosses Sphagnum capillifolium and Polytrichum strictum to nitrogen deposition in a
bog: height growth, ground cover, and CO 2 exchange
Sari Juutinen 1,2, Tim R. Moore 3, Anna M. Laine 2, Jill L. Bubier 1, Eeva Stiina Tuittila 4, Allison De
Young 3 and Mandy Chong 3
1Environmental Studies Department, Mount Holyoke College, 50 College Street, South Hadley, MA
01075, USA. [email protected]
2Department of Forest Sciences, University of Helsinki, P.O. Box 27, FI 00014 Helsinki, Finland.
[email protected], [email protected]
3Department of Geography and Global EnvironmentalDraft & Climate Change Centre,
McGill University, 805 Sherbrooke St. W., Montreal, QC H3A 0B9, Canada. [email protected],
[email protected], [email protected]
4School of Forest Sciences, University of Eastern Finland, P.O Box 111, FI 80101 Joensuu, Finland.
eeva [email protected]
Corresponding author: Sari Juutinen, [email protected], Department of Forest Sciences,
University of Helsinki, P.O. Box 27, FI 00014 Helsinki. Tel. +358 50 357 4277
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Abstract
Previous studies have shown atmospheric (N) deposition to be detrimental to Sphagna, a genera important for carbon (C) cycling in northern peatlands. Little is known about species interactions, such as relative responses of tall moss Polytrichum strictum and Sphagna. We studied the effects of N deposition on height growth, abundance, and CO 2 exchange of moss species Sphagnum capillifolium and Polytrichum strictum in an experiment at a temperate bog. Sphagnum height growth and cover decreased significantly in the high N treatment (6.4 g N m 2yr 1) in the 4 th and 5 th treatment years, while those of Polytrichum increased, relative to the control. Net CO 2 exchange, gross photosynthesis (Pg) and dark respiration (R) in the intact moss cores, measured in the 5 th treatment year, were elevated in the high N treatment relative to the control,Draft associated with enhanced Polytrichum abundance. The moss cores where Polytrichum was removed, however, had increased mass based R in the high N treatment. Our results showed that S. capillifolium at Mer Bleue may be close to N saturation as 5 years of high N loading (6.4N+ background) was harmful to this species, possibly as a result of increased respiratory cost. P. strictum had a competitive advantage, at least in short term, allocating excess N to growth. This change in moss layer composition deserves further attention as a shift to more easily decomposable litter, without corresponding increases in plant production, can decrease the C sequestration of the bog.
Keywords: peatland, photosynthesis, chlorophyll fluorescence, respiration, vegetation change, moss
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Introduction
Atmospheric nitrogen (N) deposition has drastically increased N availability in the industrialized world
during the Anthropocene (e.g. Galloway et al. 2008). Excess N from deposition threatens many natural
ecosystems and their functioning by impacting species composition and biogeochemistry through
increasing competitive ability of some species and making the conditions unfavorable for others (e.g.
Bobbink et al. 2010). Peat mosses (Sphagnum spp.) are key species in northern nutrient poor peatlands,
but sensitive to atmospheric N deposition (Limpens et al. 2011). The decrease in Sphagnum abundance
can markedly impact litter quality and decomposability, surface structure and water retention capacity,
all of which affect the important carbon (C) sequestration capacity of peatlands (e.g. Moore et al. 2007;
Straková et al. 2010 ; Larmola et al. 2013).
Sphagnum mosses can increase theirDraft growth and production under increased nutrient
availability, but the responses depend on species sensitivity, dose and temporal scale of loading (e.g.
Rochefort et al. 1990; Vitt et al. 2003; Gunnarsson et al. 2004). Critical loading of atmospheric N,
associated with reduced growth, is considered to be ~0.5 to 1.5 g N m 2 yr 1 for Sphagnum mosses
(Gunnarsson and Rydin 2000; Vitt et al. 2003; Bragazza et al. 2004; Granath et al. 2014), currently
exceeded in parts of Europe, North America, southern China, and south and southeastern Asia
(Bobbink et al. 2010). Recognized factors contributing to the negative responses of Sphagnum mosses
to increased N availability are the respiratory cost of storing excess N (Limpens and Berendse 2003;
Manninen et al. 2011), competition for light and space with vascular plants (van der Heijden et al.
2000b; Berendse et al. 2001), increase in parasitic infections (Limpens et al. 2003), decrease in other,
most importantly phosphorus (P), elements (Aerts et al. 1992; Jauhiainen et al. 1998), and greater
sensitivity to drying (van der Heijden et al. 2000a; Manninen et al. 2011; Fritz et al. 2012). In addition,
experiments indicate that Sphagnum populations adapted to higher background N loading are less
sensitive to additional N input than mosses from low deposition backgrounds (Wiederman et al. 2009a;
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Granath et al. 2012). A recent meta analysis on responses of Sphagnum mosses to experimental N addition found that tissue N concentration exceeding ~ 1% predicted a decrease in Sphagnum production, but noted that temperature, wetness, species composition and species interactions are likely site factors modulating the responses of Sphagnum mosses to N addition (Limpens et al. 2011).
While many studies have measured Sphagnum responses to N deposition, less in known about the responses of co existing plant species through competition or facilitation. Plant species differ in their ability to use the excess nutrients and maintain homeostatic stability, i.e. maintain tissue N concentration relative to input (e.g. Shaver and Laundre 2007; Wiederman et al. 2007;Elser et al.
2010). Generally excess N from atmospheric deposition becomes available to vascular plants only when Sphagnum layer becomes saturated with N, after which vascular plants are able to allocate N to increased growth and thus gain competitiveDraft advantage compared to peat mosses (e.g. Malmer et al.
2003). Polytrichum strictum is a moss species often co occurring with Sphagnum mosses, but is typically more abundant in drier microhabitats, intermediate in its N requirement, and found to benefit from low levels N and P additions compared to Sphagnum species (Vitt et al. 1990; Gunnarsson and
Rydin 2000; Berendse et al. 2001; Mitchell et al. 2002; Bubier et al. 2007; Sottocornola et al. 2007; Bu et al. 2011). P. strictum is a pioneering species and considered to help Sphagnum establishment at peatland restoration sites (e.g. Robert et al. 1999), but can outcompete Sphagna with increases in abundance (Gonzales et al. 2013). However, studies have shown that P. strictum abundance declines under high N doses (Bubier et al. 2007; Bu et al. 2011). Therefore, it is important to understand the effects of N deposition on different peatland plants and the possible interactions between the species.
In this study we examined the effects of simulated N deposition on growth and abundance of S. capillifolium (Ehrh.) Hedw. and P. strictum Menzies ex Brid. during the first five experimental years in a fertilization experiment at the temperate ombrotrophic peatland Mer Bleue bog. We also examined the impact of excess N on moss CO 2 exchange capacity after five years of fertilization. We
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hypothesized that 1) height growth of S. capillifolium would decrease but P. strictum would increase
with N addition, 2) CO 2 exchange and chlorophyll fluorescence measurements would indicate negative
impacts of N deposition on S. capillifolium and positive impacts on P. strictum .
Materials and methods
Study Site
The study was conducted at the Mer Bleue peatland, near Ottawa, Canada (46°N, 75.5°W), which has a
mean annual temperature of 6.6 oC and an average rainfall of 756 mm per year (Canadian Climate
Normals 1981 2010 ). Nitrogen fertilization was applied to randomly assigned triplicate 3 × 3 m plots
per treatment, separated by 1 m wide buffer zones. Nitrogen was given as NH 4NO 3 dissolved in seven
2 mm applications from May to August 2005–2009.Draft The N addition rates were 0, 3.2 and 6.4 g N m 2
yr 1, termed 0N (control), 3.2N and 6.4N; control plots received only distilled water and total amounts
2 1 of NH 4NO 3 in N treatments were 9.2 and 18.5 g m yr . The region receives background N deposition
of ~0.6 – 0.8 g N m 2 yr 1 (Turunen et al. 2004).
Height Growth and Species Abundance
Moss height growth was measured by the cranked wire method (Clymo 1970). Plastic covered, 2 mm
diameter metal rods were about 10 cm above the peat surface and extended downwards into the peat by
about 25 cm (see also Moore 1989). The distance between the tips of the rod and the surrounding S.
capillifolium and P. strictum was measured in early April and late October each year from 2005 to
2009, capturing most of the typical growing season (daily mean temperature above 0°C, see also
Moore et al. 2002). We acknowledge that moss growth may occur outside of this period, but believe
that these measurements can capture the relative treatment effects. The growth of S. capillifolium and P.
strictum is synchronous according to our observations at this site and the cranked wire method should
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capture the vertical height increment of both species (Moore 1989 and references therein). There were
10 replicate rods in each plot (30 per treatment) and readings per species in each treatment varied from
4 ( P. strictum in 0N, in 2008) to 29 ( S. capillifolium in 0N, in 2009).
Frequency of the moss species was determined in 60 × 60 cm gas flux measurement collars in each plot in mid July of years 2007, 2008 and 2009. A point intercept frame was set on top of the collar and hits to a pin were recorded in 61 grid points in each collar. Hits to the vascular plants were recorded similarly.
Exchange of CO 2 and Chlorophyll Fluorescence
To determine the effect of five years of treatments on photosynthesis and respiration of the mosses we measured CO 2 exchange of moss cores fromDraft which all vascular plants were removed (height~2 cm, diameter 7.2 cm) and, separately, in Sphagnum capitula and Polytrichum shoots. The moss cores were sampled in October 2009 when the plots had been treated with water or fertilizer for 5 years. Two cores with diameter of 7.2 cm, containing top 2 cm of the Sphagnum stems including capitula and taller P. strictum shoots growing between S. capillifolium , were sampled from each plot (3 treatments × 3 replicates × 2 cores). One core was kept intact and only vascular plants and visible detritus were removed, while in the other core P. strictum shoots were also removed. Samples were watered by spraying distilled water and placed into a growth chamber under the following summer conditions: 16 h light with photosynthetically active photon flux density (PPFD) ~ 500 µmol m 2 s 1 at the moss surface, 8 h darkness; 18°C day and 10°C night; CO 2 concentration 380 ppm; relative humidity 70%.
Each sample pot was measured for net CO 2 exchange (NE) under light levels of 0, 500 and 700
PPFD µmol m 2 s 1 moss water content being ~1000–2000% for S. capillifolium, optimal light
(Marschall and Proctor 2004) and moisture (Chong et al. 2012) conditions for the photosynthesis of S. capillifolium . There were 6 observations per treatment for intact and S. capillifolium cores (each core
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was measured twice). The measurements were made using a small chamber (10 cm diameter, 10 cm
high) covering the pot containing mosses, set on a small platform with a water filled groove to ensure
a gas tight seal. Headspace CO 2 concentrations were recorded every 15 s over a ~3 min period using an
EGM4 (PP Systems, UK) infrared gas analyzer. PPFD in the chamber, temperature and sample weight
were determined for each measurement. The CO 2 exchange rate was calculated from the linear change
in CO 2 concentration over the measurement period, correcting flux for gas chamber volume and
temperature. Each observation was checked for leaks or saturation. Gross photosynthesis (Pg) at light
2 1 levels of 500 and 700 µmol m s was estimated by summing net CO 2 exchange (NE) under light and
dark conditions, the latter representing respiration (R). CO 2 exchange rate was calculated per unit
ground area and per unit moss dry mass.
In order to compare Pg and R ratesDraft of the two moss species directly and test whether the Pg and
R, and quantum yield efficiency of photochemistry (ratio between variable fluorescence and maximum
fluorescence, Fv/Fm) after dark acclimation period were affected by the excess N, another moss sample
was collected in April 2010 after snow thaw. Chlorophyll fluorescence analysis is used to study the
efficiency of photosynthetic light reactions, and to gain information about the physiological state of a
plant (Maxwell and Johnson 2000; Laine et al. 2011). Cores similar as described above were collected
from treatment plots (altogether 3 × 3 cores) and the moistened samples were shipped to the University
of Helsinki, Finland, where the measurements were conducted. Measurements were made using an
open, fully controlled, flow through gas exchange fluorescence measurement system (GFS 3000, Walz,
Germany). We used a standard conifer cuvette equipped with a net in the lower jaw. A uniform layer of
S. capillifolium capitula or top 2 cm segments of P. strictum were placed on the net, so that ambient air
was allowed to flow freely above and below the sample. CO 2 exchange was measured at PPFD 600 and
1300 µmol m 2 s 1. The sample was allowed to adjust to each light level for 5 min before
measurements. After this the sample was darkened and the respiration rate was recorded after 5 min.
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Chlorophyll fluorescence (Fv/Fm) was measured after 25 min darkening. Other environmental conditions in the cuvette were maintained at a constant level during the measurement period: the CO 2 concentration of the incoming air was 500 ppm and the flow rate was 600 µmol s 1. The relative humidity inside the cuvette was controlled and temperature was set at 20 °C. Samples were weighed before and after measurements.
Upon completion of the measurements, the moss samples were oven dried (60°C) to determine dry biomass and moisture content. Subsamples from both moss samplings were ground and analyzed for C and N concentrations using a Carlo Erba TM Elemental Analyzer, model NC2500.
Homeostatic Regulation Coefficients
We compared species ability to maintain tissueDraft N concentration relative to input. Strength of N homeostasis was assessed on the basis of homeostatic regulation coefficients ( H) calculated on the basis of their tissue N concentration and N inputs (atmospheric and fertilization) as in Sterner and Elser
(2002):
equation 1 y = cx 1/ H
where y is the N content (%) in the moss, x is the input of N (combined ambient atmospheric N deposition and fertilization, g m 2 yr 1), c is a constant, and H is the homeostatic regulation coefficient.
In order to compare the moss species against the dominant bog shrub species at our site we calculated
H values for leaves of Chamaedaphne calyculata , Rhododendron groenlandicum , and Vaccinium myrtilloides from the same experiment. Leaf N concentrations for vascular plants are published also in
Bubier et al. (2011).
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Statistical Analyses
Treatment and species effects on moss height growth and cover were analyzed by two way ANOVA
for each year separately. Data were checked for normality and equality of variances. When there was a
significant species × treatment interaction the species were analyzed separately. Plot means of growth
were used in order to balance the number of observations per treatments (treatment n = 3). Vascular
plant abundance was used as covariate for moss frequency (hit count) for the years where data were
available (3 rd , 4 th , and 5 th treatment years).Treatment effects on area and mass based NE, Pg, and R
were tested using one way ANOVA, or using Kruskal Wallis test when variances were not equal,
running analyses separately for intact and S. capillifolium cores. Impacts of moss species and N
addition treatments on moss N concentration, Pg, R and Fv/Fm were tested using two way ANOVA.
Due to significant species × treatment effects,Draft the species were tested separately. Relationships of R, Pg,
and Fv/Fm to N content were examined using linear regression analyses.
Results
Height growth and abundance
Nitrogen addition treatments had no significant effect on moss height growth in the first three years
2005, 2006, and 2007 (Figure 1a b, Table 1), when the seasonal height growth varied from 7.9 to 18.3
mm in P. strictum and from 8.5 to 22.3 mm in S. capillifolium . Both species had the smallest height
increment in the year 2007. Significant species and treatment interaction appeared in the 4 th and 5 th
experimental years (2008 and 2009) when the height growth of S. capillifolium was significantly
smaller in 6.4N treatment than in the control treatment (Fig. 1a), the difference between treatment
means being large, ~15 mm. The N addition treatments had no significant effect on height growth of P.
strictum on an annual scale though there was a transient positive trend of higher mean values in 3.2N
and 6.4N treatment than in 0N treatment in the 2 nd , 3 rd and 4 th experimental years (Fig. 1b and d).
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The year to year variability in absolute growth rates should be treated with caution, because the length of the period between spring and autumn measurements was not exactly the same each year. The
5 year total height increments was 15 mm higher in S. capillifolium than in P. strictum in the 0N treatment, while the cumulative height increment of P. strictum exceeded that of S. capillifolium by 32 and 61 mm in 3.2N and 6.4N treatments, respectively.
Sphagnum capillifolium had significantly higher frequency than P. strictum in the initial year
(Figure 1e f, Table 1), but the difference diminished by the 3 rd year (no data for the 2 nd year). Similar to height growth, significant species and treatment interaction appeared in the 4 th and 5 th experimental years (2008 and 2009, Fig. 1e). The frequency of S. capillifolium was significantly smaller in 6.4N treatment than in the control treatment in the 4th treatment year (23 vs. 53 hits) and P. strictum abundance was on larger in N addition treatmentsDraft compared to control (43 and 49 vs. 26 hits ) in the 5th treatment year. Vascular plant abundance had no significant effect on S. capillifolium frequency when tested as covariate and the 6.4N treatment plots seem to have distinctively low S. capillifolium abundance relative to vascular plant abundance (Fig. 2). Vascular plant cover was, however, a significant for P. strictum frequency in the 4 th treatment year ( p=0.04). Thus there was no clear relationship between vascular plant and moss abundance
Exchange of CO 2 and chlorophyll fluorescence
Net exchange of CO 2 (NE) and its component fluxes dark respiration (R) and gross photosynthesis (Pg) in the intact moss cores were significantly higher in 6.4N treatment than in 0N treatment (NE 2.3 vs 4.5,
R 2.4 vs 3.9, and Pg 4.7 vs 8.4 mmol m 2h 1) expressed per unit area (Figure 3a c, Table 2). In the cores with S. capillifolium only ( P. strictum removed), R was significantly higher and NE was significantly decreased in 6.4N treatment relative to control (R 1.1 vs. 1.7 and NE 1.6 vs 0.32 mmol m 2h 1). There were no significant treatment effects in the intact cores when the fluxes were expressed per unit moss
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dry mass. In turn, mass based R was significantly increased and mass based NE significantly decreased,
in the 6.4N treatment compared to control in the cores with S. capillifolium only (R 4.2 vs 1.6 and NE
2.7 vs 5.1 µmol m 2h 1) (Fig. 3d f). As a result mass based Pg was negatively (R2=0.23, p=0.026)
related to the moss N concentration, while the relationship between the mass based R and N
concentration was positive (R2=0.63, p<0.001) in S. capillifolium only cores.
No treatment effects on moss CO 2 exchange were detected when S. capillifolium capitula and P.
strictum shoots were measured separately (Fig. 3, Table 3). Polytrichum strictum had significantly
higher Pg and R than S. capillifolium and had thus higher photosynthetic efficiency relative to tissue N
concentration. Maximum quantum yield efficiency of PSII, F V/F M, was low and similar in both species
and among treatments: the treatment means varied between 0.49 and 0.65 in P. strictum and between
0.58 and 0.62 in S. capillifolium and indicatedDraft no stress related to N treatments.
Response of tissue concentration to N treatment
The N treatments affected moss tissue N concentration with a doubling in S. capillifolium (0.7 to 1.4%)
and a 50% increase in P. strictum (0.8 to 1.2%) from the control to the 6.4 N treatments (Fig. 4). The
resulting homeostatic regulation coefficient (H) was 3.56 and 6.80 for S. capillifolium and P. strictum ,
respectively. The H values of the two moss species were not significantly different (at p < 0.05), but
those were significantly smaller than for three shrub species (Fig. 4). The H values indicate that S.
capillifolium accumulates excess N more than P. strictum , which resembled the shrubs growing in the
same site regarding the change in tissue N concentration under changing N inputs.
Discussion
The simulated and background levels of N deposition
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Background N deposition rate at Mer Bleue falls between the deposition values in central and northern
Europe (e.g. Berendse et al. 2001; Solberg et al. 2009), but is close to 1 g N m 2 yr 1 considered harmful for Sphagnum growth and overall biodiversity of ecosystems in a natural state (e.g. Bobbink et al. 2010,
Granath et al. 2014). The lower N application level of this study (3.2 g N m 2 yr 1) is similar to highest deposition rates measured in parts of Western Europe and the higher application level (6.4 g N m 2 yr 1) corresponds only to observed deposition around point sources (Galloway et al. 2008). These N application levels are common in peatland experiments (e.g. Jauhiainen et al.1998; Gunnarson and
Rydin 2000; Manninen et al. 2011; Granath et al. 2012).
Moss growth and abundance
As expected, the N addition decreased the Draftheight growth of S. capillifolium , but only in the 6.4N treatment (Fig. 1) and there was a trend that P. strictum height growth increased, though this may be a transient phase as the differences between the control and N treatments became smaller again after 5 years. Moss abundance responded in a similar manner to height growth (Fig. 1 e f). This negative growth response to very high N addition and lack of marked positive growth responses to smaller N addition indicate that S. capillifolium is not likely to be N limited at Mer Bleue, but excess N caused N saturation (Berendse et al. 2001) when limitation of other nutrients may impact the growth patterns
(Aerts et al. 1992; Jauhianen et al. 1998; Hoosbeek et al. 2002; Bragazza et al. 2004). P and NP co limitation occurs in many plant functional groups at Mer Bleue bog (Wang and Moore 2014), supported by stronger responses in vascular plant growth and moss cover in N and P treatments than in
N only treatments (Larmola et al. 2013).
Similar to results by Gunnarsson et al. (2004), the impact of N addition increased over time in the 6.4N treatment (Fig. 1a and c). Both small and large N additions have stimulated growth of
Sphagnum in the short term (Bonnett et al. 2010; Granath et al. 2012), and Sphagnum species differ in
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their response (e.g. Gunnarsson et al. 2004). Jauhiainen et al. (1994) found that N addition of 1 3 g N
m 2yr 1 increased growth of S. fuscum but 10 g m 2 yr 1 led to immediate reduction in biomass
production and elongation in a 4 month experiment with about ~0.2 g m 2yr 1 background deposition.
Gunnarsson and Rydin (2000), however, showed that even small N deposition rates (<1 g m 2 yr 1)
saturate Sphagnum mosses in a few years leading to growth reduction. It seems that both N addition
treatments in our study caused reduction in growth and abundance, though it was not significant under
the lower loading level. Changes in the growth and abundance can be a net effect of competition
between species and internal changes in physiology. The increase in P. strictum frequency in this study
reached a level where it was found to outcompete Sphagnum species in a peatland restoration site.
Competition for light can be a factor behind the response (Gonzales et al. 2013).
The enhanced N supply was used byDraft P. strictum for increased growth (Fig. 1b and d) in the
short term, which agrees with an earlier finding that its distribution in peatlands is N limited (Vitt et al.
1990). Responses of P. strictum tend to be positive in the initial phase as significant increase in height
growth occurred with 3 g N m 2 yr 1 applied over 3 growing seasons at a former cut away peatland
where P. strictum and S. fallax coexisted (Mitchell et al. 2002). On the other hand, ultimate reduction
in growth was detected after 2 years with N addition of 2 g m 2 yr 1 in a fen (Bu et al. 2011) and 6.4 g
m 2 yr 1 N with P and K reduced drastically abundance in the fertilization experiment at Mer Bleue
(Bubier et al. 2007). Bu et al. (2011) found that P. strictum performed better in the presence of
Sphagnum , suggesting the overall decrease in moss coverage may be harmful to P. strictum.
Moss CO 2 exchange
We hypothesized that N addition would impact photosynthesis and dark respiration of these mosses.
However, we found no significant treatment effects on CO 2 exchange rates of separated Sphagnum
capitula and P. strictum shoots (Fig. 3 g h). Moreover no differences in stress between the species or
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treatments were indicated by the measurements of maximum quantum efficiency of PSII. However, in the Sphagnum only cores R measured per unit dry mass was increased in the 6.4N treatment relative to control (Fig. 3 e f) and R was positively related to tissue N concentration. Part of the respiration can be due to decaying of the lower parts; moreover the removal of P. strictum likely loosened the cores, especially in the 6.4N treatment, thus potentially decreasing its extracellular water transport capacity affecting the distribution of water in the core (Schipperges and Rydin 1998; Marshal and Proctor 2004).
There was also a slight negative relationship between photosynthesis and Sphagnum N concentration in the cores with P. strictum removal, but no significant treatment differences.
Increased respiration is a sign of building and maintaining N rich compounds acting as N storage, for example production of detoxifying amino acids (Baxter et al. 1992; Reich et al. 1998;
Limpens and Berendse 2003; Koranda et al.Draft 2007). Increased dark respiration as response to N fertilization has also been found in dwarf shrub leaves (Heskel et al. 2012; Bui 2013). Response of photosynthesis to different tissue N concentrations has been negligible, positive or negative, in other studies. Granath et al. (2009ab; 2012) showed variation from positive to unimodal response of photosynthesis to N concentration among species S. balticum , S. fallax , and S. fuscum . Among others
Fritz et al. (2012) discussed the possibility that allocation of excess N into chlorophyll can lead to photo inhibition of photosynthesis. A decrease in photosynthesis of S. recurvum was detected when capitulum N concentration reached about 1.5%, and associated with reduced water content and necrosis
(van der Heijden et al. 2000a). These factors may contribute to the slight negative relationship of photosynthesis to tissue N content in our data.
Reduced water content can result from a change in growth pattern and lower the bulk density of the moss canopy due to excess N, which may be reflected as a decrease in photosynthetic capacity of
Sphagnum mosses (e.g., Fritz et al. 2012). Moreover, S. capillifolium , exposed to ammonium N with total load equaling our 6.4 g N m 2yr 1, allocated the excess N to cell wall proteins, which caused
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decreasing cross sectional area of hyaline cells and smaller water holding capacity (Manninen et al.
2011). It has been noted that N addition has a stronger negative impact on Sphagnum performance
during dry conditions (Gerdol et al. 2008; Limpens et al. 2011). We have observed that S. capillifolium
has reappeared in patches in 6.4N (plus PK) plots fertilized since 2001, where the surface has become
wetter and the shrub canopy has opened giving support to the idea that the response of S. capillifolium
to excess N can be modulated by the moisture environment
Maximum quantum efficiency of PSII (Fv/Fm) is considered to be indicative of plant stress (e.g.
Maxwell and Johnson 2000). The values measured in this study were low compared to what are
considered non stressed vascular plants (0.83) (e.g. Björkman and Demmig 1987) and measured for
Sphagna in shaded habitats (0.72 0.82) (Kangas et al. 2014), but in a similar range to several other
moss studies in open peatlands with no N treatmentsDraft (Murray et al. 1993; Granath et al. 2009a; Hájek
et al. 2009; Laine et al 2011).
Our data gave no clear indication that vascular plant presence played a significant role in
changes in growth and abundance of S. capillifolium and P. strictum within the five year time span.
This may be due to the fact that the changes in vascular plant canopy stayed moderate in this N only
experiment. Decrease in light penetration to the moss surface is an alternative hypothesis to explain
decrease in Sphagnum/ moss coverage in association of increased vascular plant coverage under
enhanced N availability (e.g. Bubier et al. 2007, Wiederman et al. 2009b). Drastic shading caused by
nets (PPFD <40 µmol m 2s 1) was found to decrease production of S. capillifolium (Bonnett et al. 2010).
The expansion of P. strictum, however, must cut part of the available irradiation on the S. capillifolium
surface, and it is possible that the shading by vascular plants allow first P. strictum to expand, which is
followed by reduction in S. capillifolium abundance.
Nitrogen and Ecosystem Dynamics
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The N treatments affected the moss N concentrations (Fig. 4), with a range similar to deposition gradient and simulated deposition studies from 0.1 − 4 g m 2yr 1 (e.g. Aerts et al. 1992; Jauhiainen et al.
1998; Bragazza et al. 2005). Sphagnum capillifolium tissues accumulated N while P. strictum better regulated its tissue N concentration under different N deposition doses thus having stronger N homeostasis (H) (Fig. 4). The weak H of S. capillifolium (3.6) in our study agrees with the global data set that gives an H value of 3.2 for hummock Sphagnum capitulum (Limpens et al. 2011). In turn, the
H value of P. strictum (6.80) is closer to that of the bog shrubs of Mer Bleue, 9.3 to 10.1 (Fig. 4). In a grassland study, Yu et al. (2010) found these high H values to be typical for species tolerating changes in N input.
Seemingly, P. strictum was released from nutrient deficiency by our N addition and was able to utilize the increased availability, initiating Draftand expanding new photosynthetic tissue. Our study suggests that its photosynthetic rate is not changing, but owing to an increase in biomass and higher photosynthetic capacity of P. strictum compared to S. capillifolium (Fig. 3, Proctor 2005), photosynthetic capacity of the moss layer in the 6.4N treatments increased (Fig. 3a−c). The increase in
P. strictum abundance and growth following enhanced N supply seems to be transient (Fig. 1b, Bubier et al. 2007; Juutinen et al. 2010; Larmola et al. 2013). It is possibly related to the strong increase in dwarf shrub abundance and decrease in light reaching the moss canopy (Chong et al. 2012). In addition, the conditions associated with the overall decrease in moss cover may inhibit its growth (see also Bu et al. 2011)
Changes in light and water conditions may be important factors to the growth of the moss layer and responses to excess N. The loss of Sphagnum mosses with their slow rates of decomposition, compared to P. strictum and shrub leaves and stems (Moore and Basiliko 2006), means that the ability of peatlands to accumulate C can decrease without corresponding increases in C inputs.
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Acknowledgements
We thank Mike Dalva (Geography) and Mark Romer (Biology) of McGill University for their help in
the field and Phytotron, respectively. The National Science Foundation (DEB1019523) and the Natural
Sciences and Engineering Research Council provided funding to JLB and TRM, respectively, and the
National Capital Commission gave access to Mer Bleue. SJ, AML, and E ST were funded by the
Academy of Finland (project 140863).
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Table 1. Results of analysis of variance for moss height growth and frequency (hit count) in 1st −5th treatment years (years 2005 2009). Degrees of freedom ( Df ) for species, treatment, species × treatment, and error were 1, 2, 2, and 12, respectively. For significant differences between treatments see Fig. 1.
2 Dependent Source MS F p R adj. Model p Growth 1st yr Species 1 0.018 0.894 0.14 0.707 Treatment 30 0.973 0.406 Species × treatment 15 0.497 0.620 Error 31 Growth 2nd yr Species 3 0.024 0.879 0.00 0.452 Treatment 128 1.182 0.340 Species × treatment 145 1.338 0.299 Error 108 Growth 3rd yr Species 46 2.669 0.128 0.36 0.061 Treatment 85 4.908 0.028 Species × treatment 17 0.993 0.399 Error 17 Growth 4th yr Species 980Draft 19.072 0.001 0.64 0.003 Treatment 5 0.096 0.909 Species × treatment 396 7.706 0.007 Error 51 Growth 5th yr Species 216 18.172 0.001 0.73 0.001 Treatment 98 8.238 0.006 Species × treatment 90 7.549 0.008 Error 12 Frequency Species 34672 1313.9 <0.001 0.99 <0.001 1st yr Treatment 17 0.632 0.549 Species × treatment 39 1.474 0.268 Error 26 Frequency Species 174 0.637 0.440 0.01 0.437 3rd yr Treatment 67 0.244 0.787 Species × treatment 559 2.045 0.172 Error 274 Frequency Species 80 0.611 0.450 0.39 0.049 4th yr Treatment 188 1.433 0.277 Species × treatment 800 6.093 0.015 Error 131 Frequency Species 47 0.181 0.678 0.28 0.150 5th yr Treatment 245 0.948 0.415 Species × treatment 1024 3.971 0.047 Error 258
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Table 2. Results of one way ANOVAs for net CO 2 exchange (NE), respiration (R) and gross
photosynthesis (Pg) per unit area (area) and of Kruskal Wallis test for CO2 fluxes per unit mass (mass).
Differences between treatments were assessed by using Dunett’s test. Degrees of freedom ( Df ) between,
within, and total, were 2, 15, and 17, respectively.
Core type Dependent F p Treatment differences
Intact NE (area) 5.416 0.017 6.4N > 0N, p=0.010
Intact R(area) 8.528 0.003 6.4N > 0N, p=0.002
Intact Pg (area) 8.016 0.004 6.4N > 0N, p=0.002
S. capillifolium NE (area) 5.275 0.018 6.4N < 0N, p=0.012
S. capillifolium R(area) 4.3 0.033 6.4N > 0N, p=0.024
S. capillifolium Pg (area) 3.307 0.065
Intact NE (mass) Draft 3.661 0.160 Intact R(mass) 2.217 0.330 Intact Pg (mass) 3.661 0.160
S. capillifolium NE (mass) 8.035 0.018 6.4N < 0N, p=0.017 S. capillifolium R(mass) 7.328 0.026 6.4N > 0N, p=0.028 S. capillifolium Pg (mass) 6.222 0.045 6.4N < 0N, p=0.052
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Table 3. Results of analysis of variance for moss N concentration, quantum efficiency of PSII (Fv/Fm), photosynthesis (at PAR 600 and 1300 µmol m 2 s 1), and dark respiration (R) in the fifth experimental year. Degrees of freedom ( Df ) for species, treatment, species × treatment, and error were 1, 2, 2, and 9, respectively.
Dependent Source MS F p R2adj. Model p [N] Species 3.416 239.792 <0.001 0.96 <0.001 Treatment 0.274 19.251 0.001 Species × treatment* 0.056 3.916 0.06 Error 0.014 Fv/Fm Species 0.002 0.426 0.53 0.13 0.308 Treatment 0.009 2.339 0.152 Species × treatment 0.005 1.295 0.32 Error 0.004 Pg600 Species 1951 34.782 <0.001 0.70 0.005 Treatment 165 2.942 0.104 Species × treatment Draft 107 1.906 0.204 Error 56 Pg1300 Species 267 13.261 0.005 0.38 0.09 Treatment 13 0.623 0.558 Species × treatment 25 1.215 0.341 Error 20 R Species 2326 40.014 <0.001 0.73 0.003 Treatment 84 1.452 0.284 Species × treatment 126 2.172 0.17 Error 58
*Treatments affected N concentration of S. capillifolium , Tukey HSD: 0N <3.2N ( p = 0.004), 0N< 6.4 (p < 0.001)
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Figure Captions
Fig. 1. a−b) Seasonal height growth (April October) of S. capillifolium and P. strictum in treatments
relative to control (treatment means, n=3), and c−d) frequency (hit count) of S. capillifolium and P.
strictum in 60 × 60 cm collars in treatments relative to control (treatment means, n=3) in the first five
treatment years (2005–2009). Treatment differences were assessed for the years with significant
species × treatment interactions (Table 1) and p values are shown for the treatments significantly
different from control treatment.
Fig. 2. Frequency of a) S. capillifolium and b) P. strictum in relation to vascular plant abundance in 60
× 60 cm collars within the treatment plots.Draft Data points represent treatment means of point intercept
hits (±SE, n=3) for years 2007, 2008, and 2009, i.e. 3 rd , 4 th and 5 th experimental years.
Fig. 3. Treatment means of CO 2 exchange (± SE, n=6) of the intact and S. capillifolium (P. strictum
removed) cores expressed per unit area (a c) and per unit dry mass (d f). All vascular plants were
removed from the cores. Treatment means (± SE, n=3) of net exchange, respiration and photosynthesis
of S. capillifolium capitula and P. strictum shoots (g i).Treatments significantly different from control
are indicated with p values (see Tables 2 and 3 for ANOVA results).
Fig. 4. Concentration of N in P. strictum shoots and S. capillifolium capitula after 5 years of
fertilization in relation to the annual N input (fertilization plus estimated 0.6 g N m 2 yr 1 of ambient
atmospheric deposition). Data are treatment means (± SE). The power relationship between N
concentration and N input is shown along with the homeostatic regulation coefficient ( H). Nitrogen
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concentration data for K. angustifolium , R. groenlandicum , and C. calyculata are from Bubier et al.
(2011).
Draft
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a) S. capillifolium b) P. strictum 2.5 l o r , t
h n 2.0 t o w C o : r t g 1.5 n t e h g i m t e
a 1.0 H e r T 0.5 p=0.002 p=0.03 0.0 c) S. capillifolium d) P. strictum 2.5 l o r
t p=0.019
n 2.0 y o c C n : e :
t 1.5 u n
q p=0.035 e e r m t F
a 1.0 e r T 0.5 p=0.005 p=0.04 0.0 2005 2006 2007 2008Draft2009 2005 2006 2007 2008 2009 Year Year
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t a)a i h ( y c n
e 40 u q 0N, -07 e r f 0N, -08
m 0N, -09 u i l 20 o f i l l i 3.2N, -07 p a 3.2N, -08 c
. 3.2N, -09
S 0
) b) # t i h ( y c 40 n e u q e r f m 20 u t c i r
t 6.4N, -07 s 6.4N, -08 . Draft P 6.4N, -09 0 120 160 200 240 280 320 Vascular plant frequency (hit #)
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10
) a) c) Photosynthesis Net exchange b) Respiration 2 1 1 0 - 2
8 0 0 h 0 . . 2 0 0 0 . - = = 4 0
6 p p m 2 = l 0 p . 2 o 0 4 1 = m 0 . p m 0 ( 2 = p 0
10 8 d) 0N e) 2 f) 0 2 .
3.2N 5
8 0 0 e =
6.4N . ) g p 1 0 -
n 6 = h a p 1 - 7 h g c 4 1 l x 0 . o e
2 0 m 2 = µ p O ( 0 C -2 Intact S. capillifolium Intact S. capillifolium Intact S. capillifolium core core core core core core g) h) i) ) 1
- 80 h 1 -
g 60 l o
m 40
µ Draft ( 20 0 P. strictum S. capillifolium P. strictum S. capillifolium P. strictum S. capillifolium shoot capitulum shoot capitulum shoot capitulum
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1.8 C. calyculata y = 1.08x0.104, R2 = 0.23, H = 9.30 R. groenlandicum y= 1.12x0.099, R2 = 0.25, H = 10.10 1.6 V. myrtilloides y = 1.18x0.105, R2 = 0.35, H = 9.53
V. myrtilloides C. calyculata R. groenlandicum 1.4 ) % ( n o i t 1.2 a r t n e c n o c 1.0 N e u P. strictum s s i T 0.8 Draft S. capillifolium
0.6 S. capillifolium y = 0.81x0.281 , R2 = 0.76, H = 3.56 P. strictum y = 0.90x0.147, R2 = 0.49, H = 6.80 0.4 0 2 4 6 8 N input (g m-2 yr-1)
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