Direct physiological effects of nitrogen on Sphagnum: a greenhouse experiment

Authors

  • Gustaf Granath,

    Corresponding author
    1. Department of Plant Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden
      Correspondence author. E-mail: Gustaf.Granath@ebc.uu.se
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  • Joachim Strengbom,

    1. Department of Ecology, Swedish University of Agricultural Sciences, Box 7044, SE-750 07 Uppsala, Sweden
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  • Håkan Rydin

    1. Department of Plant Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden
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Correspondence author. E-mail: Gustaf.Granath@ebc.uu.se

Summary

1. Bogs are nutrient-poor peatland ecosystems that are sensitive to nitrogen (N) deposition. Production of peat mosses (i.e. the peat-forming genus Sphagnum) is known to decrease under elevated N deposition, but the causal mechanisms are poorly understood.

2. It is predicted that increased N deposition will cause changes in Sphagnum species composition, with fast-growing species benefiting from increased N availability in contrast to slow-growing species. Knowledge of species-specific responses to N availability can help us to understand interspecific competitive relationships.

3. We investigated the direct effects of N application on plant physiology in three Sphagnum species by exposing shoots to a range of N doses (corresponding to depositions of 0–5·6 g m−2 year−1), over 5 months, in a greenhouse experiment. The species investigated included one that grows high above the water-table (Sphagnum fuscum) and two that grow lower down (Sphagnum balticum and Sphagnum fallax). S. fuscum and S. balticum originate from ombrotrophic and S. fallax from minerotrophic environments. To estimate N responses, we measured the performance and light-capture kinetics of the photosynthetic apparatus (maximum photosynthetic rate and Fv/Fm), biomass production, shoot formation, and N and phosphorus (P) concentrations in the tissue.

4. Tissue nitrogen concentration generally increased with N application rate, and photosynthetic rate increased with N concentration, although S. balticum exhibited a unimodal response. With respect to production, a negative response to N application rate was found in S. fallax and S. fuscum (weak), while production in S. balticum was unrelated to application rate. S. fallax was the fastest-growing species, producing two to three times more biomass per shoot compared with the other species.

5. The mismatch between photosynthetic capacity and production could partly be explained by an increased N : P ratio following N application. Phosphorus limitation may not negatively affect photosynthetic capacity, but may hamper production.

6. The fast-growing species S. fallax is considered to benefit from increased N deposition, but we found a negative physiological response, suggesting stoichiometric constraints. Thus, we conclude that responses to N deposition cannot be predicted in a simple way from physiological traits related to growth rate without considering local environmental factors.

Introduction

Terrestrial ecosystems are mainly nitrogen (N) limited, and N addition generally increases biomass production (LeBauer & Treseder 2008). The addition of N also affects competition among plants, resulting in subsequent loss of species diversity. This is most pronounced in nitrogen-poor ecosystems, where fast-growing plants will be favoured over slow-growing nutrient efficient plants (Strengbom et al. 2001; Stevens et al. 2004). Species’ competitive ability under different N conditions can be attributed to underlying morphological or physiological characteristics. Hence, understanding how species traits are related to N allocation and N use is essential to allow us to predict plant community composition under changing N availability (e.g. N deposition, Diekmann & Falkengren-Grerup 2002), and how such changes will influence ecosystem functions (e.g. Suding et al. 2008).

Sphagnum can be viewed as an ecosystem engineer that forms peatlands (i.e. the accumulation of peat) mainly through its production of recalcitrant litter and its acidifying capability, which makes the environment hostile for vascular plants. Sphagnum-dominated mires that have been disconnected from the minerotrophic groundwater (i.e. bogs) are dependent on atmospheric deposition of nutrients (e.g. via rain, dry deposition, dust and fog) and are thus extremely nutrient-poor ecosystems (Rydin & Jeglum 2006). Experimental data have demonstrated that under increased N input, Sphagnum production decreases, while vascular plants are favoured and can shade out Sphagnum by a denser canopy or increased litter (Berendse et al. 2001). Earlier studies indicate that N uptake capacity and N allocation patterns differ among Sphagnum species (Jauhiainen, Vasander & Silvola 1998; Jauhiainen, Wallén & Malmer 1998). These responses may be related to physiological differences between functional types of plants as well as among species, resulting in altered competitive relationships. There is some evidence that increased N availability alters competition among mosses (e.g. Twenhöven 1992; Gunnarsson, Granberg & Nilsson 2004; Paulissen et al. 2005), but most studies that report differences in N response among moss species have compared responses based on relatively few N addition treatment levels (e.g. low and high) and implicitly assume linear responses to increased N availability. One exception is Salemaa, Mäkipää & Oksanen (2008), who fitted growth response curves for three forest mosses and found distinct differences in N optima among them. Under continuous N deposition, such interspecific differences may lead to changes in species composition, resulting in an increased abundance of species adapted to high N availability and potentially decreased diversity by competitive exclusion of other moss species. Such long-term effects of increased N input are often referred to as indirect effects because they are mediated by changes in interspecific interactions (Manning et al. 2006). Direct effects on the other hand can, in the context of plant communities, be viewed as the physiological effects on the individual plant in the absence of other organisms (Manning et al. 2006). To gain information about interspecific differences that mediate responses to N deposition in peatland communities, species-specific response curves of Sphagnum are needed.

When plants experience elevated N availability, more nitrogen can be allocated to the photosynthetic apparatus (pigments and Rubisco), facilitating an increase in the rate of photosynthesis. In a recent article based on two field studies, we presented a photosynthesis–N concentration (tissue) response curve for Sphagnum balticum (Granath, Wiedermann & Strengbom 2009). We found a unimodal response with a N concentration optimum for photosynthetic capacity at around 1·3%. This contrasts with vascular plants, which show a linear, or slightly curvilinear, relationship (monotonically increasing) between foliar N concentration and photosynthetic rate (Evans 1983; Hikosaka 2004). In Sphagnum, a change in photosynthetic capacity may not always translate into a production response (Granath, Wiedermann & Strengbom 2009; Granath et al. 2009), because the latter depends also on other factors coupled to N addition, for example P limitation (Aerts, Wallén & Malmer 1992; Limpens, Berendse & Klees 2004). Nevertheless, the expected unimodal production response to N availability (e.g. ter Braak & Prentice 1988; Austin 2002) seems to hold for Sphagnum, as indicated in a recent meta-analysis (Limpens et al. 2011). However, despite the vast amount of publications, information about species-specific differences in response to a wide range of N doses and knowledge of the underlying physiological mechanisms are still sparse.

Our aim was to explore the direct effects of N on the physiology of Sphagnum species by investigating functional traits associated with plant growth. Large differences among species could explain changes in species composition under increased N deposition (directly or via altered competitive ability). To isolate the direct effects of N application from indirect effects (i.e. those mediated by intra- and interspecific interactions), we performed a greenhouse experiment using individual shoots as experimental units. Intraspecific interactions in Sphagnum are important for some species to stay hydrated by densely packed shoots, often forming the microtopography in peatlands (hummocks). By focusing on individual shoots that were kept hydrated, we avoided indirect effects mediated by the microtopography (e.g. risk of desiccation). This approach allowed us to test and quantify the direct effects of increased N application on plant production. To assess whether the response to N could be linked to growth-related traits, we measured how N accumulation in apical tissue, growth rate, shoot formation (i.e. number of new shoots formed) and photosynthetic capacity responded to eight different N application levels. Photosynthetic capacity was evaluated on the basis of maximum photosynthetic rate (NPmax, i.e. the performance of the whole photosynthetic apparatus) and the maximum efficiency of photosystem II (PSII) [variable fluorescence (Fv)/maximum fluorescence yield (Fm), i.e. the light-capture kinetics of the photosynthetic apparatus]. The N : P ratio was also evaluated as a measure of potential P limitation when N availability increases.

The species studied were Sphagnum fallax (Klinggr.) Klinggr., S. balticum (Russ.) C. Jens. (both section Cuspidata) and Sphagnum fuscum (Schimp.) Klinggr (section Acutifolia) (Fig. 1). These species are common and widespread on bogs (S. fuscum, S. balticum) and fens (S. fallax) in northern Europe, North Asia and North America (Daniels & Eddy 1990). S. fallax has recently increased in abundance in NW Europe (Limpens, Tomassen & Berendse 2003) and is considered to benefit from high N availability because of its high growth rate compared with other Sphagnum species (Twenhöven 1992; van der Heijden, Verbeek & Kuiper 2000). Similarly, Gunnarsson, Granberg & Nilsson (2004) found that the minerotrophically growing species Sphagnum lindbergii increased at the expense of the ombrotrophic S. balticum in N-treated plots. Fast-growing species are, compared with slow-growing ones, usually less conservative in their use of resources and co-limitation of nutrients other than nitrogen is therefore more common for fast growers (Aerts & Chapin 1999). This response pattern is supported by several studies, suggesting that S. fallax is more likely to suffer from phosphorus limitation than other Sphagnum species (Kooijman & Kanne 1993; Limpens, Tomassen & Berendse 2003). Hence, increased P availability may both enable an N-tolerant species to profit from increased N deposition and alleviate negative effects on N-sensitive species (Limpens et al. 2011).

Figure 1.

 Photograph of the species studied (a) Sphagnum fuscum, (b) Sphagnum balticum and (c) Sphagnum fallax. Photograph: G. Granath.

Based on existing information regarding the aforementioned traits, we expected S. fallax (minerotrophically growing and expanding) to have the highest growth rate potential, the strongest positive response to N addition and the highest N optimum, and S. fuscum (ombrotrophically growing, low growth rate) to exhibit the slowest growth and lowest N optimum. S. balticum was expected to have an intermediate response (minerotrophically to ombrotrophically growing, intermediate growth rate).

Materials and methods

Species and Collection Site

Many Sphagnum species occupy narrow niches with respect to the level of the water-table, pH and nutrient availability gradients (Rydin 1993). The species studied (S. fallax, S. balticum and S. fuscum; Fig. 1) differ with respect to both growth position above the water-table and affinity to ombrotrophic conditions (S. fuscum S. balticum S. fallax). S. fuscum is a hummock-dwelling species that grows high above the water-table in an ombrotrophic environment (i.e. with no influence from minerotrophic groundwater). S. balticum grows closer to the water-table (in hollows) and can be found in both ombrotrophic and minerotrophic environments. S. fallax is generally restricted to minerotrophic environments and grows close to the water-table.

Samples of the three species were collected on 10 June 2008 from Degerö Stormyr, a mire located in the mid-boreal zone at the Kulbäcksliden Research Park of the Vindeln Experimental Forests (64°11′N, 19°33′E; 270 m a.s.l.), northern Sweden. The collection site is a mixed mire containing fen and bog habitats, and the vegetation is dominated by S. balticum, S. lindbergii Schimp., Sphagnum majus (Russ.) C. Jens. and Sphagnum papillosum Lindb. in the lawns, and S. fuscum on the hummocks. S. fallax can also be found, but is confined to the poor fen lawns of the mire. The site has a mean (1961–1990) annual temperature of 1·2 °C (July, 14·7 °C; January, −12·4 °C) and a mean annual precipitation of 523 mm. It has a low ambient N deposition of 0·2 g N m−2 year−1. As a comparison, areas in central and western Europe may receive up to 5 g N m−2 year−1. A low ambient N deposition at the collection site was crucial because our aim was to capture the effects of N over a wide range of N levels. Had there been a high initial foliar N concentration, we would not have been able to elucidate the true response at lower N levels (Limpens et al. 2011). This is particularly important when comparisons between species are being made, because responses to N deposition may be species specific.

After collection, the samples were taken to Uppsala and kept moist with distilled water in a greenhouse under natural light conditions and at a temperature between 15 and 25 °C, until the start of the experiment on 25 June 2008.

Preparation of Plant Material and Experimental Design

Sphagnum shoots were cut to a length of 30 mm. Of each species, we randomly selected twenty shoots that were oven-dried (70 °C, 48 h) and used to determine the pre-experimental mass for capitula (top 0·5–1 cm) and stems. Capitulum mass was divided by capitulum area (calculated as the area covered by tissue using digital images of the capitula taken from above) to determine the pre-experimental mass per unit area. The capitulum area ratio among the species, S. fuscum S. balticum S. fallax, was 1 : 1·6 : 2·0. These figures were used to calculate how much N to add per shoot to give each species the same amount per unit area to avoid that N was diluted in the biomass of the larger shoots (which may otherwise be a problem in experiments based on individual shoots).

Each individual Sphagnum shoot was put in a hole of a plug tray (used for growing seedlings in greenhouses, but turned upside down for our purpose), which was placed in another, larger, water-filled tray. Each N level (8) and species (3) was represented by three shoots, amounting to 72 shoots per tray. Each tray was considered a complete block within the experiment. A block design was chosen to control for the environmental heterogeneity in the greenhouse. The complete set-up contained five trays (n = 5) and a control tray in which the shoots only received the additional N-free nutrient solution. The treatments were not randomized within a tray because there was a risk of leakage (e.g. the highest N dose adjacent to the zero treatment could lead to indirect N addition to the zero treatment). Thus, we arranged the treatments, so that the low N treatments were located close to the zero N treatments and the highest N level furthest away from the zero treatment. We avoided possible systematic environmental effects within each block (e.g. light conditions) by rotating the blocks during the experimental period.

The experiment lasted 153 days, and the three species were exposed to eight levels of N concentrations (0, 33, 66, 99, 131, 164, 197 and 230 mg N L−1, added as NH4NO3), corresponding to a range of N applications between 0 and 5·6 g N m−2 153 days−1. The duration of the experiment corresponds to approximately one growing season at the collection site, and the N application rates therefore roughly correspond to g N m−2 year−1. N solutions were applied 1–2 times per week with at least 3 days between applications, amounting to a total of 31 applications. The nitrogen solutions were applied to the capitula of each individual shoot using a pipette. After each N application, we sprayed the trays with an additional N-free nutrient solution (artificial rain) based on Salemaa, Mäkipää & Oksanen (2008), containing the macronutrients (mg L−1): P, 0·02; K, 0·4; Mg, 0·2; Ca, 0·7; S, 0·43; and Fe, 0·2. The solution contained very little P, corresponding to <0·001 g P m−2 over the experimental period.

The experiment was carried out in a greenhouse under natural light conditions during the summer and supplementary light (16 h of light, PPFD c. 200–250 μmol m−2 s−1) during the autumn. The temperature was generally between 18 and 25 °C (max 30 °C) during the summer and between 15 and 20 °C during the autumn. The six trays were placed adjacent to each other in two rows; their positions were shifted before every N application. The water level in the trays was kept constant, circa 2 cm below the capitula, and the short distance to the water prevented desiccation of the mosses. Growing close to the water surface mimics the natural growth conditions for S. fallax and S. balticum while, under natural conditions, S. fuscum grows higher above the water-table. However, this niche separation is not related to differences in physiological tolerance; field experiments have shown that S. fuscum grows well and maintains its photosynthetic capacity close to the water-table (Rydin & McDonald 1985; Granath, Strengbom & Rydin 2010). To avoid N accumulation, the water in each tray was slowly replaced from a 25-L vessel containing deionized water (refilled twice a week) by a hose perforated with small holes.

Response Variables

The response variables measured at the end of the experiment were biomass production per shoot (mg), number of side shoots formed, maximum photosynthetic rate (at optimal light conditions and water content), maximum quantum yield of PSII (Fv/Fm), and N, P and C concentrations.

As the plants grew longer during the experimental period, the basal senescent part of the stems were cut back on two occasions, leaving the top 3 cm intact (the cut parts were retained to be included in estimates of production). If a capitulum initiated a side shoot (i.e. divided into two capitula), the new capitulum was removed (and included in production). Total biomass production was calculated by adding together stem production, capitulum production and change in capitulum biomass. Stem biomass production was calculated as final stem mass (including the stem parts removed during the experiment) minus pre-experiment stem mass. Change in capitulum biomass was calculated in several steps. First, we multiplied the pre-experimental area of each capitulum by the pre-experimental species-specific mass per area and then subtracted the pre-experimental mass from the final mass. A negative value indicates decreased capitulum mass, and a positive value increased capitulum mass. Capitulum production was calculated as the sum of change in capitulum biomass and newly formed shoots. The sum of stem production and capitulum production gave us the total biomass production per shoot.

At the end of the experiment, before harvesting capitula for the calculation of biomass, we measured photosynthetic capacity, maximum photosynthetic rate, NPmax, and the light kinetics of the photosynthetic apparatus, Fv/Fm (maximum quantum efficiency of PSII). NPmax evaluates the potential maximum performance of the whole photosynthetic apparatus (i.e. growth potential), while Fv/Fm measurements are restricted to changes in PSII. In general, Fv/Fm works as a stress indicator and summarizes the plants health and vigour (Adams & Demmig-Adams 2004). Values below the theoretical maximum of 0·78–0·84 indicate impairment or down-regulation of PSII efficiency (Adams & Demmig-Adams 2004), although Sphagnum samples rarely reach such high values (Granath et al. 2009). To obtain Fv/Fm, where Fv was calculated as Fm– fluorescence origin, Fo (Maxwell & Johnson 2000), we measured chlorophyll fluorescence using a pulse-modulated fluorometer (Mini-PAM photosynthesis yield analyser; Walz, Effeltrich, Germany). Individual capitula were saturated with distilled water and dark-adapted for 15 min before being exposed to a saturation pulse lasting 0·8 s at an intensity of approximately 8300 μmol m−2 s−1, from a distance of 11 mm.

To measure maximum photosynthetic rate (NPmax), we used an infrared gas analyser (DC-CIRAS, PP Systems), running in differential mode, using the ambient CO2 concentration (365–395 p.p.m.) in an open system. To have enough plant material for accurate measurements, we were forced to use all capitula per N level (i.e. 3 capitula × 5 blocks = 15 capitula per N level). The capitula were placed on a nylon net in an air-tight chamber (Volume = 130 cm2) connected to the IRGA via inlet and outlet tubes. A water cooling system, together with a chamber fan, was used to ensure a temperature around 20 °C and well-mixed air. A greenhouse lamp was used for illumination with a PPFD of 500–550 μmol m−2 s−1. This light level is above light saturation, but below the level when photoinhibition occurs (Harley et al. 1989; Marschall & Proctor 2004). Samples were kept under these conditions for at least 15 min prior to the measurements. The maximum photosynthetic rate of mosses occurs at a specific water content (Schipperges & Rydin 1998). Hence, we followed the measuring protocol outlined in Granath et al. (2009), where we first wetted the capitula and then measured CO2 exchange as the capitula slowly dried out. The highest CO2 uptake was defined as NPmax. The samples were then photographed from above to estimate plant area, dried to constant weight (65 °C, 48 h), and NPmax was expressed as the rate of CO2 uptake per unit dry weight (mg g−1 h−1) at the optimal water content.

Plant Chemistry Analyses

The capitula were freeze-dried and ground to a homogenous powder. The three capitula per treatment and block were pooled. Carbon (C) and N concentrations were determined using a Costech ECS 4010 and were expressed on a dry weight basis. To determine total phosphorus (P), the powder was heated to 550 °C for 60 min, then boiled in 1 N HCl for 15 min, diluted and measured according to the method of Murphy & Riley (1962). As we had very little material left after the N and C measurements, P could only be analysed once for each N addition level and species, pooling over blocks. Consequently, the N : P ratio was calculated per N addition level per species.

Statistical Analyses

All statistical analyses were performed in R (The R development Team 2010). Block (n = 5) was treated as a random effect when included, and mixed models were analysed using the nlme package in R (Pinheiro et al. 2009). We treated the N application rate as a continuous variable in the analysis. The average capitulum area at the beginning of the experiment was used to calculate N application rate per area. There is treatment variation associated with the N application rate both because of variation in area of the capitula at the start of the experiment and because the size of individual capitula changed over the course of the experiment. However, the variation was not substantial, and the capitulum area was normally distributed within the blocks per species, without bias towards any specific treatment. In addition, the correlation between N application rate and N concentration further suggests that the error caused by the variation in capitulum area could be ignored.

General linear (mixed) models were employed to investigate the relationships between the variables; the possible presence of a nonlinear (i.e. unimodal) relationship was tested by adding a quadratic term. Because the larger plants may produce more biomass, we also fitted a model including ‘capitulum size at the start of the experiment’ as a covariate. By doing this, we investigated what proportion of the species’ differences could be explained by initial size. Because the experiment was balanced, we performed an F-test to examine the statistical significance of fixed effects in the mixed models (Bolker et al. 2009). To test the effect of N application on shoot formation rate, we applied a generalized linear mixed model (GLMM) with Poisson errors, including all shoots (i.e. not pooling over three shoots as for other variables). Here, we conducted a Wald test to investigate the statistical significance of the fixed effects (Bolker et al. 2009). The significance of factors was assessed in the full model; models were thereafter simplified to illustrate any interesting effects, and these are presented in figures and tables. Standard residual analyses were used to check normality and homogeneity of variance. For some of the mixed models, the variance changed with the predictor (indicating heteroscedasticity), thus violating the requirement for normally distributed residuals with constant variance. To avoid the need for transformation, we used an exponential variance structure (varExp in the R package nlme) to handle the increasing spread of residuals (Zuur et al. 2009). For background information and meta-analysis purposes, treatment means and their standard deviation for N concentration and production are presented in the supplemental material (Table S1).

Results

Nitrogen Accumulation and N : P

The control tray did not contain lower N concentrations than the zero N application treatment. Hence, the moss shoots did not receive any measurable N through leakage from nearby N treatments. Capitulum N concentration increased with increasing N addition in all the species, but levelled off at higher N addition levels, as indicated by a significant quadratic term (F1,111 = 4·9, P = 0·03; Fig. 2). Controlling for production in the analyses did not change the results. Species-specific linear and quadratic terms associated with N addition did not differ significantly from each other (P > 0·3), but overall N concentration did differ among species (F2,111 = 25·6, P < 0·0001); S. fuscum and S. fallax had the highest concentrations, and S. balticum the lowest [means (mg g−1): S. fallax, 11·3; S. balticum, 9·7; S. fuscum, 12·0]. These results suggest that initial differences in N concentration among species from different microhabitats were retained. The increase in N resulted in a decrease in C : N ratio with N addition.

Figure 2.

 Relationship between tissue (capitula) N concentration (dry mass basis) and N application over 153 days in three Sphagnum species. The modelled shape of the curves is the same for all species (N concentration = intercept + 1·3 × N application rate − 0·10 × N application rate2) but the species-specific intercepts differ: S. fuscum = 9·4, S. fallax = 8·8 and S. balticum = 7·2. See Table 1 for model coefficients and their SEs. For readability, the data points of different species are slightly separated within each N application level.

Table 1.   Summarized regression models for capitulum (i.e. apical tissue) N concentration, shoot production and length increment per shoot as functions of N application rate (g m−2 153 days−1) illustrated in Figs 2 and 5. The models have been simplified to better illustrate the important (and significant) terms that were identified in the full model. Species effects are compared with the intercept which is set to that for Sphagnum balticum
 CoefficientSEd.f.tP
N concentration
 Intercept (S. balticum)7·240·2711127·0<0·001
 Sphagnum fuscum2·170·331116·5<0·001
 Sphagnum fallax1·560·311115·0<0·001
 N application rate1·300·241115·3<0·001
 N application rate2−0·100·047111−2·20·03
Production
 Intercept (S. balticum)9·282·141094·3<0·001
 S. fuscum−1·131·39109−0·80·42
 S. fallax18·731·4410913·0<0·001
 Initial size0·040·021092·20·03
 N application rate−0·030·29109−0·10·91
 N application rate × S. fuscum−0·410·42109−1·00·33
 N application rate × S. fallax−1·040·42109−2·50·01
Length increment
 Intercept (S. balticum)2·581·901091·40·18
 S. fuscum−2·051·07109−1·90·06
 S. fallax13·171·551098·5<0·001
 Initial size0·090·021095·5<0·001
 N application rate0·150·241090·60·55
 N application rate × S. fuscum−0·670·32109−2·10·04
 N application rate × S. fallax−0·960·44109−2·20·03

The N : P ratio increased with N addition in all species, and no species-specific slopes were detected (F2,18 = 1·18, P = 0·33). S. fallax had the lowest average N : P value (36); S. balticum was intermediate (43); and S. fuscum had the highest value (60) (species effect: F2,20 = 27·1, P < 0·001; and pairwise comparisons: P < 0·05 except S. fallax vs. S. balticum where P = 0·09). Samples collected at the field site also showed that S. fallax had the lowest N : P ratio (S. fallax, 13; S. balticum, 20; S. fuscum, 22).

Capacity and Efficiency of the Photosynthetic Apparatus

Analyses of NPmax indicated a curvilinear relationship with N application, where the highest NPmax was observed around 4 g N m−2 (Fig. 3; N2: F1,15 = 4·6, P = 0·04). The model fitted S. fuscum and S. balticum rather well while it could not capture the large variation in S. balticum at the higher treatment levels. When NPmax was regressed against N concentration, data suggested species-specific quadratic terms (F2,15 = 4·3, P = 0·03). To simplify interpretation, we analysed each species separately. S. balticum exhibited a unimodal relationship. The interpretation should, however, be viewed with some caution as the pattern is affected by a few influential data points (quadratic term: t5 = 1·998, P = 0·10; Fig. 4). The other two species (S. fuscum and S. fallax) showed a clear linear relationship between photosynthesis and N concentration, with a tighter relationship and greater effect of N concentration for S. fuscum (Fig. 4; slope for N concentration; S. fuscum, 2·1; S. fallax, 0·6; t12 = 4·2, P = 0·001). Moreover, this translated into a lower photosynthetic nitrogen use efficiency (PNUE; NPmax divided by N concentration) in S. fallax with increasing N concentration (slope = −0·76, t12 = 3·6, P < 0·01), while S. fuscum exhibited similar PNUE over the N concentration range, and S. balticum, a unimodal pattern (species-specific quadratic term, F2,15 = 5·6, P = 0·02).

Figure 3.

 The relationship between maximum photosynthetic rate (NPmax; CO2 uptake per gram, on a dry mass basis, per hour) and N application over 153 days in three Sphagnum species. The modelled shape of the curves is the same for all species (NPmax = intercept + 0·33 × N application rate − 0·04 × N application rate2) but the species-specific intercepts differ: S. fuscum = 1·8, S. fallax = 1·2 and Sbalticum = 1·3. All five replicates per N treatment level were used to analyse NPmax; therefore, no error bars are presented.

Figure 4.

 The relationship between maximum photosynthetic rate (NPmax; CO2 uptake per gram, on a dry mass basis, per hour) and tissue (capitula) N concentration (dry mass basis) for each species. A positive linear relationship was obtained for Sphagnum fuscum (NPmax = −0·28 + 0·21 × N concentration) and Sphagnum fallax (NPmax = 0·98 + 0·058 × N concentration), but with significantly different slopes, and a unimodal relationship was obtained for Sphagnum balticum (NPmax = −12 + 2·9 × N concentration −0·15 × N concentration2). The two influential data points for S. balticum are the N treatments zero (to the left) and 4·8 g m−2 153 days−1 (to the right). All five replicates per N treatment level were used to analyse NPmax; therefore, no error bars are presented.

We found no significant effect of N application on Fv/Fm, but there was a species effect, indicating lower Fv/Fm values (7% lower, 0·68 vs. 0·73) for S. fuscum compared with the other two species (N × Species: F2,110 = 0·3, P = 0·73; N: F1,110 = 0·12, P = 0·73; Species: F2,110 = 25·8, P < 0·001).

Biomass Production and Length Increment

Biomass production was the greatest in S. fallax, and S. balticum and S. fuscum had similar production levels (not significantly different from each other at zero N application). Production was associated with a species-specific linear response to N addition (N × Species, F2,106 = 3·2, P = 0·05; Fig. 5). Production decreased in S. fallax and S. fuscum with N addition while S. balticum was relatively unaffected. The effect of initial capitulum size was statistically significant, but explained <5% of the overall differences between the species. Further analyses revealed that reduced stem production was responsible for the decreased production in S. fallax and S. fuscum (N × Species, F2,106 = 3·2, P = 0·05, with effects similar to the production model; Fig. 4b), while we found no significant effect of N application on capitulum mass (N × Species: F2,106 = 2·2, P = 0·12; N application: F2,106 = 2·1, P = 0·15; model coefficients for N application: S. fuscum, 0·11; S. balticum, −0·22; S. fallax,−0·20).

Figure 5.

 (a, c, e) Relationship between production per shoot and N application over 153 days, with a negative response in Sphagnum fallax and Sphagnum fuscum. (b, d, f) Relationship between length increment and N application, illustrating that a decrease in stem production is responsible for the reduction in biomass in S. fallax and S. fuscum at higher N applications. For model coefficients and their SEs, see Table 1.

There were species-specific relationships between production and the N : P ratio (species × N : P, F2,18 = 5·0, P = 0·02). Production was associated with a negative relationship for S. fallax and S. fuscum but not for S. balticum (Fig. 6).

Figure 6.

 The relationship between production per shoot and N : P ratio. (a) Non-significant relationship for Sphagnum fuscum (slope: −0·06); (b) significant negative relationship for Sphagnum fallax (slope: −0·27) and (c) non-significant relationship for S. balticum (slope: 0·04). All five replicates per N treatment level were used to analyse P; therefore, no error bars are presented.

Shoot Formation Rate

We detected an interaction effect between N application and species (Wald’s test-statistics = 6·8, d.f. = 2, P = 0·03, data not shown). While shoot formation rate in S. balticum was not greatly affected by N application (coefficient = 0·05), N application stimulated shoot formation in S. fallax (coefficient = 0·16), but suppressed it in S. fuscum (coefficient = −0·09). However, the main effect (i.e. mean shoot formation pooled over all treatments) of species was that S. fuscum produced most new shoots, and S. fallax least (new shoots formed per shoot: S. fuscum = 0·65, n = 119, S. balticum = 0·38, n = 111, S. fallax = 0·21, n = 116; Wald statistics, all comparisons, P ≤ 0·05).

Discussion

A large number of articles have been published on the effects of N application on the growth of Sphagnum mosses. Our article contrasts the majority of these articles as it is a study that explores the direct effects of N on the underlying physiology of Sphagnum species by investigating functional traits associated with plant growth. Our study demonstrates that maximum photosynthetic rate, biomass production, length increment and shoot formation rate exhibit species-specific responses to N application. The hypothesis that variation in intrinsic traits (e.g. potential growth rate) among species determines responses to N (species with high growth rate potential should respond more positively to N) could, due to stoichiometric constraints, not be supported. Capitulum N accumulation rates and changes in N : P ratio were, however, similar among the three species.

N accumulation

As fast-growing species generally have high tissue N concentration but are less conservative with N (e.g. Aerts & Chapin 1999), we expected our species to differ in their N accumulation. Fast-growing species loose more N through senescing tissue, and they can potentially lower the N concentration by dilution in the active tissue. However, N accumulation rates did not differ significantly among the species investigated, and our results were not a simple effect of N dilution. Although we did not follow the fate of added N, and thus we cannot quantify the loss, retention or N uptake, N accumulation rate may be viewed as the sum of these processes, of which some are known to be species specific (e.g. Jauhiainen, Wallén & Malmer 1998). Keeping this in mind, all three species exhibited a curvilinear relationship between N application and capitulum concentration, indicating N saturation at high addition levels. The final N concentrations were similar to those reported from field studies across Europe, exhibiting N concentrations between 10 and 15 mg g−1 at intermediate to high N deposition levels (Lamers, Bobbink & Roelofs 2000; Bragazza et al. 2004). The curvilinear N accumulation response is in accordance with previous data (Lamers, Bobbink & Roelofs 2000; Bragazza et al. 2004; Limpens et al. 2011).

Differences in N uptake may affect the N concentration, and there are reports that N uptake ability is species specific (Jauhiainen, Wallén & Malmer 1998). However, Wiedermann et al. (2009) found no major differences in N uptake between S. fuscum and S. balticum. Our study supports this finding, because it suggests that variation in N uptake response to N deposition is limited among Sphagnum species. If correct, this contrasts with a study on forest mosses, which reported distinct species-specific N accumulation patterns (Salemaa, Mäkipää & Oksanen 2008). Potentially, the greater diversification among forest bryophytes may stem from the more heterogeneous environment in forests than in peatlands.

Photosynthetic Capacity

In contrast to our hypothesis, the fast-growing species did not increase its photosynthetic rate more rapidly than the slow-growing species in response to N addition. No effects of N application on the light kinetics of the photosynthetic apparatus (Fv/Fm) were detected, although such effects have been reported in field experiments investigating the interaction between moisture stress and N addition (Carfrae et al. 2007). In general, the three species showed a similar response pattern, with increasing maximum photosynthetic rate with increasing capitulum N concentration. This was expected as previous studies have reported similar response in bryophytes (Skre & Oechel 1979; Granath, Wiedermann & Strengbom 2009; Granath et al. 2009). In bryophytes, higher N availability leads to more chlorophyll and consequently to a higher photosynthetic rate (Granath, Wiedermann & Strengbom 2009; Granath et al. 2009). An unexpected result was that PNUE decreased in S. fallax at higher N concentrations, suggesting that this species respond to increased N availability by allocating more N to processes other than photosynthesis (e.g. accumulation of amino acids) compared with S. fuscum and S. balticum. Alternatively, it may be related to toxicity or change in cellular pH at high N concentration (e.g. Limpens & Berendse 2003).

The indication that S. balticum responded differently in NPmax than the other species is supported by an earlier study (Granath, Wiedermann & Strengbom 2009). Even if we cannot present a mechanistic explanation for this, the distinctive response of the species may explain why Rice, Aclander & Hanson (2008) could not find a relationship between N concentration and photosynthetic rate in a study combining data from ten species. Studies including more species are needed to clarify the mechanisms underlying differences among Sphagnum species.

Growth and Production

According to resource gradient theory, species abundance and growth exhibit a unimodal response to environmental variables (ter Braak & Prentice 1988; Austin 2002), and a unimodal pattern has been reported for the growth response along a N addition gradient in forest mosses (Salemaa, Mäkipää & Oksanen 2008). However, in Sphagnum, both field and greenhouse studies have mainly reported negative relationships between production and N application, rather than an optimum, when N responses have been examined over a wide range of N levels (Gunnarsson & Rydin 2000; Paulissen et al. 2005; see Vitt et al. 2003 for unimodal response). In our study, we found no support for a unimodal response, and our predicted responses based on the growth rate trait for the three species were not supported. N addition resulted in reduced biomass production and length increment in fast-growing S. fallax, a species we expected to benefit from N addition, and slow-growing S. fuscum (although a smaller reduction), while S. balticum was quite unaffected. The fact that differences in biomass production were related to differences in length increment suggests that elevated N conditions result in a more compact growth form. Earlier studies have also reported effects mainly on length increment (Gunnarsson & Rydin 2000; Bragazza et al. 2004) while a recent study also noted effects on capitulum weight (Manninen et al. 2011).

In our experiment, we solely tested the direct effects on production while performance in the field also incorporates indirect effects and interactions with environmental variables. Thus, our study shows that direct physiological effects of N addition differ among the species investigated. One interaction (that we deliberately avoided by keeping the mosses hydrated) is that N addition may affect the plant structure and thereby their water-holding capacity (Aldous 2002; Carfrae et al. 2007). Although our experimental conditions are controlled and therefore somewhat unrealistic, our experiment can provide information on the importance of direct effects on the physiology of Sphagnum under N addition that cannot be revealed by field experiments.

Surprisingly, no effect of N application was found in S. balticum, a species that has previously been found to be sensitive to N deposition in field experiments (Gunnarsson, Granberg & Nilsson 2004). Our result indicates that S. balticum is less sensitive to N deposition than previously believed, at least in the short term. Similarly, a field study reported that S. balticum was the only Sphagnum species that exhibited a positive response to N application (Berendse et al. 2001), and a greenhouse study found no effects of N addition on its production (van der Heijden et al. 2000). The reported sensitivity in S. balticum to N deposition may, thus, be more related to indirect effects and interactions with other environmental variables, rather than directs effects of high N load.

Although field data have suggested that S. fallax can benefit from N additions (Twenhöven 1992; Risager 1998; Gunnarsson & Flodin 2007), there are several field studies showing strong negative effects on production following N application (Berendse et al. 2001; Mitchell et al. 2002). The explanation may be that the response in S. fallax is partly related to P limitation (Kooijman & Kanne 1993; Limpens, Tomassen & Berendse 2003). Our study supports the idea of co-limitation between N and P, as production in S. fallax was strongly negatively related to an increasing N : P ratio, again emphasizing the importance of direct physiological effects. The sensitivity to high N : P ratios was also supported by data from the collection site that showed much lower N : P ratio for S. fallax than for the other species. For Sphagnum, the critical N : P ratio (i.e. when plant growth becomes P limited) has been suggested to be between 14 (Aerts, Wallén & Malmer 1992) and 30 (Bragazza et al. 2004), and many field experiments have reported that P deficiency probably limited the growth response (e.g. Williams & Silcock 1997).

The response in S. fallax followed our prediction if nutrient imbalance is taken into account. Fast-growing species are less conservative with nutrients, and stoichiometric constraints should appear faster in such species (Sterner & Elser 2002). For example, N enrichment on P-poor soils mainly favours low productivity species over typical ‘nitrophilous’ species (Güsewell 2004). Moreover, in a P-limited grassland, slow-growing bryophytes were less sensitive to N addition than fast-growing ones (Arroniz-Crespo et al. 2008). P limitation on peatlands is common in Europe, where N : P ratios above 35 are found at a N deposition > 2·0 g−2 year−1 (Bragazza et al. 2004; Jirousek, Hájek & Bragazza 2011). Lower deposition rates occur in northern Europe, resulting in much lower N : P ratios (Aerts, Wallén & Malmer 1992). Thus, future expansion of fast-growing species like S. fallax under elevated N conditions may depend on P availability.

As found in an earlier field study (Granath, Wiedermann & Strengbom 2009; Granath et al. 2009), NPmax was not correlated with production. In nature, such a mismatch may be linked to many environmental variables affecting production (e.g. water availability, light, Gunnarsson 2005), but in a controlled greenhouse experiment, we expected a stronger correlation. The indications of P limitation and possibly of other nutrients such as potassium (Hoosbeek et al. 2002) may explain the results. In vascular plants, there is some evidence that the photosynthetic machinery is not greatly affected by P deficiency, while tissue-forming processes are (Foyer & Spencer 1986; Lynch, Lauchli & Epstein 1991). If bryophytes respond similarly, this could explain the mismatch. We measured net photosynthesis, but there is no reason to believe that the results should be caused by an effect on respiration rather than on gross photosynthesis. Previous research did not indicate increased dark respiration in N-treated plots (G. Granath unpublished data; Bubier, Moore & Bledzki 2007).

Conclusions

Our experiment demonstrates direct negative physiological effects of N on Sphagnum that may be as important as competition with vascular plants in explaining their decline under current N deposition in nature. These effects vary with species – although less than expected and not all in the directions expected; the fast-growing S. fallax was negatively affected by N application, bringing into question the idea that increased N deposition is the reason for its recent expansion in NW Europe. The effects will vary with the local environmental conditions regarding the availability of other nutrients (potassium, phosphorus), and stoichiometric constraints may be more detrimental to fast-growing Sphagnum species. Such interactions between species and the environment may explain why it has been difficult to identify a common feature of Sphagnum species that are favoured by high N input (e.g. Twenhöven 1992; Gunnarsson & Rydin 2000; Gunnarsson, Granberg & Nilsson 2004; Paulissen et al. 2005; Breeuwer et al. 2009; Bu, Rydin & Chen 2011). Our study stresses that nutrient availability should be taken into account if growth rate is used to predict N-sensitivity in peat mosses.

Acknowledgements

We wish to thank Juul Limpens and Rien Aerts for comments on the manuscript. The study was supported by Formas and VR.

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