Can small-scale experiments predict ecosystem responses? An

Oikos 000: 000
000, 2008 doi: 10.1111/j.1600-0706.2008.17129.x, # 2008 The Authors. Journal compilation # 2008 Oikos Subject Editor: Wim van der Putten. Accepted 23 September 2008

Can small-scale experiments predict ecosystem responses? An example from peatlands Magdalena M. Wiedermann, Urban Gunnarsson, Mats B. Nilsson, Annika Nordin and Lars Ericson M. M. Wiedermann ([email protected]) and L. Ericson, Dept of Ecology and Environmental Science, Umea˚ Univ., SE
901 87 Umea˚, Sweden.
U. Gunnarsson, Evolutionary Biology Centre, Dept of Plant Ecology, Uppsala Univ., Villava¨gen 14, SE
752 36 Uppsala, Sweden.
M. B. Nilsson, Dept of Forest Ecology and Management, Swedish Univ. of Agricultural Sciences, Petrus Laestadius va¨g, SE
901 83 Umea˚, Sweden.
A. Nordin, Umea˚ Plant Science Center, Dept of Forest Genetics and Plant Physiology, Swedish Univ. of Agricultural Sciences, SE
901 83 Umea˚, Sweden.

Oligotrophic, Sphagnum-dominated peatlands have been regarded as long-term stable ecosystems that function as carbon sinks. As a result of environmental perturbations, particularly anthropogenic N deposition, this view is now increasingly questioned. We examined whether small-scale field experiments can predict the direction and magnitude of ecosystem responses to increased N supply. We, therefore, compared data from a 10-year field experiment (involving deposition of 2, 15 and 30 kg N ha 1 year1) with data from a gradient associated with increased N deposition (2, 8 and 12 kg N ha 1 year 1). We chose to compare: (1) the physiological response of Sphagnum balticum, measured in the form of N accumulation as free amino acids (NAA); and (2) changes in the total Sphagnum cover, the cover of S. balticum, and vascular plant cover. In all cases we found a highly significant correlation between the two data sets. We attribute the high correspondence between the two data sets to the key function of the dominant group of organisms, the Sphagna, that monopolize N availability and control the water balance, creating an environment hostile to vascular plants. Thus the key role of Sphagna as ecosystem engineers seems to supersede the role of other, scale-dependent processes. We also conclude that NAA is a sensitive indicator that can be used to signal the slow and gradual shift from Sphagnum to vascular plant dominance.

A major issue in ecology is the extrapolation of experimental results in both space and time (Gardner et al. 2001, Englund and Cooper 2003). Since most manipulative field experiments are, for practical reasons, of limited size and duration, evaluating whether the direction and strength of observed responses is scale-dependent, is crucial for future predictions. For example, a small experimental area is likely to exclude organisms that naturally occur in low numbers which in turn will result in less complex food chains (Pimm 1991). Because treatment effects are likely to be confounded with the effect of any scale-dependent process that influences the response variable, a growing body of literature has questioned the relevance of small-scale experiments for extrapolations (Norby and Luo 2004, Rustad 2006). Another possible approach, often used in global change scenarios, is to use observations across altitudinal and latitudinal gradients as a way to understand ecosystem responses to changes in temperature, moisture or N deposition (Phoenix and Lee 2004, Rustad 2006). In this study we applied this space-fortime substitution to validate the outcome of small-scale field experiments in peat-forming, Sphagnum-dominated wetlands. Peatlands are important sinks for atmospheric carbon (Moore 2002). Although they cover only 3% of the global

land area, they are a dominant landscape element in boreal and sub-arctic regions of the northern hemisphere, where 95% of all peat reserves are found, and which store about 25% of the global soil carbon pool (Gorham 1991, Turunen et al. 2002, Smith et al. 2004). In addition, they are also the source of 20
30% of the annual methane emissions from natural sources (Mikaloff Fletcher et al. 2004). Although generally considered to be long-term stable ecosystems (Gunnarsson et al. 2002), a number of studies both in northwestern Europe (Malmer and Walle´n 2004, Bragazza et al. 2006, Franze´n 2006) and in eastern North America (Bubier et al. 1999, 2007) have suggested that boreal peatlands may be undergoing a regime shift from carbon sinks to carbon sources. Of key importance for forecasting how these ecosystems will respond to environmental perturbations is understanding how the dominant group of organisms, the Sphagna, respond to changes in temperature, precipitation, evapotranspiration, and nutrient availability; these are factors that set the limits for both autogenic and allogenic changes (Heijmans et al. 2008). Such responses are controlled by the functional morphology of the Sphagna. As a result of the extensive leaf area and extensive network of capillary spaces, this genus is an effective ion exchanger, which can Early View (EV): 1-EV

monopolize the acquisition of airborne nutrients, and has the ability to transport water to the continuously-growing shoot apex, the capitulum (Clymo and Hayward 1982, van Breemen 1995). Sphagna are certainly ecosystem engineers: they drive the formation of peatlands and create an environment that is hostile to most vascular plants (van Breemen 1995). During the last two decades, several manipulative experiments, designed primarily to examine the effects of increased N deposition and/or temperature on peatland vegetation, have reported a reduction in the amount of Sphagnum, paralleled by an increase in the abundance of vascular plants (Gunnarsson and Rydin 2000, Heijmans et al. 2002, Wiedermann et al. 2007). However, these experiments have been of short duration (from one to three years, with the exception of Wiedermann et al. (2007) who reported results after eight years of experimental treatment) and also utilized N addition doses far exceeding atmospherically derived N input. Nonetheless, the results of the few retrospective studies available also report vegetation shifts from Sphagnum to vascular plant dominance (Risager 1998, Frankl and Schmeidl 2000, Gunnarsson et al. 2002). A shift from Sphagnum dominance to vascular plant dominance will impact on the system’s C sink capacity (Malmer and Walle´n 2004), since C accumulation is greater in Sphagnum-dominated peatlands than in peatlands dominated by vascular plants (Verhoeven and Toth 1995, Dorrepaal et al. 2005). Thus, for predictive purposes, it is essential to evaluate whether Sphagnum responses to increased N supply in the more drastic experimental manipulations mirror the responses in areas subjected to atmospheric N deposition. In order to address this issue, we compared both physiological responses of one Sphagnum species (S. balticum) and the plant species coverage in an ongoing small-scale field experiment with three levels of N (2, 15 and 30 kg N ha1 year 1), initiated in 1995 and located in an area with low background N deposition (Wiedermann et al. 2007), with responses across a natural N deposition gradient (2, 8 and 12 kg N ha1 year 1). We chose to focus on soluble amino acid N content as a suitable physiological parameter. Our rationale for this choice was that several experimental studies based on manipulating N supply have demonstrated that Sphagnum tissue concentrations of total N (NTot), as well as of N in free amino acids (NAA), reflect the N addition rate (Nordin and Gunnarsson 2000, Limpens and Berendse 2003). Due to species-specific N demands among the Sphagna (Jauhiainen et al. 1998), we consider that measuring soluble amino acids in their function as N storage compounds (Baxter et al. 1992, Heeschen et al. 1997) provides a better indicator of excess N than values of NTot. Furthermore, Tomassen et al. (2003) conclude in their study that NAA can be used as a good indicator of future N saturation of the Sphagnum layer. This N saturation of the Sphagnum layer in turn leads to a major vegetation change from Sphagnum dominance to vascular plant dominance (Berendse et al. 2001, Limpens et al. 2003, Malmer et al. 2003). That also raises the question whether NAA accumulation may function as a suitable indicator signaling an incipient regime shift (Carpenter et al. 2008). We used Sphagnum balticum, a 2-EV

common, widespread lawn species of nutrient-poor mires, which dominates our experimental site, to test whether physiological responses in the manipulative field experiment differed from responses in the gradient study. In particular, we examined whether the results from the small-scale field experiment mirrored the results from the gradient study.

Material and methods Study species Sphagnum balticum belongs to the section Cuspidata and is a common species of lawn communities in oligotrophic fens and ombrotrophic bogs (Malmer 1988). It has a circumpolar distribution and is found in northern Europe, Greenland, North America and northern Asia (Frahm and Frey 1992). Study sites Gradient study

For this study we chose three mires, representing a gradient of decreasing inorganic (NH4 and NO3) N wet deposition across Sweden. The study sites (Fig. 1, Table 1) ranged

N wet deposition -1

-1

(kg ha yr ) 0–3 3–6 6–9 9 – 12

LD

MD HD 0

100 200

400 Kilometer

Figure 1. Map of Sweden, showing the location of the studied peatlands: a low N deposition site (LD), a mid N deposition site (MD), and a high N deposition site (HD). The map also shows N (NOxNHx) wet deposition (kg ha 1 year 1). Data were measured and modeled by SMHI (Swe. Meteorol. Hydrol. Inst.) and represent averaged values over a 10-year period (1995
2005).

Table 1. List of investigated peatlands with abbreviations in parenthesis, geographic position (lat. and long.), altitude, climatic data (1961
1990), nitrogen wet and dry deposition, and discharge. Study sites Degero¨ Stormyr (LD) ˚ khultmyren A (MD) ¨ resjo¨mossen O (HD)

Geographical coordinates 64811?N, 19833?E 57806?N, 14832?E 57803?N, 12851?E

Altitude Mean annual Mean annual N wet deposition N dry deposition Mean annual (m a.s.l.) precipitation (mm) temperature (8C) kg ha 1 year1 kg ha 1 year1 discharge (mm) 270

523

1.2

2

1


300

225

939

6.0

8

3


300

150

1095

6.4

12

4

400

Meteorological data from the Swe. Meteorol. Hydrol. Inst. (SMHI) (Alexandersson et al. 1991). Nitrogen wet deposition data (NO3 and ˚ khultmyren and O ¨ resjo¨mossen from the nearby IVL measuring stations Aneboda and Boa Berg, respectively NH4 contributing equally) for A (IVL Svenska Miljo¨institutet AB: Bhttp://www.ivl.se/miljo/projekt/ned_net/) and averaged for the period 1995
2003. N wet deposition at Degero¨ Stormyr was measured at the nearby field station (Granberg et al. 2001). Nitrogen dry deposition data (NO3 and NH4 ) for all sites from SMHI (Persson et al. 2004). Mean annual discharge for LD was taken from Nilsson et al. 2008 and for MD and HD from a map in Sjo¨rs (1999; based on data from Tamm 1959).

¨ resjo¨ mossen from high to low N deposition, as follows: O (HD) is a slightly raised ombrotrophic bog (mire type terminology follows Ruuhija¨rvi 1983). Here the lawn communities in the upper part of the hollows are dominated by Sphagnum balticum together with S. magellanicum. The most important species in the vascular plant layer are (in decreasing abundance) Rhynchospora alba, Erica tetralix, Calluna vulgaris and Andromeda polifolia. A˚khultmyren (MD) is an eccentric ombrotrophic bog. Here S. balticum occurs in lawn communities and co-dominant species are S. rubellum, S. magellanicum and S. tenellum. The vascular plant layer mainly consists of Trichophorum caespitosum, Calluna vulgaris, Erica tetralix and Rhynchospora alba. Degero¨ Stormyr (LD) is an aapa mire with topogenous nutrient-poor fens and ombrotrophic hummocks. Here the bottom layer of the lawn communities is dominated by S. balticum, while S. lindbergii and S. majus both occur in small amounts. The vascular plant layer is sparse and consists of Eriophorum vaginatum, Andromeda polifolia and Vaccinium oxycoccos. The surface peat water pH was around 3.9 at all three locations. The annual wet N deposition for the three sites amounted to 12, 8 and 2 kg ha1, respectively (Table 1). Additionally, for all sites approximate values of N-dry deposition are given in Table 1. However, accurate values of the contribution of N-dry deposition for a particular small patch are for several reasons difficult to estimate. Generally N-dry deposition is patchy and we believe that for all sites, dry deposition is probably of less importance as they are located in remote, forested areas and the ratio between dry and wet N deposition decreases with increased distance from sources (Fowler 1980). Further, even on a small scale dry deposition is very variable. For example, due to structural differences in the vegetation, hummocks receive higher influx of N-dry deposition than hollows (Malmer and Walle´n 2005). Thus we decided not to speculate on N-dry deposition and only use data on N-wet deposition for our models. The three selected mires differ with regard to mire type which in each case represents the dominant mire type present in each biotic region (temperate, hemiboreal, and middle boreal; Tuhkanen 1984). Following the Scandianvian mire terminology the studied lawn communities at the two southern sites are ombrotrophic bogs, while the northern site is an extremely poor fen (mostly rheotrophic) (Sjo¨rs and Gunnarsson 2002). However, from a floristic

point of view these two community types are very similar; hence it has been suggested to merge them into one unit (Wheeler and Proctor 2000). Field experiment

The small-scale field experiment was conducted at Degero¨ Stormyr (Fig. 1, Table 1). Details of the experimental set up are given in Granberg et al. (2001) and Wiedermann et al. (2007). The field experiment consisted of a factorial design involving N and sulfur (S) addition treatments, as well as a warming treatment. The field experiment started in 1995, and has three levels each of N (2, 15 and 30 kg ha1 year 1) and of S (3, 10 and 20 kg ha1 year 1) treatment applied to plots measuring 22 m. Note that the ambient deposition is 2 kg ha1 year 1 for N and 3 kg ha1 year 1 for S, respectively. Each experimental combination was replicated once, except for mid-point treatments that were replicated twice. There were two control plots. Each year, N in the form of ammonium nitrate (NH4NO3) and S in the form of sodium sulfate (Na2SO4) dissolved in 10 l of surface mire water were added each month during the growing season; application was via watering cans. Control plots received the same amount of mire water. To prevent horizontal movement of added elements, all plots were surrounded by a polyvinyl chloride frame (0.5 m deep). There were two levels of temperature (control and warmed). Warming-chambers used for the temperature treatment during the snow-free period of the year raised the mean air temperature by 3.68C, measured 0.25 m above the ground layer. Field sampling Gradient study

In the gradient study, S. balticum was sampled in June 2006. At each site we selected five 0.5 0.5 m plots. In order to allow a comparison with data from the field experiment, we stratified our sampling on the basis of the following criteria: plots should represent lawn communities and be dominated by S. balticum, and should be at least 20 m apart. For each of these plots we estimated the percentage cover separately for the bottom layer (total Sphagnum cover, cover of S. balticum and cover of bare peat) and the vascular plant layer. After vegetation recording, a sub-sample (0.2 0.2, and 0.15 m deep) of Sphagnum was collected from the center of each plot for 3-EV

the chemical analyses. These samples were placed in plastic trays and transported to the laboratory. From each of these samples we randomly picked out at least 20 capitula, which were stored in a deep freezer for about two months until analyzed. Field experiment

About 20 capitula of S. balticum were randomly sampled from across each of the experimental plots in both October 1997 and October 2004, about eight weeks after the last fertilizer application of the year. The shoots were immediately frozen and taken to the laboratory where they were stored in a deep freezer until analyzed. Scoring of the vegetation (percentage cover estimates), in 0.5 0.5 m plots, was performed in September 2004 and followed the same method as used in the gradient study.

significant factors, according to the ‘model simplification’ protocol described by Crawley (2002). For linear regression analyses of the vegetation data of the gradient study, with N wet deposition as the single explanatory variable, the software R.2.2.1 was used (ISBN 3-900051-07-0, URL Bhttp://www.R-project.org. ). Analyses of covariance (in R.2.2.1) were used to compare the slopes of regression lines from the field experiment in 1997 and 2004 and for comparing data from the field experiment in 2004 with the gradient data (Table 4). Sphagnum amino acid N concentrations were used as the response variable. The two explanatory variables were N supply as a continuous covariable and either year (Table 4a) or site (Table 4b) as categorical variables.

Results Chemical analyses

Vegetation cover

The Sphagnum samples from the gradient study and the field experiment were analyzed for concentrations of total N (NTot) and N stored as soluble amino acids (NAA). Frozen samples of the capitula were ground for 30 s using a ballmill. After drying the samples (at 608C for 24 h), NTot concentrations were determined using an elemental analyzer. Amino acids were extracted from the frozen and ground plant material using 10 mM HCl and analyzed on a HPLC capable of detecting nanomolar concentrations of 18 standard amino acids (Nordin and Gunnarsson 2000). Statistical analyses To explore data of the field experiment at Degero¨ Stormyr, multi linear regression (MLR) models (incorporating the experimentally manipulated factors N, S and Temp., as well as vascular plant cover as explanatory variables) were fitted using ‘Modde 7’ experimental design evaluation software (Umetrics, Umea˚, Sweden). To obtain the simplest significant models, model reduction was achieved by stepwise removal of non-significant factor interactions starting with the three way interactions, followed by removal of non-

In the field experiment, N addition caused a sharp decline in the amount of S. balticum, from an average cover of 89% in the non-N treated plots to 14% under the high N treatment (Fig. 2), leaving up to 42% of bare peat and vascular plant litter (data not shown; cf. also Plate 1 in Wiedermann et al. 2007). MLR analyses testing the effects of the manipulated factors and the effect of vascular plant cover in the field experiment revealed that N remained the only factor explaining the observed S. balticum decline (Table 2). Similarly total Sphagnum cover decreased in response to N addition, whereas total vascular plant cover showed the opposite response (Table 2, Fig. 2). However, the MLR analyses also revealed a negative effect of enhanced temperature on total Sphagnum cover and a positive effect on total vascular plant cover (Table 2, Fig. 2). In the gradient study, we found a significant negative response to increased N deposition for both total Sphagnum cover and S. balticum cover (Table 2, Fig. 2). Total vascular plant cover responded positively to enhanced N supply along the N deposition gradient (Table 2, Fig. 2).

Percent cover

Experiment

Gradient

100

100

80

80

60

60

40

40

20

20 0

0 2 kg

15 kg

30 kg

2 kg

8 kg

12 kg

Figure 2. Percentage cover (mean and SE) of total Sphagnum (dark grey), Sphagnum balticum (light grey), and vascular plants (white), in the field experiment (in 2004, after nine years of experimental treatment) and the gradient study. Data from 0.5 0.5 m plots. For the field experiment, n8, 4 and 8 for plots without N addition, 15 kg N and 30 kg N ha 1 year 1, respectively. For the gradient study, n5 for all sites. For species included among vascular plants see description of study sites in Material and methods.

4-EV

Table 2. Statistical results from (multiple) regression analyses using data on vegetation cover from the field experiment and the gradient study, as response variables. Note that the analysis of the field experiment is based on the full, factorial design. Statistical differences are indicated by: ‘***’ p B0.001; ‘**’ pB0.01; ‘*’pB0.05. DF

F

Model signif.

R2

Incl. model factors

Field experiment total Sphagnum Sphagnum balticum Total vascular plants

F2,17 F1,18 F2,17

43.9 93.3 7.6

p B0.001 p B0.001 p 0.004

0.84 0.84 0.47

N*** T* N*** N** T*

Gradient study total Sphagnum Sphagnum balticum total vascular plants

F1,13 F1,13 F1,13

7.3 30.3 20.3

p 0.017 p B0.001 p 0.001

0.36 0.70 0.61

N* N*** N***

Sphagnum chemistry Both NTot (Table 3) and NAA (Fig. 3) tissue concentrations increased with increasing N deposition, both in the field experiment and in the gradient study. Results from the linear regression analyses demonstrated significant and positive relationships between N deposition and N accumulation in soluble amino acids (NAA) in S. balticum tissue, both for the field experiment from 1997 and 2004 and in the gradient study (Fig. 3). The slopes of the regression lines for data from the field experiment in 2004 [slope 0.085 (F1,19 89.3; pB0.001; R2 0.82)] and the gradient study [slope 0.087 (F1,13 152.6; pB 0.001; R2 92)] are close to identical (not significantly different; Table 4). The slopes of the regression lines for the field-experimental data in 1997 [slope 0.054 (F1,19  45.2; p B0.001; R2 0.70)] and in 2004 were significantly different (Table 4).

Discussion The major results of this study are the analogous patterns shown by the small-scale field experiment and the gradient study. In both data sets we found linear relationships between N deposition and Sphagnum NAA tissue content, as well as similar trends towards a decreased Sphagnum cover and an increased cover of vascular plants. We have also demonstrated that the physiological responses of S. balticum were analogous between the small-scale field experiment, that experienced increased N deposition over a nine-year period, and the gradient study where increased N deposition has occurred over a much longer period (Galloway and Cowling 2002, Bergstro¨ m et al. 2005), although with lower Table 3. Total tissue N for S. balticum specimens originating from the field experiment after nine years of N addition, and the gradient study including three peatland sites differing in levels of N deposition. Site Field experiment Gradient study

N wet-deposition kg ha 1 year1 2 15 30 2 8 12

Total tissue N (mg g 1 DW) 4.490.2 9.190.6 12.890.8 5.590.2 9.490.2 10.990.5

annual doses (Table 1). This suggests that the chronic lowlevel N deposition in southern Sweden, which has increased by about 30
50% since the 1950s (Malmer and Walle´n 2005), has resulted in physiological responses similar to those found in the more drastic field experiment. This study and earlier data from our field experiment underline the importance that experimental manipulations have been run for a sufficiently long time period. For our field experiment this is evidenced by observed time lags in methane emissions (Granberg et al. 2001), and vegetation responses (Wiedermann et al. 2007). Here, we also demonstrate that physiological responses have an associated time lag, as the slopes for the linear relationships between N deposition and Sphagnum NAA tissue content in 1997 and 2004 were significantly different (Table 4), at a time when no vegetation change had been detected. Later, in 2003 after eight years of N application, the Sphagnum carpet had all but disappeared under the high N treatment (Wiedermann et al. 2007; see also Fig. 2 for data from 2004). The following year, 2004, the first signs of a Sphagnum decline were also observed in the mid N treatment (Fig. 2). Interestingly, the elevated NAA levels in 1997, at a time when no vegetation change had been detected, show that NAA can be used as a sensitive indicator of incipient ecosystem change (Tomassen et al. 2003). 4

NAA in mg g-1 DW

Variable

3

2

1

0

5

10

15

20

25

30

35

N deposition in kg ha-1 yr-1 Figure 3. The relationship between soluble amino acid N tissue content (mg g 1 DW) and N deposition (kg N ha 1 year 1) for S. balticum samples from the gradient study (filled triangles, dashed line), and from the field experiment in 1997 (open circles, thin line) and 2004 (filled circles, solid line).

5-EV

Table 4. Results from ANCOVAs testing the effect of N addition on soluble amino acid N tissue concentration in Sphagnum balticum: (a) for the field experiment, comparing differences in N response between the years 1997 and 2004; model values: [F3,38 47.8, R2  0.79, pB0.001]; and (b) for the field experiment in 2004 and the gradient study; model values: [F3,32 62.1, R2 0.85, p B0.001]. Source

Df

F

p-values

(a) N years (1997/2004) Nyears

1 1 1

132.80 3.93 6.51

pB0.001 p0.055 p0.015

(b) N Site (experiment/gradient) Nsite

1 1 1

186.10 0.28 0.00

pB0.001 p0.604 p0.966

The earlier response of Sphagnum NAA tissue content, compared with any observed Sphagnum decline, or increase in vascular plant cover, also points towards the key role of Sphagnum mosses for the vegetation change in response to N. The mechanisms of N induced Sphagnum decline have been extensively debated during recent years and include both direct and indirect factors, among the former mainly an unbalanced nutrient accumulation (Jauhiainen et al. 1998, Gunnarsson and Rydin 2000, Gerdold et al. 2007) causing a shift in primary element limitation from N to phosphorus (Malmer et al. 2003, Bragazza et al. 2004, Malmer and Walle´n 2005, Gerdold et al. 2007) or carbon (Baxter et al. 1992). Carbon limitation may also involve a possible decrease in the concentration of secondary metabolites (Ha¨ttenschwiler and Vitousek 2000), which may affect defense towards natural enemies (Limpens et al. 2003) and enhance decomposition processes (Scheffer et al. 2001). Enhanced light competition as a result of increased growth of vascular plants should also be mentioned among suggested indirect factors (Heijmans et al. 2002). As with the data on physiological responses, our data on vegetation change indicated that the field experiment and gradient study followed the same trends with respect to N. Utilizing results from the factorial designed field experiment allowed us to extract N as the only important factor inducing NAA accumulation, and by far the most important factor inducing Sphagnum decline and increase in vascular plant cover. However, the gradient study was also characterized by marked differences in other abiotic parameters such as temperature, precipitation, and discharge (Table 1), which raises the question whether the experimental data can be extrapolated in space. In the experiment, increased temperature had a similar, though weaker, effect on total Sphagnum cover and vascular plant cover as did increased N. This was likely attributable to drier conditions in the high temperature experimental plots, giving an advantage to vascular plants over Sphagna. In general, we would expect increasing precipitation to favor growth of Sphagnum mosses (Hajek and Beckett 2008, Heijmanns et al. 2008), and in fact during a series of consecutive wetter than average years (1999
2004), Gunnarsson and Flodin (2007) documented an increase of Sphagnum on ombrotrophic bogs in areas with high N deposition including our HD site. The southward increase in humidity in our study would thus rather suggest an increase in Sphagnum cover than the observed decline at the HD site compared to the other two

6-EV

sites with less N deposition, which strengthens the view that N plays an important role for the observed pattern. In addition, the observed trends in our study of a Sphagnum decline along with a vascular plant cover increase are in accordance with the results of a detailed re-inventory of our mid N deposition site (MD), A˚ khultmyren. The vegetation changes between 1954 and 1997, after 40 years of continuous enhanced N deposition, mirrored both a change to a drier mire surface, increased N availability, and a decline in Sphagnum cover (Gunnarsson et al. 2002, Malmer and Walle´n 2004). A similar increase in the cover of nutrient demanding vascular plants has also been documented for ombrotrophic bogs in the area of our high N deposition site (HD) (Gunnarsson and Flodin 2007). Most terrestrial ecosystems are characterized by a multitude of reticulate food webs, where top
down and bottom
up effects will vary depending upon the experimental scale. Although Sphagnum-dominated systems may not differ with regard to organizational complexity, it is striking to see that the importance of trophic interactions are virtually absent in modern text books (Charman 2002, Rydin and Jeglum 2006). Furthermore, in contrast to the immediate responses observed following experimental N additions in other N limited ecosystems (Strengbom et al. 2005, 2006), the long time delay in the present field experiment is striking. We attribute this to the key role of the Sphagnum species, which monopolize acquisition of the naturally limited nutrients (Malmer et al. 2003). This may reflect the fact that in these N-limited ecosystems such interactions will not start to affect species composition and community structure until N deposition starts to exceed Sphagnum uptake (Limpens et al. 2003, Wiedermann et al. 2007). As long as N deposition does not exceed uptake by Sphagnum, the genus will maintain its role as the ecosystem engineer (Lawton and Jones 1995). Thus, not until N deposition exceeds Sphagnum uptake will there be a gradual vegetation shift. In areas with moderately elevated N levels, this vegetation shift will be a slow and gradual process, difficult to detect without detailed retrospective data (Gunnarsson et al. 2002). From this perspective, our finding that NAA concentration functions as a sensitive indicator of a future regime shift is valuable, since this measure could be used in monitoring programs as an early warning system.

Acknowledgements
We are grateful for economic support from Stiftelsen Anna och Gunnar Vidfeldts fond (to MMW) and FORMAS (to LE, AN and MN, respectively), which made this study possible. We also thank Margareta Zetherstro¨ m for assistance with amino acid analyses.

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