1Litter decomposition can be limited by nitrogen or phosphorus, but conditions under which either nutrient is limiting remain uncertain. We investigated whether this depends on nutrient limitation during plant growth, on N : P ratios of the litter, or on activities of C-, N- and P-mineralizing enzymes.
2Nine herbaceous species were grown at N : P supply ratios (N : Ps) of 5–160 (mass-based). Fresh leaf litter was incubated in the laboratory for 5–10 weeks, partly with N or P fertilization, to determine mass loss and activities of extracellular enzymes.
3Both plant growth and litter decomposition were N-limited for plants grown at N : Ps < 20, and P-limited for those grown at N : Ps ≥ 40. Litter N : P ratios varied widely among species and did not predict which nutrient limited decomposition, except that litter with N : P > 22 always had P-limited decomposition.
4The activities of β-glucosidase, chitobiase and phosphatase differed widely among species but were poorly related to litter nutrient concentrations and mass loss. Within some species, phosphatase activity increased towards high litter N : P ratios, suggesting P limitation of decomposers.
5We conclude that there is no unique critical N : P ratio discriminating between N- and P-limited decomposition because this critical N : P ratio is species-dependent and may also depend on the physical conditions under which plants were grown.
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Changes in the type of nutrient limitation may have implications for litter quality and decomposition: if litter produced by P-limited plants has a wide N : P ratio, its decomposition is likely to be P-limited (Smith 2002). However, while P-limited decomposition has often been found in tropical wetlands (Qualls & Richardson 2000; Rejmánková 2001), fertilization experiments in northern wetlands have usually failed to reveal P limitation for decomposition (Aerts et al. 1995; Aerts et al. 2001; Aerts, de Caluwe & Beltman 2003). There may be two reasons for this: either decomposition is P-limited only for litter with an extremely high N : P ratio; or the type of nutrient limitation is not directly related to litter N : P ratios. For example, the decomposition of litter types with similar N : P ratio but differing structure or carbon quality can respond differently to nutrient addition because microbes growing on these litters have contrasting energy and nutrient requirements (Bosatta & Berendse 1984; Ågren, Bosatta & Magill 2001). Until now, however, no experiment has directly tested for such differences.
Inconsistent relationships between litter N : P ratios and nutrient limitation of litter decomposition might further result from the fact that nutrient limitation can be assessed in various ways, each of which has some drawbacks (Hobbie 2000; Bridgham & Richardson 2003). A frequent approach consists in comparing the decomposition of litter with differing chemical composition; an element is considered to be limiting if its concentration correlates positively with decomposition rate (Aerts 1997; Aerts et al. 2001). The drawback is that nutrient concentrations often correlate with each other and with the C chemistry of the litter, so that their effects on decomposition are confounded (Vitousek 1998). Alternatively, the litter or its environment can be fertilized with mineral nutrients, which allows us to vary the availability of individual elements separately (Robinson & Gessner 2000; Rejmánková 2001). However, osmotic effects or changes in pH due to fertilization can inhibit decomposers (Bolan, Currie & Baskaran 1996).
As a further possible approach, assays of enzyme activity may indicate the nutrient demands of microbial decomposers (Sinsabaugh et al. 1993; Sinsabaugh & Moorhead 1994), as microbes seem to regulate their production of C-, N- and P-mineralizing enzymes, respectively, to balance their acquisition of these elements (Olander & Vitousek 2000). The enzyme approach is attractive in that it directly addresses the responses of decomposers to the quality of their resource. In previous studies, activities of N- and P-mineralizing enzymes have been related to N and P availability in water or soil (Sinsabaugh et al. 1993; Sinsabaugh & Moorhead 1994; Olander & Vitousek 2000). It has not yet been tested whether there is a correspondence between activities of N- and P-mineralizing enzymes on fresh litter and the litter N : P ratio or the response of its decomposition to nutrient addition.
We investigated the relationships between nutrient limitation during plant growth, litter N : P ratios, and the nutrient limitation of litter decomposition in order to test three main hypotheses: (1) N or P limitation of plant growth is reflected by litter N : P ratios; (2) litter N : P ratios determine whether decomposition is N- or P-limited; (3) the activities of C-, N- and P-mineralizing enzymes on decomposing litter reflect litter N : P ratios and/or the nutrient limitation of its decomposition. Our experiment included nine herbaceous wetland species to examine whether the hypothesized relationships are consistent across species. Assuming that our hypotheses would be confirmed, our aim was to determine the ‘critical N : P ratio’ discriminating between N and P limitation of litter decomposition.
Nine wetland plant species were included in the experiment (nomenclature: Lauber & Wagner 1996): five grass species (Agrostis canina, Anthoxanthum odoratum, Alopecurus pratensis, Molinia caerulea, Phalaris arundinacea), two sedge species (Carex flava, Carex panicea) and two forb species (Centaurea angustifolia, Mentha aquatica). Further forb species were included initially but did not survive. In addition to representing three functional groups, the nine species were also selected because their typical habitats range from nutrient-rich, N-limited sites (A. pratensis, P. arundinacea) to nutrient-poor, P-limited sites (M. caerulea, C. flava, C. panicea). Plants were grown in spring 2003 from seeds (A. canina, A. odoratum, A. pratensis, C. angustifolia) or cuttings (M. aquatica, P. arundinacea, C. flava, C. panicea, M. caerulea) collected in wet areas near Zurich. When seedlings had two to three leaves or when cuttings started to form new roots, they were planted in 0·4 l pots (1 l pots for the tall species P. arundinacea) filled with quartz sand (0·1–0·7 mm; Carlo Bernasconi, Zürich) and placed on trays in a garden in Zurich at 450 m a.s.l.
Nutrient treatments were applied weekly from May until August 2003 and fortnightly in September 2003. There were 12 treatments, which combined six N : P supply ratios (N : Ps) with two supply levels in a factorial design. Treatments were defined by the total amounts of N and P (mg) supplied per plant during the growing season, which were calculated as:
with L, the supply level (geometric mean of N and P supply) being either 18 mg (high supply) or 6 mg (low supply), and N : Ps, the mass ratio of N to P supplied, forming a geometric series from 5 to 160. There were six replicates per species and treatment at high supply, and four replicates at low supply. Nitrogen was supplied as solutions of KNO3, and P as solutions of KH2PO4; KCl was added as required so that all plants received at least 250 mg K; the maximal K supply was 632 mg (as KNO3, at high supply with N : P = 160). Other essential nutrients (Ca, Mg, Fe, S, microelements) were supplied every 3 weeks to provide non-limiting amounts. Plants were watered daily or as required to keep the sand moist.
All dead leaves were removed a the end of June. Subsequently, freshly senesced leaves were collected every 2 or 3 weeks and stored air-dry in paper envelopes. As the amount of litter produced by individual plants was often small, the litter from replicate plants was pooled for the decomposition experiments, yielding 108 litter types (nine species × 12 treatments). The air-dry material was cut in pieces ≈ 0·5 cm long to obtain homogeneous samples for incubations; the length of pieces corresponded to the (unchanged) width of leaves (mostly 0·2–0·5 cm).
We harvested two replicates of each plant grown at high supply in October 2003 to determine their total biomass and nutrient concentrations of the above-ground living tissues (leaves and culms). The other plants were kept for further measurements in a second growing season. Agrostis canina was removed from the experiment during the summer because its stolons grew into neighbouring pots while its tiny leaves were difficult to harvest. The litter already collected was still used for enzyme assays.
For nutrient analyses, a 150 mg subsample of each litter type was dried at 75 °C for 24 h to determine the mean water content of the air-dry material (6%). This subsample was then digested with a modified Kjeldahl procedure (1 h digestion at 420 °C with 98% H2SO4 and a copper sulphate-titanium oxide catalyst). Concentrations of N and P in digests were determined colorimetrically on a flow-injection analyser (FIA, Tecator, Höganäs, Sweden). The same procedure was used for living tissues.
Litter was incubated in Petri dishes (6 cm diameter) on 18 g quartz sand covered with a disk of polyethylene mesh (300 µm). Eleven Petri dishes, each containing 150 mg air-dry litter, were prepared with each litter type if enough material was available. Two Petri dishes served for enzyme assays (see below), and nine to determine the initial rate of mass loss as well as its response to N or P addition (three treatments with three replicates). If the amount of material was insufficient, we prepared as many Petri dishes as possible and attributed them to incubations in the following sequence: (1,2) enzyme assays; (3) mass loss without nutrient addition; (4,5) mass loss with addition of N or P; (6,7) replicated mass loss without nutrient addition; (8–11) replicated mass loss with nutrient addition. For example, if seven Petri dishes could be prepared with a litter type, two were used for enzyme assays, three for mass loss without nutrient addition, and two for mass loss with addition of N or P, respectively. Enzyme assays were carried out with all litter types (no missing data). Mass loss could be determined with 45 of the 54 litter types from plants grown at the high supply level; only these were included in the data analysis as there were few data from plants grown at the low supply level.
The litter in Petri dishes was wetted with 8 ml of a microbial inoculum prepared by mixing 1 kg fresh topsoil from a wetland where seven of the species co-occurred with 3 l of deionized water. After 12 h with repeated stirring, the slurry was filtered (coarse filter paper, LS171/2, Schleicher & Schuell, Dassel, Germany), and the filtrate (pH 7) was used to inoculate the samples. Nutrient amendment consisted of either 8 mg N (as NH4NO3) or 1·5 mg P (as KH2PO4) per Petri dish; these nutrients were dissolved in the soil filtrate before wetting the litter. To ensure equal K supply in all treatments, 1·5 mg K as KCl was added to the control and +N treatments. Control incubations with cellulose as substrate confirmed that a good microbial inoculation was achieved with the filtrate (rapid decomposition when N + P were added), while the nutrient concentrations in the filtrate were negligible (almost no decomposition without nutrient addition, or when N or P was added alone).
Petri dishes were incubated in boxes at 22 °C. The litter was sprinkled with deionized water every 2 weeks to replace the evaporated moisture and to simulate leaching by rainfall. After 5 weeks (half the period over which mass loss was determined), the litter samples intended for microbial assays were removed from the Petri dishes with tweezers and kept in aluminium foil at 5 °C. After 10 weeks the litter was removed from the other Petri dishes and dried for 24 h at 70 °C to determine the remaining dry mass and thus percentage mass loss. The artificial conditions meant that mass loss was a measure of litter decomposability rather than actual decomposition; they were chosen here because we needed a nutrient-free control treatment and small error variation to be able to assess nutrient limitation for decomposition.
microbial respiration and enzyme assays
Microbial respiration and enzyme activities were assayed within 6 days of removing the litter from Petri dishes. To measure microbial respiration, litter samples were placed in 10 ml glass vials in darkness at 20 °C. After 24 h a 5 ml gas sample was drawn, and its CO2 concentration was analysed on a gas chromatograph (AMS Model 92, Analytical Measuring Systems, Cambridge, UK). The CO2 concentration of the gas was converted into CO2 production per initial g litter dry mass and hour of incubation. Using mass loss data (if available) we also estimated CO2 production per g litter present at the time of the measurement, but do not report these values here as they showed virtually the same species and treatment effects.
Enzyme activities were determined following Sinsabaugh et al. (1991); Kang & Freeman (1999); Kourtev et al. (2002). After respiration measurements, the litter was mixed for 60 s with 10 ml deionized water in a laboratory blender (Stomacher Type 80, Seward, UK). No buffer was used because species- or treatment-related differences in pH and their possible influence on enzyme activities were of interest here. Immediately after mixing, six 1 ml aliquots of the extracts (without large litter pieces) were transferred to six Eppendorf reaction vials. Three enzymes were assayed in duplicate by adding 0·5 ml of the appropriate substrate solutions to each of two vials. Substrate solutions were 400 µm 4-methylumbelliferyl (MUF)-β-d-glucoside (Sigma M-3633) for the activity of β-glucosidase; 400 µm 4-MUF-N-acetyl-β-d-glucosaminide (Sigma M-2133) for the activity of chitobiase; and 200 µm 4-MUF-phosphate (Sigma M-8883) for the activity of acidic and alkaline phosphatases (Sigma-Aldrich Chemie, Steinheim, Germany); each substrate was first dissolved in 20 ml cellosolve (2-ethoxyethanol) and then diluted with water to obtain a final cellosolve concentration of 2%. After 2 h incubation at 20 °C, the vials were centrifuged at 10 000 r.p.m. for 4 min, and the fluorescence of the supernatant was measured immediately on a microplate reader (FLUOstar Galaxy, BMG Labtechnologies, Offenburg, Germany) at 460 nm emission and 355 nm excitation wavelength. For the calibration curve, 0·5 ml extract from each litter sample in the current assay (24 samples per batch) were pooled and centrifuged for 4 min at 10 000 r.p.m. The supernatant was used to dilute a stock solution of 1000 µm 4-methylumbelliferone in 2% cellosolve to six concentrations between 0 and 80 µm, so that the calibration curve would account for possible interactions between MUF and other compounds in the litter extracts. The incubation period and substrate concentrations were based on preliminary assays; control assays with enzyme substrates added to deionized water showed no fluorescence. Enzyme activities were expressed as µmol of substrate converted per h and per g initial dry mass of litter. The pH of all litter extracts was measured with a glass electrode (Model 720A, Orion Research, Boston, MA, USA).
Most variables were log-transformed before statistical analysis to obtain normally distributed error terms with homogeneous variance (plant biomass, pH and percentage mass loss were analysed untransformed). The effects of plant species, N : P supply ratios and supply level on microbial activity after 5 weeks’ decomposition were analysed with three-way fixed-factor anova. To obtain a balanced design, means of the two replicates were used in data analysis, and the three-way interaction was used as error term. For response variables that were determined only at the high supply level (plant biomass, litter mass loss), two-way anova (factors species and N : P supply ratios) was used. As some species–treatment combinations were missing for mass loss after 10 weeks, the interaction term was removed; the analysis used type III sums of squares, which account for an unbalanced design.
To determine if decomposition was limited by N or P, we calculated for each litter type the difference between the mean dry mass loss with N or P addition and the mean dry mass loss without nutrient addition. For some litter types differences could not be tested statistically as there were only one or two replicates per treatment. We therefore regarded as significant a difference in mass loss of 5%, which corresponds to the least significant difference for litter types that had two or three replicates per treatment (Dunnett test; α = 0·10). This threshold was a compromise between power (ability to detect nutrient limitation when it existed) and significance (avoid inferring nutrient limitation from random differences), as both were equally important to assess if nutrient limitation was related to N : P ratios.
Correlations among enzyme activities, microbial respiration and pH were quantified with Pearson's correlation coefficients (r) in three ways: (1) overall correlations based on individual samples; (2) interspecific correlations based on species means across treatments; (3) intraspecific correlations based on individual samples after adjusting for species means. Correlations between microbial activity after 5 weeks and mass loss after 10 weeks or initial litter chemistry were calculated in the same way, but based on means per species and treatment, as only these could be matched. All analyses were carried out with the statistical package jmp ver. 3·2·2 (SAS Institute, Cary, NC, USA).
nutrient limitation of plant growth and litter decomposition
Plant biomass at the end of the growing season showed a unimodal dependence on N : Ps, with maximal biomass at N : Ps = 10 (A. odoratum), 20 (A. pratensis, P. arundinacea, C. flava, C. panicea, C. angustifolia, M. aquatica) or 40 (M. caerulea; Fig. 1). The N concentration of green leaves and litter produced during the growing season varied considerably among species (Fig. 2a,b); it was also positively related to the N : P supply ratio, with two- to threefold differences between N : Ps = 5 and N : Ps = 160. The N concentration of litter was generally 50–70% lower than that of green leaves (Fig. 2a,b). The P concentration of green leaves also varied strongly among species at low N : Ps, but at N : Ps ≥ 40 interspecific variation was small (Fig. 2c). Within species the P concentration was minimal at intermediate N : Ps (Fig. 2c). The P concentration of litter was similar to that of green leaves and varied remarkably little among species or treatments (Fig. 2d).
Plant litter lost, on average, 29·7% of its initial mass during 10 weeks of decomposition without nutrient addition (Fig. 3a); mass loss differed among species (F = 21·6, P < 0·001) but not among N : Ps (F = 1·1, P > 0·05). Nitrogen addition during the incubation slightly increased the mass loss of litter produced at N : Ps = 5 but had no effect on the decomposition rate of the other litter types (Fig. 3b). Phosphorus addition strongly increased the mass loss of litter produced at N : Ps = 80 and 160. When P was added, there was a positive relationship between N : Ps during plant growth and litter mass loss, causing high-N : Ps ratio litter with added P to decompose faster than low-N : Ps ratio litter with added N (Fig. 3b).
The effects of N or P fertilization on litter mass loss could be evaluated for 32 individual litter types (Fig. 4). In 20 cases, N or P addition (or both) increased litter mass loss by > 5% of initial mass. The type of nutrient limitation was consistently related to N : Ps: N was always limiting with litter produced at N : Ps = 5–10, and P was always limiting with litter produced at N : Ps = 40–160 (Fig. 4). Litter with N : P ratio > 22 always had P-limited decomposition, whereas litter with N : P ratio < 22 could show any type of response to nutrient addition (Fig. 4).
respiration and enzyme activities during decomposition
Microbial respiration on the litter after 5 weeks’ decomposition correlated positively with mass loss after 10 weeks (overall r = 0·43, P < 0·001, n = 45). This correlation was primarily determined by similar responses of both variables to N : Ps during plant growth (intraspecific r = 0·59, P < 0·001), whereas interspecific differences in the two variables were unrelated (interspecific r = 0·29, P > 0·05). Respiration was generally greatest with grasses and smallest with forbs (Tables 1 and 2). Nutrient treatments during plant growth had a small influence on microbial respiration (Fig. 5a; Table 1).
Table 1. anova results for effects of plant species and nutrient treatments during plant growth (N : P ratio and overall level of supply) on microbial respiration after 5 weeks’ incubation at 22 °C, activities of three extracellular enzymes (β-glucosidase, chitobioase, phosphatase), and pH of litter extracts used for enzyme assays
Percentage of total variation explained by each factor based on effects sums of squares.
Significance levels: ***P < 0·001; **P < 0·01; *P < 0·05; no symbol, P > 0·05.
Table 2. Microbial activity on decomposing litter of nine species after five weeks’ incubation at 22 °C: microbial respiration (volume of CO2 produced in 24 h per g initial litter), activities of β-glucosidase, chitobiase and phosphatase (enzyme substrate converted in 2 h per g initial litter mass), and pH of litter extracts used for enzyme assays
Data are means and SE across 12 treatments; for respiration and enzymes statistics were calculated from log-transformed data; back-transformed means and positive SE are given.
1·46 + 0·12
4·6 + 0·3
3·3 + 0·2
1·8 + 0·2
5·8 ± 0·04
1·82 + 0·14
4·8 + 0·3
4·0 + 0·3
1·4 + 0·1
5·7 ± 0·03
0·54 + 0·05
5·4 + 0·5
5·1 + 0·5
1·7 + 0·2
5·5 ± 0·09
1·22 + 0·10
1·4 + 0·1
1·3 + 0·1
0·7 + 0·1
5·7 ± 0·03
0·95 + 0·08
2·8 + 0·2
1·7 + 0·1
1·1 + 0·1
5·7 ± 0·04
1·48 + 0·15
1·0 + 0·1
0·5 + 0·0
0·6 + 0·1
6·4 ± 0·04
0·73 + 0·06
1·4 + 0·1
1·1 + 0·1
0·6 + 0·1
6·0 ± 0·03
0·79 + 0·06
2·3 + 0·2
1·9 + 0·1
1·1 + 0·1
5·7 ± 0·05
1·32 + 0·10
2·8 + 0·2
1·8 + 0·1
1·5 + 0·1
5·9 ± 0·04
Enzyme activities in litter extracts differed among plant species and partly among nutrient treatments during plant growth (Tables 1 and 2). Most of the variation was among species (33·8–69·8% of total), enzyme activities being greatest on grass litter and smallest on forb litter (Table 2). The activities of β-glucosidase and chitobiase varied little in relation to N : Ps (Fig. 5b), whereas phosphatase activity increased towards high N : Ps (Fig. 5b). Activities of the three enzymes correlated positively with each other and with microbial respiration across the entire data set and among the nine species; correlations with the pH were weaker and negative (Table 3). Within species, the activity of β-glucosidase correlated positively with that of chitobiase (r = 0·68, P < 0·001) and phosphatase (r = 0·60, P < 0·001); no other intraspecific correlation was significant (not shown).
Table 3. Correlations among microbial respiration, enzyme activities during decomposition (β-glucosidase, chitobiase, phosphatase), and pH of litter extracts calculated across the entire data set (upper part of table) and among species means (lower part of table)
Data are Pearson's correlation coefficients and pairwise significance levels calculated from log-transformed data.
Significance levels: ***P < 0·001; **P < 0·01; *P < 0·05; no symbol, P > 0·05.
Enzyme activities correlated only weakly with nutrient concentrations of the litter and litter mass loss; all significant correlations were positive (Table 4). Relationships with N and P depended on the type of comparison: among species, enzyme activities were mainly correlated with the P concentration of the litter (although not significant due to small n), whereas within species (among treatments), enzyme activities correlated with the N concentration and N : P ratio of the litter; these correlations were strongest for phosphatase activity (Table 4).
Table 4. Correlations among enzyme activities (β-glucosidase, chitobiase, phosphatase), litter nutrient concentrations, and litter N : P ratios calculated across individual litter types; among species means; and across treatments within species
Across litter types (n = 45)
Among species (n = 8–9)
Within species (n = 45)
Data are Pearson's correlation coefficients and pairwise significance levels calculated from log-transformed data.
Significance levels: ***P < 0·001; **P < 0·01; *P < 0·05; no symbol, P > 0·05.
nutrient limitation of plant growth poorly reflected by litter n : p ratios
The wetland plants used to produce litter for our decomposition experiments were grown at a broad range of N : P supply ratios (N : Ps = 5–160). Our particular design of nutrient treatments suggested that across the range of N : Ps, maximal biomass would be produced by plants that were equally limited by N and P (Güsewell 2005c). Therefore relationships between plant biomass and N : Ps (Fig. 1) suggest that plant growth was generally N-limited at N : Ps = 5 and 10; either P-limited or colimited by N and P at N : Ps = 20 and 40; and P-limited at N : Ps = 80 and 160.
Our first hypothesis was that N vs P limitation of plant growth would be reflected by the N : P ratios of green leaves and litter. These N : P ratios did increase across the range of N : Ps, but they also varied considerably among species (Fig. 4). At N : Ps = 20, when most species were colimited by N and P, their green leaf N : P ratios ranged from 18 to 40, which is much higher than biomass N : P ratios of colimited plants in other studies (Koerselman & Meuleman 1996; Tessier & Raynal 2003). Litter N : P ratios at N : Ps = 20 ranged from 5 to 22, which is unusually low for colimited plants. Together with the large interspecific variation, this means that litter N : P ratios did not consistently reflect the nutrient limitation of plant growth.
Nutrient concentrations of plant material in this experiment differed considerably from previous experiments with similar nutrient treatments (Güsewell 2005a; S.G., unpublished data). The N concentration of green leaves was generally higher in 2003 than in previous years (Fig. 6a). In contrast, the N concentration of litter was similar in all years (Fig. 6b). The small variation in P concentration found in 2003 differs markedly from previous years, when P concentrations decreased strongly towards high N : Ps (Fig. 6c,d). The average litter P concentrations at high N : Ps in 2003 were nearly 10-fold those found in 1999–2001 (Fig. 6d). The N : P ratios of green leaves and litter varied comparatively little among treatments in 2003 (Fig. 6e,f), and litter N : P ratios of P-limited plants (high N : Ps) were much lower in 2003 than in 1999–2001 (Fig. 6f). Because P concentrations differed little among treatments, variation in N : P ratios was largely determined by N concentrations. This is atypical for herbaceous species, whose N : P ratios are usually mainly determined by P concentrations (Güsewell 2004).
The absence of difference in P concentration between green leaves and litter for most plants grown at N : P ≥ 10 indicates that little P was resorbed during senescence, as any P resorption must have been compensated by a decrease in leaf mass during senescence, which represents less than 30% of initial mass in wetland plants (Güsewell 2005b). This contrasts again with the 80–90% P resorption found in earlier experiments for plants grown at N : Ps ≥ 5 (Güsewell 2005a). Efficient P resorption is typical of graminoids growing in P-poor fens or bogs (Aerts, Verhoeven & Whigham 1999; El-Kahloun et al. 2000; Van Heerwaarden, Toet & Aerts 2003), and P is normally resorbed more efficiently than N in herbaceous wetland plants (Aerts et al. 1999). The opposite was found here: unlike P, the N concentration was 50–70% lower in litter than in green leaves, suggesting considerable N resorption. Phosphorus resorption can be inhibited specifically by unusually high P supply or by micronutrient deficiency (Marschner, Kirkby & Cakmak 1996). If either of these factors had played a role in our experiment, the P concentration of green leaves and litter should have increased towards low N : P supply ratios (Teng & Timmer 1990). The absence of such a pattern argues against a role of P excess or micronutrient deficiency in preventing P resorption.
While nutrient treatments were similar for all experiments compared in Fig. 6, exposure of plants to sunlight differed. In 1999–2001 plants were grown in an open greenhouse with 40–50% daylight, whereas in 2003 they were grown outdoors in full light. This might explain the contrasting nutrient concentrations. Strong sunlight reduces plant growth because of photoinhibition, to which nutrient-deficient plants are particularly sensitive (Marschner et al. 1996; Steyn et al. 2002). Both strong P deficiency and photoinhibition reduce the utilization of starch and the export of assimilates from leaves, increasing concentrations of starch or sugars (Qiu & Israel 1992). Reduced export of assimilates can affect nutrient resorption during senescence (Feild, Lee & Holbroock 2001), leading to greater P concentrations in litter. Some P-deficient plants convert excess starch into amino acids, thereby reducing the accumulation of sugars (Nanamori et al. 2004). Whether this could explain the relatively efficient N resorption found here remains an open question. Our tentative explanation suggests that specific effects of growth conditions on N and P resorption may contribute to inconsistent relationships between nutrient limitation of plant growth and litter N : P ratios.
nutrient limitation of decomposition inconsistently related to litter n : p ratios
Litter decomposition was accelerated by the addition of mineral N or P in two-thirds of the cases for which this could be tested (Fig. 4). For 12 litter types, the separate addition of N or P did not accelerate decomposition. This mostly concerned litter produced at N : Ps = 20 or 40, when plant growth was (nearly) colimited by N and P. The decomposition of this litter was possibly also colimited by N and P, meaning that it would have been accelerated by the joint addition of N and P. This treatment was not included in our experiment for lack of material.
Which element limited litter decomposition depended more consistently on N : P supply ratios during plant growth than on litter N : P ratios. Litter with N : P > 22 always had P-limited decomposition, but litter with lower N : P ratios could show any type of response to nutrient addition. For example, litter from P-limited M. caerulea (N : Ps = 40) had a low internal N : P ratio (9), but its decomposition was nevertheless P-limited (Fig. 4). This contrasts with a study using wetland plant litter collected in the field, in which decomposition was P-limited only if the litter N : P ratio exceeded 25 (S.G., unpublished data). Our second hypothesis, that litter N : P ratios would determine whether decomposition is N- or P-limited, was therefore only partly confirmed. We could not establish a critical litter N : P ratio discriminating between N- and P-limited decomposition, as this threshold appeared to differ for each species in this experiment. Furthermore, the fact that the species-specific critical N : P ratios were lower in this experiment than for litter collected in the field suggests that growth conditions can also influence the critical N : P ratio, although the nature of this influence remains to be determined.
The strong effect of P addition on the decomposition of litter from plants grown at N : P supply ≥ 40 (Fig. 3b) and the fast decomposition of these litter types after P addition (40–50% mass loss in 10 weeks) suggest a high proportion of labile C sources. Labile compounds are mainly consumed by fast-growing microbes with high P requirements, growth of which may have been stimulated by P addition even on relatively P-rich litter (Henriksen & Breland 1999; Findlay et al. 2002). In an earlier experiment with plant material from an alpine pasture (Güsewell, Jewell & Edwards 2005), P fertilization accelerated the decomposition of a P-rich substrate (3 mg P g−1, N : P = 10·6) with 33% water-soluble (labile) material, but not the decomposition of a P-poor substrate (0·6 mg P g−1, N : P = 15·2) of which only 9% was water-soluble (Güsewell et al. 2005). In the present study, high contents of labile compounds in the litter from P-limited plants could have resulted from the accumulation of assimilates in leaves under strong sunlight and P limitation, as proposed above.
Based on the model of Sinsabaugh & Moorhead (1994), we hypothesized that enzyme activities would reflect the N : P ratio of the litter and the element limiting its decomposition. Phosphatase activity was indeed positively related to N : Ps, supporting the assumption that microbes produce and release more phosphatases when they are P-limited (Sinsabaugh & Moorhead 1994). Increased phosphatase activity on high-N : Ps litter was not associated with decreasing activity of the two other enzymes in our study: there was no trade-off in the resource allocation of microbes to these three exoenzymes (as suggested by Sinsabaugh & Moorhead 1994). The extremely weak effect of N : Ps on chitobiase activity possibly reflects the weakness of N limitation for litter decomposition in this study: N limitation was found only for four litter types.
Although significant, the increase in phosphatase activity towards high N : Ps was small and was not consistent across species. This contrasts with some field studies showing stronger relationships between phosphatase activity and P availability in soils (Olander & Vitousek 2000; Colvan, Syers & O'Donnell 2001). Phosphatase activity in soils results from both microbial enzymes and root enzymes; root phosphatases may be more responsive than microbial phosphatases to P limitation (Colvan et al. 2001). Furthermore, the activity of phosphatases is inhibited mainly by their product, orthophosphate (Colvan et al. 2001), whereas the substrate, organic P compounds, stimulates phosphatase activity (Gressel & McColl 2003). In an earlier study we found that decomposing litter released orthophosphate during the first 10 weeks of incubation only if its initial P concentration exceeded 2 mg g−1 (S.G., unpublished data). As all litter P concentrations were below this value in the present experiment, phosphatase activity could hardly be inhibited by orthophosphate.
Interspecific differences accounted for most of the variation in microbial activity, as found elsewhere (Kourtev et al. 2002). The small differences in pH of litter extracts (Table 2) were unlikely to be the cause, as all pH values were close to 6 and were negatively correlated with microbial activity. Enzyme activities correlated positively with each other and with microbial respiration among the nine plant species, but hardly with the litter N and P concentrations (Tables 3 and 4). These contrasting correlations suggest an overall control of microbial activity by species-specific differences in the availability of labile C (Sinsabaugh 1994), which corroborates the opinion that species identity has a greater influence on litter decomposition than variations in nutrient concentrations caused by differing growth conditions (Aerts et al. 2003).
Our laboratory incubations have revealed a correspondence between N or P limitation of plant growth and N or P limitation of litter decomposition. However, there was no consistent relationship between the type of nutrient limitation, N : P ratios of biomass or litter, and enzyme activities. We propose that there is no universal critical litter N : P ratio discriminating between N- and P-limited decomposition because the critical N : P ratio depends on species-specific litter properties (not measured here). Our results from 2003 and the literature about photoinhibition have led us to speculate that the quality of litter produced by P-limited plants might also be modified by strong sunlight. Further decomposition studies with litter from P-limited plants would help to verify our suggestions.
We thank B. Hoorens, K. Küffer, H. Olde Venterink, D. Ramseier, and two referees for comments on successive drafts of the manuscript, Tino Fotsch for support with plant cultivation, Irene Handke and Sarah Omlin for collecting the litter, Tim Jones for assistance with the enzyme assays, Rose Trachsler for chemical analyses, and the European Science Foundation for a short-term exchange grant to S.G.