Interactive effects of elevated CO 2, P availability and legume presence on calcareous grassland: results of a glasshouse experiment

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1 Functional Ecology 1999 ORIGINAL ARTICLE OA 000 EN Interactive effects of elevated CO 2, P availability and legume presence on calcareous grassland: results of a glasshouse experiment J. STÖCKLIN and CH. KÖRNER Institute of Botany, University of Basel, Schönbeinstr. 6, CH-4056 Basel, Switzerland Summary 1. We investigated the interactive effects of elevated CO 2, supply of phosphorus (P) and legume presence in model communities of calcareous grassland. Half of the communities contained six graminoids and eight non-legume forb species. In the other half, four non-legume forbs were replaced by legumes. 2. Ecosystem responses. Above-ground phytomass (> 5 cm) hardly responded to elevated CO 2 alone. However, when P and legumes were combined, the CO 2 effect on above-ground community phytomass in year two was a stimulation of 45% (P < 0 001). Below-ground community dry matter was stimulated by elevated CO 2 alone by + 36% (P < 0 01), but was only + 20% (P < 0 05) when legumes were present and P was added. At the final (late season) harvest the mean effect of elevated CO 2 on total above- and below-ground phytomass was + 23% (P < 0 001) and revealed no significant interactions among treatment combinations, because above- and belowground effects of CO 2 enrichment had opposite directions. 3. Functional group responses. When legumes were absent, graminoids increased their total above- and below-ground phytomass in elevated CO 2 by 60% (P < 0 001) but there was no increase when legumes were present. The response of forbs to CO 2 was not significant, irrespective of co-treatment. Legumes, however, were significantly stimulated by P supply and their CO 2 response was much larger when P was added (+ 55%, P < 0 01 vs + 25%, NS). 4. Species responses. CO 2 effects on species ranged from highly positive (+ 143%) to moderately negative ( 43%). 5. Our results demonstrate that the effect of CO 2 enrichment in such natural grassland communities will be low on above-ground phytomass and largely below-ground if no additional nutrients are provided. N 2 -fixing legumes appear to be crucial for the community response to elevated CO 2 but legume responsiveness is largely controlled by P availability. Key-words: Biodiversity, carbon dioxide, fertilization, nutrients, phosphorus Functional Ecology (1999) Ecological Society Introduction Plant community responses to changes in atmospheric CO 2 concentration include complex interactions which may influence species diversity as well as productivity. The community response to CO 2 enrichment depends on the presence of responsive species (Körner & Bazzaz 1996). Plant plant interactions in grassland communities may become more intense under elevated CO 2 (Stewart & Potvin 1996) and may be amplified when nutrients are supplied (Schenk et al. 1997). In studies with unfertilized calcareous grassland above-ground biomass responses have always been much lower than expected from responses of isolated species (Navas et al. 1995; Wolfenden & Diggle 1995; Roy et al. 1996; Leadley & Körner 1996; Stöcklin, Leadley & Körner 1997). Several studies have even reported negative species responses (Leadley & Stöcklin 1996; Roy et al. 1996; Stöcklin et al. 1997). Many plants take little advantage of elevated CO 2 when nutrient availability is low (Zangerl & Bazzaz 1984; Conroy, Milham & Barlow 1992; Newbery & Wolfenden 1996). While the most realistic picture of plant responses to elevated CO 2 will be found in situ, a disadvantage of this approach is that the mechanisms of CO 2 effects will be obscured by a multitude of unknown interactions. In addition large spatial heterogeneity often 200

2 201 P, legumes and CO 2 responses in grassland constrains detection of species responses in the field. The current project attempts to bypass this problem by studying carefully designed artificial communities growing in natural soil in a glasshouse. Manipulations included legume presence or absence and phosporus availability. Our reference site is a semi-natural calcareous grassland utilized in a long-term CO 2 -enrichment project in the field (Körner 1995; Leadley & Körner 1996). Such grasslands are of special conservation interest because of their high species diversity (Zoller 1954; Fischer & Stöcklin 1997). The study of low fertility ecosystems is of general significance because of the well known nutrient CO 2 interactions (Körner 1996). It has been shown that a moderate P fertilization can maintain species richness in calcareous grassland (Bobbink 1991) while at the same time fertilization enhanced overall productivity via legume stimulation (Cole & Heil 1981; Crews 1993). On the other hand, it has been reported that legumes with symbiotic N 2 fixation can fix more carbon than non-legumes when exposed to elevated CO 2 under agronomic conditions (Overdieck 1986; Pitelka 1994; Zanetti et al. 1997). The main advantage of using designed communities is that differential responses of functional groups of species (graminoids, non-legume forbs and legumes) and their interactions can be tested while maintaining overall species diversity constant. The specific objectives of this study were: (1) to test whether a stimulation of plant biomass production at elevated CO 2 would be modified by community composition (i.e. the presence or absence of legumes) or (2) by changes in resource availability (i.e. P fertilization), and (3) whether such a stimulation would, in the longer term, change community compositon through either species-specific or functional group specific responses to elevated CO 2. Materials and methods MODEL COMMUNITIES AND PLANT SPECIES We simulated communities of the endangered Mesobromion -type of calcareous grassland in NW Switzerland (Fischer & Stöcklin 1997). Tillers of Festuca ovina, Festuca rubra and Carex flacca were collected in the field immediately before planting. All other species were grown from seeds (collected at natural field sites) in a mixture of field soil and calcareous sand in a cool glasshouse several weeks before the start of the experiment. We constructed two types of model communities, each composed of 14 species and equal density with 60 individuals (100%) planted in totally randomized positions. The first community type contained only two functional groups: graminoids (Bromus erectus 18 3%, Anthoxanthum odoratum 8 3%, Brachypodium pinnatum 8 3%, C. flacca 8 3%, Festuca ovina 8 3%, F. rubra 8 3%, a total of 60% of all individuals), and non-legume forbs (Betonica officinalis 5%, Centaurea jacea 5%, Leucanthemum vulgare 5%, Hieracium pilosella 5%, Prunella grandiflora 5%, Prunella vulgaris 5%, Salvia pratensis 5%, Sanguisorba minor 5%, a total of 40% of all individuals). In the second community type four non-legume forbs were replaced by four legume species (Hippocrepis comosa 5%, Lotus corniculatus 5%, Trifolium medium 5%, Trifolium repens 5%). Nonlegume forb species to be replaced by legumes were selected from the pool of eight species in such a way that all species were represented equally in each treatment combination. Thus, in this second community type, three functional groups were present, graminoids (60%), non-legume forbs (20%) and legumes (20%). We established 32 model communities in plastic containers that were 36 cm 26 cm 26 cm (length width depth) with a drainage mat (2 cm deep) and holes in the bottom. Containers were first filled to 19 cm deep with a 1:1 mixture of calcareous marl and sieved top soil, both from the foothills of the Jura Mountains in NW Switzerland. The top 5 cm consisted of undiluted top soil. The soil belongs to the para-rendzina type naturally forming 5 20 cm thick layers over calcareous debris or marl (ph 6 5, siltyloamy texture). Seedlings or rooted tillers were planted on a hexagonal grid with a 4 3 cm spacing between planting positions. In total, 60 individuals per container were planted between 20 and 24 April Four randomized planting schemes for each community type were replicated in such a way that in each treatment combination all four planting schemes were represented once. Plantlets had similar size (on a fresh mass basis) within species and were selected so that differences in size between species were as small as possible. Total dry mass of plantlets per container was between 2 and 2 5 g. TREATMENTS AND GROWTH CONDITIONS The experiment began on 24 April 1995 and lasted for two growing seasons (1995 and 1996). Sixteen model communities (eight of each community type) were exposed to ambient (daytime average c. 360 µl litre 1 ) and 16 elevated (c. 600 µl litre 1 ) CO 2. Eight of the communities at each CO 2 level received a low dose of phosphorous fertilizer of 1 gpm 2 a 1, and eight remained unfertilized. The fertilizer was provided as dry superphospate (Triple-Superphospate 46%, Landor AG, Birsfelden, Switzerland) in five equal portions during the growing season. In summary, there were eight treatment combinations resulting from the crossing of community types and fertilization treatments, replicated four times at both CO 2 levels (in total 32 replicates). Model communities assigned to these treatment combinations were then allocated to four naturally lit, air-conditioned glasshouse chambers (two for each CO 2 level). In each chamber, containers were placed on trolleys which were rotated within greenhouses weekly. Positions on each trolley were randomized

3 202 J. Stöcklin & Ch. Körner monthly and trolleys were switched between greenhouses every 2 months to minimize potential effects of environmental variation within and between chambers. All model communities were watered equally, but only every 3 7 days (depending on weather) with 5 10 mm of rain water. This watering regime led to short-term top-soil desiccation as is typical of these grasslands during summer. We used rain water because we wanted to simulate the conditions of a parallel CO 2 -enrichment experiment in the field. The temperature regime in the glasshouses was maintained close to conditions outside the greenhouse using air conditioners. The reduction of solar radiation in the greenhouse was partly compensated by additional light provided by two 1000 W daylight halogen lamps mounted above each 7 m 2 chamber which were activated automatically when ambient photon flux density dropped below c. 180 µmol m 2 s 1 during the natural photoperiod. At canopy level photon flux densities were µmol m 2 s 1 at midday during sunny days. Pesticide had to be applied several times in all treatments to control white flies and, once in year two, against mildew. Spontaneously emerging weeds were removed. During winter, from 20 November 1995 to 14 March 1996, the model communities were placed in an outdoor soil bed and experienced natural midwinter climate, including freezing. No growth occurred during this time and the swards were brown. HARVESTING Twice per year (15 16 June and October 1995, June and October 1996) above-ground phytomass was harvested 5 cm above soil level for every species individually so that functional groups and species could be analysed separately. The two harvests per year corresponded to the time of mowing or grazing in the field. Plant material was separated into green leaves, stems, inflorescence and standing dead. After two growing seasons (October 1996) the below-ground organs (roots and rhizomes) and stubble (stems and leaf bases of the bottom 0 5 cm layer) were harvested for each species. Fine roots which could not be sorted by species (c. 30% of total root mass) were treated as a bulk sample for each container and were included in analysis of community phytomass. Roots were separated from the soil by wet sieving. All samples were dried at 80 C for 48 h. DATA ANALYSIS Each model community was considered a replicate (n = 4 in each of the four treatment combinations at both CO 2 levels). Results are analysed separately for community phytomass, for phytomass of functional groups and for phytomass of individual species. Components of above-ground phytomass (leaves, stems, inflorescence and standing dead) were also analysed separately but this provided little additional information (not shown). Standing dead material was always less than 5% of total and is included in phytomass; subtraction of standing dead plant mass did not change the results of the statistical analysis. For both years we present annual sums for above-ground community phytomass above 5 cm from the June and October harvest. From the final harvest (October 1996) we present stubble, below-ground and total above- (including stubble) and below-ground community phytomass. For functional groups and individual species total above- and below-ground phytomass values from the final harvest are presented. Harvest data for year one and year two were analysed separately using the ANOVA procedure of JMP, version 3 1 (SAS Institute, Cary, NC, USA) to assess the effects of CO 2, P fertilization and the presence or absence of legumes. We calculate CO 2 responses as percentage increase or decrease at elevated CO 2 compared to ambient CO 2. Significant differences in responsiveness among species (or functional groups) were tested using ANOVA with log-transformed dry mass as CO 2 species interaction (or CO 2 functional group interaction) against the residual error. A priori contrasts were used to test for the effects of elevated CO 2 within each treatment combination separately for above-ground and below-ground data and are indicated in figures. Results COMMUNITY PHYTOMASS ABOVE 5 CM In the first year (6 months after planting in October 1995) above-ground phytomass production (> 5 cm) of model communities pooled for all treatment combinations was 292 g m 2 in ambient and 312 g m 2 (+ 6 7%, P = 0 05) in elevated CO 2 (Fig. 1). Phosphorus addition pooled for all treatment combinations resulted in an almost 10% increase in phytomass (P < 0 01). However, P addition or legume presence had no effect on above-ground CO 2 responsiveness. Establishment and growth of legumes was slow in the first year. By the end of the second year (1996) above-ground community phytomass (> 5 cm) was 328 g m 2 for the least productive systems (ambient CO 2, minus P, without legumes) and 631 g m 2 (+ 92%) for the most productive systems (elevated CO 2, with P and including legumes). All treatments and their interactions had significant effects on phytomass production (> 5 cm) (Fig. 1, Table 1). Treatment combinations were decisive for CO 2 effects. The effect of elevated CO 2 alone on above-ground community phytomass (> 5 cm) was only + 8 8% (NS). When P was added or legumes were present the effects of elevated CO 2 on aboveground community phytomass (> 5 cm) increased and was + 16% (P < 0 05) and + 14% (P < 0 05), respectively. The combination of P fertilization and legume presence was most effective and above-ground community phytomass (> 5 cm) increased by 45% (P < 0 001) in elevated CO 2. P fertilization alone had

4 Fig. 1. Community phytomass above 5 cm in year one (1995) and in year two (1996) for two CO 2 levels (ambient A, elevated E), P fertilization (0 or 10 kg ha 1 year 1 ) and two community types (minus legumes, plus legumes). Values are means for plant dry mass with standard error bars from pooled harvests in June and October. Percentage differences and P values owing to CO 2 level (a priori contrasts) within treatment combinations are indicated (P values above 0 1 are indicated as NS). no significant effect for phytomass production (> 5 cm) of model communities, but in communities with legumes above-ground phytomass (> 5 cm) was increased by 26% (P < 0 01) independently from P supply or CO 2 level. BELOW-GROUND COMMUNITY BIOMASS At the final harvest (October 1996) below-ground community phytomass was 431 g m 2 for the least productive systems (ambient CO 2, minus P, without legumes) and 704 g m 2 (+ 63%) for the most productive systems (elevated CO 2, with P and including legumes). There were no significant interactions with either P fertilization or community type (Table 1). The effect of elevated CO 2 on below-ground community phytomass at the final harvest in the absence of P fertilization and legumes was + 36% (P < 0 01). When P was added or legumes were present the effects of elevated CO 2 were + 28% (P < 0 05) and + 30% (P < 0 01), respectively. When legumes were present and P was added, the CO 2 effect on roots was only 20% (P < 0 05). P fertilization had no effect on belowground dry matter, but in communities with legumes it was increased by + 18% (P < 0 01) compared to those without legumes. Above-ground and below-ground dry matter were affected differently by the combination of CO 2 enrichment and P fertilization (Table 1). For below-ground community dry mass the effect of elevated CO 2 was always larger when P was absent. While aboveground CO 2 effects were largest when P fertilization and the presence of legumes were combined (Fig. 1), effects on below-ground dry matter were largest without P fertilization and without legumes. COMMUNITY PHYTOMASS AT THE FINAL HARVEST At the final harvest accumulated stubble phytomass (0 5 cm) was larger than community regrowth > 5 cm between June and October As usual in such grasslands the late season harvests (> 5 cm) yielded only 30 50% of the June harvest (Table 2). In elevated CO 2, stubble phytomass (accumulated over the whole duration of the experiment) in communities without legumes increased by 19% (NS) without P and by 44% (P < 0 01) when P was added, but when legumes were present the CO 2 effect on stubble was small and not significant (Table 2). These differences in stubble phytomass reflect the CO 2 induced increase of graminoids in the absence of legumes, an effect not observed when Table 1. F values and significance levels from ANOVA with community phytomass above 5 cm from the first (1995) and second year (1996), and with community phytomass from the final harvest (October 1996) separately for stubble, below-ground organs and the total above- and below-ground phytomass. Sources include the three treatments CO 2 level (350, 600 µl 1 ), phosphorous fertilization ( P, + P), community type (without legumes vs legumes present) and their interactions First year 1995 Second year 1996 Final harvest Final harvest Final harvest Community Community Stubble Below-ground Total above- and dry mass dry mass community community below-ground Source above 5 cm above 5 cm dry mass dry mass community dry mass CO 2 level (CO 2 ) 4 1 * 52 4 *** 4 6 * 27 0 *** 30 8 *** P fertilization (P) 7 9 ** 13 2 ** 0 8 NS 1 9 NS 3 3 NS Community type (community) 2 6 NS 168 *** 0 1 NS 12 7 ** 26 2 *** CO 2 P 0 1 NS 11 9 ** 1 1 NS 0 3 NS 0 1 NS CO 2 community 0 5 NS 14 5 *** 3 8 NS 0 1 NS 0 3 NS P community 0 1 NS 18 5 *** 0 1 NS 0 2 NS 0 1 NS CO 2 P community 0 1 NS 6 5 * 0 3 NS 0 1 NS 0 2 NS Levels of significance: * P 0 05, ** P < 0 01, *** P <

5 legumes were present (see below). Absolutely, the CO 2 -induced difference in stubble phytomass in communities without legumes remains small compared to the large CO 2 effect on phytomass above 5 cm when legumes were present and P was added. Total above- and below-ground phytomass at the final harvest was 706 g m 2 for the least productive system and 1118 g m 2 (+ 58%) for the most productive sytem (Table 2). Total phytomass responses revealed no significant interactions among treatment combinations (Table 1), because above- and belowground effects owing to CO 2 enrichment and P fertilization had opposite directions. At the final harvest the mean effect across treatment combinations on total phytomass was % (P < 0 001) for elevated CO 2, + 21% (P < 0 001) for legume presence/absence, and + 7 0% (P = 0 08) for P fertilization. CO 2 RESPONSE OF FUNCTIONAL GROUPS Fig. 2. Above-ground phytomass (including stubble) and below-ground biomass of three functional groups (graminoids, non-legume forbs, legumes) from the final harvest (October 1996, means for plant dry mass with standard error bars). Percentage differences and P values owing to CO 2 level (a priori contrasts) within treatment combinations are indicated separately for above- and below-ground data (P values above 0 11 are indicated as NS). By the end of year two (final harvest, October 1996, Fig. 2) CO 2 enrichment increased total above- and below-ground phytomass in graminoids by 60% (P < 0 001) in communities without legumes and had no effect when legumes were present. In non-legume forbs the CO 2 response did not depend on the presence or absence of legumes, and the total above- and below-ground phytomass increase was 15% (P = 0 07). Legumes increased their total dry mass by 40% (P < 0 002) in elevated CO 2. These differences in CO 2 responsiveness were statistically nonsignificant when tested across functional groups. (P = 0 2 for functional group CO 2 interaction with log transformed biomass). That is, the relative contribution of functional groups to community composition was not significantly different between CO 2 concentrations despite some significant group specific responses. P fertilization did not significantly influence bulk biomass of graminoids and forbs at either ambient or Table 2. Mean community dry mass (g m 2 ± SE) from harvests in 1996 for two CO 2 levels (ambient A, elevated E), P fertilization (0 or 10 kg ha 1 year 1 ) and two community types (minus legumes vs plus legumes). Significant differences owing to CO 2 level (a-priori contrasts) within treatment combinations are indicated with * P 0 05, ** P < 0 01, *** P < Communities without legumes Communities with legumes minus P plus P minus P plus P A E A E A E A E Harvest > 5 cm (June 1996) 249 ± ± ± ± 12* 276 ± ± 26* 263 ± ± 11*** Final harvest (October 1996) Phytomass > 5 cm (a) 80 ± 8 99 ± 9 79 ± ± ± ± ± ± 24 Stubble 0 5 cm (b) 180 ± ± ± ± 23** 199 ± ± ± ± 14 Below-ground phytomass (c) 431 ± ± 18* 491 ± ± 46** 537 ± ± 48* 586 ± ± 26* Final harvest (October 1996) Total above- and below-ground 692 ± ± 4* 741 ± ± 68** 873 ± ± 48* 963 ± ± 16** (a+b+c)

6 205 P, legumes and CO 2 responses in grassland elevated CO 2. Legumes, however, increased their biomass significantly by 29% (P = 0 01) when P was added, and their above-ground biomass response to elevated CO 2 was much larger with P fertilization (+ 55% vs + 22%, Fig. 2). As for the community level, above-ground and below-ground CO 2 responses of functional groups were affected differently by P fertilization. For graminoids and for legumes, positive CO 2 responses in above-ground phytomass were larger when P was added, below-ground CO 2 responses in all functional groups were much larger when no P was added (Fig. 2). CO 2 RESPONSE OF INDIVIDUAL SPECIES By the end of year two (final harvest, October 1996, Table 3) species differed greatly in their contribution to total above- and below-ground community dry mass (P < 0 001) and also in their response to elevated CO 2. Hence, species composition was different at ambient and elevated CO 2 (P < for species CO 2 interaction with log-transformed phytomass). Species responses ranged from highly positive in communities without legumes (+ 143%) to moderately negative in communities with legumes ( 43%). The response of graminoid and forb species was strongly dependent on community type (Fig. 3, Table 3). In communities without legumes total aboveand below-ground dry mass in four of six graminoids (A. odoratum, C. flacca, F. rubra, F. ovina) and in one of eight forbs (C. jacea) was significantly increased under elevated CO 2 (Table 3, Fig. 3). The dominant grass species (B. erectus) and the most abundant forb species (S. minor) showed no significant response to elevated CO 2. In the other community type, legume species profited much more from CO 2 enrichment than the other two functional groups, particularly when P was added (Fig. 2, Fig. 4). In communities with legumes the CO 2 responsiveness of graminoids and C. jacea was largely reduced compared to the community type without legumes. There was even a significant reduction in phytomass of B. pinnatum, F. ovina and S. minor under elevated CO 2 when legumes were present (Table 3). Hence, the greater vigour of legumes in elevated CO 2 had negative effects on CO 2 responsiveness of several non-legumes. Consequently, graminoid and forb species in communities without legumes, and legumes in the other community type replaced each other in their contribution to the increase of total community dry mass in elevated CO 2. As for the community and the functional group data, above-ground species responses to elevated CO 2 were always larger when P was added, and opposite to below-ground responses, which were larger when no P was added (Figs 3 and 4). Table 3. Mean dry mass (g m 2 ± SE) of calcareous grassland species grown in two types of model communities at ambient CO 2 (both fertilization treatments within each community type pooled) and their response (% increase or decrease) when exposed to elevated CO 2. Values are total above- and below-ground phytomass from the final harvest (October 1996) Communities without legumes Communities with legumes Dry mass at Response at Dry mass at Response at ambient CO 2 elevated CO 2 ambient CO 2 elevated CO 2 (g m 2 ± SE) %, P value (g m 2 ± SE) %, P value GRAMINOIDS Bromus erectus 78 2 ± ± Carex flacca 76 8 ± *** 77 4 ± Anthoxanthum odoratum 34 4 ± *** 64 9 ± Brachypodium pinnatum 42 2 ± ± * Festuca rubra 19 6 ± *** 25 0 ± Festuca ovina 10 3 ± * 18 9 ± NON-LEGUME FORBS Sanguisorba minor 68 1 ± ± Centaurea jacea 59 4 ± * 68 6 ± Leucanthemum vulgare 49 3 ± ± Hieracium pilosella 27 2 ± ± Salvia pratensis 24 2 ± ± Prunella vulgaris 15 8 ± ± Prunella grandiflora 8 5 ± ± Betonica officinalis 7 1 ± ± LEGUMES Trifolium medium 135 ± Lotus corniculatus 69 5 ± Hippocrepis comosa 41 4 ± Trifolium repens 4 4 ± Significant (P 0 05) or marginally significant (0 1 P 0 05) biomass responses are printed in bold. Significant levels: *** (P < 0 001), ** (P < 0 01), * (P < 0 05), + (P 0 1).

7 206 J. Stöcklin & Ch. Körner Discussion Our results clearly demonstrate that the effect of CO 2 enrichment on above-ground community phytomass will be low and largely below-ground if no additional nutrients are provided. In the absence of P fertilization or legumes the stimulation of community phytomass regrowth above 5 cm at elevated CO 2 in year two was quite small (9%). However, when P, legumes and CO 2 enrichment were combined, there was a 45% increase in community phytomass (> 5 cm) compared to untreated control, suggesting that growth limitations by either P or N were overcome. If accumlated stubble phytomass at the end of our study is taken into account the picture remains essentially the same. CO 2 RESPONSE AND NUTRIENT AVAILABILITY Fig. 3. Above-ground (including stubble) and below-ground phytomass of the four most responding non-legume species (Anthoxanthum odoratum, Carex flacca, Festuca rubra, Centaurea jacea) from the final harvest (October 1996, means for plant dry mass with standard error bars). Percentage differences and P values owing to CO 2 level (a priori contrasts) within treatment combinations are indicated separately for above- and below-ground data (P values above 0 12 are indicated as NS). The small effect of CO 2 enrichment alone on aboveground phytomass is in accordance with generally low community responses in undisturbed vegetation (Körner 1996) and unfertilized calcareous grassland communities in particular (Navas et al. 1995; Wolfenden & Diggle 1995; Roy et al. 1996; Stöcklin et al. 1997). In combination with P fertilization and legume presence we found much larger biomass responses to elevated CO 2. The combination of CO 2 enrichment with only P fertilization or with only legumes present somewhat enhanced the effect of CO 2 enrichment alone, but neither treatment combination produced the dramatic increase in aboveground phytomass which resulted when elevated CO 2, P addition and legume presence were combined. Our results suggest a co-limitation of above-ground response to elevated CO 2 by P and symbiotically fixed N in this grassland community. When either P or legumes were missing the increase in below-ground organs in elevated CO 2 was always two to three times larger compared to above-ground responses, i.e. the allocation of extra carbon gains to roots was larger than the allocation to shoots, in line with what had been found for a number of natural ecosystems (Koch & Mooney 1996). Below-ground biomass responses are difficult to ascertain in the field, because of small scale heterogeneity, great rooting depth, large turnover in fine roots and limited sampling procedures (Norby et al. 1992). At our reference field site above-ground responses to elevated CO 2 exposure built up over 2 years, and have stabilized around + 20% up till now (P. Leadley & P. Niklaus, unpublished data). Our model communities responded in the same way, the phytomass response to elevated CO 2 was much stronger in year two. This delayed CO 2 response suggests that strong initial responses to a step increase of CO 2 may not universally be observed. One reason why aboveground community responses are delayed in such low fertility soils is that responsive species first allocated carbon gains to roots, particularly when no additional P was available. P fertilization alone affected only shoot to root allocation. However, when P fertilization was combined with CO 2 enrichment the above-ground community CO 2 response was enhanced. In the case of legumes the combined effect of P and CO 2 enrich-

8 207 P, legumes and CO 2 responses in grassland Fig. 4. Above-ground (including stubble) and belowground phytomass of the three most responding legumes (Lotus corniculatus, Hippocrepis comosa, Trifolium repens) from the final harvest (October 1996, means for plant dry mass with standard error bars). Percentage differences and P values owing to CO 2 level (a priori contrasts) within treatment combinations are indicated separately for above- and below-ground data. Trifolium medium is not shown; regrowth of this species after the June harvest was small. ment was remarkable. P fertilization alone already stimulated legume growth but when combined with elevated CO 2 this functional group increased extraordinarily. Nodule development, and therefore legume growth, require adequate phosphorus (Bordeleau & Prévost 1994), but the energydemanding N 2 fixation itself may be limited by available carbon (Schulze, Adgo & Schilling 1994). Stimulation of symbiotic N 2 fixation at elevated CO 2 and under fertile agronomic conditions has been reported earlier (Zanetti et al. 1997) and stimulated growth of N 2 fixers (Hebeisen et al. 1997). Our results suggest that this stimulation is faciliated by P supply. In calcareous grasslands as in many other ecosystems, legume growth appears to be P limited (Williams 1978; Van Hecke, Impens & Behaeghe 1981; Willems, Peet & Bik 1993) and only the combination of P supply and CO 2 enrichment induced strong legume growth. INDIVIDUAL SPECIES RESPONSES TO ELEVATED CO 2 There was a considerable variation among individual species in their responses to elevated CO 2. The legumes included in this experiment were the only plant functional group which behaved as a group consistently positive under elevated CO 2. Graminoids and non-legume forbs showed no consistent behaviour as functional groups; this result confirms earlier findings (Bazzaz 1990; Leadley & Stöcklin 1996). It is hardly possible to denote common traits of the responsive species. In the context of calcareous grassland Anthoxanthum, Centaurea and F. rubra may be characterized as relatively competitive, fast-growing species; but C. flacca and F. ovina are slow-growing, not very competitive graminoids. Similarly, non-responsive or even negatively responding species do not share traits that might explain their behaviour. The trend observed by Poorter et al. (1996) for faster growing species to respond much more strongly than slower growing species was not found in our experiment. Also the hypothesis of Hunt et al. (1991, 1993) that the position of a species in the strategy triangle of Grime (1979) might forecast species behaviour under elevated CO 2 was not supported. In particular, negative responses in a community context probably result from relative competitive weakness and not from plant physiology or growth pattern under elevated CO 2. Bromus erectus and S. minor, exhibiting no response or a negative one in our communities, have previously been reported to show positive growth responses when grown in isolation (Hunt et al. 1991, 1993; Poorter et al. 1996). The results of our glasshouse study are largely in accordance with available data from the field experiment and confirm findings from other model community studies (Leadley & Stöcklin 1996; and unpublished work). First, the dominant grass B. erectus and the most abundant forb S. minor do not show a positive response to elevated CO 2 if grown under conditions close to those experienced in the field. Second, and somewhat surprisingly, C. flacca, a subdominant sedge, is probably the most important winning species of calcareous grassland when CO 2 is increasing. Third, mesophytic species like A. odoratum, C. jacea and F. rubra might be at a competitive advantage, possibly because elevated CO 2 reduces soil moisture depletion during drying cycles (Owensby et al. 1993; Jackson et al. 1994; Field, Jackson & Mooney 1995; Lauber & Körner 1997). THE COMMUNITY CONTEXT Community responses resulted from large differences in CO 2 responses among species and functional groups, including negative ones for several species. These responses were dependent on the community context, i.e. the competitive performance relative to that of the other species present. In communities with

9 208 J. Stöcklin & Ch. Körner legumes their large response to elevated CO 2 reduced or in some cases even prevented the increase of otherwise responsive graminoids and forbs. This was most spectacular for C. flacca and A. odoratum, but also occurred in Festuca ssp. and C. jacea (Fig. 3). Legumes thus gained much more in competitive strength with elevated CO 2 compared to responsive graminoids or non-legume forbs, especially when P was supplied. It seems obvious that this is related to their easy access to symbiotically fixed N 2 which was introduced into the communities and which was not available for nonlegume species within the 2 years of our experiment. N content in communities with legumes was c. 30% higher at elevated CO 2 (P. Niklaus, unpublished data). Hence, increased nutrient availability will not only determine the responsiveness to elevated CO 2 at the level of biomass production but will also determine the direction of shifts in community structure that occur as a consequence of differences in species response to elevated CO 2. The long-term consequence of such shifts among species for community composition is, however, difficult to predict from a 2 year experiment. Over time part of the additional N fixed by legumes may become available to other species and the predominance of the legume response might be weakened once the system has reached a new (quasi) steady state. Conclusion This controlled environmental study revealed a remarkable chain reaction, with P-priming legumes which in turn produced a strong above-ground community biomass response to elevated CO 2. 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