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Increasing concern about the ecological consequences of rising atmospheric CO2 concentrations, a key component of global change (Houghton et al., 2001), has boosted research to assess its effects on virtually all aspects of plant biology (Poorter, 1993; Wand et al., 1999; Poorter & Navas, 2003). A substantial amount of this research has been conducted in controlled environments where plants have been grown under homogeneous soil conditions (Poorter, 1993). In the natural world, spatial heterogeneity in the availability of soil-based resources (hereafter termed soil nutrient heterogeneity) is the norm, rather than the exception, in most ecosystems (Jackson & Caldwell, 1993; Cain et al., 1999; Farley & Fitter, 1999). At the spatial scale of the root system, soil nutrient heterogeneity promotes a suite of plant physiological and morphological responses, including changes in biomass allocation, root morphology, longevity and growth, and in nutrient uptake patterns (Robinson, 1994; Huber-Sannwald & Jackson, 2001; Hodge, 2004). These responses determine the competitive ability and survival of individual plants within assemblages (Hodge, 2004), and thus soil nutrient heterogeneity has the potential to modify assemblage composition and productivity (Bliss et al., 2002; Wijeshinge et al., 2005; but see Casper & Cahill, 1996).
During the past decade, elevated CO2 research has developed towards more complex experiments that evaluate the joint effects of elevated CO2 and other environmental factors on plant performance and ecosystem processes (Stöcklin et al., 1998; Zavaleta et al., 2003; Reich et al., 2004). These experiments have demonstrated that plant responses to elevated CO2 at both species and assemblage levels are often dependent on the availability of soil nutrients such as nitrogen and phosphorus (Berntson & Bazzaz, 1997; Stöcklin et al., 1998; Bassirirad et al., 2001). However, it is unknown whether such responses are modified by other nutrient attributes, including their spatial distribution, because interactions between soil nutrient heterogeneity and elevated CO2 on plant assemblages have barely begun to be explored (Arnone, 1997). At the species level, interactions between elevated CO2 and soil nutrient heterogeneity may occur because soil nutrient heterogeneity promotes plant responses, such as changes in biomass allocation and nutrient uptake patterns, that are also a consequence of elevated CO2 (Bassirirad et al., 2001; Poorter & Navas, 2003; Hodge, 2004). At the assemblage level, such interactions may occur because co-occurring plants often differ in their ability to profit from this heterogeneity (Bliss et al., 2002; Wijesinghe et al., 2005), and in the direction and magnitude of their responses to elevated CO2 (Berntson et al., 1998; Grünzweig & Körner, 2001). Given the expectation that nutrients are usually distributed heterogeneously in soils, the potential for soil nutrient heterogeneity to interact with elevated CO2 to determine plant species and assemblages responses is large.
To our knowledge, no previous study has evaluated the joint effects of elevated CO2, overall soil nutrient availability and nutrient heterogeneity on plant performance, at either the species or assemblage level. To address this need, we conducted a microcosm experiment to evaluate the joint effects of these variables on the response of an assemblage formed by Lolium perenne L., Plantago lanceolata L., Anthoxantum odoratum L., Holcus lanatus L. and Trifolium repens L. These species commonly co-occur in seminatural temperate grasslands (Joshi et al., 2000) and show differences in the magnitude of their responses to elevated CO2, soil nutrient heterogeneity and nutrient availability (Staddon et al., 1999; Goverde et al., 2002; Hodge, 2004). We used natural soil and organic material (labeled with 15N), rather than growing medium and inorganic fertilizer, because they are relevant materials that permit interpretation of plant responses to varying soil nutrient availability and heterogeneity in a more realistic context (Hodge, 2004). We tested the following hypotheses.
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We found interactive effects of elevated CO2 and soil nutrient heterogeneity or availability, respectively, on a number of assemblage characteristics (biomass, foliar N concentration, root foraging responses). Notably, the only responses that were consistent with our first hypothesis (three-way interactions between nutrient heterogeneity, CO2 and nutrient availability) were observed for variables related to the patterns of N uptake by the assemblages. The fact that N uptake patterns were affected cautions that the responses of plant assemblages to joint increases in the concentration of CO2 and in nutrient availability are likely to be influenced by nutrient heterogeneity. If the interactions we found stand in field ecosystems, then predicting responses of plant assemblages to elevated CO2 by considering only observed responses will not be informative (Körner, 2003). Indeed, such responses will be an outcome of both interactive effects of other global change drivers, and interactions between these drivers and intrinsic ecosystem features such as soil nutrient heterogeneity.
As in previous studies conducted with grassland species (Stöcklin et al., 1998; He et al., 2002; Grünzweig & Körner, 2003), significant interactions between CO2 and nutrient availability were found for assemblage attributes such as above-ground biomass, below : above-ground ratio and the amount of N captured from the added organic material. The increase in nutrient availability increased growth and N capture responses to elevated CO2, suggesting that CO2 effects on these assemblage attributes were constrained by low nutrient availability. To our knowledge, this study is the first to report a CO2 × nutrient heterogeneity interaction when estimating plant performance using variables such as biomass and N content. In the only previous study that evaluated the joint effects of soil nutrient heterogeneity and CO2 on plant performance, Arnone (1997) found that precision of root foraging measured with fine root length (his response variable) increased under elevated CO2 69 d after the addition of the nutrient patches. Such a response was, however, not observed in our study, as indicated by the lack of a significant CO2 × nutrient heterogeneity interaction when analysing precision in root foraging data. Notably, the positive effects of elevated CO2 on above-ground biomass were observed only under homogeneous conditions. Thus our results suggest that benefits that plants obtain when growing in a CO2-enriched atmosphere, such as reduced water requirements, and increases in soil water availability and N mineralization (Poorter, 1993; Niklaus & Körner, 2004), do not interact additively with those that plants obtain from soil nutrient heterogeneity (an increase in nutrient uptake resulting from precise root foraging into nutrient patches). More studies are needed to evaluate the generality of our findings, and to test if similar responses are observed with species belonging to other functional and community types.
Soil nutrient heterogeneity interacted with nutrient availability to determine root foraging responses. Under heterogeneous conditions, the proliferation of roots into the nutrient patches by the assemblages was greater with higher nutrient availability. The increasing contrast between the nutrient patch and background soil, a necessary result of increasing the amount of nutrients in a patch, potentially stimulated this proliferation. The contrast in nutrient content between a nutrient patch and the background soil is an important feature of heterogeneous soil environments that has received little attention to date (Hodge, 2004). If the nutrient demand in the background soil is not met, root proliferation in the nutrient patches should increase with increasing contrast between these patches and the background soil (Lamb et al., 2004). However, previous studies conducted with single species suggest that the increase in root proliferation with increasing contrast between the patch and the background soil becomes saturated at high contrast levels (Wijesinghe & Hutchings, 1999; Lamb et al., 2004). As discussed by Hodge (2004), root proliferation responses are only relevant for plant performance when different plant species compete for organic patches containing a limited local supply of different N sources, as such responses are critical to determine their competitive success. Thus, in a competitive setting such as that evaluated here, we would expect that: (a) root proliferation by the assemblages under heterogeneous conditions increases with increasing contrast between the patch and the background soil; and (b) a saturating response of root proliferation by the assemblages, if any, should occur at higher contrast levels than those promoting a saturating response in species grown individually. However, our results do not provide definitive evidence for this affirmation, which should be tested in future studies.
Consistent with the findings of Day et al. (2003) and Wijesinghe et al. (2005) (but see Casper & Cahill, 1996), higher assemblage biomass was observed when nutrients were supplied heterogeneously (Fig. 1). These biomass responses were associated with precise root foraging in response to heterogeneous nutrient supply (Fig. 2), suggesting that root foraging precision is a key driver of the assemblage biomass responses. This assertion appears to be supported by the fact the assemblages captured more N from the organic material added when it was supplied heterogeneously (Table 1), and their total biomass increased linearly with increased foraging precision under these conditions (Fig. 3). Physiological changes in nutrient uptake induced by soil heterogeneity (Hodge, 2004) may also have contributed to our results, but we did not assess this.
Our second hypothesis – that elevated CO2, soil nutrient heterogeneity and overall nutrient availability, alone and in combination, will promote changes in the composition of assemblages – was partially supported by our results. Overall, CO2 and nutrient heterogeneity did not singly influence the species biomass responses. However, significant or marginally significant CO2 × nutrient heterogeneity interactions were found for all species except A. odoratum. When they occurred, the direction of these interactions was species-specific (Fig. 4). Species-dependent differences in the response of above-ground biomass to elevated CO2 were also evident when evaluating the results of the WinRatio index (Fig. 5). While the dominant species, H. lanatus, was the species that responded most to elevated CO2 under homogeneous conditions at the medium and highest nutrient availability conditions, under heterogeneous conditions the subordinate species were those that responded most to elevated CO2. In a review of competition experiments, Poorter & Navas (2003) found that C3 grasses and N2-fixing species were the groups of species that benefited most from elevated CO2 (values of WinRatio index significantly >1) under high- and low-nutrient conditions, respectively. Interestingly, and contrary to this general pattern, we found that the N2-fixer T. repens was a ‘winner’ under high nutrient availability conditions, but only when the nutrients were supplied heterogeneously. Previous studies have found that T. repens responds to both increases in soil nutrient availability and heterogeneity by increasing biomass growth (Turkington et al., 1991; Hutchings et al., 1997), but the joint effects of both variables on this species when growing in competition have not been evaluated before. Our results are consistent with these studies, and suggest that a plastic response of T. repens to the joint presence of increased nutrient availability and heterogeneity may be nonadditive under elevated CO2. As legumes can transfer the N fixed from the atmosphere to other species, they are of paramount importance as drivers of community-wide responses to elevated CO2 (Navas et al., 1997; Warwick et al., 1998; Stöcklin & Körner, 1999). Thus the evaluation of the role of soil nutrient heterogeneity as a driver of legume responses to joint increases in the atmospheric concentration of CO2 and nutrient availability merits further study.
Consistent with the findings of Berntson et al. (1998) for a model, annual grassland community, the enhancement of N uptake was linearly related to biomass production. This suggests that the ability of plants to increase N uptake may be an important determinant of which species in an assemblage will be able to respond to increased CO2 levels with increased biomass production. Notably, the greatest enhancement of both biomass and nutrient uptake under elevated CO2 was found for the subordinate species under conditions of high nutrient availability and heterogeneous nutrient supply. Others (Navas et al., 1997; Berntson et al., 1998; Stöcklin & Körner, 1999) have also found that subordinate species are the most responsive to an increase in the concentration of atmospheric CO2. Our results add to this literature by emphasizing that such responses may be dependent on both soil nutrient heterogeneity and availability. If the patterns observed in our experiment hold true, species composition is likely to change under elevated CO2 conditions, in a manner that is dependent on nutrient availability and spatial pattern. Changes in species composition under elevated CO2 have been detected in both field and microcosm studies (Berntson et al., 1998; Joel et al., 2001; He et al., 2002), but none of these studies has explicitly considered soil nutrient heterogeneity as a factor in the outcome of these changes.
In the field, heterogeneity in resource distribution arises as a result of organic inputs (derived from leaf litter, dead roots, animal corpses, etc.) and their subsequent decomposition. Given that we used natural soil and organic material, the decomposition of the organic material in our experiment may resemble that observed in the field (Hodge, 2004). However, our approach is not without limitations. For example: (a) growing assemblages in PVC piping may alter root foraging responses because of physical restriction of lateral root growth (Fransen et al., 1999); (b) patterns of patch heterogeneity and degree of contrast to the background soil may not reflect those found in the field; and (c) standardized climatic conditions (light, temperature, humidity, water supply) may amplify plant responses to soil nutrient availability and heterogeneity over those that may be observed in the field. The latter limitation is applicable to many controlled-environment studies and, when taken together with the first two limitations, it is clear that extrapolation of our results to the natural world should be done with caution. What we demonstrate are potential plant responses to simultaneous changes in elevated CO2, nutrient heterogeneity and nutrient availability (Jones et al., 2000).
Given the multifactorial nature of global change (Houghton et al., 2001), there is a need to include additional environmental factors in experiments devoted to understanding or predicting the biological effects of CO2 enrichment (Körner, 2001). Despite the large number of studies conducted to assess the ecological consequences of soil nutrient heterogeneity, it has been an overlooked experimental factor in elevated CO2 research. Recently, it has been emphasized that the interpretation of plant responses to elevated CO2 should exclusively be in the context of the nutrient availability experienced by the species or assemblages under study (Körner, 2003). We strongly support this recommendation, and suggest that soil nutrient heterogeneity may also be of paramount importance in determining plant responses to elevated CO2. Future field studies should consider explicitly how nutrient heterogeneity may influence the responses of plant species and assemblages to elevated CO2 and nutrient availability. To achieve this, two different approaches may prove useful: (a) experimental addition of nutrient patches to field soils; and/or (b) consideration of the intrinsic nutrient heterogeneity within experimental plots as a covariate. Such studies will undoubtedly increase our ability to predict how species and ecosystems will respond to ongoing increases in atmospheric CO2 concentration and nutrient availability in the real, and highly heterogeneous, world.