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. 2006 Jul;98(1):227–235. doi: 10.1093/aob/mcl093

Nutrient Availability and Atmospheric CO2 Partial Pressure Modulate the Effects of Nutrient Heterogeneity on the Size Structure of Populations in Grassland Species

FERNANDO T MAESTRE 1,*, JAMES F REYNOLDS 1,2
PMCID: PMC2803555  PMID: 16705000

Abstract

• Background and Aims Size-asymmetric competition occurs when larger plants have a disproportionate advantage in competition with smaller plants. It has been hypothesized that nutrient heterogeneity may promote it. Experiments testing this hypothesis are inconclusive, and in most cases have evaluated the effects of nutrient heterogeneity separately from other environmental factors. The aim of this study was to test, using populations of Lolium perenne, Plantago lanceolata and Holcus lanatus, two hypotheses: (a) nutrient heterogeneity promotes size-asymmetric competition; and (b) nutrient heterogeneity interacts with both atmospheric CO2 partial pressure (PCO2) and nutrient availability to determine the magnitude of this response.

• Methods Microcosms consisting of monocultures of the three species were grown for 90 d in a factorial experiment with the following treatments: PCO2 (37·5 and 70 Pa) and nutrient availability (NA; 40 and 120  mg of N added as organic material) combined with different spatial distribution of the organic material (NH; homogeneous and heterogeneous). Differences in the size of individual plants within populations (size inequality) were quantified using the coefficient of variation of individual above-ground biomass and the combined biomass of the two largest individuals in each microcosm. Increases in size inequality were associated with size-asymmetric competition.

• Key Results Size inequality increased when the nutrients were heterogeneously supplied in the three species. The effects of NH on this response were more pronounced under high nutrient supply in both Plantago and Holcus (significant NA × NH interactions) and under elevated PCO2 in Plantago (significant PCO2 × NA × NH interaction). No significant two- and three-way interactions were found for Lolium.

• Conclusions Our first hypothesis was supported by our results, as nutrient heterogeneity promoted size-asymmetric competition in the three species evaluated. Nutrient supply and PCO2 modified the magnitude of this effect in Plantago and Holcus, but not in Lolium. Thus, our second hypothesis was partially supported.

Keywords: Lolium perenne, Plantago lanceolata, Holcus lanatus, elevated CO2, nutrient heterogeneity, nutrient availability, below-ground competition, size inequality

INTRODUCTION

In most ecosystems, the distribution of soil nutrients is spatially heterogeneous at scales ranging from individual root systems to the landscape (e.g. Jackson and Caldwell, 1993; Cain et al., 1999). Heterogeneity of nutrient supply (hereafter called nutrient heterogeneity), occurring at the scale of the root system, induces many physiological and morphological plant responses, proliferation of roots in the nutrient patches and changes in nutrient uptake rates being two of the most important (Hodge, 2004). These responses are ecologically relevant, as they may determine the competitive ability of competing individual plants (Hodge et al., 1999), the structure of populations (Day et al., 2003a) and the productivity of communities (Maestre et al., 2005, 2006; Wijesinghe et al., 2005).

An aspect that has received increased attention in recent years is the potential influence of nutrient heterogeneity on plant co-existence via its effects on the relationship between a plant's size and its competitive effect (Weiner, 1990; Schwinning and Weiner, 1998). Size-symmetric competition occurs when individuals compete in proportion to their size, with large plants causing a large decrease in the growth of smaller plants, and small plants causing a small (but proportionate to their size) decrease in the growth of the larger plants (Schwinning and Weiner, 1998). In contrast, size-asymmetric competition occurs when larger plants have a disproportionate advantage (for their relative size) in competition with smaller plants (Schwinning and Weiner, 1998). Increases in the differences in size of individual plants within populations (hereafter referred to as size inequality) are associated with size-asymmetric competition (Casper and Cahill, 1998; Schwinning and Weiner, 1998; Nagashima et al., 2003). It is often assumed that competition for nutrients is size-symmetric (Weiner, 1990; Cahill and Casper, 2000). However, it has been hypothesized that, with heterogeneous nutrient supply, large plants can have a disproportionately large competitive effect if they can reach and exploit nutrient patches faster than smaller plants, a process that would promote size-asymmetric competition (Schwinning and Weiner, 1998). Experiments both support (Fransen et al., 2001; Day et al., 2003a; Rajaniemi, 2003) and reject (Casper and Cahill, 1998; Blair, 2001; Day et al., 2003b) this prediction. Most have, however, used artificial substrates and inorganic fertilizer to simulate nutrient heterogeneity. In the field, this heterogeneity arises from deposition of organic matter (e.g. leaf litter) and its subsequent microbial decomposition. Such decomposition releases inorganic nutrients, which plants capture, in a manner which is more complex than that resulting from placing a patch of inorganic nutrients directly into the growing medium (Hodge, 2004). In addition, most of our knowledge on how plants respond to small-scale nutrient heterogeneity has been derived from studies evaluating responses to this heterogeneity isolated from other environmental factors (Robinson, 1994; Hutchings and John, 2004).

Evaluating how joint changes in nutrient heterogeneity and global change drivers, such as atmospheric CO2 partial pressure (PCO2) and nutrient availability, affect the relationship between a plant's size and its competitive ability is relevant for several reasons. First, this relationship strongly influences plant community structure and dynamics (Newman, 1973). Secondly, there is a pressing need to increase understanding of the consequences of global change for plant individuals, populations and communities (Houghton et al., 2001). Thirdly, nutrient heterogeneity may potentially interact with nutrient availability and PCO2 to determine plant population responses. Maestre et al. (2005) evaluated the effects of PCO2, and nutrient availability and heterogeneity on the responses of model communities of Lolium perenne, Plantago lanceolata, Anthoxanthum odoratum, Trifolium repens and Holcus lanatus. They showed that nutrient heterogeneity interacted with PCO2 and nutrient availability to determine different aspects of the productivity and the nutrient uptake patterns of communities. Nagashima et al. (2003) investigated the effects of PCO2 and nutrient availability on size inequality within populations of Chenopodium album. They found that size inequality increased at elevated PCO2, and that the magnitude of this response was modulated by nutrient availability.

We conducted a microcosm experiment to evaluate, for what we believe to be the first time, the effects of PCO2, nutrient availability and nutrient heterogeneity on individual size differences within populations of Lolium, Plantago and Holcus. These species commonly occur together in semi-natural temperate grasslands (Joshi et al., 2000), but differ in the magnitude of their responses to PCO2, nutrient heterogeneity and availability (Poorter, 1993; Kirkham et al., 1996; Grime and Mackey, 2002). We tested the following hypotheses: (a) nutrient heterogeneity promotes size-asymmetric competition (Schwinning and Weiner, 1998); and (b) nutrient heterogeneity interacts with both PCO2 and nutrient availability to determine the magnitude of such a response (Nagashima et al., 2003; Maestre et al., 2005).

MATERIALS AND METHODS

Experimental design

The experiment, conducted in the Duke University Phytotron between January 3 and April 4, 2005, had four treatments: two atmospheric PCO2s (37·5 Pa and 70 Pa), two fertilization applications (40 and 120 mg of N added as organic material), two spatial distributions of the organic material (homogeneous and heterogeneous) and three species (Lolium, Holcus and Plantago). Microcosms consisted of PVC pipe (length 38 cm, internal diameter 10 cm) filled with, from the base, 5 cm of gravel (for drainage), and then 28 cm of a 50 : 50 mixture of sandy-loam soil and sand, as described in Maestre et al. (2005). On top of this was a 2 cm layer of a 50 : 50 mixture of organic soil : peat to resemble the typical accumulation of organic matter in the topsoil of temperate grasslands. To recreate realistic microbial communities, all the microcosms were irrigated with 200 mL of a fresh soil solution and with 100 mL of a solution derived from root macerations (Maestre et al., 2005).

The two nutrient availabilities were 1·036 and 3·108 g of finely cut (2 mm) Trifolium shoots (3·9% N, 10·8 C : N) in the low and high nutrient availability treatments, respectively. These are equivalent to adding 40  and 120 mg of N per microcosm, respectively. Within each of these nutrient applications, the Trifolium shoots were added homogeneously (homogeneous treatment) or as a patch (heterogeneous treatment). In the homogeneous treatment, we thoroughly mixed the organic material with the soil before filling the PVC pipe. In the heterogeneous treatment, the organic material was placed within discrete 31 cm3 volumes of soil. To create one of these patches, 25 cm3 of soil was mixed with the organic material and placed into a 31 cm3 plastic cylinder (length 75 mm; internal diameter 23 mm) consisting of a light mesh with square pores 5 × 10 mm in size. We refer to this as the patch cylinder. A second (control) cylinder, filled only with background soil, was placed 2 cm away from and parallel to the patch cylinder. Cylinders were located 12 cm below the surface of the organic soil. In the homogeneous treatments, both cylinders were filled with the mixture of background soil and organic material.

Seeds from the three species were obtained from commercial suppliers (Lolium seeds were provided by Granite Seed Company, Lehi, UT; Plantago and Holcus seeds by V & J Seed Service, Woodstock, IL, USA). These were germinated in a growth chamber (20 °C temperature and PAR of 350 μmol m−2 s−1 with a 14 h photoperiod) on different days to ensure that all the species had a similar size (two-leaf stage) at the start of the experiment. Each microcosm contained six seedlings of a single species. The planting positions of the six seedlings were allocated at random, but were the same in all microcosms. Seedlings that died during the first week of the experiment were replaced. After that period, no further mortality was observed. We established four replicated microcosms for each of the 24 treatment combinations, resulting in 96 microcosms in total. The microcosms were placed in four walk-in growth chambers (two for each PCO2), within which atmospheric temperature and PCO2 were independently controlled. For each PCO2, half of the microcosms per combination of treatments were randomly assigned to one of the chambers (24 microcosms per chamber), and then were randomly grouped on two trolleys. To minimize possible chamber effects, the PCO2 levels and trolleys were rotated between chambers every week, and the position of the microcosms within the chamber was randomized after every rotation. Strictly speaking, this does not eliminate the problem of pseudoreplication, but we are confident that any chamber effect was negligible because the PCO2 and trolleys were switched 12 times during the experiment. At harvest, all the microcosms had spent the same amount of time in each chamber. The chambers were maintained at a day/night air temperature of 27/20 °C, a 15 h photoperiod and an average relative humidity of 70 % during the day and 85 % during the night. To prevent seedlings being exposed to high-light shock, PAR was maintained at 500 μmol m−2 s−1 during the first week of the experiment, 750 μmol m−2 s−1 during the second week and at 1000 μmol m−2 s−1 thereafter. These conditions included a simulated dawn and dusk period, each of 2 h duration, where temperature, radiation and humidity were gradually increased or decreased. Each microcosm was irrigated daily with 30 ml of distilled water during the first 2 weeks of the experiment, and with 50 ml thereafter. This irrigation scheme resulted in appropriate conditions for plant growth, as indicated by the lack of mortality during the course of the experiment. Because of low overall soil fertility, all the microcosms were watered with 50 mL of a nutrient solution containing 35 mg of Ca (added as CaCl2·2H2O) and 29 mg of Mg (added as MgSO4.7H2O) twice during the course of the experiment (1 February and 1 March).

Measurements and harvest

After 90 d of growth, the plants were harvested. Each individual plant was clipped at the soil surface, and dried at 60 °C to constant mass. Once the above-ground biomass was removed, volumetric soil moisture (0–12 cm depth) was measured in all the microcosms using a HydroSense probe (Campbell Scientific, Logan, UT, USA). After these measurements, the soil was carefully removed from the microcosm unit and the roots were harvested and dried as above. Roots were so entangled that it was not possible to separate those belonging to each individual. We harvested the roots within control and patch cylinders separately from the bulk soil. To standardize measurement of root proliferation (defined here as the biomass of roots per unit of soil volume) into nutrient patches, the index RII was used (Armas et al., 2004). RII was calculated as (RBp − RBc)/(RBp + RBc), where RBp and RBc are the root biomass in the patch and control cylinders, respectively. In the homogeneous treatment, the cylinder placed in the same location as the patch cylinder in the heterogeneous treatment was treated as the patch cylinder for the purposes of calculating the RII index. RII ranges from –1 to +1: a value of zero indicates equal root growth in nutrient patches and background soil.

Size inequality within each population was estimated using two variables: the coefficient of variation of above-ground biomass per microcosm (CVABO; Weiner, 1990) and the combined biomass of the two largest individuals per microcosm (Casper and Cahill, 1998). The combined biomass of the two smallest individuals per microcosm was also calculated. Under size-asymmetric competition, one would expect that the largest plants would get larger at the expense of the smallest plants, which would, in absolute terms, therefore get smaller. Thus, increases in CVABO and/or in the biomass of the largest individuals, accompanied by a decrease in the biomass of the smallest individuals in the latter case, would indicate an increase in size inequality within the population (Casper and Cahill, 1998; Nagashima et al., 2003). If such an increase occurs in response to nutrient heterogeneity, it would indicate that this factor promotes size-asymmetric competition, according to our first hypothesis.

Statistical analyses

The effects of PCO2, nutrient availability (NA) and nutrient heterogeneity (NH) on CVABO, the combined biomass of the two largest and smallest individuals and soil moisture were evaluated separately for each species with a three-way analysis of variance (ANOVA) model. In this model, all the factors were considered fixed, and all main effects and interactions were tested against the residual error. To evaluate if the initial distance to the nutrient patch (on a horizontal scale) affected plant growth in heterogeneous treatments, above-ground biomass data were also analysed separately for each species using a four-way nested ANOVA model. The model included PCO2 and NA as fixed between-plots factors, planting position as a fixed within-plot factor and microcosm as a random factor nested within PCO2 and NA. In this model, the effects of PCO2 and NA, and their interaction, were tested against the random effect of the microcosm. The main effect of planting position and the two- and three-term interactions involving this factor were tested against the interaction microcosm × planting position (Quinn and Keough, 2002). Relationships between root proliferation and the estimators of size inequality were evaluated in the heterogeneous treatment using linear and non-linear (quadratic, logarithmic, power and exponential) functions. Among those models that significantly fitted the data, we selected the model that minimized the second-order Akaike's information criterion (Sugiura, 1978), after examining the residuals for normality and homoscedasticity. Prior to statistical analyses, the CVABO and the biomass of the two largest and smallest individuals of Lolium were log-transformed to meet the homogeneity of variances assumption of ANOVA. Where appropriate, Tukey's HSD test was used for pairwise post hoc comparisons. Although we conducted a large number of statistical tests, P-values were not adjusted for multiple testing as this approach is considered overly conservative (Gotelli and Ellison, 2004). All the statistical analyses were performed using SPSS 10.0 (SPSS Inc., Chicago, IL, USA).

RESULTS

When the organic material was supplied heterogeneously, roots of all the species proliferated in the nutrient patches (Fig. 1). The magnitude of this response was not modified by PCO2 or NA for either Plantago or Holcus (ANOVA, P > 0·215 in all cases). A marginally significant effect of NA on this variable was found for Lolium (ANOVA, F1,12 = 4·09, P = 0·066; no interactions), indicating a trend towards greater root proliferation in the high NA treatment (Fig. 1).

Fig. 1.

Fig. 1.

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Proliferation of roots into the nutrient patches, as measured with the RII index, in soil microcosms differing in nutrient availability, heterogeneity and CO2 partial pressure. Values are means ± 95 % confidence intervals (n = 4). Significant root proliferation is indicated by confidence intervals that do not overlap 0.

Both the CVABO and the biomass of the largest individuals increased in the three species when nutrients were heterogeneously supplied (Fig. 2). Significant PCO2 × NA × NH interactions, as well as PCO2 × NH and NA × NH interactions modulated these responses in Plantago (Table 1), showing that the effect of NH on size inequality within the population was more pronounced under high nutrient supply, especially at elevated PCO2 (Fig. 2). No significant two- or three-way interactions were found when analysing the data from Lolium, but the biomass of the largest individuals increased under high NA conditions. The NH × NA interaction for this variable was significant in Holcus (Table 1); as with Plantago, the effect of NH on the biomass of the largest individuals was more pronounced under high nutrient supply. Biomass was also increased under elevated PCO2 for Plantago (Table 1, Fig. 2). The biomass of the smallest individuals increased under high nutrient supply in the three species (Fig. 2), but was not affected by NH (Table 1). Elevated PCO2 also increased it for Holcus.

Fig. 2.

Fig. 2.

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Biomass of the two largest (A) and smallest (B) individuals and coefficient of variation of above-ground biomass (C) in soil microcosms differing in nutrient availability, heterogeneity and CO2 partial pressure. Data are means ± 1 s.e. (n = 4).

Table 1.

Results of analysis of variance showing the effects of CO2 partial pressure (PCO2), nutrient heterogeneity (NH), nutrient availability (NA) and their interactions on the biomass of the two largest (BMAX) and smallest (BMIN) individuals, and on the coefficient of variation of above-ground biomass (CVABO)

BMAX
 BMIN
 CVABO
Species Source of variation F1,24 P F1,24 P F1,24 P
Plantago lanceolata PCO2 4·10 0·054 0·15 0·701 0·24 0·628
NH 63·25 <0·001 0·05 0·819 42·65 <0·001
NA 86·11 <0·001 22·55 <0·001 11·53 0·002
PCO2 × NH 5·45 0·028 0·81 0·376 1·75 0·199
PCO2 × NA 2·23 0·148 8·09 0·009 5·54 0·027
NH × NA 24·07 <0·001 1·31 0·264 5·46 0·028
PCO2 × NA × NH 4·61 0·042 4·18 0·052 7·39 0·012
Lolium perenne PCO2 0·44 0·514 1·30 0·265 1·13 0·297
NH 43·45 <0·001 0·12 0·734 16·90 <0·001
NA 74·85 <0·001 45·88 <0·001 1·04 0·318
PCO2 × NH 0·21 0·655 1·22 0·280 0·03 0·867
PCO2 × NA 0·01 0·935 0·08 0·781 <0·01 0·970
NH × NA 0·24 0·628 <0·01 0·979 0·14 0·713
PCO2 × NA × NH 0·20 0·656 0·10 0·754 0·14 0·709
Holcus lanatus PCO2 4·30 0·049 4·93 0·036 0·26 0·616
NH 38·28 <0·001 1·39 0·250 7·07 0·014
NA 114·50 <0·001 31·77 <0·001 0·12 0·734
PCO2 × NH 1·94 0·177 0·02 0·889 1·40 0·249
PCO2 × NA 0·99 0·331 0·21 0·654 0·15 0·702
NH × NA 10·50 0·003 2·28 0·144 1·69 0·206
PCO2 × NA × NH 1·71 0·204 0·07 0·793 0·42 0·524
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Results for Lolium are shown for log10-transformed data. P-values below 0·05 are in bold.

In the heterogeneous treatments, seedlings of Plantago and Holcus planted nearest to the nutrient patch had more biomass than those furthest away (Fig. 3). No significant interactions between planting position and the remaining factors were found in any species (ANOVA, P > 0·105 in all cases). Root proliferation in the heterogeneous treatments was positively and exponentially related to the biomass of the two largest individuals and to the CVABO of Lolium and Holcus, respectively (Fig. 4). Soil moisture increased under elevated PCO2 in Plantago and Holcus microcosms (Fig. 5; Plantago, F1,24= 6·33, P = 0·019; Holcus, F1,24 = 11·27, P = 0·003), and decreased under high NA conditions in the latter (F1,24 = 30·68, P < 0·001). A significant NA × NH interaction was found for this variable in Plantago microcosms (F1,24 = 12·07, P = 0·002). Soil moisture in Lolium microcosms was not affected by any of the factors evaluated.

Fig. 3.

Fig. 3.

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The dependence of above-ground biomass of individual plants in the heterogeneous nutrient treatment on the distance each seedling was planted from the nutrient patch. For clarity, the data from the low and high nutrient availability treatments were pooled. Data are means ± 1 s.e. (n = 8). Lower case letters denote significant differences among planting positions (P < 0·05, Tukey's HSD test after four-way nested ANOVA). Post hoc results for Plantago lanceolata are shown for log10-transformed data. Note the different scales on the y-axes.

Fig. 4.

Fig. 4.

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The relationship between root proliferation in the heterogeneous nutrient treatment, as measured with the RII index, and the measures of differences in individual plant size within the population employed: biomass of the two largest individuals (upper panels) and coefficient of variation of individual above-ground biomass (lower panels). Each point represents the value obtained in a single microcosm. Root proliferation is measured in the microcosm, and reflects the summed response of all the individual plants within the microcosm. Only significant relationships (P < 0·05) are drawn.

Fig. 5.

Fig. 5.

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Soil moisture (0–12 cm depth) at harvest for Plantago lanceolata, Lolium perenne and Holcus lanatus grown in microcosms with different nutrient availability, heterogeneity and CO2 partial pressure. Data represent means ± s.e. (n = 4).

DISCUSSION

Our first hypothesis, that nutrient heterogeneity promotes size-asymmetric competition, was supported by the results. This heterogeneity increased both the CVABO and the biomass of the largest individuals in the three species evaluated. Greater nutrient availability also increased the biomass of both the largest and smallest individuals within each microcosm, but the biomass of the smallest individuals remained unaffected by nutrient heterogeneity. The latter result was unexpected. In our experiment, the total biomass of the microcosms increased under heterogeneous nutrient supply (Supplementary Information), a response commonly observed with plant populations and communities (Day et al., 2003a; Maestre et al., 2005, 2006). Thus, it is likely that the reduction in size of the smallest plants is outweighed by increases in total microcosm biomass because of the more efficient extraction of nutrients from patches in heterogeneous environments (Jackson and Caldwell, 1996; Maestre et al., 2005).

It has been suggested that small-scale nutrient heterogeneity is a requisite for below-ground size-asymmetric competition to occur (Rajaniemi, 2003). However, this has not been fully supported by experiments, which have produced conflicting results (e.g. Casper and Cahill, 1998; Blair, 2001; Fransen et al., 2001; Facelli and Facelli, 2002). This is not surprising given the importance of both the experimental context and the nature of interactions (intra- or interspecific) when evaluating plant responses to small-scale nutrient heterogeneity (Wijesinghe et al., 2001; Maestre et al., 2006). In this regard, it is interesting that experiments to evaluate the effect of nutrient heterogeneity on the relationships between a plant's size and its competitive effect have used nutrient solutions, commercial potting mixtures or fertilizer pellets to simulate nutrient heterogeneity. Thus, previous results are not directly comparable with those presented here, where such heterogeneity was simulated using organic material.

We were unable to elucidate the relative importance of plasticity in morphological vs. physiological factors responsible for the increase in size inequality promoted by nutrient heterogeneity. However, the positive relationships found between root proliferation into the nutrient patches and the biomass of the largest individuals and the CVABO observed under heterogeneous conditions for both Lolium and Holcus suggest that this proliferation was an important mechanism driving the responses. Our results suggest that feedbacks between plant size and root proliferation are crucial in determining the effects of nutrient heterogeneity on the size symmetry of competitive interactions. Studies comparing groups of species have often found a positive relationship between plant size/growth rate and root proliferation into nutrient patches (Einsmann et al., 1999; Rajaniemi and Reynolds, 2004; Kembel and Cahill, 2005). Similar comparative studies on intraspecific variability in growth and size, and in root proliferation are lacking, but it is reasonable to argue that the largest individuals were those with the greatest proliferation of roots in nutrient patches. Despite having a similar initial size, some seedlings may have encountered the nutrient patch sooner than others. Individual plants quickly proliferate roots when they encounter a nutrient patch (Hodge, 2004), which increases nutrient uptake and overall plant growth (Hodge et al., 1999; Maestre et al., 2005). This, in turn, further stimulates root proliferation via the relationship between this process and growth rate (Fransen et al., 1999), in a positive feedback that increases growth until the supply of nutrients is exhausted (Fransen et al., 2001). The fact that individuals of Plantago and Holcus close to the nutrient patch were larger than those further away supports this view.

Nutrient supply and PCO2 modified the magnitude of the effects of nutrient heterogeneity in Plantago and Holcus, but not in Lolium. Thus, our second hypothesis, that nutrient heterogeneity interacts with both nutrient availability and PCO2 to determine the magnitude of size-asymmetric competitive responses, was partially supported by the results. In terms of biomass production, the three species evaluated have been shown to respond positively to elevated PCO2 (Poorter, 1993), nutrient heterogeneity (Grime and Mackey, 2002) and increases in nutrient supply (Kirkham et al., 1996). However, they differ in the magnitude of these responses, as well as in key attributes such as relative growth rate (RGR; Lolium has a lower RGR than Holcus and Plantago; Grime and Hunt, 1975). These differences may help to explain why interactions among the evaluated factors were not observed in all of the species. For both Plantago and Holcus, increases in size inequality promoted by nutrient heterogeneity were more pronounced where more nutrient was available, possibly caused by the increase in plant size with fertilization. Under nutrient-limiting conditions, such as experienced by the plants growing with little nutrient, addition of nutrients typically increases plant growth, and such an increase would foster the positive feedback between growth rate and root proliferation. However, other mechanisms not accounted for in this study, such as changes in nutrient uptake rates associated with changes in nutrient availability (Robinson, 1994), could also explain the results. Elevated PCO2 accentuated the combined effects of nutrient availability and heterogeneity on the differences in individual plant size within Plantago populations. This result is in line with observations from Nagashima et al. (2003), who found that elevated PCO2 increased size inequality in populations of Chenopodium album, and that this effect was larger under high nutrient availability conditions. Our results cannot provide a fully mechanistic explanation for this synergistic effect, but it could have been mediated by PCO2-induced changes in nutrient availability and use. Despite having higher biomass, soil moisture at harvest in Plantago microcosms was significantly higher under elevated than under ambient PCO2 conditions, a result probably caused by the increase in the water use efficiency of this species commonly observed under elevated PCO2 (Klus et al., 2001). In our experiment, as in the field, the release of nutrients from added organic material depends on mineralization by the soil microbial and faunal populations, which typically increases with increasing water availability (Ebersberger et al., 2003). Thus, the increased mineralization could have increased nutrient availability for plants under elevated PCO2, which would have increased the feedback between growth rate and foraging precision described above. However, this is speculative as mineralization rates were not measured. Regardless of the underlying mechanisms, our results suggest that the combined effects of nutrient heterogeneity, PCO2 and increased nutrient availability on the differences in the size of individual plants within populations may be non-additive.

Our results must be interpreted with some caution because the experiment did not separate above- and below-ground competitive interactions. Both operate and interact in the field, and the direction and magnitude of such interaction may depend on factors such as species traits and nutrient availability (Cahill, 1999, 2002). Within microcosms, plants probably competed for light, and below- and above-ground competition occurred, influencing each other, especially at later stages of growth and with ample nutrients. However, such interaction did not obscure the effects of nutrient heterogeneity on the differences in individual plant size within the population. Competition for above-ground resources such as light is usually size-asymmetric (Weiner, 1990; Schiwinning and Weiner, 1998). If the differences in size were the consequence of such competition, and not of changes in below-ground competition induced by nutrient heterogeneity, we should have not observed differences between homogeneous and heterogeneous treatments, as both have the same soil resources. However, competition for light could have magnified the effect of nutrient heterogeneity, as differences in size among neighbours resulting from nutrient heterogeneity may have been accentuated by larger plants reducing the light available to smaller neighbours. Detailed analyses of the effects of nutrient heterogeneity on the interactions between above- and below-ground competition are lacking, and should be addressed.

Our first hypothesis was supported by our results, as nutrient heterogeneity promoted size-asymmetric competition in the three species evaluated. They also indicate that the proliferation of roots into nutrient patches was an important driver of this response. As predicted by our second hypothesis, nutrient supply and PCO2 modified the magnitude of the effects of nutrient heterogeneity in Plantago and Holcus, but not in Lolium. We suggest that these differences are caused by species-specific differences in attributes such as relative growth rate. Our results add to studies evaluating the joint effects of nutrient heterogeneity and global change drivers on grassland communities (Maestre et al., 2005, 2006), and suggest that this heterogeneity may be a key environmental factor when interpreting and assessing plant population responses to global change.

SUPPLEMENTARY INFORMATION

The following Supplementary Information is available online at http://www.aob.oxfordjournals.org/. Figure A1: total biomass in soil microcosms differing in nutrient availability, heterogeneity and CO2 partial pressure. Table A1: results of a three-way analysis of variance showing the effects of CO2 partial pressure, nutrient heterogeneity and availability on total microcosm biomass.

Acknowledgments

We thank María D. Puche, José Luis Quero, Anne Rosenbarger and the Duke Phytotron staff for their help during the different phases of this study, and anonymous referees for comments on the manuscript. F.T.M. was supported by a Fulbright fellowship (FU2003-0398), a Ramón y Cajal contract from the Ministerio de Educación y Ciencia, and by the CEFEMED (URJC-RNT-063-2) grant, funded by Comunidad de Madrid and Universidad Rey Juan Carlos. This research was partially supported by USDA Specific Co-operative Agreement #58-1270-3-070, NSF-DEB-02-12123, NSF-SBR-9521914 (Subcontract # 538819-55801 from Carnegie Mellon University).

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