Ecology, 80(3), 1999, pp.

873–881 ᭧ 1999 by the Ecological Society of America

INTERACTIONS BETWEEN PLANT SPECIES AND EARTHWORM CASTS IN A CALCAREOUS GRASSLAND UNDER ELEVATED CO2
JOHANN G. ZALLER1
AND

JOHN A. ARNONE III2

Botanisches Institut, Universita ¨ t Basel, Scho ¨ nbeinstrasse 6, CH-4056 Basel, Switzerland

Abstract. We tested the hypothesis that the spatial proximity of a plant species to nutrient-rich earthworm casts (e.g., 100% more ammonium and 30% more phosphate than in adjacent soil) is an important determinant of a plant’s responsiveness to elevated atmospheric CO2. In 1995 we mapped the location of both earthworm surface casts and plants in each of 16 1.2-m2 plots in a species-rich calcareous grassland in northwestern Switzerland. Eight plots have been maintained under current ambient CO 2 concentrations (350 ␮L CO2/ L), and eight have been maintained at elevated CO2 (600 ␮L CO2/L) since March 1994. In addition, total ramet production of each species, as a measure of performance, and cumulative cast production at each location (cell) were recorded at peak community biomass in 1995. Plant species within functional groups (graminoids, non-legume forbs, and legumes) differed markedly in their degree of association with casts; however, after two growing seasons elevated CO2 had no effect on plant species or functional group associations with casts. No statistically significant relationship could be demonstrated between plantspecies response (i.e., ramet production) to elevated CO 2 and the degree of association with casts within any of the functional groups. However, a positive relationship was observed between the mean response of graminoid species to elevated CO 2 (measured as the percentage change in mean total ramet production of graminoid species, relative to mean total ramet production at ambient CO2) and their mean degree of association (%) with surface casts at ambient CO2. Thus, graminoid species more frequently associated with casts (e.g., Anthoxanthum odoratum and Carex caryophyllea) produced more ramets per square meter at elevated CO2 than those less frequently associated with casts (e.g., Agrostis tenuis and Danthonia decumbens). These results, along with the strong and significant positive correlations observed between ramet production and associated cumulative cast mass across CO2 treatments for most plant species in all functional groups demonstrate: (1) that plant species differ significantly in their degree of association with nutrient-rich earthworm surface casts, regardless of the relative abundance of plant species in the community; (2) that graminoid species that are more highly associated with casts may respond more strongly to rising CO2 than those less highly associated with casts; and (3) that nutrient-rich earthworm casts stimulate the growth (ramet production) of most plant species in these grassland communities, even at current levels of atmospheric CO2. The data further suggest that these species-specific relationships between plants and casts have helped define the current structure of these highly diverse grassland communities and will likely influence their future structure as global CO2 levels continue to rise.
Key words: CO2 enrichment; earthworm activity; earthworm surface casts; elevated CO2, plant species responses; fertile soil microsites, role of earthworm casts; global warming, plant species responses; Lumbricidae; plant–animal interactions; plant–cast spatial distribution; soil ecology; spatial distribution of plants, earthworm effects.

INTRODUCTION Numerous studies suggest that rising atmospheric CO2 will alter the competitive interactions among plant species and thus lead to changes in the structure and composition of plant communities (e.g., Bazzaz 1990,
Manuscript received 10 November 1997; revised 6 April 1998; accepted 13 April 1998; final version received 18 May 1998. 1 Present address: Department of Rangeland Resources and The Ecology Center, Utah State University, Logan, Utah 84322-5230 USA. E-mail: jgz@cc.usu.edu 2 Present address: Biological Sciences Center, Desert Research Institute, P.O. Box 60220, Reno, Nevada 89506 USA.

Ko ¨ rner and Bazzaz 1996). These interactions are influenced strongly by soil fertility, with species in lowfertility ecosystems responding more slowly than those in high-fertility systems (Ko ¨ rner 1996, Reynolds 1996). However, low nutrient supply may not necessarily preclude pronounced effects on plant community structure in the long term (Leadley et al. 1998). In the grassland communities we studied, causes for changing species abundance are numerous but may ultimately derive from species-specific gas-exchange responses to elevated CO2 (Lauber and Ko ¨ rner 1997). However, indirect effects of elevated CO2, such as increases in soil moisture (Niklaus et al. 1998) and greater inputs of carbon to the rhizosphere (e.g., Hungate et al. 1997,

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Meier et al. 1997) may be more important determinants of competitive outcome because of their secondary effects on soil heterotrophs. Indeed, increased soil water content under elevated CO2 appears to be the major cause of the 35% increase in earthworm activity (5.7 ϫ 103 kg more surface-cast dry mass produced per hectare measured over one year) we observed in calcareous grasslands (Zaller and Arnone 1997). This increased activity corresponded to a 30% increase in total N and C egested by earthworms at our site, 70% of which are known surface-casting species (Zu ¨ rcher 1994; J. Zaller, unpublished data). These casts often contain higher concentrations of plant-available nutrients (e.g., Puh 1941, Aldag and Graff 1975) than does the surrounding soil, and may represent nutrient-rich microsites that plants can exploit. Casts may even contain plant growth substances (Gavrilov 1963, Nielson 1965, Tomati et al. 1988). In many plant communities, nutrient-rich soil microsites represent a valuable source of mineral nutrients (Chapin 1980, Grime et al. 1986, Caldwell et al. 1991). A plant’s ability to exploit such spatially and temporally heterogeneous soil resources, either through enhanced fine-root proliferation (e.g., Drew and Saker 1975, St. John et al. 1983, Crick and Grime 1987, Jackson and Caldwell 1989, Jackson and Caldwell 1992, Bilbrough and Caldwell 1997) or improved uptake kinetics (Jackson et al. 1990, Jackson and Caldwell 1991) can affect the competitive balance among species in a community (e.g., Jackson and Caldwell 1992, Bilbrough and Caldwell 1997). Actually, fine roots have been observed to proliferate extensively in earthworm casts (Springett and Syers 1979, Spiers et al. 1986, J. Zaller, personal observation), and at our grassland site the distribution of surface casts and plant species are spatially quite heterogeneous at scales of less than 1 m. These patterns led us to test several hypotheses related to the spatial relationships between plants and casts that may determine species’ responses to elevated CO2. We tested the hypothesis that plant species more highly associated with surface casts would respond more positively to elevated CO2 by producing more ramets, than species less associated with casts, because of greater nutrient availability in casts relative to adjacent soil. The objectives of our study were to determine: (1) whether plant species or functional groups in these grassland communities differ in their association with surface casts at current ambient atmospheric CO2 concentrations, (2) whether elevated CO2 alters these associations, (3) if species and functional groups more closely associated with casts produce more ramets under elevated CO2 than those less closely associated, and (4) how well production of ramets among plant species and functional groups is related to the associated cast mass. MATERIALS AND METHODS Study site, CO2 enrichment, and experimental design We conducted this study in a species-rich calcareous grassland in the Jura Mountains located near Basel,

Switzerland (520 m; 47Њ27Ј N, 7Њ34Ј E) on a southwestfacing slope (20Њ). Annual precipitation averages 900 mm, with mean annual air temperatures between 8.5Њ and 9.0ЊC (Ogermann et al. 1994). The grassland is dominated by the tussock grass Bromus erectus but contains Ͼ75 other vascular plant species (HuovinenHufschmid and Ko ¨ rner 1998). Until 1993 the area was managed mainly as cattle pasture. Since then it has been mown annually in early summer and early fall. Mean aboveground net primary productivity is ϳ400 g·mϪ2·yrϪ1, 70% of which is represented by graminoid species (Huovinen-Hufschmid and Ko ¨ rner 1998, Leadley et al. 1998). Soils are classified as a transition Rendzina with a well-developed, stone-free, loamy topsoil (Ah horizon: pH 6.5, bulk density of 1.1 g/cm3, organic carbon content 3.2%, C:N ratio ϳ12) and a rapid transition near 15 cm depth to the underlying scree material (Ogermann et al. 1994). Since March 1994 eight plant communities on 1.2m2 hexagonal experimental plots have been maintained at current ambient CO2 concentrations (‘‘A,’’ 350 ␮L CO2/L) and eight at elevated CO2 (‘‘E,’’ 600 ␮L CO2/ L) using the Screen Aided CO2 Control technology (SACC, Leadley et al. 1997). Each SACC unit consisted of a 50-cm-tall ϫ 1.2-m-diameter (hexagonal) transparent plastic screen (open bottom and open top), and a pipe at the base of the screen through which CO2enriched air is directed into the unit. Plots were arranged over an area of 1400 m2 in a randomized complete block design with eight blocks (i.e., one A and one E plot per block). CO2 treatments were maintained continuously, except during the winter months (December to March) when CO2 enrichment was stopped.

Plant species spatial distribution and ramet production
We determined the presence or absence of vascular plant species in each of 100 3 ϫ 3 cm cells in a 30 ϫ 30 cm area in each experimental plot from 19 July to 1 August 1995 (Fig. 1, top). The frequency of each species was expressed as the percentage of the cells in which any part of a plant of that species was rooted (i.e., rooted frequency, Greig-Smith 1983). The number of ramets of each plant species in each cell also was counted as an indicator of performance and growth potential of that species. The response of each plant species to elevated CO2 was calculated for each block as the difference between the total number of ramets observed in the 30 ϫ 30 cm area at ambient CO2 and the total number observed at elevated CO2 divided by the total number observed at ambient CO2, expressed as a percentage. Each plant species was assigned to one of three functional groups based on taxonomy—graminoids (Poaceae, Cyperaceae and Juncaceae), nonleguminous forbs, and leguminous forbs—to assess whether relationships with casts and responses to elevated CO2 occurred at this more aggregated level of plant community organization. Only plots in which a

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FIG. 1. Schematic diagram of method used to collect plant and cast data (grid cell size: 3 ϫ 3 cm). Ramets of each plant species were counted if its roots were present in a grid cell. Cumulative earthworm surface cast production was calculated for each cell over the 10-wk measurement period.

species occurred were used to assess the response of ramet production to elevated CO2. All data are expressed on an areal basis. Nomenclature for plant species follows Binz and Heitz (1990).

density near the study site censused using the electrocapture method averaged 150 worms/m2 (see Zaller and Arnone 1998).

Earthworm community and spatial distribution of earthworm surface casts
On the same 30 ϫ 30 cm grid we also measured earthworm surface-cast production in each cell every 9 d (see Zaller and Arnone 1997) from 5 April to 19 July 1995. Cumulative cast production over this period was calculated for each of the 100 grid cells (Fig. 1 bottom). The overall size of the 30 ϫ 30 cm grid and individual cells were chosen to enable the tracking of individual plants (Huovinen-Hufschmid and Ko ¨ rner 1998) and casts (see below). Individual surface casts were weighed in the field, and several subsamples from each plot were taken at each sampling date to calculate fresh mass/dry mass ratios. After weighing, each cast was slightly deformed (flattened to prevent recounting) and returned immediately to its original position in order to maintain the natural pattern of cast distribution. Methodological details of cast sampling are provided in Zaller and Arnone (1997). A total of nine earthworm species was recorded (nomenclature follows Bouche ´ 1972) near the study site, representing three ecological groups (Bouche ´ 1977): anecics (Lumbricus terrestris L., Nicodrilus longus Ude., and N. nocturnus Ev.), endogeics (Allolobophora chlorotica Sav., A. rosea Sav., N. caliginosus Sav., and Octolasion cyaneum Sav.) and epigeics (Dendrobaena mammalis Ger. and L. castaneus Sav.). Total earthworm

Plant species–cast association
To check whether our selection of a 3 ϫ 3 cm cell size was statistically appropriate for our questions, we calculated the dispersion index (Krebs 1989) for worm casts and for each plant species in each 30 ϫ 30 cm area in the ambient-CO2 plots. The dispersion index describes whether the spatial distribution of surface casts or of an individual plant species, is random, uniform, or aggregated. Goodness-of-fit of each dispersion index to a random Poisson distribution was tested using chi-square (Zar 1996). We tested cell sizes of 3 ϫ 3 cm, 6 ϫ 6 cm, and 9 ϫ 9 cm. To calculate the plant– cast association of each species, we used the cell size that produced an aggregated dispersion pattern of that plant species and the casts. For 22 of the 28 plant species considered, a 3 ϫ 3 cm cell size was most appropriate. The remaining six species (four non-legumes, two legumes) would have required larger grid sizes than the largest (i.e., 9 ϫ 9 cm) that could be considered in our experiment. Despite this situation we included all six in the plant species–cast association analyses. The degree to which a plant species was associated with casts was calculated as the percentage of the total number of cells in which both that species and casts were present divided by the total number of cells in which the plant species was present.

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Plant species performance vs. cumulative cast production
To assess plant species’ growth responses to casts, we took the total number of ramets of each plant species from individuals growing in association with casts in each 30 ϫ 30 cm sampling area on each plot, and plotted this as a function of the total cumulative cast mass associated with each species (both expressed on an areal basis). This was done at peak plant community biomass.

Available nutrients in casts and adjacent soil
On 5 June and 11 July 1996, when surface cast production was high, we collected three fresh surface casts (not older than 3 d) and adjacent topsoil (to a depth of 1.5 cm from areas with no casts present) from each experimental plot. Samples from each plot were pooled and stored in plastic vials overnight on ice. Material from each vial was mixed and sieved (0.7-mm mesh) to remove fine roots. To determine ammonium (NH4ϩN) and nitrate (NO3Ϫ-N) nitrogen concentrations, 2.0 g of fresh mass was extracted with 20 mL of 2 mol KCl/L solution by shaking for 1 h. The same procedure was used to extract phosphate-P (PO4Ϫ-P), except that 0.5 mol NaHCO3/L was used as extractant. Filtered extracts were analyzed on a Flow Solution segmented flow analyzer (Perstorp Analytical, Perstorp, Sweden). NH4ϩ-N was determined using the indophenol method (Keeney and Nelson 1982). NO3Ϫ-N was reduced to nitrite (NO2Ϫ) by Cd then determined using the GriessIlosvay method (as described in Keeney and Nelson 1982). PO4Ϫ-P was determined using the molybdateascorbic acid method (Olsen and Sommers 1982). Subsamples of fresh cast and adjacent soil were dried at 80ЊC to calculate water content and allow expression of nutrient concentration on a dry-mass basis.

duction and the mass of the associated casts for each species and CO2 level. To test for differences in slope between CO2 treatments, we used two-way ANOVAs (with slopes calculated from the linear regressions for each plot and species as dependent variables, and CO2 concentration and block as factors). The final analysis at the species-within-functional-group level was a twoway ANOVA to test for effects of elevated CO2 on ramet production, with CO2 and block as factors. Analogous analyses were conducted at the level of the functional group. Nutrient concentrations in casts and adjacent soil for the two sampling dates were tested using three-way repeated-measures ANOVAs with CO2, block, and substrate type (i.e., cast or soil) as factors. All analyses were conducted with plot as the experimental unit. We considered P Ͻ 0.05 to be ‘‘significant’’ and 0.05 ϽP Ͻ 0.10 to be ‘‘marginally significant’’. All analyses were performed using SYSTAT (Wilkinson et al. 1992). RESULTS

Plant species association with casts
Plant species within each of the three functional groups differed significantly in their association with earthworm casts at ambient CO2 (Table 1, Table 2a). Often plant species that were less abundant were associated to a greater degree with casts than were the more abundant plant species. Mean associations ranged from 43 to 66% for graminoid species, from 48 to 77% for non-legume forbs, and from 8 to 83% for legumes (Table 1). Elevated CO2 had no effect on the degree of association of plant species with casts within each of the three functional groups (Table 2b). However, nonlegume forbs and legumes differed in their association with casts under ambient and elevated CO2 (i.e., significant interactions, Table 2b: P ϭ 0.036, and P ϭ 0.081, respectively), with some species exhibiting reductions and others increases. At the level of the functional group no significant differences were observed in association with casts at either ambient or elevated CO2 (Table 2a and b).

Statistical analyses
Although we recorded a total of 60 plant species among all 16 A and E plots, our analyses included only those species (28) present on at least two plots per CO 2 treatment. To evaluate whether plant species within each of the functional groups differed in their degree (expressed as %) of association with casts at ambient CO2 (n ϭ 8 replications), we used a two-way ANOVA with species and block as factors. The effects of elevated CO2 on plant species–cast association within each functional group was tested using a three-way ANOVA with CO2 concentration, species, and block as factors. All percentage data were arcsin transformed prior to statistical analysis (Sokal and Rohlf 1981). Linear regression analysis was used to assess the relationship between plant species’ responses to elevated CO2 and the degree of association with casts within each of the functional groups (using differences of treatment means). We also used linear regression analysis to evaluate the relationship between plant species’ ramet pro-

Plant species responses to elevated CO2 as a function of association with casts and cumulative cast production
Total ramet production was not related to the degree that a species, or functional group, was associated with casts at either CO2 level (Table 2c). We did, however, see a positive (P ϭ 0.007, r ϭ 0.789) relationship between the response of graminoid species to elevated CO2 (measured as the percentage change in mean total ramet production of graminoid species, relative to mean total ramet production at ambient CO2) and their mean degree of association (in percentage) with surface casts at ambient CO2 (Fig. 2, Table 3). Thus, graminoid species more frequently associated with casts (e.g., Anthoxanthum odoratum and Carex caryophyllea) pro-

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TABLE 1. Degree of plant species association with earthworm surface casts, and the frequency of plant species in grassland plots maintained at ambient (Amb., 350 ␮L CO2/L) and elevated (Elev., 600 ␮L CO2/L) atmospheric CO2 (data are means Ϯ 1 SE). Plant species within each of the three functional groups are ranked by their degree of association with casts at ambient CO2. No. plots† Functional group Graminoids Carex caryophyllea Dactylis glomerata Anthoxanthum odoratum Carex flacca Danthonia decumbens Bromus erectus Cynosurus cristatus Festuca rubra Agrostis tenuis Luzula campestris Non-legume forbs Cerastium fontanum Betonica officinalis Hieracium pilosella Sanguisorba minor Prunella vulgaris Linum catharticum Plantago lanceolata Thymus serpyllum Leucanthemum vulgare Legumes Medicago lupulina Trifolium medium Trifolium spp.‡ Lotus corniculatus Amb. CO2 8 8 5 4 8 5 8 8 8 8 6 8 5 3 4 7 8 6 3 4 3 8 3 5 6 4 Elev. CO2 8 8 5 6 8 8 8 6 8 8 2 8 2 3 3 7 7 5 3 2 3 8 2 2 6 5 Plant species–cast assoc. (%) Amb. CO2 56.7 65.7 65.0 64.6 58.4 56.0 54.8 52.9 52.8 52.1 43.3 59.2 76.7 66.7 62.5 61.9 55.3 52.1 50.0 50.0 48.3 55.6 83.3 66.0 64.6 8.3
Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

Plant species frequency (%) Amb. CO2 81.4 18.1 1.1 2.0 9.4 5.9 61.8 4.5 15.1 28.0 2.3 19.8 2.0 0.9 1.3 2.1 7.8 3.8 0.9 1.0 2.0 7.8 0.5 2.1 5.5 0.9
Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

Elev. CO2 52.5 50.7 74.3 56.5 51.8 36.3 53.2 50.4 60.4 54.5 75.0 57.7 75.0 22.2 25.0 59.7 57.7 55.0 70.0 100.0 83.3 32.9 75.0 0.0 40.6 20.0
Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

Elev. CO2 90.3 29.5 2.5 5.4 12.1 5.1 62.1 5.8 8.9 25.6 0.4 18.4 0.5 1.0 0.6 4.6 8.1 1.1 1.1 0.4 1.5 5.4 0.4 0.3 2.6 1.6
Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

2.0 8.7 8.1 3.3 4.1 6.4 1.7 6.2 10.3 1.6 12.9 2.1 11.5 10.2 7.5 2.8 10.5 4.5 5.8 17.7 9.3 9.0 10.2 14.9 10.1 5.9

2.9 7.8 9.8 7.1 12.1 7.2 3.2 11.3 7.9 4.6 12.5 6.2 12.5 13.6 12.5 8.7 9.6 18.1 15.8 0.0 10.2 9.3 12.5 0.0 13.1 9.7

7.3 4.0 0.6 0.8 1.5 3.4 2.7 1.0 4.2 4.9 0.8 4.4 0.8 0.5 0.6 0.7 2.2 1.6 0.4 0.7 1.3 2.7 0.3 0.9 2.3 0.4

2.7 5.7 0.9 1.5 3.6 1.2 3.3 1.9 1.9 4.4 0.3 3.9 0.3 0.7 0.5 1.3 3.2 0.5 0.5 0.3 0.9 1.5 0.3 0.2 0.7 0.8

† Number of plots where each plant species occurred. ‡ Trifolium spp. include: Trifolium campestre, T. montanum, T. ochroleucon, T. pratensis, and T. repens.

duced more ramets per square meter at elevated CO2 than those less frequently associated with casts (e.g., Agrostis tenuis and Danthonia decumbens). Total ramet production of species associated with

casts increased significantly with increasing cast mass in 9 of the 10 graminoid species (Fig. 3), 5 of the 9 non-legume forb species (r values ranged from 0.80 to 0.98; P values for linear regression from Ͻ0.001 to

TABLE 2. Results from ANOVAs for (a) differences between plant species–earthworm cast associations at ambient CO2, (b) effect of elevated CO2 on plant–cast associations, and (c) effect of elevated CO2 on ramet production. Within functional groups Source of variation Graminoids df Non-legume df Legumes df 3 7 11 3 1 7 3 18 3 1 7 3 16 Between functional groups, FG

P

P

P
0.021 0.055

Source of variation FG Block Error FG CO2 Block FG ϫ CO2 Error FG CO2 Block FG ϫ CO2 Error

df 2 7 13 2 1 7 2 33 2 1 7 2 35

P
0.868 0.446

a) Plant species–cast associations at ambient CO2 Species 9 0.046 8 0.086 Block 7 0.007 7 0.946 Error 49 25 b) Effects of elevated CO2 on plant–cast associations Species, Spp. 9 0.020 8 0.160 CO2 1 0.162 1 0.851 Block 7 0.001 7 0.057 Spp. ϫ CO2 9 0.347 8 0.036 Error 106 50 c) Effects of elevated CO2 on production of ramets per m2 Species, Spp. Ͻ0.001 9 8 0.017 CO2 0.925 1 1 0.719 Block 0.916 7 7 0.069 Spp. ϫ CO2 0.794 9 8 0.589 Error 106 50

Ͻ0.001 0.188 0.031 0.081

0.283 0.285 0.234 0.498

0.341 0.503 0.630 0.823

Ͻ0.001 0.807 0.588 0.973

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DISCUSSION Our results demonstrate that plant species differ significantly in their degree of association with nutrientrich earthworm surface casts, regardless of the relative abundance of plant species in the community. However, dramatically increased carbon supply to communities maintained at elevated CO2 (Stocker et al. 1997) did not affect the degree to which plant species are associated with casts (Table 1). Clearly casts had a profound effect on ramet production for most of the species occurring in the communities when only plants associated with casts were considered; i.e., increasing total ramet production with increasing cumulative cast production per mass (Fig. 3). This effect most likely was due to higher nutrient availability in larger casts. We and others (Springett and Syers 1979, Spiers et al. 1989) have observed fine-root proliferation into casts, indicating that plants can exploit these nutrients. Differences in the abilities of species to exploit these nutrient patches (sensu Jackson and Caldwell 1992, Bilbrough and Caldwell 1997), especially in the spring when casting activity (Zaller and Arnone 1997) and plant growth and nutrient demand are greatest, may alter the competitive
TABLE 3. Number of ramets for each plant species in plots maintained at ambient (Amb., 350 ␮L CO2 / L) and elevated (Elev., 600 ␮L CO2 / L) atmospheric CO2. Plant species within functional groups are ranked by number of ramets at ambient CO2 (data are means Ϯ 1 SE). See Table 1 for details about individual species performance. Number of ramets/m2† Functional group Amb. CO2 3756 2082 647 303 285 224 125 97 75 51 36 274 90 89 72 39 38 37 30 28 27 86 48 44 22 15
Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

FIG. 2. Response of graminoid species to elevated CO2 (measured as the percentage change in mean total ramet production of graminoid species, relative to mean total ramet production at ambient CO2) as a function of their mean degree of association with surface casts at ambient CO2. Plant species key: Agr ten ϭ Agrostis tenuis, Ant odo ϭ Anthoxanthum odoratum, Bro ere ϭ Bromus erectus, Car car ϭ Carex caryophyllea, Car fla ϭ Carex flacca, Cyn cri ϭ Cynosurus cristatus, Dac glo ϭ Dactylis glomerata, Dan dec ϭ Danthonia decumbens, Fes rub ϭ Festuca rubra, Luz cam ϭ Luzula campestris. See Table 1 for details about individual species performance. The line was fitted by linear regression.

Elev. CO2 3762 1988 518 499 263 85 163 139 96 27 67 219 113 44 24 15 22 17 37 33 59 53 42 17 29 17
Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

0.02), and 2 of the 4 legume species across both CO 2 treatments (r values ranged from 0.58 to 0.95; P values for linear regression from Ͻ0.001 to 0.05). However, elevated CO2 had no effect on the positive response to increasing cast mass (data only shown for graminoid species; Fig. 3).

Surface cast production and nutrient concentrations
Cumulative surface cast production measured between April and August 1995 (as both grams dry mass per square meter and number of casts per square meter) and average area of soil covered by casts were unaffected by elevated CO2 (Table 4). The concentrations of ammonium, nitrate, and phosphate were also not affected by elevated CO2 in either of the two sampling dates (NH4ϩ-N: P ϭ 0.470; NO3Ϫ-N: P ϭ 0.578; PO4ϪP: P ϭ 0.257). However, per unit dry mass the casts contained 100% more NH4ϩ-N (9.05 Ϯ 1.39 mg/g vs. 4.44 Ϯ 0.49 mg/g for casts vs. soil, respectively), almost 30% more PO4Ϫ-P (2.27 Ϯ 0.20 mg/g vs. 1.79 Ϯ 0.19 mg/g for casts vs. soil, respectively), but similar amounts of NO3Ϫ-N (8.11 Ϯ 1.13 mg/g vs. 7.32 Ϯ 0.61 mg/g for casts vs. soil, respectively) than adjacent topsoil, averaged across sampling dates and CO2 treatments.

Graminoids Bromus erectus Agrostis tenuis Carex caryophyllea Festuca rubra Danthonia decumbens Carex flacca Anthoxanthum odoratum Cynosurus cristatus Luzula campestris Dactylis glomerata Non-legume forbs Prunella vulgaris Leucanthemum vulgare Linum catharticum Plantago lanceolata Cerastium fontanum Thymus serpyllum Betonica officinalis Hieracium pilosella Sanguisorba minor Legumes Trifolium spp.‡ Trifolium medium Lotus corniculatus Medicago lupulina

203 173 115 81 91 134 22 30 20 10 12 76 27 51 31 6 10 26 13 7 8 32 28 17 6 4

230 124 141 117 75 22 47 24 31 6 19 48 42 17 8 4 0 6 21 22 14 19 9 6 11 6

† Only plots in which a plant species occurred were included in the calculations. ‡ Trifolium spp. include: T. campestre, T. montanum, T. ochroleucon, T. pratensis, and T. repens.

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FIG. 3. Total ramet production of graminoid species as a function of associated total cumulative surface cast production in grassland communities maintained at ambient (350 ␮L CO2 / L; ⅜) and elevated (600 ␮L CO2 /L; ⅷ) atmospheric CO2. Lines indicate linear regressions across CO2 treatments; results of linear regression analyses across CO2 treatments for each species are given on the figure. Note varying scales on both axes.

balance among plant species in these communities. The lack of an apparent correlation between cast mass and ramet (tiller) production of Bromus erectus, the most dominant species in these communities (43% of aboveground biomass, Huovinen-Hufschmid and Ko ¨ rner 1998), may have been due to its relative inability to respond to nutrient additions (Ellenberg 1986). The absence of a statistically significant relationship between the degree of association with earthworm casts and responses to elevated CO2 for plant species in any of the functional groups (e.g., Fig. 2 for graminoids) was puzzling but may simply be due to the fact that many plant species in these communities had too little time to respond to increased carbon (i.e., elevated CO2; see also Leadley et al. 1997) and nutrient availability (i.e., in casts). Also, the significant (P ϭ 0.007) positive correlation between the mean degrees of association with casts and the mean responses to elevated CO2 of graminoid species (Fig. 2), indicates that plant species were in fact beginning to respond to both elevated CO2 and increased nutrient availability in the second growing season of CO2 enrichment. Further, cast-associated ramet production in some species (e.g., Anthoxanthum odoratum, Carex caryophyllea, C. flacca, and Cynosurus cristatus) clearly tended to be greater under el-

evated CO2 when plants were near larger casts (Fig. 3). Although we focused on the role of casts as a nutrient source for plants, other properties of earthworm casts, such as possible greater water-holding capacity than neighboring soil (e.g., Edwards and Bohlen 1996), may have contributed to the effects we observed on plants. We also cannot rule out the possibility that the presence of certain plant species may influence casting patterns. For example, root architecture and distribution have been shown to influence patterns of earthworm burrowing (Edwards and Lofty 1980, Springett and Gray 1997), and root exudate quantity and quality may play a role in attracting earthworms (Graff 1977). Based on our observations, it also seems that earthworms deposit more casts near plant species with a compact root system near the soil surface, and near plant species that provide some form of shelter to worms (shade, protection from birds) by virtue of their tussock growth form (e.g., Anthoxanthum odoratum, Carex spp., Dactylis glomerata, Cerastium fontanum). It should be noted that, although we observed no effects of elevated CO2 on cumulative surface-cast production during the study period (April to August 1995), we saw a dramatic stimulation in subsequent measurements through April

TABLE 4. Cumulative earthworm surface-cast production between 5 April and 5 July 1995 in a calcareous grassland community maintained at ambient (350 ␮L CO2 / L) and elevated (600 ␮L CO2 / L) atmospheric CO2 (data are means Ϯ 1 SE, n ϭ 8 replicates). Cumulative cast production Dry mass (g/m2) Number of casts/m2 Plot area covered by casts (%) Ambient CO2 492 Ϯ 4 999 Ϯ 41 58 Ϯ 2 Elevated CO2 594 Ϯ 58 1075 Ϯ 77 60 Ϯ 3

P†
0.221 0.484 0.563

† Effect of elevated CO2; P values are for ANOVA with CO2 level and Block as independent variables.

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1996 (Zaller and Arnone 1997). However, it should be further noted that the locations of surface cast deposition in each 30 ϫ 30 cm grid remained similar up to April 1996. Thus, our results demonstrate that (1) plant species differ significantly in their degree of association with nutrient-rich earthworm surface casts, regardless of the relative abundance of plant species in the community— often plant species that were less abundant were associated to a greater degree with casts than were more abundant plant species; (2) graminoid species that are more highly associated with casts may respond more strongly to rising CO2 than those less highly associated with casts; and (3) nutrient-rich earthworm casts stimulate the growth (ramet production) of most plant species in these grassland communities, even at current levels of atmospheric CO2. The data further suggest that these species-specific relationships between plants and casts have helped define the current structure of these highly diverse grassland communities and will likely influence their future structure as global CO2 levels continue to rise.
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