This experiment was conducted at Cedar Creek Natural History Area, located on a glacial outwash sandplain in east-central Minnesota (Tilman 1988; http://www.lter.umn.edu). The 33 C3 grasses, C4 grasses, forbs, legumes and woody species surveyed in this study were established in fall 1992 on an abandoned agricultural field that had the top 60–80 cm of soil removed with a bulldozer (LTER experiment E111). On average, soils (0–20 cm) contained 0·46% C, 93% sand, 3% clay and 4% silt. To exclude mammalian herbivores, a 1·8-m-tall above-ground fence and a 1·2-m-deep below-ground fence was installed at the time of establishment.
Four replicate monocultures of each species were established at a seeding rate of 12 g m−2 with seed obtained from a local supplier (Prairie Restorations, Inc., Princeton, MN). Species names and functional classifications of the species used at Cedar Creek are given in Table 1. Authorities for species follow Moore (1973). Plots were 2·4 × 1·5 m for most species, and 1·2 × 1·5 m for others. Plot size was not an important determinant of any of the patterns discussed. Adjacent plots were separated by 25 cm deep in-ground sheet metal dividers. Each year, plots were weeded to maintain monoculture status and watered weekly as necessary during the 1997 growing season to ensure at least 2·5 cm weekly precipitation. Any plots that had experienced disturbance by gophers or had poor initial establishment were not sampled. In total, 114 plots were sampled with at least two replicates per species.
Table 1. Species list, number of plots sampled, functional groups and species scores for each axis of the principal component analysis (PCA)
|Species||n||Functional||Grass/Forb||Axis 1||Axis 2||Axis 3||Axis 4|
|Agropyron repens||4||C3 grass||Grass||0·15||−0·52||−0·48||1·82|
|Agrostis scabra||2||C3 grass||Grass||−0·22||−1·54||−0·80||2·13|
|Koeleria cristata||4||C3 grass||Grass||0·47||−0·11||−0·76||−0·47|
|Poa pratensis||3||C3 grass||Grass||0·43||−0·06||−1·26||1·17|
|Stipa spartea||4||C3 grass||Grass||0·06||0·07||−0·29||−0·49|
|Andropogon gerardi||4||C4 grass||Grass||0·21||1·62||−0·50||0·67|
|Bouteloua curtipendula||4||C4 grass||Grass||0·30||0·77||−0·83||1·32|
|Calamovilfa longifolia||4||C4 grass||Grass||−0·65||1·38||−0·15||−0·28|
|Panicum virgatum||4||C4 grass||Grass||−0·08||0·74||−0·22||0·81|
|Schizachyrium scoparium||4||C4 grass||Grass||0·26||1·87||−0·41||1·41|
|Sorghastrum nutans||4||C4 grass||Grass||0·58||0·76||−0·82||0·63|
All parameters are referred to by a unique table-specific reference number. For example, parameters 101–162 are contained in Table 2; whereas parameters 201–225 are contained in Table 3. A superscript of ns following the reference number signifies that the relationship was not significant, though the trend may have been in the direction predicted.
Table 2. Component loadings for the PCA. A coefficient whose absolute value is >0·33 is equivalent to a correlation with the axis at P < 0·05
| || ||Axis 1||Axis 2||Axis 3||Axis 4|
|108||Jul AG C : N||0·17||0·73||−0·29||−0·06|
|109||Jul BG C : N||0·17||0·87||−0·11||−0·08|
|110||Aug AG C : N||0·39||0·79||−0·16||0·05|
|111||Aug BG C : N||0·23||0·84||−0·08||0·08|
|115||Jul root 0–10||0·11||0·71||0·09||0·29|
|116||Jul root 10–20||−0·26||0·78||0·31||0·01|
|117||Jul root 20–40||−0·74||0·38||0·16||−0·22|
|118||Jul root 40–60||−0·78||0·20||0·38||0·14|
|119||Jul root 60–80||−0·69||0·15||0·49||0·08|
|120||Jul root 80–100||−0·93||0·02||0·12||0·02|
|124||Aug 0–20 Coarse||−0·14||0·34||0·65||0·25|
|125||Aug 0–20 Fine||0·22||0·66||−0·32||0·37|
|137||Aug %Coarse 0–20||−0·53||0·16||0·16||0·33|
|138||Aug %Fine 0–20||0·33||0·30||−0·66||0·25|
|158||N min AM||−0·40||−0·25||−0·33||0·11|
|159||N min MJ||−0·78||−0·39||−0·02||0·12|
|160||N min JJ||−0·69||−0·21||0·04||0·23|
|161||N min JA||−0·69||−0·33||0·43||0·16|
|162||N min AO||−0·52||−0·35||0·48||0·41|
Table 3. Other correlations (r) with PCA axes 1–4 (legumes were excluded from the analysis of correlations with axis 2)
| || || ||Axis 1||Axis 2||Axis 3||Axis 4|
| || ||n||r||P||r||P||r||P||r||P|
|202||Fine root longevity||33||0·25||>0·1||0·43||<0·05||−0·31||<0·1||0·33||<0·1|
|204||Max total biomass||33||−0·59||<0·001||0·83||<0·001||0·55||<0·01||0·02||>0·1|
|205||Max total plant N||33||−0·78||<0·001||0·65||<0·01||0·50||<0·01||0·12||>0·1|
|206||July R : S||33||0·48||<0·01||0·57||<0·01||−0·38||<0·05||0·34||<0·1|
|207||August R : S||33||−0·23||>0·1||0·48||<0·05||−0·49||<0·01||0·49||<0·01|
|208||C : N senesced leaves||15||0·88||<0·001||0·53||<0·1||−0·08||>0·1||0·26||>0·1|
|209||Aug C : N reproductive||24||−0·15||>0·1||0·31||>0·1||−0·18||>0·1||0·15||>0·1|
|210||Aug C : N leaves||28||0·07||>0·1||0·78||<0·001||−0·09||>0·1||0·25||>0·1|
|211||Aug C : N stems||28||0·36||<0·1||0·48||<0·01||−0·07||>0·1||−0·04||>0·1|
|212||Aug C : N fine||28||−0·21||>0·1||0·70||<0·001||−0·08||>0·1||0·20||>0·1|
|213||Aug C : N coarse||24||−0·04||>0·1||0·76||<0·001||0·30||>0·1||0·04||>0·1|
|214||Jul C : N reproductive||27||0·19||>0·1||0·42||>0·1||0·08||>0·1||−0·07||>0·1|
|215||Jul C : N leaves||13||0·39||>0·1||0·75||<0·001||0·18||>0·1||−0·13||>0·1|
|216||Jul C : N stems||28||0·18||>0·1||0·31||>0·1||−0·06||>0·1||0·13||>0·1|
|217||Jul C : N 0–20||28||−0·23||>0·1||0·89||<0·001||0·33||<0·1||0·00||>0·1|
|228||Jul C : N 20–40||25||−0·41||<0·05||0·79||<0·001||0·43||<0·05||−0·33||>0·1|
|219||Jul C : N 40–100||27||−0·45||<0·05||0·62||<0·001||0·12||>0·1||0·13||>0·1|
|220||Annual total productivity||33||−0·81||<0·001||0·69||<0·001||0·47||<0·01||−0·03||>0·1|
|221||Total plant N uptake||33||−0·90||<0·001||0·11||>0·1||0·27||>0·1||0·02||>0·1|
|222||Plant N loss rate||33||−0·90||<0·001||0·06||>0·1||0·26||>0·1||−0·01||>0·1|
|223||Total above-ground production||33||−0·61||<0·001||0·42||<0·05||0·71||>0·001||−0·05||>0·1|
|224||Total below-ground production||33||−0·90||<0·001||0·77||<0·001||0·03||>0·1||0·01||>0·94|
A suite of metrics of ecophysiology and organ morphology [specific leaf area (SLA); specific root length (SRL); fine root specific respiration rate (FSRR); photosynthesis per unit mass (Ps); stomatal conductance (gs); leaf respiration per unit mass (Rs); and average root diameter] (101–107) were measured on the species in this experiment (M. Tjoelker, unpublished). We determined light-saturated rates of leaf net photosynthesis in the field using a portable photosynthesis system (CIRAS-1, PP Systems, Hitchin, UK). We conducted measurements on clear sunny days (25 June and 7, 21, 28 August 1997) at light-saturating conditions between 10·30 and 14·00 h CDT. We measured two to four mature leaves from the top of the canopy in each of two to four replicate plots for a species. Leaf area for SLA was determined with a video image analysis system (AgVision, Decagon Devices, Inc., Pullman, WA). Net photosynthesis rates were calculated on the basis of leaf mass.
To determine specific respiration rates of leaves and roots, we harvested intact shoots, including stems and leaves, from plots on the mornings of 17 and 18 June. Samples were transferred to a controlled-environment chamber (Conviron E15, Controlled Environments, Inc., Winnipeg, Canada) to measure dark respiration at a standard temperature (26·1 ± 0·6 °C) and CO2 concentration (381 ± 15 µmol mol−1 CO2). Rates of net CO2 efflux were determined using infrared gas analysers (IRGA) and cuvettes (LCA-3 and PLC-C, Analytical Development Co. Ltd, Hoddesdon, UK), operated in an open configuration.
Aggregate root samples from soil cores (5 cm diameter, 20 cm depth) were collected for each of up to four plots per species (minimum of two plots for three species) across three dates (18, 20 and 21 August). Roots were washed from soil cores and kept moist at 26 °C prior to measurement, typically within 2·5 h of harvest. Net CO2 efflux was determined on the fine root fraction at a standard temperature (25·7 ± 0·4 °C) and atmospheric CO2 concentrations (366 ± 13 µmol mol−1 CO2) using IRGA and cuvettes as described above. We determined root lengths using a digital image analysis system (WinRhizo, Régent Instruments, Inc., Québec City, Canada). Root length and oven-dry mass measures were used to calculate specific root lengths.
Leaf longevity (201) was determined previously by Craine et al. (1999). Plant biomass and other associated measures were determined in early July and mid-August of 1997 by clipping to soil level all above-ground biomass in a previously unclipped 2·3 × 0·10 m strip (or two 1·15 × 0·10 m strips for 1·2 m wide plots). The previous years’ litter was removed from above-ground biomass samples, and above-ground biomass was separated into leaves, stems and reproductive parts (flowers, seeds and associated stems) (112–114, 121–123).
Below-ground biomass was sampled at both harvests. In July, three soil cores 5 cm in diameter and 100 cm deep were taken per plot and divided 0–10, 10–20, 20–40, 40–60, 60–80 and 80–100 cm (115–120). In August, three cores per plot were taken for a depth of 0–20 cm. For both harvests, all cores in a plot for a given depth interval were composited and then washed over a 1·3 mm screen. In August, roots were also separated into fine roots (<2 mm) and coarse below-ground biomass (roots and rhizomes >2 mm, crowns, corms) (124 125). All biomass was dried at 60 °C for a minimum of 96 h, weighed, and ground in a Wiley mill. Roots from the July harvest were composited 0–20, 20–40 and 40–100 cm and then ground. Tissue C and N concentrations for each fraction were determined with a Carlo-Erba NA1500 analyser. As tissue N concentrations can be biased by contamination with mineral soil, particularly for roots, we report tissue C : N ratios of biomass fractions (209–219). The N content of recently senesced leaves of 15 species was determined in August (208) in a manner similar to the live biomass.
Fine root production (139–141) was estimated with an ingrowth technique. At three times during the year (May, July and August) a soil core 5 cm in diameter was taken from a random location within the plot to a depth of 25 cm. Each hole was then refilled with a common root-free soil taken from an area of the garden that underwent the same initial treatment as all the plots and kept unvegetated for the entire experiment. The ingrowth cores were resampled after 41, 40 and 56 days by removing a 20 cm deep, 3·75 cm diameter core from the centre of the ingrowth core. The roots in the core were washed free of soil, coarse roots (>2 mm) removed, and the root biomass dried and weighed in the same manner as other root samples.
An exponential decay constant for the dependence of root biomass on depth (133) was computed for each species by fitting an exponential function to cumulative biomass and soil depth data (Jackson et al. 1996). We calculated maximal total biomass (204) as the sum of below-ground biomass to 100 cm measured in July and above-ground biomass measured in August, except for Lupinus perennis, for which July above-ground biomass was used due to the earlier phenology of L. perennis. Maximal total plant N (205) was calculated as the sum of the calculated N content of each biomass fraction in August, except for L. perennis for which the July data were used.
Total productivity (220) and relative production of different biomass fractions (143–147) required calculating the productivity for each of the fractions. Annual fine root production was calculated as the sum of the fine root production in the 0–20 and 20–100 cm strata. Annual fine root production at 0–20 cm was equal to the sum of fine root production in the ingrowth cores. The production of fine roots deeper than 20 cm was calculated by assuming that the root biomass below 20 cm had the same relative proportion of coarse and fine material as calculated in August at 0–20 cm, and that these roots had the same turnover rates as fine roots at 0–20 cm. The longevity of fine roots 0–20 cm (202) was calculated as the ratio of August fine root biomass at 0–20 cm to fine root production from ingrowth cores (139–141).
With no data on turnover or production of coarse below-ground biomass, we conservatively calculated annual production of coarse below-ground biomass by assuming equal coarse below-ground biomass production among years; no turnover of coarse below-ground biomass; and scaled coarse below-ground biomass to 100 cm assuming equivalent coarse : fine ratios in the 20–100 cm stratum and the 0–20 cm stratum. Leaf and reproductive biomass production were assumed to be equivalent to the greater of the July or August biomasses for leaves and reproductive parts (112, 113, 121, 122), respectively. Stem production (142) was calculated in a similar manner, except for the two woody species where, as with coarse below-ground biomass, stem biomass was divided by five to equalize production among years. Relative production of a biomass fraction or set of fractions was calculated as the ratio of production of a biomass fraction to total biomass production (143–147, 225).
Annual net N uptake (221) was calculated as the sum of the amounts of N incorporated into new production (N demand) for each biomass fraction. The N demand for a fraction was calculated as the product of the N concentration as measured in August and the annual production of each fraction, except for leaves for which senesced leaf tissue N was estimated from August green leaf N concentrations (%N senesced leaves = −0·510 + 1·032 × August %N leaves; r2 = 0·90, based on 15 species for which %N of senesced leaves was measured; July %N leaves was used for L. perennis). Plant N-loss rate (222) was calculated in a similar manner to annual N uptake, except that the N incorporated into stem or coarse below-ground biomass production was not considered to be lost from the plant.
All statistical analyses were performed in jmp 3·2 (SAS Institute, Carey, NC). We analysed 62 traits by principal component analysis (PCA): seven ecophysiological/morphological parameters (101–107); four measurements of plant tissue C or N of above-ground or below-ground biomass (108–111); 18 measurements of biomass pools or production rates of different plant fractions (112–125, 139–142); 18 measurements of the relative size of these plant biomass pools or their production relative to total production (126–138, 143–147); and 15 measurements of N associated with soils (148–162). The PCA weights all traits equally, and does not presuppose directional or causal relationships among traits. The first four resultant axes were determined to be the most biologically significant and then rotated using the Varimax rotation protocol to strengthen contrasts and aid in interpretation. For a parameter of a given rotated axis, a coefficient of absolute value >0·33 corresponds to a probability of P < 0·05 that a parameter is significantly correlated with the resultant axis.
As discussed below, the second axis primarily differentiates non-legumes in their trait relationships. Axes 1 and 3 account for most of the variation associated with legumes; scores on axis 2 of legumes are all close to zero. In order to provide the strongest analysis of how non-legumes differ in their trait relationships, we removed legumes from the data set and re-examined the correlations between the variables used in the PCA with the original scores of species on axis 2. This provides an average absolute value of the eigenvector coefficients that is approximately equal to the original coefficient (about 0·08 higher on average, or the equivalent of significance changing from P < 0·05 to P < 0·01). Yet individual variables that are also strongly associated with legumes and axis 2 had much higher correlations, such as August coarse below-ground biomass (124) (r = 0·32, P < 0·07 vs r = 0·66, P < 0·001). Overall, the loadings of variables on the PCA and their subsequent correlation coefficients were well correlated (r = 0·96, P < 0·001), but 10 more variables came to be considered as significant and one lost significance.
In addition to the 62 variables of the PCA, two other types of variable were examined as part of the relationship of traits among species. The first data type is metrics that were not measured on all of the species [leaf longevity (201); N content of senesced leaves (208); coarse below-ground biomass respiration rates (203); tissue C : N of certain biomass fractions (209, 211, 213, 214, 216)] and therefore could not be included in the PCA. The average C : N of above- and below-ground biomass for each harvest (108–111) were included in the PCA, while the C : N ratios of all individual fractions (209–219) were later correlated with the PCA axes. Other organ-level measures (202), whole-plant biomass and relative biomass measures (204–207), and measures of productivity and N utilization (220–225) were not included in the PCA, as these were calculated from metrics included in the PCA and their inclusion would have unnecessarily emphasized constituent factors.
For both measured and calculated parameters not included in the PCA, a pairwise correlation coefficient and the significance of the correlation were determined for each trait and PCA axes 1, 3 and 4 to determine if these traits and axes were associated with one another. Legumes were removed from the data set when examining the relationship between additional metrics and axis 2. For both PCA and additional correlations, we generally consider a trait to be a part of a given suite of traits if it is statistically significant (P < 0·05), although in particular circumstances we relax this condition.