Materials and Methods Online
Experimental Design. The BioCON (Biodiversity, CO2 and N) experiment includes 296 2 x 2 m plots arranged in six circular 20-meter diameter rings, located at the Cedar Creek Natural History Area in Minnesota, USA. Plots were established on a secondary successional grassland on a sandy outwash soil after removing the previous vegetation. The experimental treatments were arranged in complete factorial combination of CO2 (ambient or 560mmol mol-1), N level (ambient and enriched), and species number (1, 4, 9, and 16). Each plot was planted in 1997 with 12 g m-2 of seed partitioned equally among all species planted in a plot. The design consisted of a split-plot arrangement of treatments in a completely randomized design. CO2 treatment is the whole-plot factor and is replicated three times among the six rings. The subplot factors of species number (hereafter called diversity) and N treatment were assigned randomly and replicated in individual plots among the six rings. For each of the four combinations of CO2 and N levels, pooled across all rings, there were 32 randomly assigned replicates for the plots planted to 1 species, 15 for those planted to 4 species, 15 for 9 species, and 12 for 16 species. Beginning in 1998, the plots assigned to the enriched N treatment were amended with 4 g N m-2 yr-1, applied over three dates each year. This N addition is comparable or slightly larger than the average annual net N mineralization rate in similar secondary grasslands on these soils. Beginning in 1998, a free-air CO2 enrichment system was used during each growing season to maintain the CO2 concentration at an average of 560 µmol mol-1 in elevated treatments during all daylight hours from spring (early April) to fall (late October to mid-November) each year. Three ambient CO2 rings were treated identically but without additional CO2.
The 16 species used in this study were all native or naturalized to the Cedar Creek Natural History Area. They include four C4 grasses (Andropogon gerardii, Bouteloua gracilis, Schizachyrium scoparium, Sorghastrum nutans), four C3 grasses (Agropyron repens, Bromus inermis, Koeleria cristata, Poa pratensis), four N-fixing legumes (Amorpha canescens, Lespedeza capitata, Lupinus perennis, Petalostemum villosum) and four non-N-fixing herbaceous species (Achillea millefolium, Anemone cylindrica, Asclepias tuberosa, Solidago rigida), and all are referred to by genus elsewhere. Monocultures of all species were replicated twice at all CO2 and N levels. The 4- and 9-species plots were random selections from all species. Plots were regularly weeded to remove unwanted species.
Biomass sampling and biogeochemistry measurements. In each year we assessed above- and below-ground biomass, plant C and N, and soil N in every plot. In June and August of each year, aboveground biomass was harvested in every plot by clipping a 10 x 100 cm strip just above the soil surface. All biomass was collected, sorted to live material and senesced litter, dried and weighed. Live material was considered as current plant biomass. Total belowground biomass (fine roots, coarse roots and crowns) was sampled in every plot within days of aboveground harvests in June and August at 0-20 cm depth using three 5 cm dia. cores in the area used for the aboveground biomass sampling. Roots were thoroughly washed, sorted into fine (<1 mm diameter) and coarse classes and crowns, dried and weighed. Any given area was sampled only once during the six years of this study. All biomass from August harvests was ground and analyzed separately for aboveground and belowground components for C and N concentrations using a CHN analyzer (Carlo-Erba Strumatzione, Milan, Italy). Total plant N pools for the August harvests were estimated by multiplying total live plant biomass by the N concentration. Soil net N mineralization was measured in each plot every year with a semi-open core (2.5 cm diameter) technique using one-month in situ incubations at 0-20 cm depth during midsummer. We sieved (2 mm) incubated soil cores, as well as an equal number of soil cores taken at the start of each incubation, and extracted them with 1 M KCl. Extracts were analyzed for NO3- and NH4+ on an Alpkem auto-analyzer. We measured soil moisture content on a sub-sample by oven drying (48 h, 105°C). We calculated net N mineralization by subtracting initial from final total inorganic N (NO3- + NH4+).
Statistical analysis. We examined results for the entire 1998-2003 period, using a repeated measures analysis of variance (JMP Statistical Software 5.0.1a) to test for main effects and interactions of the treatments, and whether these changed over time (contrasting across years). Given the nature of both the hypothesized progressive N limitation and the observed patterns over time, year was considered a continuous rather than a nominal factor. The repeated measures procedure accounts for the non-independence among multiple measures of plots over time. To do this, variance among plots nested within CO2, N and diversity levels was used as a random effect such that measures that co-vary across time (years) were not counted as fully independent. The F statistic for the main effects of N and diversity used the nested effect of plot within CO2, N and diversity treatments. The F statistic for year and for treatment x year effects used the residual error term. The F statistic for CO2 used the nested effect of ring within CO2.