BioCON: Biodiversity, CO2, and Nitrogen
Experiment Id

Many ecosystems around the world are experiencing simultaneous increases in atmospheric CO2 levels and N deposition, and decreases in biodiversity. The BioCON project addresses basic scientific questions about coupled biogeochemical cycles, biodiversity, and other issues while also providing information relevant to society about the implications of these global change variables.

BioCON focuses on 4 key questions: (1) Do CO2 and N interact at physiological, whole plant, multitrophic, community and/or biogeochemical scales, on short- and long-term time horizons? (2) Do plant species and/or functional group diversity and composition influence responses to CO2 and N? (3) Are there linear or non-linear temporal changes in effects of treatments on individual, community, or ecosystem metrics? (4) What mechanisms (physiological, biotic interaction, biogeochemical, etc.) explain the patterns observed in addressing questions 1-3? In other words, how does the integration of plant, consumer, mutualist, and decomposer interactions at multiple temporal scales lead to the responses observed at tissue to ecosystem scales across various time scales?

TeRaCON warming and rainfall reduction treatments in the BioCON plots. 

Experiment Design 

from: (BioCON): Reich, P. B., D. Tilman, J. Craine, D. Ellsworth, M. G. Tjoelker, J. Knops, D. Wedin, S. Naeem, D. Bahauddin, J. Goth, W. Bengtson, and T. D. Lee. 2001. Do species and functional groups differ in acquisition and use of C, N and water under varying atmospheric CO2 and N availability regimes? A field test with 16 grassland species. New Phytologist 150:435-448. doi:10.1046/j.1469-8137.2001.00114.x

(TeRaCON):  Reich, P. B., S. E. Hobbie, T. D. Lee, R. Rich, M. A. Pastore, and K. Worm. 2020. Synergistic effects of four climate change drivers on terrestrial carbon cycling. Nature Geoscience 13:787-793.doi:10.1038/s41561-020-00657-1

The main BioCON experiment is 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 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 (2 replicates per species), 15 for those planted to 4 species, 15 for 9 species, and 12 for 16 species. This arrangement applies to the “main” experiment which utilizes 296 plots. 

Sub Experiment-Functional Diversity 

There is also a sub experiment within BioCON’s framework in which functional group and species assignments were not completely random; functional group diversity was controlled thereby limiting the choices for species composition. The spatial distribution of plots within the rings was still randomly chosen. The 296 main experiment plots are still utilized in analyses for this part of the study; the species assignments for these plots were necessary to complete the factorial design for functional vs. species diversity analyses. In total there are 371 plots in the BioCON experiment: 296 plots in the main randomly assigned experiment, 63 additional plots for controlling functional group diversity, and 12 bare ground plots, void of any plant species.

Initial Seeding and Biodiversity Treatment

Plots were planted in 1997. Each plot was planted with 12g/m2 of seed, split equally among the planted species. In 1997 the plots were watered regularly to ensure germination and establishment. The plots were not watered after 1997. The 16 species in the experiment were chosen from the following criteria: A species had to (1) be native or naturalized to the area; (2) have a proven track record of establishment in previous experiments at Cedar Creek; (3) belong to 1 of 4 functional groups: leguminous forbs, non-leguminous forbs; C3 grasses, and C4 grasses.  

SpeciesSeed source
Andropogon gerardi, Asclepias tuberosa, Amorpha canescens, Bouteloua gracilis, Schizachyrium scoparium, Sorghastrum nutans, Solidago rigidaPrairie Restorations, Inc. (Princeton location)
Anemone cylindrica, Koeleria cristata, Lespedeza capitata, Lupinus perennis, Petalostemum villosumPrairie Moon Nursery
Achillea millefoliumStock Seed Farms
Agropyron repensV & J Seed Farms
Bromus inermis, Poa pratensisStock Seed Farms (source uncertain)
Carbon Dioxide Treatment

Elevated CO2 treatment rings 1, 3 and 5 receive air with a target CO2 concentration set at 180 ppm above ambient conditions. Elevated CO2 treatments are applied seven days per week, during daylight hours, for the full growing season (roughly May 1 to October 15).

Nitrogen Treatment and 15N

N enriched plots receive nitrogen addition 3 times per growing season: mid-May, mid-June, and mid-July. We used 4 g NH4NO3/ M2/year and 0.474g 15NH415NO3 /plot/year.


Species composition is controlled by hand weeding 2-4 times per growing season.


From 2000-2012 BioCON was burned approximately every other spring. Since 2013 BioCON has been burned every year in the fall.

TeRaCON Sub Experiment Addition

TeRaCON was established in spring of 2012 as a sub experiment within the BioCON experiment. TeRaCON is composed of 48 plots chosen with a stratified random approach (to balance the treatments and locations among experimental blocks) from 64 plots within the greater BioCON experiment that were initially planted with nine perennial grassland species. The TeRaCON experiment is a complete factorial design with two temperature (ambient and about +2.5 °C soil and surface warming), two growing-season rainfall (ambient and about –30% April through September), two atmospheric CO2 (ambient and +180 ppm) and two soil N (ambient and +4 g N m–2 yr–1) treatments. 

The rainfall reduction treatment began in 2007 and is administered to half of the 48 plots that compose the TeRaCON experiment. Portable 2 × 2 m rainout shelters are used to intercept select rain events; intercepted rain is moved away from the plots via gutters.  The goal of the rainfall treatment is to intercept select rain events during the middle period of the growing season to achieve a growing season rainfall reduction of ~30%. 

The warming treatment began in 2012 and is also administered to half of the 48 plots that compose the TeRaCON experiment. Heating is applied prior to, during and after the growing season, but not in winter. Belowground, distributed resistance pins heat soils down to 80cm. Aboveground, an array of infrared lamps heat plant surfaces. The 2 degrees C increase in plant and soil temperature represents the minimum warming predicted over the next century for central North America, assuming moderate to high fossil fuel emissions increases (IPCC 2007). Plots are warmed 24 h/d from roughly May 1 to October 15. This timing closely matches eCO2 treatment timing. Four ceramic infrared lamps (1000 W 240V, 245 mm long by 60 mm wide) are used above each warmed plot (HTelv). Lamp wattage output is regulated continuously by a proportional integrative derivative (PID) controlled dimmer system to maintain the plus 2 degrees C treatments and feedback control. Soil warming is accomplished through soil vertical warming pins. Twelve 0.81 m pins are installed vertically in each HTelv plot. Soil temperature has PID feedback control on an individual plot basis based on maintaining treatment temperature differential between plot warmed treatments and nearby ambient control plots fitted with dummy soil heating pins. In-ground thermo-couples at various depths in the 0-30 cm zone in the soil horizon connect via multiplexer to a Campbell Scientific controller to regulate a PID control that turns pins up or down. Temperature is monitored in close proximity to warming pins. The system uses the soil’s inherent thermal diffusivity and mass to maintain treatment.

Treatment Tables

Download treatment tables (zip file)


View data in the Data Catalog

Select Publications: 

Do species and functional groups differ in acquisition and use of C, N and water under varying atmospheric CO2 and N availability regimes? A field test with 16 …
PB Reich, D Tilman, J Craine, D Ellsworth, MG Tjoelker, J Knops, ...
2001 New Phytologist 150 (2), 435-448

Plant diversity enhances ecosystem responses to elevated CO2 and nitrogen deposition
PB Reich, J Knops, D Tilman, J Craine, D Ellsworth, M Tjoelker, T Lee, ...
2001 Nature 410 (6830), 809-810

Unexpected reversal of C3 versus C4 grass response to elevated CO2 during a 20-year field experiment
PB Reich, SE Hobbie, TD Lee, MA Pastore
2018 Science 360 (6386), 317-320

Sensitivity of grassland carbon pools to plant diversity, elevated CO2, and soil nitrogen addition over 19 years
MA Pastore, SE Hobbie, PB Reich
2021 Proceedings of the National Academy of Sciences 118 (17), e2016965118

Synergistic effects of four climate change drivers on terrestrial carbon cycling
PB Reich, SE Hobbie, TD Lee, R Rich, MA Pastore, K Worm
2020 Nature Geoscience 13 (12), 787-793