University of Minnesota
University of Minnesota
College of Biological Sciences

Experiment 001 - Long-Term Nitrogen Addition to Undisturbed Vegetation




In 1982, Experiment 001 was established by David Tilman to examine the long-term effects of low-level nitrogen addition on undisturbed nitrogen-limited grassland ecosystems. For 20 years plots were sampled annually for above-ground biomass (sorted by species). However, because plot data have shown few changes in recent years, and because treatment responses have converged among the various fields, sampling is now periodic and a new treatment has been imposed in one of the fields. Soil chemistry (NH4, NO3, Ca, Mg, P, and K), belowground biomass, insect abundances, light penetration, and small mammal densities have been sampled intermittently.
The 207 experimental plots are divided among three successional grassland fields (Fields A, B and C - 54 plots each) and a savanna prairie opening (Field D - 45 plots). In each locale, nine treatments are imposed on sets of replicate plots. Two of the treatments are controls, neither of which receives ammonium nitrate fertilization, but one of which receives other nutrients (P, K, Ca, Mg and trace metals). The seven remaining treatments add ammonium nitrate at varying rates as well as the other nutrients. Starting in 2005, the successional grassland plots have been burned annually. The savanna plots have been burned every two out of three since 1987.
In 1986, aluminum flashing was buried between the individual plots to prevent plants from spreading by vegetative reproduction. In 1982, the plot grids were enclosed by fencing to exclude large mammalian herbivores, however in 2004 this was removed. At the same time, in one of the successional grasslands, half of the replicate plots were individually re-fenced. Gophers are trapped and removed from all experiment plots.

Key Results


Using data from the first 25 years of the N addition experiment, Clark and Tilman (2008) found that chronic low-level N addition (10 kg ha-1 yr-1) reduced plant species numbers by 17% relative to controls receiving ambient N deposition (Fig. 2). Moreover, species numbers were reduced more per unit of added N at lower addition rates, suggesting that chronic, low-level N deposition may have a greater impact on diversity than previously thought. Clark et al. (2009) found that net N mineralization rates remained elevated in plots that had ceased receiving N 12 years earlier. Although these grassland ecosystems had not retained a high portion of the deposited N, the effects of this N retention were surprisingly long-lasting.  
Because soils are the largest active terrestrial sink of C, the potential effects of elevated N deposition on soil C stores is of great interest. Earlier CDR work suggested that N deposition did not impact soil C stores (Wedin and Tilman 1996). However, we have recently found that 27-years of chronic N addition to prairie grasslands strongly increased the C sequestration in mineral soils (Fornara and Tilman 2012 Ecology). A key mechanism was an N-induced increase in root mass accumulation with a shift to C3 grasses, which despite their lower N-retention ability still acted as important soil C sinks.  
We found that although chronic nutrient enrichment initially increased productivity, it also led to loss of plant species, including initially dominant species, which then caused substantial diminishing returns of productivity from nitrogen fertilization. Our results support the hypothesis that the long-term impacts of global changes on ecosystem functioning can strongly depend on how such drivers gradually decrease biodiversity and restructure communities. Isbell et al. 2013 PNAS 
We found that the stability of ecosystem productivity was only changed by anthropogenic drivers that altered biodiversity, with a given rate of plant species loss leading to a quantitatively similar decrease in ecosystem stability regardless of which driver caused the biodiversity loss. These results suggest that changes in biodiversity caused by anthropogenic drivers may be a major factor determining how global changes affect ecosystem stability. Hautier et al. 2015 Science

Experiment 001 has provided insights into causes of successional dynamics (e.g., Tilman 1987 , 1988, 1990 ), effects of N deposition on C and N storage (Wedin and Tilman 1996), causes of diversity differences along productivity gradients (Tilman 1990 , 1993 , 1996a), impacts of diversity on ecosystem stability (Tilman and Downing 1994, Tilman 1996a, 1999a, Tilman et al. 1998, Lehman and Tilman, 2000), impacts of climatic variation on biodiversity (Tilman and El Haddi 1992, Tilman 1996a), and long-term dynamics after a major drought (Haddad et al. 2002).

Experiment 001 was the first multi-decadal experiment to examine the impacts of chronic, experimental nitrogen addition as low as 10 kg of N per hectare per year above ambient atmospheric nitrogen deposition (6 kg of N per hectare per year). This total input rate is comparable to terrestrial nitrogen deposition in many industrialized nations. Chronic low-level nitrogen addition rates were found to reduce plant species numbers by 17% relative to controls receiving ambient N deposition (Fig. 1, Clark and Tilman 2008). Moreover, species number were reduced more per unit of added nitrogen at lower addition rates, suggesting that chronic but low-level nitrogen deposition may have a greater impact on diversity than previously thought (Fig. 2, Clark and Tilman 2008).


Figure 1. Relative species number versus time. The treatment-specific average annual relative species numbers (+/- one s.e.m.) through time averaged over the three fields are shown. Dashed lines correspond to annual standard errors in control plots, and arrows indicate the year of first significant (P<0.01) detection of relative species loss for a particular nitrogen addition treatment rate using MANOVA over three-year intervals (middle year highlighted). For clarity, only three of five nitrogen addition treatments are shown. (Clark and Tilman, 2008)
Figure 2. Proportional species loss versus nitrogen input rate for (a) 2002-2004 and (b) 1983-1985. Plot averages for each field over the three year-period fitted to a logarithmic curve excluding controls (95% confidence curves included). P values correspond to the significance of the nitrogen input term (N input = experimental N addition + atmospheric N deposition) in a model of the proportional loss of species regressed on the natural logarithm of the nitrogen input rate, Field, and their interaction. Dashed lines correspond to linear interpolation between the mean effect at the highest nitrogen addition rate and controls. (Clark and Tilman 2008)

The resistance of total plant biomass to drought was significantly greater at higher diversity in Experiment 001 plots, even after controlling for numerous potentially confounding variables (Fig. 4; Tilman and Downing 1994). For all non-drought years, interannual variation in total plant biomass was greater at lower diversity (Fig. 5; Tilman 1996, 1999). Results of both papers support the diversity-stability hypothesis.

Figure 4. Relationship between drought resistance of grassland plots and plant species richness (SR86) preceding a severe drought. Mean, standard error and number of plots with a given species richness are shown. Drought resistance was measured as dB/Bdt (per year), that is, as 0.5 (ln[biomass(1988)/biomass(1986)]; left had scale. The right-hand scale shows the proportionate decrease in plant biomass associated with the dB/Bdt values. (Tilman and Downing 1994)
Fig 5. The dependence of the coefficient of variation (cv) of total community biomass on plant species diversity, based on the data for Experiment 001. The cv measures the extent of year-to-year variation in total plant biomass within a plot (relative to mean biomass). The lower coefficients of variation of the more diverse plots mean that total community biomass is stabilized by diversity. As shown in Tilman (1996), abundances of individual species are destabilized by diversity. Coefficients of variation of each field were adjusted for differences in intercepts as determined by a GLM regression. (Tilman 1999)
Figure 6. The dynamics of total plant biomass before and after drought in Experiment 001. (Haddad et al. 2002)
(A) Mean and SE of total aboveground biomass in the unfertilized control plots for the three successional fields of Experiment 001. Note relative consistency before the drought and the 2 year or 3 year cycle after the drought. These fields are unburned. 
(B) Similar dynamics for the periodically burned savanna plots. Note that, after the drought, low points correspond with years when plots were unburned.

Nitrogen addition impacts ecosystem carbon and nitrogen stores in Experiment 001 via effects on species composition and thus on litter C:N (Wedin and Tilman 1996). At higher N addition, diversity is lower, C4 grasses are less abundant, and litter and root C:N ratios are lower (Fig. 7A-D). The nitrogen-dependent shift to low C:N species corresponds with decreased ecosystem retention of added nitrogen (Fig. 8A) and to lower carbon storage (Fig. 8B), likely because of immobilization and decomposition effects (Fig. 8C).

Figure 7. Ecosystem C and N and vegetation responses to 12 years of N addition in Experiment 001. Points represent treatment means (6 replicates per N addition level, 12 for controls) for each of three fields. (Wedin and Tilman 1996)
(A) Number of vascular plant species found in 0.3-m2 vegetation samples.
(B) Biomass of grasses with the C4 photosynthetic pathway as a proportion of aboveground live biomass at mid-growing season. One species, Schizachyrium scoparium, contributed >95% of the C4 biomass in the plots.
Biomass C:N ratios for (C) litter, i.e. aboveground dead biomass (both recent and old) and (D) belowground root biomass, both alive and dead root fragments.
Figure 8. (A) Nitrogen dynamics after 12 years of N addition. Net N retention after 12 years estimated as the change in total system N (relative to controls) divided by the sum of experimental N additions. (B) Net C storage per unit experimentally added N after 12 years. Because C storage rates (g C/g N) did not differ significantly between Fields B and C (34), overall treatment means for the two C4-dominated fields are presented. (C) The relationship between soil NO3- and the C:N ratio of plant biomass (aboveground dead biomass plus belowground biomass). Vertical line represents a biomass C:N ratio of 32. (Wedin and Tilman 1996)

Shifts in species abundances in response to nitrogen addition did not support predictions of Hubbell's (2001) neutral theory (Fig. 9; Harpole and Tilman 2006).

Figure 9. Relationship between R* and abundance changes along an experimental N addition gradient: species ranking switches from dominance of good N competitors (low R* values) to poor N competitors (high R* values) with increasing rates of N addition. Each point is the treatment-level mean correlation of R* indexes with species abundances. Dashed lines indicate the bootstrapped mean (short dash) and 95% confidence region.

Data from this experiment have also been extensively shared with and used by non-CDR researchers, and been included in a variety of publications (Gough et al. 2000; Gross et al. 2000; Knapp and Smith 2001; Johnson et al. 2003ab, 2005; Pennings et al. 2005; Suding et al. 2005).

Methods for e001


Datasets for e001: Long-Term Nitrogen Deposition: Population, Community, and Ecosystem Consequences

Dataset IDTitleRange of Years (# years with data)
avne001Average soil nitrogen1985-1985 (1 year)
abue001Field C Microplot Arthropod Sweepnet Sampling1995-1996 (2 years)
lpe001Percent light penetration1982-2004 (11 years)
nbe001Plant aboveground biomass carbon and nitrogen2009-2009 (1 year)
ple001Plant aboveground biomass data1982-2020 (36 years)
rcne001Root Carbon and Nitrogen2009-2009 (1 year)
rbe001Root biomass data1987-2013 (16 years)
mse001Small mammal abundance1982-1985 (4 years)
cae001Soil Calcium1982-1982 (1 year)
nhe001Soil ammonium1986-1988 (3 years)
care001Soil carbon1982-2011 (5 years)
mge001Soil magnesium1982-1982 (1 year)
nohe001Soil nitrate and ammonium1985-2013 (14 years)
ne001Soil nitrogen1982-2011 (7 years)
phe001Soil pH1982-2010 (10 years)
pe001Soil phosphorous1982-1982 (1 year)
ke001Soil potassium1982-1982 (1 year)

Selected Recent Publications

Avolio, M. L., Komatsu, K. J., Collins, S. L., Grman, E., Koerner, S. E., Tredennick, A. T., Wilcox, K. R., Baer, S., Boughton, E. H., Britton, A. J., Foster, B., Gough, L., Hovenden, M., Isbell, F., Jentsch, A., Johnson, D. S., Knapp, A. K., Kreyling, J., Langley, J. A., Lortie, C., McCulley, R. L., McLaren, J. R., Reich, P. B., Seabloom, E. W., Smith, M. D., Suding, K. N., Suttle, K. B., & Tognetti, P. M. (2021) Determinants of community compositional change are equally affected by global change. Ecology Letters. doi: 2021 e001 e002 e141

Cusser, S., Helms Iv, J., Bahlai, C. A., & Haddad, N. M. (2021) How long do population level field experiments need to be? Utilising data from the 40-year-old LTER network. Ecology Letters. doi: 2021 e001 e002

Proulx, R. (2021) On the general relationship between plant height and aboveground biomass of vegetation stands in contrasted ecosystems. PLOS ONE, 16(5), e0252080. doi:10.1371/journal.pone.0252080 2021 e001 e002

Avolio, M. L., Wilcox, K. R., Komatsu, K. J., Lemoine, N., Bowman, W. D., Collins, S. L., Knapp, A. K., Koerner, S. E., Smith, M. D., Baer, S. G., Gross, K. L., Isbell, F., McLaren, J., Reich, P. B., Suding, K. N., Suttle, K. B., Tilman, D., Xu, Z., and Yu, Q. (2020) Temporal variability in production is not consistently affected by global change drivers across herbaceous-dominated ecosystems. Oecologia, 194(4), 735-744. doi:10.1007/s00442-020-04787-6 2020 e001 e001 e141

Han, Y., Feng, J., Han, M., & Zhu, B. (2020) Responses of arbuscular mycorrhizal fungi to nitrogen addition: A meta-analysis. Global Change Biology, 26(12), 7229-7241. doi:10.1111/gcb.15369 2020 e001 e002 e052 e120 e141

Jochum, M., Fischer, M., Isbell, F., Roscher, C., van der Plas, F., Boch, S., Boenisch, G., Buchmann, N., Catford, J. A., Cavender-Bares, J., Ebeling, A., Eisenhauer, N., Gleixner, G., Holzel, N., Kattge, J., Klaus, V. H., Kleinebecker, T., Lange, M., Le Provost, G., Meyer, S. T., Molina-Venegas, R., Mommer, L., Oelmann, Y., Penone, C., Prati, D., Reich, P. B., Rindisbacher, A., Schafer, D., Scheu, S., Schmid, B., Tilman, D., Tscharntke, T., Vogel, A., Wagg, C., Weigelt, A., Weisser, W. W., Wilcke, W., & Manning, P. (2020) The results of biodiversity???ecosystem functioning experiments are realistic. Nature Ecology & Evolution, 4(11), 1485-1494. doi:10.1038/s41559-020-1280-9 2020 e001 e002 e014 e093 e120

Prather, R. M., Castillioni, K., Kaspari, M., Souza, L., Prather, C. M., Reihart, R. W., & Welti, E. A. R. (2020) Micronutrients enhance macronutrient effects in a meta-analysis of grassland arthropod abundance. Global Ecology and Biogeography, 29(12), 2273-2288. doi:10.1111/geb.13196 2020 e001 e004 e141 e247

Seabloom, E. W., Borer, E. T., & Tilman, D. (2020) Grassland ecosystem recovery after soil disturbance depends on nutrient supply rate. Ecology Letters, 23(12), 1756-1765. doi:10.1111/ele.13591 2020 e001 e002

Song, J., Ru, J., Zheng, M., Wang, H., Fan, Y., Yue, X., Yu, K., Zhou, Z., Shao, P., Han, H., Lei, L., Zhang, Q., Li, X., Su, F., Zhang, K., & Wan, S. (2020) A global database of plant production and carbon exchange from global change manipulative experiments. Scientific Data, 7(1), 323. doi:10.1038/s41597-020-00661-5 2020 e001 e002 e014 e026 e097 e141 e247

Isbell, F., Tilman, D., Reich, P. B., & Clark, A. T. (2019) Deficits of biodiversity and productivity linger a century after agricultural abandonment. Nature Ecology & Evolution, 3(11), 1533-1538. doi:10.1038/s41559-019-1012-1 2019 e001 e054

Schlatter, D. C., Song, Z., Vaz-Jauri, P., & Kinkel, L. L. (2019) Inhibitory interaction networks among coevolved Streptomyces populations from prairie soils. PLOS ONE, 14(10), e0223779. doi:10.1371/journal.pone.0223779 2019 e001

Song, J., Wan, S., Piao, S., Knapp, A. K., Classen, A. T., Vicca, S., Ciais, P., Hovenden, M. J., Leuzinger, S., Beier, C., Kardol, P., Xia, J., Liu, Q., Ru, J., Zhou, Z., Luo, Y., Guo, D., Adam Langley, J., Zscheischler, J., Dukes, J. S., Tang, J., Chen, J., Hofmockel, K. S., Kueppers, L. M., Rustad, L., Liu, L., Smith, M. D., Templer, P. H., Quinn Thomas, R., Norby, R. J., Phillips, R. P., Niu, S., Fatichi, S., Wang, Y., Shao, P., Han, H., Wang, D., Lei, L., Wang, J., Li, X., Zhang, Q., Li, X., Su, F., Liu, B., Yang, F., Ma, G., Li, G., Liu, Y., Liu, Y., Yang, Z., Zhang, K., Miao, Y., Hu, M., Yan, C., Zhang, A., Zhong, M., Hui, Y., Li, Y., & Zheng, M. (2019) A meta-analysis of 1,119 manipulative experiments on terrestrial carbon-cycling responses to global change. Nature Ecology & Evolution, 3(9), 1309-1320. doi:10.1038/s41559-019-0958-3 2019 e001 e002 e014 e026 e097 e141 e247

Snell Taylor, S. J., B. S. Evans, E. P. White and A. H. Hurlbert (2018). "The prevalence and impact of transient species in ecological communities." Ecology 99(8): 1825?1835. 2018 e001

Ratajczak, Z., D`Odorico, P., Collins, S. L., Bestelmeyer, B. T., Isbell, F. I., and Nippert, J. B. (2017). The interactive effects of press/pulse intensity and duration on regime shifts at multiple scales. Ecological Monographs, accepted article. doi:10.1002/ecm.1249 2017 e001 e002 e097

Wilcox, K. R., A. T. Tredennick, S. E. Koerner, E. Grman, L. M. Hallett, M. L. Avolio, K. J. La Pierre, G. R. Houseman, F. Isbell, D. S. Johnson, J. M. Alatalo, A. H. Baldwin, E. W. Bork, E. H. Boughton, W. D. Bowman, A. J. Britton, J. F. Cahill, S. L. Collins, G. Du, A. Eskelinen, L. Gough, A. Jentsch, C. Kern, K. Klanderud, A. K. Knapp, J. Kreyling, Y. Luo, J. R. McLaren, P. Megonigal, V. Onipchenko, J. Prevey, J. N. Price, C. H. Robinson, O. E. Sala, M. D. Smith, N. A. Soudzilovskaia, L. Souza, D. Tilman, S. R. White, Z. Xu, L. Yahdjian, Q. Yu, P. Zhang and Y. Zhang (2017). "Asynchrony among local communities stabilises ecosystem function of metacommunities." Ecology Letters 20(12): 1534-1545. DOI 10.1111/ele.12861 2017 e001 e002

Delgado-Baquerizo, Manuel; Maestre, Fernando T.; Reich, Peter B.; Trivedi, Pankaj; Osanai,Yui; Liu, Yu-Rong; Hamonts, Kelly; Jeffries, Thomas C.; Singh, Brajesh K. (2016). Carbon content and climate variability drive global soil bacterial diversity patterns. Ecological Monographs, 86(3), 373-390. doi:10.1002/ecm.1216 2016 [Full Text] e001

Ladwig, L. M., Ratajczak, Z. R., Ocheltree, T. W., Hafich, K. A., Churchill, A. C., Frey, S. J. K., Fuss, C. B., Kazanski, C. E., Munoz, J. D., Petrie, M. D., Reinmann, A. B. and Smith, J. G. (2016). "Beyond arctic and alpine: the influence of winter climate on temperate ecosystems." Ecology 97(2): 372-382. 2016 [Full Text] e001 e014 e080

Hautier, Y.; Tilman, D.; Isbell, F.; Seabloom, E. W.; Borer, E. T.; Reich, P. B.; Anthropogenic environmental changes affect ecosystem stability via biodiversity. Science, 2015, 348, 6232, 336-340 DOI:10.1126/science.aaa1788 2015 [Full Text] e001 e002 e003 e012 e098 e120 e141 e245 e247 e248

Schlatter, Daniel C. and Linda L. Kinkel. Do tradeoffs structure antibiotic inhibition, resistance, and resource use among soil-borne Streptomyces? BMC Evolutionary Biology 2015, 15:186 doi:10.1186/s12862-015-0470-6 2015 [Full Text] e001

Hallett, Lauren M.; Hsu, Joanna S.; Cleland, Elsa E.; Collins, Scott L.; Dickson, Timothy L.; Farrer, Emily C.; Gherardi, Laureano A.; Gross ,Katherine L.; Hobbs, Richard J.; Turnbull, Laura; Suding, Katharine N.; Biotic mechanisms of community stability shift along a precipitation gradient; Ecology, 2014, 95, 6, 1693-1700 2014 [Full Text] e001

Myrold, David D., Lydia H. Zeglin, and Janet K. Jansson. "The potential of metagenomic approaches for understanding soil microbial processes." Soil Science Society of America Journal 78, no. 1 (2014): 3-10. 2014 [Full Text] e001

Schlatter, D. C.; Kinkel, L. L.; 2014, Global biogeography of Streptomyces antibiotic inhibition, resistance, and resource use. FEMS microbiology ecology, 88(2), 386-397. 2014 [Full Text] e001

Symstad, A.; Jonas, J.; Using Natural Range of Variation to Set Decision Thresholds: A Case Study for Great Plains Grasslands; In book: Application of Threshold Concepts in Natural Resource Decision Making, Publisher: Springer, Editors: Glenn R. Guntenspergen, pp.131-156 DOI: 10.1007/978-1-4899-8041-0_8 2014 [Full Text] e001

Vaz Jauri, P. and Kinkel, L. L. (2014), Nutrient overlap, genetic relatedness and spatial origin influence interaction-mediated shifts in inhibitory phenotype among Streptomyces spp. FEMS Microbiology Ecology, 90: 264?275. doi: 10.1111/1574-6941.12389 2014 [Full Text] e001

Cleland, Elsa E.; Collins, Scott L.; Dickson, Timothy L.; Farrer, Emily C.; Gross, Katherine L.; Gherardi, Laureano A.; Hallett, Lauren M.; Hobbs, Richard J.; Hsu, Joanna S.; Turnbull, Laura; Suding, Katharine N.; Sensitivity of grassland plant community composition to spatial vs. temporal variation in precipitation; Ecology, 2013, 94, 8, 1687-1696 2013 [Full Text] e001

Isbell, F.; Tilman, D.; Polasky, S.; Binder, S.; Hawthorne, P.; Low biodiversity state persists two decades after cessation of nutrient enrichment; Ecology Letters (2013) 16: 454?460 DOI: 10.1111/ele.12066 2013 [Full Text] e001 e002 e120 e141

Isbell, Forest; Reich, Peter B.; Tilman, David; Hobbie, Sarah E.; Polasky, Stephen; Binder, Seth. Nutrient enrichment, biodiversity loss, and consequent declines in ecosystem productivity. Proceedings of the National Academy of Sciences of the United States of America. 2013 110 (29):11911-11916. 2013 [Full Text] e001 e002. e120 e141

Robinson, T. M. P., La Pierre, K. J., Vadeboncoeur, M. A., Byrne, K. M., Thomey, M. L. and Colby, S. E. (2013), Seasonal, not annual precipitation drives community productivity across ecosystems. Oikos, 122: 727?738. doi: 10.1111/j.1600-0706.2012.20655.x 2013 [Full Text] e001 e080

Schlatter, Daniel C.; DavelosBaines, Anita L.; Xiao, Kun; Kinkel, Linda L.; Resource Use of Soilborne Streptomyces Varies with Location, Phylogeny, and Nitrogen Amendment; Microb.Ecol.; 2013; 1-11 2013 [Full Text] e001

Schlatter, Daniel C.; Global Biogeography and Local Adaptation of Streptomyces; 2013; Ph.D. Thesis University of Minnesota Digital Conservancy Permanent URL 2013 [Full Text] e001

Trivedi, P.; Anderson, I. C.; Singh, B. K.; Microbial modulators of soil carbon storage: integrating genomic and metabolic knowledge for global prediction; Trends Microbiol., 2013, 21, 12, 641 - 651 2013 [Full Text] e001

Fierer, N.; Lauber, C.L.; Ramirez, K.S.; Zaneveld, J.; Bradford, M.A.; Knight, R.; Comparative metagenomic, phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients; ISME J., 2012, 6, 5, 1007 - 1017 2012 [Full Text] e001

Fornara, Dario A.; Tilman, David; Soil carbon sequestration in prairie grasslands increased by chronic nitrogen addition; Ecology, 2012, 93, 9, 2030 - 2036 2012 [Full Text] e001 e002

Tilman, D.; Reich, P. B.; Isbell, F.; Biodiversity impacts ecosystem productivity as much as resources, disturbance, or herbivory; Proceedings of the National Academy of Sciences; 2012; 109, 26, 10394-10397 2012 [Full Text] e001 e002 e003 e004 e012 e062 e098 e120 e141 e172