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Methods for Experiment 141 -

Burning

From 2000-2012 BioCON was burned approximately every other spring. Since 2013 BioCON is burned every year in the fall. BioCON is burned using the National Fire Academy prescribed burning method. All plots in each ring are burned in a wind driven pass ignited on the upwind side of the ring. Plots with vegetation that does not burn well during the first burn are spot burned. Years burned: 2000, 2002, 2003, 2005, 2007, 2009, 2011, 2012, 2013, 2014, 2015, 2016, 2017 (last updated 2017).

Carbon Dioxide Treatment

Elevated CO₂ treatment rings 1, 3 and 5 receive air with a target CO₂ concentration set at 550 ppm in 1997, gradually rising to 565 ppm in 2013 to address rising carbon dioxide concentrations in the atmosphere. The ambient CO₂ treatment rings 2, 4 and 6 are ?fumigated’ with ambient air with no experimental CO₂ addition in order to protect against erroneous results caused by the FACE equipment (see FACE technology). Elevated CO₂ treatments are applied seven days per week, during daylight hours, for the full growing season (roughly May 1 to October 15).

Experiment Design

Main Experiment BioCON 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 nmber 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 plant 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. Water Experiment: Implemented in 2007 Water treatments were added to 48 of the BioCON 9-species plots. Half of these receive natural rain fall while the other half experience rain removal via portable rain shelters. The goal of this sub-experiment is to examine how inputs of water, CO₂ and N interact to influence soil water availability, soil-plant interactions that influence available N, interactions with the belowground community of decomposers and mutualists, and thus net primary production (NPP) and plant and soil C pools. Warming Experiment: Implemented spring 2012 Uses 48 of the BioCON 9-species plots. These plots now contain 6-7 species on average. Although the major focus of this project is on Warming treatment plots, understanding of the main, interactive and indirect effects of each of the global change factors (diversity, CO₂, N, temperature, rainfall) in BioCON will be gleaned from observations of responses of microbes, plants or other organisms in the other 330 plots in BioCON, each with some subset of the treatments in the full factorial experiment that is the core of this project. The warming treatment is applied pairing below and aboveground heating to elevate ecosystem temperature. The treatments continuously elevate growing season plant and soil temperatures by approximately 2 degrees C.

FACE System Schematic

FACE Technology

BioCON uses a unique Free Air CO₂ Enrichment (FACE) technology to elevate the atmospheric concentration of CO₂ in the experimental plots. The FACE system used in the BioCON experiment was developed at Brookhaven National Laboratory. It uses natural wind conditions to carry CO₂ enriched air across the vegetation. Because the plants are outside in a more natural environment, the chamber effects normally created by enclosures such as greenhouses are reduced or eliminated. Wind direction, wind velocity, and [CO₂] are measured at the center of each plot and this information is used by a computer-controlled system to adjust CO₂ flow rate to maintain the target elevated [CO₂] (FACE System Schematic). Only pipes on the upwind side of the plots release CO₂, unless wind velocity is very low, at that time CO₂ is released alternately from adjacent release points. Fast feedback algorithms avoid large overshoots in response to fluctuations in [CO₂] and provide a stable elevation of [CO₂].

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.

Initial species planted into plots

1 indicates species was planted in 1997, 0 indicates not planted

Location and site description

BioCON is located at Lat. 45N, Long. 93W. The site is on a glacial outwash sandplain and production is nitrogen limited. The experiment was set up in a secondary successional old field after the existing vegetation was cleared. BioCON consists of 371 2-meter x 2-meter plots, arranged into 6 circular areas or ?rings? (20 meter diameter), each containing 61, 62, or 63 plots.

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. First, we filled a small tin full of soil (approximately 100g) and put it in a bucket. We did this 31 times for ring one. Each ring will have a varying number of tins full of soil based on the number of plots in that ring that are receiving the nitrogen application. We then added 3.28g 15NH₄15NO₃ in the ring one bucket (each ring will have a different amount depending on the number of N addition plots in that ring). If the ring has 30 plots to receive the application 3.17g of 15NH₄15NO₃ were added. If the ring has 32 plots to receive the application 3.39 g of 15 NH₄15NO₃ were added. We mixed up the contents in the bucket and then placed a tin full of soil in each 2lb paper bag labeled with the plots for ring one. We then placed 15.69g of NH₄NO₃ in each paper bag and mixed the soil with the15N and the NH₄NO₃ as much as possible. We then repeated this with the appropriate number of tins of soil per bucket and appropriate amounts of 15N for each subsequent ring. When applying the fertilizer, we mentally created four equal squares in the plot and tried to place equal amounts of fertilizer in each of the four squares.

Warming Treatment

Started spring 2012 in 48 of the 9 species plots. 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 for approximately 8 months per year (roughly the time of year when ambient temperatures are greater than plus/minus 3 degrees C on average). 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.

Water Treatment

Began in 2007, applied in 48 9-species plots. To implement H2O treatments we designed and built 24 portable rain-out shelters that remove almost all rainfall (approximately 95-98%) from a given plot when they are in place The rainout shelters cover entire individual plots and consist of removable shelters that fit over permanent frames situated around the outside of each plot. The shelters have a slanted roof and four individual sidewalls. There are gaps below the roof and above and below the sidewalls to allow considerable air flow. To prevent runoff into adjacent plots, intercepted rain is channeled using gutters, and moved well outside each ring. The protocol is to put the shelters into place immediately prior to 45% (on average) of significant rainfall events between May 10-August 15 and remove them after the completion of these events. In the case of predicted nighttime precipitation, shelters are left in place until sunrise when they are removed. Shelters are in place much less than 1% of the time (nearly always under cloudy conditions and rarely during midday) and cumulatively reduce integrated PPFD (mid-May to mid-August) by approximately 0.05% (1/2000th) pools.

Weeding

Species composition is controlled by hand weeding 2-4 times per growing season. Species distribution cards are used to identify a list of target species for each plot; all non-target species are removed. Weeding tools are used if needed to try and remove roots of each non-target plant without damaging other plants surrounding it. If soil is disturbed during the process of extracting plants, it’s smoothed out afterwards. Portable elevated weeding benches that span the plot are used to prevent stepping or kneeling in the plots during weeding.

aake141 - Lupinus Transgenerational Effects

Growth measures Transgenerational Effects Lupinus perennis

We measured plant height, leaf number and length of longest leaflet monthly throughout the growing season. After approximately 3 months of growth, all plants were harvested and aboveground biomass was weighed after drying for 2 days at 60C. Length of the longest Lupinus root was measured.

Sampling Method Transgenerational Effects

In Fall 2002, seeds were collected from a single maternal plant of each species in each of the 16 species plots in the BioCON experiment. In Spring 2006, these seeds were then planted into pots and placed into the BioCON experiment under ambient or elevated CO₂ conditions. One Lupinus seed was planted into each plot. Four Poa and Schizachyrium seeds were planted into each plot; therefore, the Poa and Schizachyrium data are mean values of the 1-4 individuals per pot (depending on how many germinated).

aale141 - Poa Transgenerational Effects

Growth measures Transgenerational Effects Poa pratensis

We measured plant height, leaf number and culm number monthly throughout the growing season. After approximately 3 months of growth, all plants were harvested and aboveground biomass was weighed after drying for 2 days at 60C.

Sampling Method Transgenerational Effects

In Fall 2002, seeds were collected from a single maternal plant of each species in each of the 16 species plots in the BioCON experiment. In Spring 2006, these seeds were then planted into pots and placed into the BioCON experiment under ambient or elevated CO₂ conditions. One Lupinus seed was planted into each plot. Four Poa and Schizachyrium seeds were planted into each plot; therefore, the Poa and Schizachyrium data are mean values of the 1-4 individuals per pot (depending on how many germinated).

aame141 - Schizachyrium Transgenerational Effects

Growth measures Transgenerational Effects Schizachyrium scoparium

We measured plant height, leaf number and culm number monthly throughout the growing season. After approximately 3 months of growth, all plants were harvested and aboveground and belowground biomass was weighed after drying for 2 days at 60C.

Sampling Method Transgenerational Effects

In Fall 2002, seeds were collected from a single maternal plant of each species in each of the 16 species plots in the BioCON experiment. In Spring 2006, these seeds were then planted into pots and placed into the BioCON experiment under ambient or elevated CO₂ conditions. One Lupinus seed was planted into each plot. Four Poa and Schizachyrium seeds were planted into each plot; therefore, the Poa and Schizachyrium data are mean values of the 1-4 individuals per pot (depending on how many germinated).

acae141 - Vac Sampling arthropod community

All Insect Vac Sampling

The data in All Insect Vav Sampling are from samples taken by John Haarstad and crew as part of a study by Dr. Mark Ritchie between 2000-2003. This dataset contains sampling data that was designed to look at the whole arthropod community in BioCON plots. Separate samplings that targeted aphids specifically were conducted in 2000-2002 and that data is contained in a different file. Sampling methods were the same as for the aphid-specific sampling and are as follows. Arthropods were sampled from eight 0.08 meter square subplots in each plot with a Vortis ? insect vacuum sampler (airflow 10L/s), frozen at -20 degrees C. Eight subsamples/plot was chosen because it allowed all plots to be sampled in a reasonable amount of time. Samples were taken 25/June/01 (rings1,2,5,6) and 3/July/01 (rings 3,4), 10/July/02, 7-11/ July/03 in all plots. Additional bare ground plots added in 2002 and 2003 to bring the total bare ground plots/ring up to 2 each were also sampled. Samples were frozen and combined when sorted. Arthropods of all kinds were removed and identified and enumerated. Sandy samples were sifted to aid sorting. Identification was generally to species or genus, but occasionally a morphological descriptive is used when identification was uncertain. Collection and identifications were done by primarily by John Haarstad.

All Insect Vac Sampling Data preparation and Irregularities

Data preparation was started by John Haarstad and completed by Colleen Satyshur after his death. John did not assign Arnett codes, so Colleen compiled his codes matched with raw data abbreviations from e120, e052, e151 and e122 in order to deduce the most accurate taxonomic information. John placed asterisks before some of his species names and Colleen kept that information in the final file. Irregularities 1. In 2003 two plots are labeled ?unl? and one plot number has an * after it. Notes on raw data sheets indicate that ?unl? stands for ?unlabeled?. Ring1: there is a 62nd plot, an extra plot, which is labeled ?unl?. This may be plot 367 which is a bare ground plot that was added to ring 2 and is listed in 2002, but not 2003. The plot is labeled ?unl? in this data set. Ring 2: In the place where plot 96 would be in the data sequence, the plot is labeled ?92*?. There is a 92 in the data sequence already. Ring 3: in the place where plot 145 would be in the data sequence, the plot is labeled ?unl?. This plot is labeled ?145u? in this data file. 2. 10 samples instead of 8 were taken from Plot 236 in 2002.

acbe141 - Vac Sampling aphids

Vac Sampling aphids

The data in this file are from samples taken by John Haarstad and crew as part of a study by Dr. Mark Ritchie between 2000-2003. This dataset contains data from from 2000-2002 sampling that specifically targeted aphids. Sampling was generally restricted to plots that contained lupine. Arthropods were sampled from eight 0.08 meter square subplots in each plot with a Vortis ? insect vacuum sampler (airflow 10L/s), frozen at -20 degrees C. Eight subsamples/plot was chosen because it allowed all plots to be sampled in a reasonable amount of time. Samples were taken 17-25 May 2000, after a warm dry spring and 20-25 June 2001 after a cool wet spring and in July of 2002 (day not recorded). Samples were frozen and combined when sorted. Aphids and Empoasca (Homoptera, Cicadellidae) were enumerated. Sandy samples were sifted to aid sorting. Collection and identifications were done by primarily by John Haarstad. Measures of lupine C, N and seed set were also taken but are not included here.

Vac Sampling aphids- Data preparation and Irregularities

Data preparation was done by John Haarstad and Mark Ritchie. Colleen Satyshur combined and formatted files from Dr. Ritchie in 2013 to create this data set. Irregularities 1) Empoasca were only counted in 2002. 2) Sometimes number of vac samples per plot varied. This is noted in the data set.

acce141 - Photosynthesis Leaf Carbon and Nitrogen

Photosynthesis Leaf CN - Instrumentation

Leaves used in gas exchange measurements are ground and analyzed for tissue nitrogen concentration with a C-N Analyzer (NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc.).

Photosynthesis Leaf CN processes

Leaves used in photosynthesis gas exchange measurements are kept cool after harvest, scanned to determine area, then oven-dried at 65 degrees C. Dried leaves are ground and analyzed for tissue nitrogen and carbon concentration. In some years individual leaves from repeated measurements of treatment/plot/species are combined into a composite sample prior to analysis.

acfe141 - Lysimeter Water Treatment Plots

Lysimeter Water Treatment Plots

The lysimeters for this experiment were installed in the fall of 2007. One lysimeter was installed in each of the 48 water treatment plots in the southwest portion of the percent cover area. The lysimeters were placed in the soil at a 64? angle from the ground. The lysimeters were sampled periodically throughout the growing season after rainfalls. We created a vacuum in each of the bottles then placed them on each lysimeter for approximately 24 hours of collection. If it was sunny outside, the bottles were wrapped with aluminum foil to decrease the amount of evaporation. The bottles were kept cool until the samples were processed at the lab. For each individual sample we ran 3 replicates and those replicates were averaged. The samples were sent to University of MN to be processed for DIN, DOC, and TDN. Chris Buyarski, from Sarah Hobbie?s lab, analyzed these samples. All of the equipment used (bottles, septa, caps etc) was washed in an acid bath then with DI water or with only DI water.

Lysimeter Water Treatment Plots - Instrumentation

Dissolved inorganic N (DIN, NO₃- +NH₄+) was done on an Lachat (Lachat QuikChem 8500, Hach Company, Loveland, CO, USA) and total dissolved N (TDN) on a Shimadzu TOC-VCPN (Shimadzu Scientific Instruments, Wood Dale, Illinois, USA).

ache141 - Total and non-hydrolyzable soil carbon and nitrogen

Total and non-hydrolyzable soil carbon and nitrogen

In August 2006, we sampled soils from the 48 16 species plots and 64 monoculture plots (total 112 plots) by taking three 2.5-cm-diameter cores (0 - 20 cm) per plot. Soils were composited by plot and immediately sieved (2 mm). Visible roots were removed by hand. We took immediate subsamples for soil C respiration incubations and gravimetric soil water content. The remaining soil was airdried, ground, and subsampled for total C and N and nonhydrolyzable C and N analyses. Resistant soil C was estimated using an acid digest procedure that hydrolyzes polysaccharides and nitrogenous material, leaving a residue consisting primarily of lignin and polyaromatic humics. Identifiable plant materials were removed from air-dried, ground soil. One-gram soil samples were refluxed for 16 hours in digestion tubes with 10 mL of 6 M hydrochloric acid solution. The remaining residue was filtered, washed with 100 mL of nanopure water, dried for 24 hours in a 60 degrees C oven, weighed, and analyzed for total C by combustion (Model ECS 4010, COSTECH Analytical, Valencia, CA). The remaining nonhydrolyzable, or chemically resistant, C represents resistant soil C, which 14C-dating indicates is much older than bulk soil. Results from this data are published in: Reid, J. P., Adair, E. C., Hobbie, S. E., & Reich, P. B. (2012). Biodiversity, Nitrogen Deposition, and CO₂ Affect Grassland Soil Carbon Cycling but not Storage. Ecosystems, 15(4), 580-590. doi:10.1007/s10021-012-9532-4

acie141 - Carbon respiration from soil incubation

Carbon respiration from soil incubation

Incubations conducted by EC Adair and J Reid in SE Hobbie lab at University of Minnesota. Incubation was conducted from August 25, 2006 - September 27, 2007 Incubations were conducted at 70% field capacity for 391 days Sampling: August 2006, we sampled soils from 112 plots; three 2.5 cm diameter cores (0-20 cm) per plot. Soils were composited by plot and immediately sieved (2 mm). Visible roots were removed by hand. We took immediate subsamples for soil C respiration incubations and gravimetric soil water content. The remaining soil was air dried, ground, and subsampled for total C and N and non- hydrolyzable C and N analyses. Cumulative respriation was calculated as: D(t-1)rate + avg[D(t-1)&D(t)]*days between samplings+D(t)rate, except t = 391 is previous t For cumulative respiration, all missing data (NA) from Di.DRR were outliers (largely negative values) and were replaced by the average respriation rate from the previous and following measurements within the same jar. This accounted for 18 of 1456 values These data were used in: Reid, J., E.C. Adair, S.E. Hobbie and P.B. Reich. 2012. Biodiversity, nitrogen deposition and CO₂ affect grassland soil carbon cycling but not storage. Ecosystems. 15:580-590. Reid, Joseph Pignatello. 2013 Non-equilibrium dynamics of ecosystem processes in a changing world. PhD Thesis University of Minnesota; Digital Conservancy Permanent URL http://purl.umn.edu/159915

acye141 - 1996 Ring soil texture, pH and Cation Exchange Capacity (CEC)

1996 Soil analyses

Soil texture analysis was done at UMN Department of Forest Resources using the Hydrometer Method-calculations were based on 30 seconds and 120 minutes. pH was measured at UMN Department of Forest Resources. Bray-P and CEC analyses done at the Research Analytical Laboratory at the University of Minnesota http://ral.cfans.umn.edu/

adue141 - Oak leaf water potential

Oak leaf water potential instrumentation

Scholander Pressure Chamber (model 600 http://www.pmsinstrument.com/products/model-600-pressure-chamber-instrument) ImageJ software (http://imagej.nih.gov/ij/) ML3 ThetaProbe Soil Moisture Sensor (Delta-T Devices) iButton temperature and humidity sensor (DS1923 http://www.maximintegrated.com/en/products/comms/ibutton/DS1923.html)

Oak leaf water potential methods

During the summer of 2012 Wright sampled predawn and midday leaf water potential of a total of 151 separate oak seedlings that were planted into BioCON as acorns in 2010 and 2012. She selected days for measurements that varied temperature and humidity. On each day she sampled predawn leaf water potential (3-5am) using a Scholander Pressure Chamber. She kept the harvested leaf and then scanned it and measured leaf area using ImageJ software. At midday on the following day (11am-1pm) she repeated this process on the same plants (to compare with predawn values). On the same day she also measured soil moisture in a circle around the sampled oak seedlings using an ML3 ThetaProbe Soil Moisture Sensor. During 2011 & 2012 she continuously measured microclimate air temperature and relative humidity in the plots using an iButton temperature and humidity sensor (DS1923). These sensors measured every five minutes and were downloaded every two weeks. All BioCON plots used in this dataset have ambient treatments for CO₂, water removal and temperature.

aege141 - Light and heavy soil fraction total N and delta 15N

Instrumentation

Light and heavy soil fractions were analysed for total total N and d15N on a mass spectrometer (ThermoFinnigan Delta Plus, Bremen, Germany).

Sampling and analyses methods

In July 2002, we sampled three soil cores (diameter 2.5 cm) to 10-cm depth from each plot. We sieved soils (2 mm) to remove roots and then separated soils into light and heavy soil fractions in a sodium iodide (NaI) solution (density 1.8 g cm)3, Gregorich & Ellert 1993). We placed 15 g of air-dry soil into 50 mL plastic centrifuge tubes and added 30 mL NaI solution. After 16 h of shaking and 1 day of settling we removed the light soil fraction floating on top of the solution. Both light and heavy fraction were filtered (through Magna 0.45 lm nylon and Whatman G/F glass fiber filter, respectively) and thoroughly rinsed with nanopure water. Light and heavy fractions were dried and ground with mortar and pestle. We calculated light and heavy soil fractions per m2 using soil bulk densities that were measured from a soil core (diameter 5 cm) to 10-cm depth taken in each plot in August 2003. The heavy soil fraction consists of clay-associated soil organic matter that is more recalcitrant while the light soil fraction is thought to contain partially degraded organic compounds that undergo further relatively rapid decomposition (Gregorich & Janzen 1995). For the N fertilized and bare plots we analysed the light and heavy soil fraction for total N and d15N. Details for sampling and analyses are reported in:Dijkstra, F. A.; Hobbie, S. E.; Knops, J. M. H.; Reich, P. B.; Nitrogen deposition and plant species interact to influence soil carbon stabilization. Ecology Letters 7:1192-1198. 2004 Method references cited: Gregorich, E.G. & Ellert, B.H. (1993). Light fraction and macro- organic matter in mineral soils. In: Soil Sampling and Methods of Analysis (ed. Carter, M.R.). CRC Press, Boca Raton, pp. 397?407. Gregorich, E.G. & Janzen, H.H. (1995). Storage of soil carbon in the light fraction and macroorganic matter. In: Structure and Soil Organic Matter Storage in Agricultural Soils (eds Carter, M.R. & Stewart, B.A.). Lewis Publishers, Boca Raton, FL, pp. 167?190.

aeie141 - Monoculture species green leaf total N and delta 15N

Monoculture species green leaf 15N

In June 2002, green leaves of all legume species and all reference plants were collected, dried, ground to a fine powder and analyzed for total N and delta 15N. is expressed as ?per mil? relative to atmospheric N2 [(Rsample/Rstandard − 1) ? 1000, where R is the ratio of 15N : 14N, and the concentration of atmospheric 15N is 0.366%]. These data permitted an estimation of the percentage of leaf N that originated from soil pools vs the amount that is obtained from atmospheric N via rhizobial N fixation (Shearer & Kohl, 1991). Our approach followed that described by Shearer & Kohl (1991, see also Handley & Scrimgeour, 1997). Non-legume species with similar growth forms growing in the same soil as the legume of interest were used to estimate delta 15Nsoil-derived N. Thus, a central assumption of this method is that the delta 15N of the non-legume species is an accurate representation of soil-derived N in the legumes. Published results from this data in: West, J.; Hille Ris Lambers, J.; Lee, T. D.; Hobbie, S. E.; Reich, P. B.; "Legume species identity and soil nitrogen supply determine symbiotic nitrogen-fixation responses to elevated atmospheric [CO₂]. New Phytologist, 167:523-530" 2005 Method references cited: Handley LL, Scrimgeour CM. 1997. Terrestrial plant ecology and N-15 natural abundance: The present limits to interpretation for uncultivated systems with original data from a Scottish old field. In: Begon M, Fitter AH, eds. Advances in Ecological Research, Vol. 27. London, UK: Academic Press Ltd, 133?212. Shearer G, Kohl D. 1991. The 15N natural abundance method for measuring biological nitrogen fixation: practicalities and possibilities. In: Flitton SP, ed. Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies. Vienna, Austria: International Atomic Energy Association, 103?115.

Monoculture species green leaf 15N - Instrumentation

ThermoFinnigan Delta Plus mass spectrometer; Kansas State University Stable Isotope Mass Spectrometry Laboratory, Manhattan, KS, USA

aeje141 - Leaf 15N isotope, total N and delta 15N from 9 species water treatment plots

Analysis instrumentation

Leaf Tissue N15 isotopic Nitrogen Analysis is done at UC Davis Stable Isotope Facility, Department of Plant Sciences Davis, California, USA. PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK).

Sampling method

As possible, 3 samples of each legume species and two samples of each reference are collected from each treatment combination [CO₂ x N x H20] in each year. Due to declining diversity within the BioCON plots, all species in all years in all combination were not able to be collected.

Sampling protocol

Plots sampled were planted in 1997 with 9 randomly selected species from the 16 BioCON species. The water treatment sub-experiment began in 2007. Upper fully expanded leaves of legumes are collected in June and August. Two non-nitrogen fixing forb species are also collected as reference species. Leaves are dried, ground and analyzed for N15.

aeke141 - Leaf delta 13C and total C from 9 species water treatment plots

Analysis instrumentation

Leaf Tissue N15 isotopic Nitrogen Analysis was done at UC Davis Stable Isotope Facility, Department of Plant Sciences Davis, California, USA. PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK).

Sampling protocol

Plots sampled were planted in 1997 with 9 randomly selected species from the 16 BioCON species. The water treatment sub-experiment began in 2007. Upper fully expanded leaves of legumes are collected in June and August.

aele141 - June aboveground 15N isotope, total N and delta 15N

Instrumentation

N15 isotopic Nitrogen Analysis is done at UC Davis Stable Isotope Facility, Department of Plant Sciences Davis, California, USA. PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK).

Sampling protocol

From 2000 to 2006 samples were collected from the June aboveground clip-strip biomass. After 2006 June clipped biomass was not done, so plants were hand collected from the June clip-strip zones within each monoculture plot. Three individuals of each species were collected. When individual plants were large, 1 stem from 2 different individuals OR when stems were really large, just 1 stem and various aged leaves from others were taken. Samples were dried and ground for analysis.

aeoe141 - Leaf net photosynthesis in CO2 x N x water treatment combinations

Measurement protocols

Leaf light saturated net photosynthesis: In situ rates of leaf net photosynthesis (A) were measured on upper fully expanded sun leaves using either CIRAS-1 portable infrared gas exchange systems (PP Systems, Hitchin, UK) operated in open configuration with controlled temperature, CO₂ concentration, and vapor pressure (2007-2008); or LICOR 6400 portable infrared gas exchange systems (Li-Cor Inc, Lincoln Nebraska) using program OPEN 6.1.3-4 equipped with a 6400-02B red/blue external light source set to PAR 1500. Volumetric soil water content: TH2O Portable Soil Moisture Probe (Dynamax, Inc. Houston, TX) with probe depth of 60 mm was inserted near base of plant being used in measurement. Leaf area: The projected areas of leaves used in gas exchange measurements were determined using a digital image analysis program (WINRHIZO 3.9, Regent Instruments, QC, Canada, SCION IMAGE or IMAGEJ, National Institutes of Health, Bethesda, MD, USA).

aese141 - Soil Organisms

Soil organisms samplings and measurements

In August 2010, i.e. 13 years after establishment of the CO₂ and N treatments, and after 4 years of summer drought treatment, we took soil samples to investigate treatment effects on soil biota. From each of the 48 plots, we took three small soil samples (diameter 2 cm, depth 6 cm) and one larger soil sample (diameter 5 cm, depth 6 cm) using steel corers. The small soil samples were pooled in a plastic bag carefully, but thoroughly homogenized and stored at 4 ?C until further processing. Large soil samples were kept intact and stored in plastic containers at 4 ?C until further processing. Soil from the small samples was subdivided into three portions of approximately 15 g of soil (fresh weight) and used to measure soil microbial biomass, protozoans, and nematodes, whereas large samples were extracted for soil microarthropods. Before measurement of soil microbial biomass, soil subsamples were sieved (2 mm) to remove larger roots, animals, and stones (Anderson & Domsch, 1978). Microbial biomass C of approximately 5 g soil (fresh weight) was measured using an O2-microcompensation apparatus (Scheu, 1992). Substrateinduced respiration was calculated from the respiratory response to D-glucose for 10 h at 22 ?C (Anderson & Domsch, 1978). Glucose was added according to preliminary studies to saturate the catabolic enzymes of microorganisms (4 mg g_1 dry weight dissolved in 400 ?l deionized water). The mean of the lowest three readings within the first 10 h was taken as maximum initial respiratory response (MIRR; ?l O2 h_1g_1 soil dry weight) and microbial biomass (lg C g_1 soil dry weight) was calculated as 38 9 MIRR (Beck et al., 1997). Total numbers of protozoa (i.e., active and encysted forms of amoebae, ciliates, and flagellates) were enumerated using a modified most-probable number method (Darbyshire et al., 1974). Briefly, 5 g fresh weight of soil was suspended in 20 ml sterile Neff?s modified amoebae saline (NMAS; Page, 1976) and gently shaken (70 rpm) for 20 min on a vertical shaker. Threefold dilution series with nutrient broth (Merck, Darmstadt, Germany) and NMAS at 1 : 9 v/v were prepared in 96- well microtiter plates (VWR, Darmstadt, Germany) with four replicates each. The microtiter plates were incubated at 15 ?C in darkness, and the wells were inspected for presence of protozoa using an inverted microscope at 9100 and 9200 magnification (Nikon, Eclipse TE 2000-E, Tokyo, Japan) after 3, 6, 11, 19, and 26 days. Densities of protozoa were calculated according to Hurley & Roscoe (1983), and related to g soil dry weight. Nematodes were extracted from 10 g soil (fresh weight) using a modified Baermann method (Ruess, 1995). After an extraction time of 30 h, nematodes were preserved in 4% formaldehyde, counted, and related to g soil dry weight. Subsequently, 10% of the individuals (but not less than 100 individuals, if possible) were additionally identified to family level, or if necessary, to genus level and assigned to the trophic groups bacterial feeders, fungal feeders, omnivores, plant feeders, and predators according to Yeates et al. (1993). Soil microarthropods were extracted by heat (Kempson et al., 1963), collected in diluted glycerol, and transferred into ethanol (70%) for storage. Soil animals were determined following Gisin (1960), Fjellberg (1980), Hopkin (2007), Krantz & Walter (2009) and Schaefer (2000), and counted (abundance m_2). Astigmatic and prostigmatic were pooled without further determination; however, for the calculation of microarthropod taxa richness, morpho-species were distinguished. Method references cited can be found in: Eisenhauer, N., Dobies, T.; Cesarz, S.; Hobbie, S. E.; Meyer, R. J.; Worm, K.; Reich. P. B.; 2013; Plant diversity effects on soil food webs are stronger than those of elevated CO₂ and N deposition in a long-term grassland experiment.; PNAS 110:6889-6894 2013 These data were used in: Eisenhauer, Nico; Cesarz, Simone; Koller, Robert; Worm, Kally; Reich, Peter B.; Global change belowground: impacts of elevated CO₂, nitrogen, and summer drought on soil food webs and biodiversity. Global Change Biology. 18:435?447, doi: 10.1111/j.1365-2486.2011.02555.x 2012 Eisenhauer, N., Dobies, T.; Cesarz, S.; Hobbie, S. E.; Meyer, R. J.; Worm, K.; Reich. P. B.; 2013; Plant diversity effects on soil food webs are stronger than those of elevated CO₂ and N deposition in a long-term grassland experiment.; PNAS 110:6889-6894 2013

aewe141 - Photosynthesis of Bromus inermis and Andropogon gerardi under treatment combinations of CO2 x Warm in 9 species plots

Instrumentation

Li-COR 6400 portable infrared gas exchange systems (Li-Cor Inc., Lincoln Nebraska) using program OPEN 6.1.3-4 equipped with a 6400-02B red/blue external light source set to PAR 1500. The projected areas of leaves used in gas exchange measurements were determined using a digital image analysis program (IMAGEJ, National Institutes of Health, Bethesda, MD, USA).

Measurements of leaf level photosynthesis and stomatal conductance

Objective: To determine how warming treatments interact with CO₂ and/or reduced precipitation to affect leaf level photosynthetic and stomatal conductance in these species. Measured leaf level gas exchange of 4 upper fully expanded sun leaves in 2 plots per each of the 4 CO₂ x warm treatments (n=8 plots, N=32 leaves) both years. All plots at ambient nitrogen. In 2013, each leaf was measured at 3 temperatures 23, 26, 29 degrees Celsius (and all from low to high temp) and in 2014, each leaf was measured at 26 degrees Celsius between 09:00 and 15:00 local time (replicates randomly measured across day and time of day) and otherwise at the environmental conditions the leaves were experiencing at the time.

afce141 - Litter biomass carbon nitrogen from water treatment plots

Protocol - Litter biomass carbon nitrogen from water treatment plots

Litter collection: Litter of 3 species (Lupinus perennis, Poa pratensis, and Schizachyrium scoparium) was collected. Lupine and poa leaf litter from the fields surrounding BioCON. Some of the schizachyrium litter we collected was also from the fields surrounding BioCON, but most of it came from a bale that Sarah Hobbie provided. Leaves were separated from the stems kept for initial litter and air dried. Making litterbags: A total of 150 litterbags were constructed: 3 species x 2 litter treatment destinations x 5 reps x 5 harvests. A fiberglass screen was used to make 15cm by 15cm square litterbags. Approximately 5 g of dried litter then placed in the litterbag, edges were sealed with a heat sealer. An aluminum tag labeled with the plot number, species type, and harvest number on it was stapled to the bag. All bags from the same plot were strung together in order to easily move the bags around in the plot before fires and during harvest. Litterbag placement and removal: The litterbags were placed in 10 ambient CO₂ and ambient nitrogen treated plots within the water experiment in October 2007. There were a total of 15 bags placed in each plot. One litter bag per plot was removed in the early summer and one in the fall each year from 2008-2010. Upon harvest, litter was dried (65 degrees C), weighed and analyzed for total C and N to determine mass loss and litter N dynamics. Initial litter carbon and nitrogen: Dried litter for each species was well mixed to homogenize. Three sub-samples from each species litter were dried (65 degrees C), weighed and analyzed for total carbon and nitrogen. Mean of sub-sample CN values were used for each species.

afde141 - Oak seedling survival

Protocol oak seedling survial

In October 2001, we collected and germinated recently fallen acorns from multiple local adult bur oak trees at the Cedar Creek Ecosystem Science Reserve. We combined and homogenized the acorns and planted three germinated seeds in each plot. Seedling were planted in a line on the south perimeter of each plot. In June 2002, we recorded oak survival and the number of leaves per plant. In August 2002, we recorded plant survival, height, and the number of leaves per plant. In August 2004, we recorded survival, height, diameter, and the number of leaves per plant and then harvested all surviving individuals.

afke141 - Photosynthetic CO2 response curves at controlled light, CO2 and temperatures

Abstract: Photosynthetic CO2 response curves at controlled light, CO2 and temperatures

These measurements are part of a 15-year dataset of photosynthetic CO₂ response curves measured in the field for 51 different plant species (mostly trees) over 20 different sites in N. America, Australia and Europe including free-air CO₂ enrichment (FACE) sites and particularly EucFACE. Ecosystem types range from arid shrubland temperate deciduous forest to open eucalypt woodland and wet sclerophyll forest. Species names are done according to the Taxonomic Name Resolution Service TRNS. The BioCON portion of this data set includes twenty eight response curves. The complete data set is available from Research Data Australia (a national e-research platform) portal. Data Citation: Ellsworth, David; Crous, Kristine (2016): A global dataset of photosynthetic CO₂ response curves measured in the field at controlled light, CO₂ and temperatures. University of Western Sydney. DOI: 10.4225/35/569434cfba16e Results from these data have been published in: Atkin et al (2015), Global variability in leaf respiration in relation to climate, plant functional types and leaf traits. New Phytologist. DOI: 10.1111/nph.13253 2015

Cedar Creek BioCON Methods

Photosynthetic CO₂ response curves (A-Ci) were measured on leaves of each plant species with a minimum of seven different CO₂ concentrations between 60 and 1500 mmol-2 mol-1 using saturating light conditions (photon flux density of 1800 mmol m-2 s-1) and controlled temperatures (leaf temperatures of 28 to 30 degrees C) in the leaf cuvette. All grass measurements were from the topmost fully expanded leaf adjacent to the flag leaf to ensure similar leaf ages. Leaves at the top of the plant canopy developed in full sun were measured typically in the morning before the onset of stomatal closure. Measurements were typically conducted on sunny days during nondrought periods and were made during the growing season at times designed to coincide with peak photosynthetic activity approximately mid-summer. Response curves were measured during 1998, 1999 and 2000 on BioCON species: Achillea millefolium (non-leguminous forb), Agropyron repens (C-3 grass), Anemone cylindrica (non-leguminous forb), Bromus inermis (C-3 grass), Lespedeza capitata (leguminous forb), Lupinus perennis (leguminous forb), Poa pratensis (C-3 grass), Solidago rigida (non-leguminous forb). All plots at ambient CO₂ treatment and with monoculture diversity, except the following four species plots: 89, 218, 340, 364, 337, 363.

Instrumentation

LICOR 6400 portable infrared gas exchange systems (Li-Cor Inc, Lincoln Nebraska). All parameters in the dataset as described in the Li-6400 instruction manual (https://www.licor.com/env/products/photosynthesis/manuals.html ).

afqe141 - Soil metagenome fungal responses to elevated CO2

Sampling and genomic methods

Sampling: 24 plots (12 from aCO₂ and 12 from eCO₂), with 16 plant species and no additional N supply, were used. Bulk soil samples were obtained in July 2009 under ambient and eCO₂ conditions for microbial community analysis, and each sample was composited from five soil cores at a depth of 0 to 15 cm. All samples were immediately transported to the laboratory, frozen and stored at minus 80 degrees Celsius for DNA extraction, PCR amplification, and 454 pyrosequencing. DNA extraction, purification, and quantification: Soil DNA was extracted by freeze-grinding mechanical lysis as described previously (39) and was purified using a low-melting-point agarose gel, followed by phenol extraction for all 24 soil samples collected. DNA quality was assessed by using ratios of 260 to 280 nm and 260 to 230 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE), and final soil DNA concentrations were quantified with PicoGreen (40) using a FLUOstar Optima (BMG Labtech, Jena, Germany). PCR amplification and 454 pyrosequencing: A total of 23 samples instead of 24 were subjected to 454 pyrosequencing due to insufficient remaining DNA for one of the samples. Amplification was performed using a fungal 28S rRNA gene primer pair with the forward primer LR3 (ACCCGCTGAACTTAAGC) and the reverse primer LR0R (CCGTGTT TCAAGACGGG), whose products are expected to be approximately 625 bp (41). A unique 8-mer barcode was added for each sample at the 5` end of the forward primer. The barcode primers were synthesized by Invitrogen (Carlsbad, CA) and used for the generation of PCR amplicons. Quadruplicate 20-ul PCRs were performed as follows: 4 ul of Promega GoTaq buffer, 0.5 ul of GoTaq DNA polymerase, 1.5 ul of Roche 25 mM MgCl2, 1 u of Invitrogen 10 mM deoxynucleoside triphosphate mix, 1 ul of each primer (10 pmol ul -1 ), 0.2 ul of New England BioLabs bovine serum albumin at 10 mg ml-1 , 1 l of template (1.43 ng DNA/ul), and 9.8 l of H2O. The cycling conditions were an initial denaturation of 94degrees C for 3 min, followed by 30 cycles of 94degrees C for 1 min, 51 degrees C for 40 s, and 72 degrees C for 1 min, and then a final extension at 72 degrees C for 10 min. Replicates were pooled and gel purified using a Qiagen gel purification kit after band excision. The products were further purified using a Qiagen PCR purification kit. After adapter ligation, amplicons were sequenced on an FLX 454 system (454 Life Sciences, Branford, CT) by Macrogen (Seoul, South Korea) using Lib-L kits and processed using the shotgun protocol. Data associated with this study were deposited in the NCBI database under BioProject accession number PRJNA232496. Results from this data are published in: Tu Q, Yuan M, He Z, Deng Y, Xue K, Wu L, Hobbie SE, Reich PB, Zhou J. 2015. Fungal communities respond to long-term CO₂ elevation by community reassembly. Appl Environ Microbiol 81:2445 to 2454. doi:10.1128/AEM.04040-14. 201 Method references cited: 39. Zhou J, Bruns MA, Tiedje JM. 1996. DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316 to 322. 40. Ahn SJ, Costa J, Rettig Emanuel J. 1996. PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res 24: 2623 to 2625. http://dx.doi.org/10.1093/nar/24.13.2623 41. Liu K-L, Porras-Alfaro A, Kuske CR, Eichorst SA, Xie G. 2012. Accurate, rapid taxonomic classification of fungal large-subunit rRNA genes. Appl Environ Microbiol 78:1523 to 1533. http://dx.doi.org/10.1128/AEM.06826-11

ahie141 - TeRaCON eight years collected species composition, productivity (NPP), soil carbon emissions and plant carbon stocks

Abstract

Disentangling impacts of multiple global changes on terrestrial carbon cycling is important, both in its own right and because such impacts can dampen or accelerate increases in atmospheric CO₂ concentration. Herein we report on an 8-year grassland experiment, TeRaCON, a subset of the BioCON experiment at Cedar Creek Ecosystem Science Reserve, that factorially manipulated temperature, rainfall, CO₂ and nitrogen deposition. Species composition, productivity (NPP), species biomass, soil carbon emissions and plant carbon stocks are measured under all sixteen combinations of two contrasting levels of temperature, rainfall, CO₂, and N.

Data collection and assembly methods

TeRaCON was established as a sub-experiment within the BioCON experiment. TeRaCON is composed of 48 plots chosen with a stratified random approach (to balance 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 approximately plus 2.5 degrees C soil and surface warming), two growing-season rainfall (ambient and approximately minus 30 percent April through September), two atmospheric CO₂ (ambient and plus 180 ppm), and two soil N (ambient and plus 4 gramsPerMeterSquaredPerYear) treatments. Aboveground biomass in each plot was sampled annually in early August, with a 10 x 100 cm clipping. We also estimated annual root production at 0-20 cm depth using in-growth root cores. We combined aboveground biomass and the in- growth root biomass as our estimate of net primary production (NPP) for every plot in every year. We also estimated total plant biomass pools by combining the aboveground data described above with measurements of belowground standing biomass (independent of the in-growth root core measurements), derived from three 5 cm diameter (0-100 cm deep) cores per plot. We also measured percent C of roots and aboveground biomass in every plot in every year from 2012-2018 and used these data to express biomass and productivity from 2012-2019 on a C rather than total biomass basis (total plant C pool and NPP, respectively). In 2016 we sampled soils in each plot in 0-10, 10-20, 20-40 and 40-60 cm horizons. We used measurements of soil bulk density and percent C to estimate total soil C pool from 0-60 cm depth. We combined 2016 soil C pool data with mean 2012-2019 total plant C pool data to estimate total ecosystem C pools. In every plot, we measured soil CO₂ flux approximately 12-15 times per year between early May and late September using a LI-COR 6400 gas exchange system with a LI-COR 6400-09 soil respiration chamber. To best match the time period during which all treatments were imposed, we used soil CO₂ flux data from May 1- July 31 herein; moreover we averaged these data to derive a single mean soil CO₂ flux value for each plot each year. Results from these data have been submitted as: Reich, P. B., Hobbie, S. E., Lee, T. D., Rich, R. Pastore, M. A., Worm, K. (2020) Synergistic effects of four climate change drivers on terrestrial carbon cycling. Nature Geoscience (accepted, in principle)

Instrumentation

Carbon analyses: ECS 4010 CHNSO Analyzer, Costech Analytical Technologies Inc., Valencia, CA, USA or NA 1500 CNS Analyzer, Carlo-Erba Instruments, Egelsbach, Germany Soil Carbon Flux: LI-COR 6400 gas exchange system with a LI-COR 6400-09 soil respiration chamber (LI-COR, Lincoln, Nebraska, USA)

ahje141 - TeRaCON eight year mean of NPP, carbon pools, and soil flux

Abstract

Disentangling impacts of multiple global changes on terrestrial carbon cycling is important, both in its own right and because such impacts can dampen or accelerate increases in atmospheric CO₂ concentration. Herein we report on an 8-year grassland experiment, TeRaCON, a subset of the BioCON experiment at Cedar Creek Ecosystem Science Reserve, that factorially manipulated temperature, rainfall, CO₂ and nitrogen deposition. Mean NPP and ecosystem carbon pools are measured under all sixteen combinations of two contrasting levels of temperature, rainfall, CO₂, and N.

Data collection and assembly methods

TeRaCON was established as a sub-experiment within the BioCON experiment. TeRaCON is composed of 48 plots chosen with a stratified random approach (to balance 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 approximately plus 2.5 degrees C soil and surface warming), two growing-season rainfall (ambient and approximately minus 30 percent April through September), two atmospheric CO₂ (ambient and plus 180 ppm), and two soil N (ambient and plus 4 gramsPerMeterSquaredPerYear) treatments. Aboveground biomass in each plot was sampled annually in early August, with a 10 x 100 cm clipping. We also estimated annual root production at 0-20 cm depth using in-growth root cores. We combined aboveground biomass and the in- growth root biomass as our estimate of net primary production (NPP) for every plot in every year. We also estimated total plant biomass pools by combining the aboveground data described above with measurements of belowground standing biomass (independent of the in-growth root core measurements), derived from three 5 cm diameter (0-100 cm deep) cores per plot. We also measured percent C of roots and aboveground biomass in every plot in every year from 2012-2018 and used these data to express biomass and productivity from 2012-2019 on a C rather than total biomass basis (total plant C pool and NPP, respectively). In 2016 we sampled soils in each plot in 0-10, 10-20, 20-40 and 40-60 cm horizons. We used measurements of soil bulk density and percent C to estimate total soil C pool from 0-60 cm depth. We combined 2016 soil C pool data with mean 2012-2019 total plant C pool data to estimate total ecosystem C pools. In every plot, we measured soil CO₂ flux approximately 12-15 times per year between early May and late September using a LI-COR 6400 gas exchange system with a LI-COR 6400-09 soil respiration chamber. To best match the time period during which all treatments were imposed, we used soil CO₂ flux data from May 1- July 31 herein; moreover we averaged these data to derive a single mean soil CO₂ flux value for each plot each year. Results from these data have been submitted as: Reich, P. B., Hobbie, S. E., Lee, T. D., Rich, R. Pastore, M. A., Worm, K. (2020) Synergistic effects of four climate change drivers on terrestrial carbon cycling. Nature Geoscience (accepted, in principle)

Instrumentation

Carbon analyses: ECS 4010 CHNSO Analyzer, Costech Analytical Technologies Inc., Valencia, CA, USA or NA 1500 CNS Analyzer, Carlo-Erba Instruments, Egelsbach, Germany Soil Carbon Flux: LI-COR 6400 gas exchange system with a LI-COR 6400-09 soil respiration chamber (LI-COR, Lincoln, Nebraska, USA)

ahke141 - Fire Biomass Loss

Fire biomass loss measurements

To measure pre- and post-fire aboveground biomass, we measured biomass in adjacent 10 by 100 cm strips to approximately 1 cm above soil level immediately before and after the burn in 2016. For pre-fire biomass, we sorted standing biomass, litter, and weeds. Biomass was dried at 40 degrees Celsius and subsequently weighed.

ahme141 - 16S DNA from BioCON dry roots

Sampling and molecular methods

Next generation sequencing was performed on DNA extracted from dry roots that were not surface sterilized, and therefore retained an intact rhizoplane community along with endophytic bacteria. Samples were collected from two FACE rings and two ambient control rings at BioCON, including the R16, R9, and R1 plots that were either unfertilized or Nenrich. Roots were collected in summer 2013 by root-coring (5 cm) to a depth of 20 cm. The roots of three replicate cores from each plot were homogenized, rinsed with tap water at Cedar Creek Ecosystem Science Reserve, and dried in solar ovens at 40 degrees Celsius. Dried roots were shipped to Northern Arizona University, and processed for DNA extraction. DNA was extracted from dry roots using the MO BIO PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) with a slightly modified protocol. Briefly, 5 stainless steel beads (2 mm) were added to each well during cellular lysis, and 96-well plates were heated to 60 degrees Celsius for 15 minutes after mechanical lysis. All other extraction procedures followed protocol. Genomic DNA was observed by NanoDrop and then purified using magnetic beads to remove potential PCR inhibitors. PCR was carried out utilizing the 515F-806R primers to amplify the V4 region of the 16S SSU rRNA (Gilbert, Jansson, and Knight, 2014). Final 16S DNA quantitation was performed using standard dsDNA quantitation for PicoGreen (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and all samples were normalized to 2ng DNA/??L prior to sequencing. Samples were paired-end sequenced using the Illumina MiSeq desktop platform (Illumina, Inc., San Diego, CA, USA). Method reference cited: Gilbert, J. A., Jansson, J. K., & Knight, R. (2014). The Earth Microbiome project: successes and aspirations. BMC Biology, 12(1), 69. https://doi.org/10.1186/s12915-014-0069-1

ahte141 - TeRaCON Leaf Gas Exchange

Instrumentation

Measurements were made using LICOR 6400 portable infrared gas exchange systems (Li-Cor Inc, Lincoln Nebraska); Portable Soil Moisture Probe (ML2x ThetaProbe, Delta-T Devices Ltd., Cambridge, UK); Elemental C-N Analyzer (ECS 4010 CHNSO Analyzer, Costech Analytical Technologies Inc., Valencia, CA, USA)

Measurement protocol

Leaf net photosynthesis and stomatal conductance were measured using LICOR 6400 portable infrared gas exchange systems (Li-Cor Inc, Lincoln Nebraska) with program OPEN 6.1.3-4 equipped with a 6400-02B red/blue external light source set to 1500 ??mol m-2 s-1 photosynthetically active radiation (PAR). Measurements were made on upper, fully expanded leaves of similar ontogenetic stage typically between 08:00 and 15:00 local time between June 12 and July 27 of 2018. Measurements were made six times within a plot for each combination of species x temperature x rainfall x CO₂ x N throughout the growing season on sunny days. Measurements of a plot were made at random times of day on separate days for different plants in order to capture potential variability. Rates were determined at light saturation near ambient humidity with cuvette block temperature set to 25C and at approximately the CO₂ concentration in which leaves were grown. Intrinsic water-use efficiency (iWUE) was calculated as the rate of leaf net photosynthesis divided by stomatal conductance. The projected areas of leaves used in gas exchange measurements were determined using IMAGEJ, National Institutes of Health, Bethesda, MD, USA). Specific leaf area (SLA) was calculated as the ratio of leaf area to dry mass. Each leaf portion for the first four measurement dates for each species was ground and analyzed for foliar C and N concentrations on an Elemental C-N Analyzer (ECS 4010 CHNSO Analyzer, Costech Analytical Technologies Inc., Valencia, CA, USA). Volumetric soil water content (VWC, m3 H2O m-3 soil) was measured instantaneously at the time of leaf gas exchange measurements for the area directly surrounding each plant used. This was done using a Portable Soil Moisture Probe (ML2x ThetaProbe, Delta-T Devices Ltd., Cambridge, UK), which measures a cylindrical ???75 cm3 volume of soil (6 cm deep with a diameter of 4 cm) and was calibrated to BioCON soil. Three instantaneous soil VWC measurements were made concurrently with each leaf gas exchange measurement surrounding the base of the plant and averaged. Results from this data published in: Pastore, M. A., Lee, T. D., Hobbie, S. E., & Reich, P. B. (2020). Interactive effects of elevated CO₂, warming, reduced rainfall, and nitrogen on leaf gas exchange in five perennial grassland species. Plant, Cell & Environment, 43(8), 1862-1878. doi:10.1111/pce.13783

bde141 - Soil bulk density

Soil Bulk Density

These samples were taken at the same depths as the 5-year archival soil samples: 0-10cm, 10-20cm, 20-40cm, and 40-60cm. A 2" diameter schedule 40 PVC root corer was used for the 0-10 and 10-20 cm depths. A 2" diameter machined metal root corer was used for the 20-40 and 40-60 cm depth. Soil was sampled when moist. We made sure we didn't knock soil into the hole when inserting the root cores, so the amount of soil in the cores accurately reflected the density in the field. We cored every plot and put the respective depth in a paper bag and put these bags in the drying room. Once dry, we sifted the soil to remove roots and rocks and then we weighed the soil. SPECIAL NOTE: This sampling was not done according to standard bulk density sampling protocols. I (Jared) conferred with Jean Knops and got our protocol approved, but never double-checked the standard protocols until after the sampling, sifting, and weighing. The first problem is the roots should not have been sifted out. However, their weight is a very small % of the total soil weight taken. I compared the bulk density sample soil weight to the June belowground harvest weight and got the following percentages: the 0-20 root weight is, on average, 0.197% of the 0-10 and 10-20 cm soil bulk density sample weights added together; the 20-40 root weight is, on average, 0.0284% of the 20-40 soil bulk density sample weight, no 40-60cm root samples were collected so I could not make a comparison at that depth. The second problem is that the volume was not determined for the rocks that were removed (this volume was supposed to be subtracted from the volume of the soil core taken). However, there are very few rocks in the soil at Cedar Creek, so hopefully this won't significantly impact the final bulk density calculation.

hoe141 - Soil moisture

Soil Moisture

In order to install the TDR tubes we cut 2? diameter PVC pipe into 1.1m lengths. We capped one end with a rubber stopper that we glued in place. These PVC tubes were inserted in the northeast corner of every plot. A metal corer with a reverse bevel was used to create the hole for the pipe. A perfectly vertical hole that was between 1.03m and 1.08m deep was created. The TDR tube was then inserted into the hole with the stoppered end down. A rubber stopper was placed on the upper end of the tube to keep the rain out. The measurements for a full sampling take a couple of days. The measurements for a sampling of the water treatment subset of plots take approximately one day. Two people were necessary to do these measurements. Since a significant amount of moisture can condense on the insides of the TDR tubes, we first had to use a long piece of 1/2" diameter PVC with a rag attached to one end to soak up this moisture. So for every tube, the rubber stopper was removed from the top and the swabby stick was pushed all the way to the bottom of the TDR tube and then pulled up. If there is standing water at the bottom of the tubes we made sure to soak all of it up so that we did not get a false high reading.

lpe141 - Percent light penetration

Percent Light Penetration

Decagon Accupar light meters were used to take light measurements. For most of the measurements we took we used three places in the plot to place the light meter, all being in the percent cover frame area. First the light meters were calibrated. There was then one measurement taken above the canopy. The next three subsequent measurements were taken underneath the plant canopy but above last year's litter (still below this year's litter). The three below canopy measurements were averaged through the electronics of the light meter. We also did a light reading before the harvest in the clip strip. We only took one above canopy measurement and one below canopy measurement.

lye141 - Lysimeter data

Instrumentation-Lysimeters sampling

Dissolved inorganic N (DIN, NO₃- +NH₄+)) was done on an Alpkem auto-analyzer (Pulse Instrumentation, Saskatoon, Saskatchewan, Canada) and total dissolved N (TDN) on a Shimadzu TOC-VCPN (Shimadzu Scientific Instruments, Wood Dale, Illinois, USA).

Lysimeters sampling - analysis

In 2003 Lysimeters were installed in all monoculture and 16 species plots in the southwest corner of the percent cover area. Lysimeters were put into the soil at a 30? angle, where the bottom of the lysimeter was 60cm deep. Sampling always occurred within 48 h after rainfall events. After collection, samples were immediately transported to the University of MN laboratory where they were split into two subsamples and frozen until analyses for dissolved inorganic N (DIN) and total dissolved N (TDN). Feike A. Dijkstra analyzed the samples and calculated dissolved organic N concentration (DON) as the difference between the TDN and DIN concentration. All of the equipment (bottles, septa, caps etc) was washed either in an acid bath then with DI water or with only DI water between each collection.

mre141 - Nitrogen mineralization rate

Nitrogen Mineralization (N Min)

1 inch PVC tubes 20 cm long with one end filed to a point are used as the nitrogen mineralization tubes. First soil cores are taken and placed in a plastic sandwich bag, this is the initial sample. At the same time n-min tubes are placed in the ground with rubber stoppers on the tops close to where the cores were taken, but not the same exact spot. The soil in these tubes is collected after one month, and this is considered the final sample. When the soil samples are taken (whether it is the initial sample or the final sample) it is then taken into the lab and two heaping scoopfuls are placed into a pre-prepared, preweighed, barcoded vial of 1M KCl, one for each plot. These are then weighed and placed on the shaker for 30 minutes. After they are finished shaking, they are placed in the fridge overnight. In the morning the contents are pipetted into smaller plastic vials that have the same label. It is important to not get any of the soil into the pipette and into the small plastic vials. These samples were then frozen until they were ready to be analyzed.

nbe141 - Plant aboveground biomass carbon and nitrogen

Plant aboveground biomass carbon/nitrogen

Biomass from annual harvests is analyzed for percent carbon and nitrogen to examine effects on plant tissue across time. Samples are dried at 40 degrees C, and then ground with a Wiley Mini-Milll using a 40 mesh screen. Ground samples are analyzed for % carbon and % nitrogen. Samples are run in this order: all ambient N followed by elevated N within each set of samples.

Plant aboveground biomass carbon/nitrogen-instrumentation

Samples are analyzed using C-N Analyzers, NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc., Valencia, CA, USA

ne141 - Soil percent nitrogen and carbon

Soil Nitrogen and Carbon - Instrumentation

Soil nitrogen and carbon analysis were run with a C-N Analyzer (NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc.).

Soil Nitrogen and Carbon sampling

We take soil samples from all plots in the BioCON experiment every 5 years. Five soil cores were taken per plot at depths of 0-10cm, 10-20cm, 20-40cm, and 40-60cm. One core was taken at each corner of the plot just outside of the buffer zone towards the middle of the plot. The fifth core was taken in the middle of the north side of the plot right outside of the north edge of the percent cover area. If, when a corer was put into an existing hole, the appropriate line was not at soil level (i.e. the metal corer?s 10 cm mark should be at soil level when taking a 10-20cm core) enough soil was removed and put outside the ring so that the soil core would begin at the right soil depth. All 5 cores for a specific depth were combined in a labeled paper bag and placed in a drying room. After the soil was dry, it was sieved to remove roots and other plant material. Some of this soil was placed in a glass scintillation vial. The rest of this soil was finely ground. The ground soil was placed in another glass scintillation vial. A sample of this ground soil was packed and analyzed for total CN. This was done for each of the 4 sample depths from each plot.

nie141 - N15 isotope in plants

N15 isotope in plants

Leaf tissue samples harvested in June were dried at 65 degrees C then finely ground and analyzed for 15N atom %. All ages of leaves were used in the samples; stem tissues were not included.

N15 isotope in plants-Instrumentation

Equipment used for analysis was a Europa Scientific Integra isotope ratio mass spectrometer, University of California at Davis, Stable Isotope Facility, Davis, Calif.

nohe141 - Soil nitrate and ammonium

Soil Ammonium and Nitrate

We completed this protocol in order to measure differences in soil NH₄ and NO₃ across treatments and across time. We prepped for this process by labeling tins, sandwich bags, and vials with the appropriate labels. We then weighed the empty tins. We made up a 0.01 M KCl solution for the extraction. We pipetted 50ml of the KCl solution into each vial. After filling all of the vials we weighed them and placed them in the fridge. Soil cores were taken in each plot after the biomass harvest. Metal soil cores (approx. 3/4 in. diameter) were used to take the cores. Cores were then taken at varying depths and placed into labeled plastic sandwich bags. If a core was needed below an existing soil layer (for example, 40-60cm is desired but is covered by 20-40cm of soil), the undesired soil depth was discarded before taking the appropriate depth. After all of the samples were brought in from the field, the soil was mixed in the bag and then two spoonfuls were placed in the vials of KCl. Soil was also placed in the tin until the bottom was covered, and then the bag, tin, and vial were scanned. The vials were weighed, placed on a shaker for 30 minutes, and then placed in the refrigerator. The tins were weighed then placed in a drying oven. After drying overnight, the tins were weighed again. This process was used for each sample. The next morning the 50 ml vials were taken out of the refrigerator and disposable pipettes were used to extract samples from each vial. The scintillation vial was filled almost to the top with KCl liquid from the vial. When the scintillation vial was filled, the scintillation vials were labeled with the label from the specimen vials. The scintillation vials were then put into the freezer until they were analyzed.

nre141 - Root carbon/nitrogen data

Root carbon/nitrogen

Root biomass from annual harvests is analyzed for percent carbon and nitrogen to examine effects on plant tissue across time. Washed root samples are dried at 40 degrees C, and then ground with a Wiley Mini-Milll using a 40 mesh screen. Ground samples are analyzed for % carbon and % nitrogen. Samples are run in this order: all ambient N followed by elevated N within each set of samples.

pce141 - Plant species percent cover data

Percent Cover

Percent cover was done in June and August before the aboveground harvest in the permanent percent cover area in the center of the plot. Palm top computers were used to enter the data. Weeding benches were used to be able to look over the plot in order to increase accuracy. Before they began to actually collect data, the two people compared a few plots and calibrated themselves to each other. Then they each measured a plot separately. Each plant species was estimated for percent cover separately with bare ground and litter also included. In each plot the total of all estimates must equal 100%. The palm top computers were downloaded at the end of each day.

phoe141 - Photosynthesis (A max, etc.)

Photosynthesis Instrumentation

In situ rates of leaf net photosynthesis (A) were measured using either CIRAS-1 portable infrared gas exchange systems (PP Systems, Hitchin, UK) operated in open configuration with controlled temperature, CO₂ concentration, and vapor pressure (1998-2008); or LICOR 6400 portable infrared gas exchange systems (Li-Cor Inc, Lincoln Nebraska) using program OPEN 6.1.3-4 equipped with a 6400-02B red/blue external light source set to PAR 1500 (2009-2010). The projected areas of leaves used in gas exchange measurements were determined using a digital image analysis program (WINRHIZO 3.9, Regent Instruments, QC, Canada, SCION IMAGE or IMAGEJ, National Institutes of Health, Bethesda, MD, USA).

Photosynthesis parameters

On sunny days between May and August measures of in situ rates of leaf net photosynthesis (A) are made on a young to mid-aged, upper canopy and fully expanded leaf of an individual plant representing each plot, typically between 09:00 and 15:00 local time. Separate plant individuals are selected for each measurement. Replicate plots representing treatment combinations are sampled three to four times each year at random time points over the day.

ple141 - Plant aboveground biomass data

Plant aboveground biomass

BioCON plots were harvested twice during the field season, usually at the end of June and in the middle of August. We marked a clip strip with flags in each plot running north to south that was 1m x 10cm. We used hedge clippers (blade width of 10cm) to clip the strip from the plots. Plants were clipped approximately 1cm above soil level. Biomass from the clip strip that was collected for sorting includes: every plant rooted in the clip strip (not things leaning over) and all litter lying on the ground within the strip. Each plot is checked off and brought into the lab to be sorted. The plots are then sorted according to the rules of the season. Some seasons we sorted all plots to species and some seasons we sorted some plots to green biomass and miscellaneous litter. In order to determine what miscellaneous litter was we followed certain guidelines. In years when BioCON was burned in the spring miscellaneous litter was considered to be anything that was unidentifiable or charred. In years when BioCON was not burned in the spring the guidelines for determining what is miscellaneous litter is as follows. Anything that was dark brown or unidentifiable was considered litter. Any plant material with a trace of green or that was connected to green biomass was considered this year?s growth. The plots were bagged and then dried so they could be weighed later. Each plot was sorted according to what was listed on the plot sheet that was made for each plot. If the sheet said ?Green Biomass? rather than species names, all of the species that were planted in the plot were sorted as one group. If there were any species that were not on the sheet, but found in the plot, they were sorted into "Real Weeds" and "16 Species Weeds". There was also a category for miscellaneous litter. "16 Species Weeds" are BioCON species that were not intentionally planted in an individual plot; "Real Weeds" are all other species.

rie141 - Root ingrowth biomass

Root Ingrowth Cores (RICs)

A root corer was used to create the root ingrowth sampling area in the soil. The core went to a depth of approximately 20 cm. A screen made of hardware cloth was molded around the root corer and inserted into the hole created by the first core. Soil from outside of the plot was sieved of rocks, live plants and root mass (which was discarded) and then placed in the hole. The soil was packed into the hole until it was level with the surrounding soil and approximately 1 cm of the hardware cloth in the hole was left above soil level. In order to sample the coring area an initial core was taken to a depth of 20 cm and the contents were put into a labeled plastic bag. The hardware cloth screen was then pulled out of the hole and a reverse corer (a corer with the inside of the bottom edge filed) was inserted in the already existing hole to 25 cm. The contents removed from the hole with the reverse corer were discarded. The screen was then put back into the hole. When we filled the sample holes back in, we used soil from within the buffer area of each plot. A root corer was pounded down approximately 40 cm in the buffer of the plot. This soil was then sieved through hardware cloth to remove plants and roots and was put into the hole that the RIC sample was taken from and packed down. This step was implemented so each subsequent core has the same amount of nitrogen as what occurs in the plot. Soil was then taken from outside of the ring, sieved through hardware cloth, and used to fill in the filler hole in the buffer. The roots were then washed. We took a root sample and placed the soil on a screen. Water was then sprayed at a medium speed over the soil (so all of the fine roots wouldn?t get blasted through the screen). If it was difficult to get all of the dirt/particles out of the sample, we put water in a small tub and floated the roots. This allowed the heavier particles, such as sand, to sink to the bottom away from the floating roots. We standardized 95% accuracy for all of the samples we took. This means that we collected roughly 95% of the roots that were found in the sample. Usually about 5% are missed because they fall through the screen, or by human error. Washed roots were placed in labeled paper bags/envelopes and placed directly into the drying room.

roote141 - Root biomass data

Belowground Biomass

Belowground harvest takes place during the week directly following the aboveground harvest. Root cores have been taken at 3 different depths. For root cores taken at a depth of 0-20cm and 20-40cm, a 2" diameter schedule 40 PVC pipe was used. Root cores at the 40-100cm depth were taken with a 2" metal corer. All of the cores from a single depth (either 0-20, 20-40, or 40-100cm) in a plot were combined. The holes that had been created in all of the plots were filled with soil from outside the ring that had been sieved through 1/4" hardware cloth. Washing. We took a root sample and placed the soil on a screen. Water was then sprayed at a medium speed over the soil (so all of the fine roots wouldn?t get blasted through the screen). This got rid of the sand and separated the roots from the soil. Oftentimes in the 0-20cm core depth, a dense mat of roots kept soil and rocks from being washed away by the water stream and so this dense mat had to be ripped apart and broken. The roots were then picked out of the remaining objects that were found on the screen. The person washing the roots had to be able to distinguish the live roots from dead roots and other miscellaneous debris. Live roots are commonly light colored, hard to break, and flexible. Dead roots are commonly flimsy, easily broken, lacking structural integrity, and are dark in color. If it was difficult to get all of the dirt/particles out of the sample, we put water in a small tub and floated the roots. This allowed the heavier particles, such as sand, to sink to the bottom away from the floating roots. We standardized 95% accuracy for all of the samples we took. This means that we collected roughly 95% of the roots that were found in the sample. Usually about 5% are missed because they fall through the screen, or by human error. A new sandwich bag was labeled with the correct plot number and the washed roots were placed in this bag. The roots were then placed in the fridge until they were sorted. The roots were then sorted (and checked for non-rooty material) into coarse, crown, and fine categories. Fine roots are thin and will usually comprise the bulk of the sample. Coarse roots are defined as having a diameter greater than 1 mm. Calipers were used to determine this width. Crowns are the points at or below the soil surface where the root joins the stem; they are generally characterized by (a) having several roots radiating out (the radiating roots are cut off and classified as either course or fine, they are not part of the crown) from a small area and/or (b) being connected to aboveground biomass (anything green is removed). Once these roots had been sorted, the crowns, coarse roots, and fine roots were put into separate paper bags/envelopes. The bags/envelopes for each plot were then all put into 1 large plot bag, which was placed in the drying oven until they were dry enough to weigh. Washed roots are placed in paper envelopes and dried at 40 degrees Celsius. Dried roots are weighed by fine roots, coarse roots and crowns at each depth collected prior to grinding. June harvest: The June harvest roots were only ground in 2001. We ground the roots from only the 0-20cm depth for all monoculture plots in BioCON. All roots from the 0-20cm depth were combined and ground to make 1 composite sample per monoculture plot. August harvest: In every plot all roots from the 0-20cm depth were combined and ground to make 1 composite sample per plot. In select years roots from 20-40cm and 40-100cm depths are combined by depth and ground.

sachmie141 - Reproduction data for Achillea millefolium

Reproductive Outputs - Achillea millefolium

For Achillea millefolium we counted the number of inflorescences that were rooted in the 0.5 x 1.0 meter percent cover quadrat of each plot. We sampled as soon as all flowering stalks had emerged and could unambiguously be identified to species, and completed sampling in one or two days.

samocae141 - Reproduction data for Amorpha canescens

Reproductive Outputs - Amorpha canescens

For both Amorpha canescens and Petalostemum villosum we counted the number of inflorescences on stalks that were rooted in a 0.25 m2 percent cover area. Only inflorescences that were greater than 1cm in length were counted. Stalks that had passed flowering, and had already gone to seed we counted only if they were greater than 1cm in length.

sasctue141 - Reproduction data for Asclepias tuberosa

Reproductive Outputs - Asclepias tuberosa

For Asclepias tuberosa we counted the number of stalks in each stage of reproductive development that were rooted within a 0.5 m2 percent cover area. The stages were, budding, flowering, seeding, and not flowering.

scfe141 - Soil carbon flux

Soil CO2 Flux (SCF)

A full sampling takes 2-3 days depending on the health of the machines. A sampling of the subset of NICCR plots takes approximately 1 day. All samplings previous to 2007 were done using a LiCor 6200. In 2007 we incorporated the LiCor 6400 into the samplings as well. All vegetation was cleared from the inside of the pvc collar located in the northwest corner of every plot. The LiCor chamber was set on top of the pvc collar. A thermometer was placed in the soil next to the pvc collar to measure the soil temperature at the time of the measurement. After all the plots were measured, the machines were then downloaded and the data were checked. Collars used for measuring were cut from 4" I.D. PVC. Each collar is 2" tall. One side was sharpened at a 45? angle on grinder. Using 10 cm clippers, a 10 cm x 10 cm square is clipped at the site of collar installation. Collars are inserted to a depth of .75" (range = 0.5" to 1").

sgrasse141 - Reproduction data for grasses

Reproductive Outputs - Grasses

All plots containing four focal grass species were surveyed. A 1 x 0.5m frame was placed around the permanent percent cover area. We counted the # of flowering stalks rooted within this area for each species.

slae141 - Specific Leaf Area

Specific Leaf Area (SLA)

We performed SLA measurements on leaves from every species in monoculture and on leaves from Achillea millefolium, Bouteloua gracilis, Bromus inermis, Koeleria cristata, Lupinus perennis, and Poa pratensis in a subset of 16 species plots in the June and August harvest. Individual leaves were taken from plants in the clip strip sample. We tried to pick anywhere from 4-8 leaves that were a similar size and from various plants in the sample. They were scanned with the WinRhizo program at a resolution of 200 dpi. Next, the leaves/grasses were put into a large coin envelope and labeled appropriately. The leaves were weighed separately from the rest of the plot.

slescae141 - Reproduction data for Lespedeza capitata

Reproductive Outputs - Lespedeza capitata

For Lespedeza capitata we measured reproduction in 2 ways. First we recorded the number of flowering stalks/unit area. We counted the number of flowering stalks rooted within a 0.5 m2 percent cover area of all plots containing lespedeza except for monocultures. In monocultures we counted the number of flowering stalks within the entire plot. Second, we recorded the number of flowering clusters/plant on a subset of stalks. In each plot, we began counting the flower clusters on the flowering stalks at the south edge of the permanent % cover area. We counted the number of flowers on the first 5 flowering stalks we came across in polycultures and the first 12 we came across in monocultures. If several stems were coming from 1 central location (indicating that all of the stems probably belonged to 1 individual), we only counted 1 of the stems, unless there weren't enough flowering stalks available in the % cover area. If the permanent % cover area didn't have 5 flowering stalks, then we had a smaller sample size for that plot. We didn't count flowers beyond the % cover area.

sluppee141 - Reproduction data for Lupinus perennis

Reproductive outputs - Lupinus perennis

We measured Lupinus perennis reproductive effort in three ways. First we counted the number of flowering stalks per unit area. We counted the number of flowering stalks rooted within a 0.25 m2 percent cover area. Then we measured the total amount of biomass allocated to seeds and pods per flowering stalk. We also counted the number of pods and seeds produced per flowering stalk. We harvested lupine seed stalks from all plots with lupine. We picked stalks with a small amount of flowers at the top and fully formed pods on the bottom; the pods were beginning the dry at the seam. To pick the seed stalks, we started looking in the northeast corner of the plot (in the rear percent cover area) and moved west from there. We picked the first 5 stalks in polycultures and the first 10 stalks in monocultures from the northeast corner. We used this method to try and eliminate visual bias as much as possible. If there was more than 1 stalk coming from the same plant, we only harvested 1 stalk/plant. If we couldn't find enough seed stalks in the rear percent cover, we started searching for stalks in the buffer, starting at the west edge of the plot and working south, then east, then north, then west. All of the stalks from each plot were put together into a plot bag and then put in the drying room. After they were dry, the pods were counted and broken apart. A "pod" was only considered a "pod" if it was longer than 1/2", if it was shorter, it was not counted. Only a subsample of the seeds were counted, dried, and weighed because many of the seeds are so tiny. We did a subsample and used that data to estimate the rest. The pod remnants were also weighed. All 5 or 10 stalks/plot were combined.

spetvie141 - Reproduction data for Petalostemum villosum

Reproductive Outputs - Petalostemum villosum

For both Amorpha canescens and Petalostemum villosum we counted the number of inflorescences on stalks that were rooted in a 0.25 m2 percent cover area. Only inflorescences that were greater than 1cm in length were counted. Stalks that had passed flowering, and had already gone to seed we counted only if they were greater than 1cm in length.

sphe141 - Soil pH

pH

We measured the pH of the samples that were taken for nitrogen mineralization. Soil samples were taken from each plot and put into a solution of 1M KCl, which was used to extract available soil nitrogen (ammonium, nitrate, nitrite). The supernatant was then pipetted off the soil/KCl mixture into vials. This solution was then analyzed for nitrogen content. The samples were then frozen until we measured pH. We take this value to be an approximation of soil pH. First we thawed the samples. All pH measurements must be taken within 1 week from the day the samples were thawed. When not measuring pH, the samples should be kept in the fridge. We spread the samples out so that they could reach room temperature before we started the measurements. First, we calibrated the pH probe using pH standards (pH4, pH7, and pH10). We recalibrated after each set of 10 sample measurements. If the machine was not drifting, we recalibrated after every 25 samples. We made sure to rinse the probe with deionized water after every measurement.

ssolrie141 - Reproduction data for Solidago rigida

Reproductive Outputs - Solidago rigida

All plots containing Solidago rigida were surveyed. A 0.5 X 0.5m frame was placed in the south .5mX.5m of the permanent percent cover area in these plots. Within this area, the number of bolts rooted within the area with flowers and the number of bolts rooted in the area without flowers were counted and recorded.

swe141 - Seed weight

Reproductive Outputs - Seed Weights

For both Amorpha canescens and Petalostemum villosum we counted the number of inflorescences on stalks that were rooted in a 0.25 m2 percent cover area. Only inflorescences that were greater than 1cm in length were counted. Stalks that had passed flowering, and had already gone to seed we counted only if they were greater than 1cm in length. For Andropogon gerardi and Sorghastrum nutans we counted the number of flowering stalks that were rooted within a 0.5 m2 percent cover area. For Asclepias tuberosa we counted the number of stalks in each stage of reproductive development that were rooted within a 0.5 m2 percent cover area. The stages were, budding, flowering, seeding, and not flowering. For Bouteloua gracilis, Koeleria cristata, Poa pratensis, and Bromus inermis we counted the number of flowering stalks rooted within a 0.25 m2 percent cover area. For Solidago rigida we counted the number of bolts with and without flowers rooted within a 0.25 m2 percent cover area. For Lespedeza capitata we measured reproduction in 2 ways. First we recorded the number of flowering stalks/unit area. We counted the number of flowering stalks rooted within a 0.5 m2 percent cover area of all plots containing lespedeza except for monocultures. In monocultures we counted the number of flowering stalks within the entire plot. Second, we recorded the number of flowering clusters/plant on a subset of stalks. In each plot, we began counting the flower clusters on the flowering stalks at the south edge of the permanent % cover area. We counted the number of flowers on the first 5 flowering stalks we came across in polycultures and the first 12 we came across in monocultures. If several stems were coming from 1 central location (indicating that all of the stems probably belonged to 1 individual), we only counted 1 of the stems, unless there weren't enough flowering stalks available in the % cover area. If the permanent % cover area didn't have 5 flowering stalks, then we had a smaller sample size for that plot. We didn't count flowers beyond the % cover area. We measured Lupinus perennis reproductive effort in three ways. First we counted the number of flowering stalks per unit area. We counted the number of flowering stalks rooted within a 0.25 m2 percent cover area. Then we measured the total amount of biomass allocated to seeds and pods per flowering stalk. We also counted the number of pods and seeds produced per flowering stalk. We harvested lupine seed stalks from all plots with lupine. We picked stalks with a small amount of flowers at the top and fully formed pods on the bottom; the pods were beginning the dry at the seam. To pick the seed stalks, we started looking in the northeast corner of the plot (in the rear percent cover area) and moved west from there. We picked the first 5 stalks in polycultures and the first 10 stalks in monocultures from the northeast corner. We used this method to try and eliminate visual bias as much as possible. If there was more than 1 stalk coming from the same plant, we only harvested 1 stalk/plant. If we couldn't find enough seed stalks in the rear percent cover, we started searching for stalks in the buffer, starting at the west edge of the plot and working south, then east, then north, then west. All of the stalks from each plot were put together into a plot bag and then put in the drying room. After they were dry, the pods were counted and broken apart. A "pod" was only considered a "pod" if it was longer than 1/2", if it was shorter, it was not counted. Only a subsample of the seeds were counted, dried, and weighed because many of the seeds are so tiny. We did a subsample and used that data to estimate the rest. The pod remnants were also weighed. All 5 or 10 stalks/plot were combined.