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Methods for Experiment 247 -

Nutrient Network Experiment design and protocols

The NutNet study is a completely randomized block (environmental gradient) design with three blocks and 10 plots per block (N = 30 total units/site). Each experimental unit is a 5 by 5 m plot that is separated by at least 1-m walkways. Each plot is divided into four equal-sized subplots: one dedicated to core sampling, one to additional site-specific or subnetwork studies and the last two for the future network-level research.

Core Sampling. In each plot, the core sampling 2.5 by 2.5 m subplot is divided into four 1 by 1 m permanent subplots, surrounded by a 0.25-m buffer. Within the core-sampling subplot, one 1-m2 subplot is permanently marked for annual plant composition sampling; the other three are used for destructive biomass sampling. Core annual sampling includes clipping of total above-ground biomass of all plants rooted within two 0.1-m2 strips (10 by 100 cm) for a total of 0.2 m2. These are sorted to live and dead (or further, e.g. forb, grass, moss at many sites), dried at 60 degrees C to constant mass and weighed to the nearest 0.01 g. Leaves and current years woody growth are collected from shrubs and subshrubs. Light availability above and at ground level below the canopy is measured in the core subplot using a linear 1-m bar (e.g. Apogee Instruments, Inc., Logan, UT, USA). Areal cover is estimated to the nearest 1% for each species rooted in the core subplot; cover estimates include woody overstorey, litter, bare soil, rock and animal activity (e.g. digging). All core measurements are collected from all plots, annually at peak biomass.

Two 2.5 cm diameter by 10 cm depth soil cores, free of litter and vegetation, are collected from each plot prior to initiation of the experiment (Y0) and 3 years after treatment initiation (Y3). Soils from each plot are composited, homogenized, air-dried and shipped to a single laboratory for analysis and long-term storage. Samples are assayed for % total C and % total N, extractable soil phosphorus, potassium and micronutrients, soil pH, soil organic matter and soil texture.

The experimental treatments are applied at the scale of the 5 by 5 m plots, as follows:
Fertilization treatments. Three nutrient treatments (N, P and K plus micronutrients), each with two levels (control, added), are crossed in a factorial design, for a total of eight treatment combinations per block, to test multiple nutrient limitation on plant composition and ecosystem function. Nutrient addition rates and sources are: 10 g N m-2 year-1 as timed-release urea [(NH2)2CO], 10 g P m-2 year-1 as triple-super phosphate [Ca(H2PO4)2], 10 g K m-2 year-1 as potassium sulphate [K2SO4] and 100 g m-2 of a micronutrient mix of Fe (15%), S (14%), Mg (1.5%), Mn (2.5%), Cu (1%), Zn (1%), B (0.2%) and Mo (0.05%). N, P and K are applied annually; micronutrients are applied once at the start of the experiment to avoid toxicity. In addition to the standard NutNet protocol, e247 includes an additional low Nitrogen gradient (1 gram Nitrogen per meter squared per year and 5 grams Nitrogen per meter squared per year in addition to the standard 10 grams Nitrogen per meter squared per year).

Fencing treatments. A fencing treatment is crossed with the control and NPK treatments to assess the interactive effects of fertilization and food web manipulation on plant composition and ecosystem function. The 230-cm-tall fences restrict access by mid-to-large-sized above-ground mammalian herbivores (>50 g). The lower 90 cm is surrounded by 1-cm woven wire mesh (hardware cloth) with a 30-cm outward-facing flange stapled to the ground to exclude digging animals (e.g. rabbits, voles), although not fully subterranean ones (e.g. gophers, moles). The upper fence is composed of four strands of tensioned wire strung at equal vertical intervals.

The experimental design and sampling are replicated at grassland sites around the world. Most sites are contributing pre-treatment and all experimental data (full experiment); however, some sites have contributed only pre-treatment data (observational), and a few are applying only the nutrient addition treatments (nutrients only).

Plots at Cedar Creek were established in 2007.

See the following for further information:
Borer, E.T. et al.; Finding generality in ecology: a model for globally distributed experiments; Methods in Ecology and Evolution; 2014; 5: 65 - 73.; doi: 10.1111/2041-210X.12125 2014

acue247 - Aboveground Standing Crop Biomass

Aboveground Standing Crop Biomass

Aboveground standing crop will be estimated destructively by clipping at ground level all aboveground biomass of individual plants rooted within a 0.2 m2 (two 10 x 100 cm) strips. Biomass will be clipped within the the 1-m2 subplots designated for destructive sampling within the core sampling subplot. Location of the quadrats should be noted or marked permanently to prevent resampling during the duration of the study. For shrubs and subshrubs rooted within the quadrat, leaves and current year?s woody growth should be collected.

Standing crop should be separated into the following categories: previous year?s dead, current year?s bryophytes, and current year?s vascular plant. If time permits, it would be highly valuable to separate biomass into the following six categories: 1. previous year?s dead, 2. current year?s bryophytes, 3. current year?s graminoid (grasses, sedges, rushes), 4. current year?s legumes, 5. current year?s non-leguminous forbs, 6. current year?s woody growth. All biomass should be dried at 60?C for 48hrs prior to weighing to the nearest 0.01 g.

acze247 - Plant Species Composition percent cover

Plant Species Composition percent cover

Prior to initiation of the experiment, percent aerial cover will be estimated in one permanently marked 1-m2 subplot, one within the core-sampling subplot. Aerial cover will be estimated for each plant species separately using a modified Daubenmire method (Daubenmire 1959), in which cover is estimated to the nearest 1% percent for each species rooted within the plot (cardboard cutouts can be used to facilitate estimation). Percent cover also should be estimated for woody overstory, litter, bare soil, animal diggings/disturbance, and rocks if present. Note that total cover will typically exceed 100% because species cover is estimated independently for each species.
Within-season sampling frequency will need to be adjusted for individual ecosystems based on the phenology of the component species in order to capture the maximum cover of each species, which will be used in subsequent analyses. For example, in the tallgrass prairie, species composition will be measured in the spring (late-May) and again in the fall (late-Aug) to capture maximum relative cover of early-season C3 forb and grass species and late-season C4 forb and grass species, respectively.

adae247 - Light Availability

Light Availability

Light availability will be measured using a light meter (e.g., 1-m length Decagon Ceptometer if possible) capable of integrated measures of photosynthetically active radiation (PAR, mmol m-2 sec-1). Light availability will be measured at the same time and in the same 1-m2 subplot used for the species composition measurements. Light readings must be taken on a cloudless day as close to solar noon as possible (i.e., 11 am to 2 pm). For each subplot, two light measurements at ground level (at opposite corners of the 1-m2 plot, diagonal to each other) and one above the canopy will be taken. Light availability will be calculated as the ratio of PAR below and above the canopy.

Note: Light was not measured at the Cedar Creek site in 2011

adbe247 - 2007 pre-treatment soils pH, nutrients, texture

2007 pre-treatment soils: pH, nutrients, texture

Prior to initiation of the experiment, soil cores will be collected during the growing season from all of the plots. For each plot, collect two to three soil cores (soil corer - 2.5 x 10 cm) from each of the 2.5 x 2.5 m subplots (in areas designated for destructive biomass sampling). Litter and vegetation should be removed from the soil surface before collecting each sample. Composite and homogenize these sub-samples into a single sample for each 5x5 m plot (total of 30 roughly 500 g samples). All soil samples should be double bagged in paper and allowed to air dry. Label each bag (with permanent marker, Sharpie preferred) with the following information: date of collection, name of collector, name of sampling site, and block/plot/treatment identification.

PERCENT CARBON and NITROGEN, by mass, as determined by COSTECH Analytical Elemental Combustion System 4010 (ESC 4010)[Valencia, CA USA]

P, K, Ca, Mg, S, Na, Zn, Mn, Fe, Cu, B as determined by Melich-3 analysis (A&L Labs, Memphis, TN USA)

pH as determined by water pH meter with soil in 1:1 soil:water suspension (A and L Labs, Memphis, TN USA)as determined by mixing known

PERCENT SAND, SILT, CLAY as determined by mixing known amount of sample with water; allowing different particle types (sand, silt and clay) to settle out over time; measuring each layer to calculate what portion it is of the whole sample. (A and L Labs, Memphis, TN USA)

PRE-TREATMENT SOIL CLASSIFICATION determined by relative fractions of sand, silt, and clay (A and L Labs, Memphis TN USA)

aeme247 - Multi-site grassland plant biomass, species richness and light (PAR)

Abstact

This dataset contains multi-year data (up to 5 years of data at each site) from 40 global Nutrient Network sites, including five USA LTER sites, published in: "Borer et. al.; Herbivores and nutrients control grassland plant diversity via light limitation; Nature.2014; 508(7497):517-520., doi:10.1038/nature13144" Corresponding author: Elizabeth T. Borer Email: borer@umn.edu

Compiled data methods

We used an experiment replicated at 40 sites on 6 continents to test the hypothesis that herbivores mediate species losses caused by nutrient addition by increasing ground-level light, particularly in eutrophic and highly productive systems. We manipulated herbivores and nutrients using a factorial experiment (nutrient addition x exclusion of herbivores > about 50 g, see Methods Borer et. al. 2014a and 2014b for details) replicated in 40 sites dominated by herbaceous plants, spanning broad environmental gradients of productivity (114 to 1,976 g per meter squared per year), precipitation (mean annual precipitation from 224 to 1,898 mm per year), temperature (mean annual temperature from 0 to 22.1 degrees C), and soil nitrogen (mean percentage of soil N from 0.018 to 1.182 percent). In each plot, we measured local-scale responses of productivity, light and the number of plant species (diversity) using standard methods (Borer et. al. 2014b). We also examined site-level covariates including precipitation, temperature, herbivory intensity, soil nitrogen and atmospheric-nitrogen deposition rates. Although most sites provided 3 years of data, a subset of sites contributed 4 years of post-treatment data, and a few sites, established later, provided only 1 or 2 years of data (Extended Data Table 1 Borer et. al. 2014a).

Method references cited:

Borer et. al.; Herbivores and nutrients control grassland plant diversity via light limitation; Nature, doi:10.1038/nature13144 2014a

Borer et. al.; Finding generality in ecology: a model for globally distributed experiments; Methods in Ecology and Evolution; 2014b; 5: 65?73.; doi: 10.1111/2041-210X.12125

Instrumentation

Soil Nitrogen analyses: COSTECH Analytical Elemental Combustion System 4010 (ESC 4010)[Valencia, CA USA]

aeve247 - Soil organic matter responses to nutrient enrichment in the Nutrient Network

Sampling and Lab methods

Soil sampling

Soils were sampled from nutrient addition plots at five participatory sites of the Nutrient Network. The Nutrient Network is a collaborative, global network of experiments established to investigate the effects of multiple nutrient additions, including N, on ecosystem processes in grasslands. Participatory sites are located across the globe and follow standard protocols for sampling and analysis (Borer et al. 2014). Soil samples were collected in July and August of 2012. Three cores (5 cm diameter and 10 cm deep) were sampled from each plot and composited across the full factorial of nutrient treatments; a fourth core was sampled from the control and N addition treatments for root analyses. Samples were kept on ice or in the refrigerator for a maximum of 6 days until processed in the lab. A subsample of composite soil from each plot was sieved to 2 mm for chemical and biological analysis and 8 mm for soil aggregate isolation. Fresh, 2 mm-sieved soil was used to mea-sure gravimetric soil moisture, microbial respiration, microbial biomass and net N mineralization. Air-dried, 2 mm-sieved soil was used to measure total soil % C and % N by combustion (Costech ESC 4010 Elemental Analyzer, Valencia, California, USA), soil pH (1:1 soil:water slurry method), and particulate organic matter (POM) C and N via density flotation (method detailed below). Additionally, soil texture was measured on air-dried, 2 mm-sieved soil from the control plots using the hydrometer method and sodium hexametaphosphate as the dispersing agent (Ashworth et al. 2001). Soil sieved to 8 mm from the control and N addition plots was air-dried and used to measure water-stable soil aggregates.

Analyses: decomposition of unoccluded SOM

A subsample of fresh, 2 mm sieved soil from each plot was placed in a 120 ml specimen cup and soil moisture was adjusted to 70 percent field capacity. field capacity was calculated separately for each site by pulling 20 kPa pressure on saturated soil. Microbial respiration rate (mg C g soil-1 day-1) was determined at least 17 times during the 380-day laboratory incubation. For each respiration rate measurement, the specimen cups were placed inside 1 L Mason jars and sealed for either 24- or 48-hour intervals. The CO2 concentration in the airtight jars was measured at the beginning and end of each interval using an infrared gas analyzer (LICOR 7000). When not being measured, specimen cups were covered with gas-permeable, low-density polyethylene film. Throughout the incubation, soil samples were maintained at 70 percent field capacity and kept at 20 degrees C in the dark. We calculated cumulative C respired (mgCgsoil-1) during the incubation by averaging the respiration rate between adjacent measurement dates and multiplying by the interval between them, then summing the amount of C respired in between each rate measurement. We assessed the effects of N addition on microbial biomass C and N at the start of the respiration incubation using chloroform fumigation extraction (Brookes et al. 1985). Briefly, replicate fresh, 2 mm-sieved soil samples were extracted with 0.5 M K2SO4 prior to and following chloroform fumigation under vacuum for 5 days. Following filtration, extracts were analyzed for total organic C and total N (Shimadzu TOC-V, Shimadzu Corpora-tion, Kyoto, Japan). Soil microbial biomass C (MC) and N (MN) were calculated as: MC = EC/kEC and MN = EN/kEN, where EC is the difference between extractable C in the fumigated and unfumigated samples, EN is the difference between extractable N in the fumigated and unfumigated samples, kEC is the C extraction efficiency coefficient, and kEN is the N extraction efficiency coefficient. We used the standard extraction efficiency coefficients of 0.45 (kEC) and 0.54 (kEN) from the literature (Brookes et al. 1985; Beck et al. 1997).

Analyses: aggregate-occluded and mineral-associated SOM

Briefly, air-dried, 8 mm-sieved soil subsamples from the control and N addition treatments only were wet sieved with a 2 mm sieve for 2 min each to isolate large macro-aggregates ([2000 lm). Soil that passed through the sieve was wet-sieved with a 250 lm sieve to isolate small macro-aggregates (2000 - 250 lm). finally, the remaining material was wet-sieved with a 53 lm sieve to isolate micro-aggregates (250 - 53 lm) and mineral-associated SOM (\53 lm). During wet sieving, floating organic matter was removed so we could test for N effects on C that was occluded within each aggregate fraction. The isolated fractions were dried at 105 degrees C for12 h, followed by 60 degrees C for 48 h. Fractions were weighed and analyzed for C and N concentration (Costech ESC 4010 Elemental Analyzer, Valencia, California, USA) and used to determine percentage of whole soil total C and N contributed by each fraction. The large macro-aggregate, small macro-aggregate, and micro-aggregate fractions were used to evaluate H2 (aggregate-occluded SOM), while the smallest size fraction informed H3(mineral-associated SOM).
Directly following collection, the additional intact core sampled from the control and N treatment plots was washed in wire mesh tubes (0.28 mm mesh) in a rotating elutriator (Wiles et al. 1996)until allsoil was removed (*3 h). Remaining material was suspended in water and roots were captured with fine sieves and hand-picking. Root crowns were not considered root biomass and removed. Once free of soil, roots were dried at 65 degrees C overnight and weighed to calculate dry root biomass per unit area. Colonization of root tissue by arbuscular mycorrhizal fungi was determined by the point intercept method. Roots were removed from soil cores by washing gently with water over a 53 lm sieve. Cleaned roots were stained with Trypan Blue and stored in a 1:1:1 (vol) solution of glycerin:lactic acid:water at 4 degrees C. Roots were spread in a petri dish marked with 13 mm square grid and examined at 940 magnification to determine presence of fungal structures (hyphae and/or vesicles) at each root-grid line intersection. One hundred intersects were counted for every sample to determine the proportion of root tissue colonized, and each sample was counted twice to ensure reproducible results. Seven root samples were not prepared for mycorrhizal analysis and, consequently, are not included in the statistical analyses.

Results from these data and full statistical analysis used for decay and respiration are presented in: Riggs, Charlotte E.; Hobbie, Sarah E.; Bach, Elizabeth M.; Hofmockel, Kirsten S.; Kazanski, Clare E.; (2015) Nitrogen addition changes grassland soil organic matter decomposition. Biogeochemistry, DOI: 10.1007/s10533-015-0123-2

Methods cited:
Ashworth J, Keyes D, Kirk R, Lessard R (2001) Standard procedure in the hydrometer method for particle size analysis. Commun Soil Sci Plant Anal 32:633 to 642. doi:10.1081/CSS-100103897

Borer ET, HarpoleWS, Adler PB et al (2014) finding generality in ecology: a model for globally distributed experiments. Methods Ecol Evol 5:65 to 73. doi:10.1111/2041-210X.12125

Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem 17:837 to 842. doi:10.1016/0038-0717(85)90144-0

Beck T, Joergensen RG, Kandeler E et al (1997) An inter-laboratory comparison of ten different ways of measuring soil microbial biomass C. Soil Biol Biochem 29:1023 to 1032. doi:10.1016/S0038-0717(97)00030-8

afje247 - Soil nutrient analysis

Soil nutrient analysis

In 2012 four soil cores, 2.5 cm in diameter and 10 cm deep were taken from the 10 by 200 cm biomass clip strip after biomass and letter were removed. Samples were sieved and homogenized prior to analysis.