Humans are creating significant global environmental change, including shifts in climate, increased nitrogen (N) deposition, and the facilitation of species invasions. A multi-factorial field experiment is being performed in an arid grassland within the Sevilleta National Wildlife Refuge (NWR) to simulate increased nighttime temperature, higher N deposition, and heightened El Niño frequency (which increases winter precipitation by an average of 50%). The purpose of the experiment is to better understand the potential effects of environmental drivers on grassland community composition, aboveground net primary production and soil respiration. The focus is on the response of two dominant grasses (Bouteloua gracilis and B eriopoda), in an ecotone near their range margins and thus these species may be particularly susceptible to global environmental change.
It is hypothesized that warmer summer temperatures and increased evaporation will favor growth of black grama (Bouteloua eriopoda), a desert grass, but that increased winter precipitation and/or available nitrogen will favor the growth of blue grama (Bouteloua gracilis), a shortgrass prairie species. Treatment effects on limiting resources (soil moisture, nitrogen availability, species abundance, and net primary production (NPP) are all being measured to determine the interactive effects of key global change drivers on arid grassland plant community dynamics and ecosystem processes.
On 4 August 2009 lightning ignited a ~3300 ha wildfire that burned through the experiment and its surroundings. Because desert grassland fires are patchy, not all of the replicate plots burned in the wildfire. Therefore, seven days after the wildfire was extinguished, the Sevilleta NWR Fire Crew thoroughly burned the remaining plots allowing us to assess experimentally the effects of interactions among multiple global change presses and a pulse disturbance on post-fire grassland dynamics.
This data set provides soil N availability in each plot of the warming experiment for the monsoon season (also see SEV176).
Our experimental design consists of three fully crossed factors (warming, increased winter precipitation, and N addition) in a completely randomized design, for a total of eight treatment combinations, with five replicates of each treatment combination, for a total of 40 plots. Each plot is 3 x 3.5 m. All plots contain B. eriopoda, B. gracilis and G. sarothrae. Our nighttime warming treatment is imposed using lightweight aluminum fabric shelters (mounted on rollers similar to a window shade) that are drawn across the warming plots each night to trap outgoing longwave radiation. The dataloggers controlling shelter movements are programmed to retract the shelters on nights when wind speeds exceed a threshold value (to prevent damage to shelters) and when rain is detected by a rain gauge or snow is detected by a leaf wetness sensor (to prevent an unintended rainout effect).
Each winter we impose an El Nino-like rainfall regime (50% increase over long-term average for non-El Nino years) using an irrigation system and RO water. El Nino rains are added in 6 experimental storm events that mimic actual El Nino winter-storm event size and frequency. From January-March, there are 4x5mm applications, 1x10mm application and 1x20mm application. For N deposition, we add 2.0 g m-2 y-1 of N in the form of NH4NO3 because NH4 and NO3 contribute approximately equally to N deposition at SNWR (57% NH4 and 43% NO3; Báez et al., 2007, J Arid Environments). The NH4NO3 is dissolved in 12 liters of deionized water, equivalent to a 1 mm rainfall event, and applied with a backpack sprayer prior to the summer monsoon. Control plots receive the same amount of deionized water.
Soil N is measured using Plant Root Simulator Probes (PRS® Probes, Western Ag Innovations, Saskatoon, Saskatchewan, Canada https://www.westernag.ca/innov).
Probes are installed in late June or early July prior to the monsoon season and removed in October each year.
Study Area Name: Warming site
Study Area Location: Within the Sevilleta, the site is located just Northeast of Deep Well meteorological station. The site can be reached by parking on the main road next to the signs for deep well and the minirhiztron study. Note that the road to Deep Well met station does not permit vehicles. Travel on foot towards deep well and look for a well-trod path off to the right shortly before the met station.
Vegetation: The vegetation is Chihuahuan Desert Grassland, dominated by black grama (Bouteloua eriopoda & B. gracilis).
Several long-term studies at the Sevilleta LTER measure net primary production (NPP) across ecosystems and treatments. Net primary production is a fundamental ecological variable that quantifies rates of carbon consumption and fixation. Estimates of NPP are important in understanding energy flow at a community level as well as spatial and temporal responses to a range of ecological processes. The NPP weight data (SEV 157) is obtained by harvesting a series of covers for species observed during plot sampling. These species are always harvested from habitat comparable to the plots in which they were recorded. This data is then used to make volumetric measurements of species and build regressions correlating biomass and volume. From these calculations, seasonal biomass and seasonal and annual NPP are determined. These sampled are then vouchered for use to do analyses of inorganic and organic components such as carbon, nitrogen, and phosphorous as well as and other macro and micro nutrients and organic components such as cellulose and lignin.
After all aboveground net primary production (ANPP) quadrat measurements are complete, plants of similar size classes are harvested outside the permanent quadrats. These samples are sorted, dried, and weighed and the resulting data (weight dataset- SEV157) is used to create regressions that estimate aboveground biomass. Then the harvest samples of all size classes, for each species, are then combined to make a voucher sample. A subsample of that combined sample is then ground up mechanically and stored in a sealed glass vial. These samples are available for quantitative chemical analysis of their inorganic and organic composition. Seasonal as well as inter-annual compositions of the various species on the Sevilleta can be derived from this material. The samples are stored at the Sevilleta Field Station. Please contact Stephanie Baker for sample access.
The purpose of this project is to: 1.) determine how biological soil crust (BSC) cover changes along an elevation gradient and across seasons, 2.) determine how carbon and nitrogen exchanges of BSC communities vary with temperature along an elevation gradient in arid and semi-arid environments and, 3.) use photosynthetic and respiration rates of BSC communities to determine how the contribution of the BSC communities to whole ecosystem carbon exchange varies across the same gradient and over seasons.
At each sampling site and sampling period a small amount of surface crust (approx. one teaspoonful per sample) was taken from each of 10 locations at approximately 1 meter intervals over a transect. Samples were transported back to the laboratory in plastic bags.
On rare occasions we removed a larger sample, 0.5 liter volume or less, at one or two sampling stations.
Study sites included: Flux tower sites, desert grassland, desert shrubland, juniper savanna, piñon-juniper woodland, ponderosa pine forest, and mixed conifer forest.
The distribution, structure and function of mesic savanna grasslands are strongly driven by fire regimes, grazing by large herbivores, and their interactions. This research addresses a general question about our understanding of savanna grasslands globally: Is our knowledge of fire and grazing sufficiently general to enable us to make accurate predictions of how these ecosystems will respond to changes in these drivers over time? Some evidence suggests that fire and grazing influence savanna grassland structure and function differently in South Africa (SA) compared to North America (NA). These differences have been attributed to the contingent factors of greater biome age, longer evolutionary history with fire and grazing, reduced soil fertility, and greater diversity of plants and large herbivores in SA. An alternative hypothesis is that differences in methods and approaches used to study these systems have led to differing perspectives on the role of these drivers. If the impacts of shared ecosystem drivers truly differ between NA and SA, this calls into question the generality of our understanding of these ecosystems and our ability to forecast how changes in key drivers will affect savanna grasslands globally. Since 2006, an explicitly comparative research program has been conducted to determine the degree of convergence in ecosystem (productivity, N and C cycling) and plant community (composition, diversity, dynamics) responses to fire and grazing in SA and NA.
Thus far, initial support has been found for convergence at the ecosystem level and divergence at the community level in response to alterations in both fire regimes and grazing. However, there have also been two unexpected findings (1) the ways in which fire and grazing interact differed between NA and SA, and (2) the rate of change in communities when grazers were removed was much greater in NA than in SA. These unexpected findings raise a number of important new questions: (Q1) Will exclusion of grazing eventually affect community structure and composition across all fire regimes in SA? (Q2) Will these effects differ from those observed in NA? (Q3) What are the determinants of the different rates of community change? (Q4) How will these determinants influence future trajectories of change? (Q5) Will the different rates and trajectories of community change be mirrored by responses in ecosystem function over time? This project is based on a large herbivore exclusion study established within the context of long-term (25-50+ yr) experimental manipulations of fire frequency at the Konza Prairie Biological Station (KPBS) in NA and the Kruger National Park (KNP) in SA. The suite of core studies and measurements include plant community composition, ANPP, and herbivore abundance and distribution at both study sites to answer these research questions.
We used comparable experimental designs and sampling procedures at both URF and KPBS. At URF we used three replicate plots (not hayed or mowed) that have been burned every 1 and 3 years in the spring, and those left unburned (N=9 plots). At KPBS, we established replicate plots in experimental watersheds burned every 1 and 4 years in the spring, and those left unburned (N=9 plots). Thus, the only difference in design between NA and SA was the intermediate burn frequency. In 2005 at both sites we established four 2x2m areas in each replicate of the 1-yr, 3-4 yr burned, and unburned plots (N=36 subplots). We then randomly selected two of the subplots for the fertilization treatment and the other two subplots served as controls (Fig. 1). Starting in 2006 at KPBS and 2007 at URF, we began adding 10 gN/m2/yr as NH4+NO3- to assess the interactive effects of fire frequency and nitrogen limitation on plant community composition, structure and dynamics.
Fig. 1. Experimental design and sampling for the proposed studies: A) the role of long-term fire regimes (without megaherbivores), B) the importance of grazing and grazing/fire interactions, and C) the role of megaherbivore diversity. Moveable exclosures (3/plot) will be used to estimate ANPP in the grazed plots. N addition subplots (2 x 2 m) will be divided into 4 1 x 1 plots, with two designated for plant species composition sampling and the other two for destructive sampling. Soil samples will be collected from areas not designated for ANPP or plant composition sampling. Note that the same annually and infrequently burned plots at Kruger and Konza will be used in (B) and (C). In addition, similar plots will be established minus the N addition subplots in the 1-yr and 4-yr burned blocks of the Buffalo enclosure for (C).
Each of the 2x2m subplots was divided into four 1x1m quadrats. Annually since 2005 (prior to nitrogen addition) canopy cover of each species rooted in each quadrat was visually estimated twice during the growing season to sample early and late season species. As a surrogate for aboveground production, we measured light availability at the end of the growing season above the canopy at the ground surface in each quadrat (N=4 per subplot) using a Decagon ceptometer.
Net primary production measurements: Prior to the 2005 growing season we established plots (13.7 m by 18.3 m) in ungrazed areas burned annually, at 3–4-year intervals, and unburned (n = 3 per fire treatment) at both KBPS and URF. Areas with trees or large shrubs were avoided as our main goal was to evaluate responses in the herbaceous plant community. ANPP was estimated from end-of-season harvests starting in 2005 (September for KBPS, April for URF). In 10, 0.1-m2 (20 cm by 50 cm) quadrats randomly located in each plot (n = 30/treatment/site), we harvested the vegetation at ground level and separated it into grass, forb, and previous year’s dead biomass. Samples were dried at 60C to a constant weight. For annually burned plots, total biomass harvested represents ANPP. For the intermediate and unburned sites, we calculated ANPP by summing all but the previous year’s dead component.
To assess the impacts of fire on ANPP in grazed areas, we established herbivore exclusion treatments in KBPS in North America and KNP in South Africa. Herbivore exclosures in grazed areas in KPBS and KNP were erected prior to the 2006 growing season. The exclosures were 7 m in diameter, 2 m tall, and constructed of diamond mesh (5-cm diameter). Seven exclosures were established in each of three blocks of the three fire treatments— annually burned, intermediate burn (3- years for KNP or 4-years for KPBS), and unburned (n = 21 exclosures/treatment/site). As our focus was on ANPP responses of the herbaceous layer, exclosures were not located beneath trees or where dense shrub patches were present. Additionally, in the Satara region of KNP is a 900-ha permanent enclosure containing 80–90 adult African buffalo (S. caffer). This enclosure was erected in 2000 and was divided into six areas (100–200 ha each), with these burned on a rotational basis including plots burned annually and plots that were unburned. We used the unburned and annually burned areas in the buffalo enclosure to provide a direct comparison for determining the effects of a single-species large grazer in KNP and KPBS, and to assess the effects of large herbivore diversity at adjacent sites in KNP. Similar exclosures were built in the African buffalo enclosure at KNP. We placed 7 exclosures in the three blocks of each fire treatment (annually burned and unburned) resulting in 21 exclosures/treatment. We sampled ANPP by harvesting plant biomass from three 0.1 m2 quadrats per herbivore exclosure at the end of the growing season starting in 2006.
Data are collected twice each year at each site. Sample periods are equivalent to spring and late summer at each study site (December/January and March/April in South Africa, May and September in North America.
Where the Data were Collected:
Ukulinga Research Farm, Pietermaritzburg, South Africa; Satara Region of Kruger National Park, South Africa; Konza Prairie Biological Station, North America
Additional Geographic Metadata:
Ukulinga Research Farm (URF), South Africa. The URF of the University of KwaZulu-Natal is located in Pietermaritzburg, in southeastern South Africa (30o 24’ S, 29o 24’ E). The site is dominated by native perennial C4 grasses, such as Themeda triandra and Heteropogon contortus, that account for much of the herbaceous aboveground net primary production (ANPP). Mean annual precipitation is 790 mm, coming mostly as convective storms during summer (Oct-Apr). Summers are warm with a mean monthly maximum of 26.4oC in February, and winters are mild with occasional frost. Soils are fine-textured and derived from shales. There has been no grazing at this site for >60 years. Long-term experimental plots were established at URF in 1950 with the objective of determining the optimal fire and/or summer cutting regime to maximize hay production. The experiment is a randomized block (three replicates) split-plot design with four whole-plot haying treatments and 11 subplot fire or mowing treatments. Subplot sizes are 13.7 x 18.3 m.
Kruger National Park (KNP), South Africa. The KNP is a 2 million ha protected area of savanna grassland that includes many of the large herbivores common to southern Africa (22º 25' to 25º 2 32' S, 30º 50' to 32º 2' E). The extant abundance and grazing intensity of herbivores in KNP is considered moderate for regional savanna grasslands. In the south-central region of KNP where our research takes place, average rainfall is 537 mm with most falling during the growing season (Oct-Apr). The dormant season is mild, dry and frost free, and summers are warm with mean monthly maximum air temperature of 28.9oC in January. Because of the importance of fire in savanna grassland ecosystems, the Experimental Burn Plot (EBP) experiment was initiated in 1954 to examine the effects of fire frequency (control-no fire, 1-, 2-, 3-, 4- and 6-yr return interval) and season [early spring (Aug), spring (Oct), mid-summer (Dec), late summer (Feb), and fall (Apr)] on vegetation communities in the park. Four blocks of 12 plots (two were later split for the 4- and 6-yr trts), each ~7 ha (370 x 180 m) in size, were established in four primary vegetation types covering the two major soil types (granites and basalts) and spanning the precipitation gradient in the park. Each plot has 50+ years of known fire history, and native herbivores have had unrestricted access, thus fire and grazing effects are combined. This research focuses on the EBPs located near Satara where precipitation, soil type, and the mix of herbaceous and woody plants are similar to KPBS. Vegetation on the blocks is co-dominated by C4 grasses, such as Bothriochloa radicans, Panicum coloratum and Digiteria eriantha, and woody plants, such as Acacia nigrescens and Sclerocarya birrea. Soils are fine-textured and derived from basalts. Adjacent to one of the Satara blocks is the Cape buffalo enclosure, erected in 2000 for veterinary purposes. The 200 ha permanent enclosure contains 65-80 animals and is divided into 4 blocks burned on a rotational basis. The grazing intensity inside is comparable to the moderate levels imposed in the park and at KPBS. Two blocks are burned annually while others are burned infrequently (approximately once every 4-yr).
Konza Prairie Biological Station (KPBS), North America. The KPBS is a 3,487 ha savanna grassland in northeastern Kansas, USA (39o 05’ N, 96o 35’ W) dominated by native perennial C4 grasses such as Andropogon gerardii and Sorghastrum nutans that account for the majority of ANPP. Scattered shrub and tree species include Cornus drummondii, Gleditsia triacanthos, and Prunus spp. Numerous sub-dominant grasses and forbs contribute to the floristic diversity of the site. The climate is continental, with mean July air temperature of 27°C. Annual precipitation is ca. 820 mm/year, with 75% falling as rain during the Apr-Oct growing season. Soils are fine textured, silty clay loams derived from limestone and shales. KPBS includes fully replicated watershed-level fire and fire/grazing treatments, in place since 1977 and 1987, respectively. Replicate watersheds (mean size ~60ha) are burned at 1-, 2-, 4-, 10- and 20-yr intervals, mainly in April, to encompass a range of likely natural fire frequencies and management practices. A subset of watersheds has not been grazed for more than 30 years. To address the role of native grazers and fire/grazing interactions, bison (~260 individuals) were reintroduced to KPBS in a 1000-ha fenced area that includes replicate watersheds burned in the spring at 1-, 2-, 4- and 20-year intervals. The overall grazing intensity is considered moderate.
Study Area 1:
Study Area Name: Ukulinga Research Farm
Study Area Location: Near Pietermaritzburg, South Africa
Elevation: 840 m above sea level
Landform: Colluvium fan
Geology: Marine shales and dolerite colluvium
Soils: Dystric leptosols, Chromic luvisols, Haplic plinthisols
Vegetation: Native grassland
Climate: Mean annual precipitation is 844 mm, Mean annual temperature 17.6C
Site history: Ungrazed since 1950
Single Point: 29o 40’ S / 30o 20’ E
Study Area 2: Kruger National Park, South Africa
Study Area Name: Satara Experimental Burn Plots and Cape Buffalo Exclosure
Study Area Location: Near Satara rest camp
Elevation: 240-320 meters above sea level
Landform: Level Upland
Soils: Rhodic nitisols, Haplic luvisols, Leptic phaeozems
Climate: Mean annual precipitation 544 mm; mean annual temperature 21.2–23.3C
Site history: Grazed by native herbivores
Single Point: 23–25o S /30-31o E
Study Area 3: Konza Prairie Biological Station
Study Area Name: Konza Prairie
Study Area Location: Watersheds N20B, N4D, N1B, N4B; 1D, 4F, 20B
Elevation: 320-444 meters above sea level
Landform: Alluvial terrace
Geology: Cherty limestone and shale
Soils: Udic argiustolls
Climate: Mean annual precipitation 835 mm; mean annual temperature 12.7C
Site history: Ungrazed watersheds (since 1971), watersheds grazed by native herbivores (since 1987)
Single Point: 39o 05.48’ N / 96o 34.12’ W
Konza-Ukulinga fire by nitrogen project: We used comparable experimental designs and sampling procedures at both URF and KPBS (Figure 1). At URF we used three replicate plots (not hayed or mowed) that have been burned every 1 and 3 years in the spring, and those left unburned (N=9 plots). At KPBS, we established replicate plots in experimental watersheds burned every 1 and 4 years in the spring, and those left unburned (N=9 plots). Thus, the only difference in design between NA and SA was the intermediate burn frequency. In 2005 at both sites we established four 2x2m areas in each replicate of the 1-yr, 3-4 yr burned, and unburned plots (N=36 subplots). We then randomly selected two of the subplots for the fertilization treatment and the other two subplots served as controls (Fig. 1). Starting in 2006 at KPBS and 2007 at URF, we began adding 10 gN/m2/yr as NH4+NO3- to assess the interactive effects of fire frequency and nitrogen limitation on plant community composition, structure and dynamics.
Konza-Kruger fire by grazing project: For this study, we are utilizing the long-term experiments at KPBS and KNP in which native megaherbivore grazers are present and fire frequency is directly manipulated. To assess the effects of grazing and fire-grazing interactions, we constructed seven sets of permanent exclosures and adjacent control plots in three blocks at both sites. The exclosures and matching paired open plots were established in 2005 in the Satara EBPs that are burned every 1 and 3 years in the spring or left unburned and at KPBS in watersheds that are burned every 1 and 4 years or left unburned. (N=63 exclosures/site; Fig. 1). Within each exclosure and paired open plot, we sample plant community composition and light availability in permanent 2x2 m subplots. We collect ANPP at the end of each growing season from each exclosure, and throughout the growing season in grazed areas adjacent to the unexclosed plots using 1x1 m moveable exclosures (Fig. 1).
Because grasses and shrubs may induce different spatial distributions of nutrients in desert soils, this study was initiated to examine the redistribution of nitrogen in grassland and shrubland soils over a long time period. The stable isotope N15 was applied to plots in grassland and shrubland, and the plots were measured annually from 1989-1993 and again in 1999, 2001, and 2002.
Plot establishment - We established four 10 X 10-m plots in grassland and shrubland habitats near the Five Points area of the Sevilleta Long Term Ecological Research Site (LTER) in the northern Chihuahuan Desert, New Mexico, USA.
In the grassland, paired plots (two plots separated by 50-m) were located at sites where Bouteloua eriopoda (Torr.) Torr. (Black grama) and B. gracilis (H.B.K.) Lag. ex Steud. (Blue grama) dominate. In the shrubland, paired plots were located in areas dominated by Larrea tridentata D.C. (Cov.)(creosotebush).
Tracer application - 15NH4Cl tracer was applied to ten 15.25 cm diameter points arranged in a stratified random design in each of the four 10 X 10 m plots in the grassland and shrubland. 0.33 g of 15NH4Cl were dissolved in 500 ml of deionized water and applied to ten sites per plot in 50 mL aliquots in July, 1989.
Sampling - The site of each spike application was sampled annually during the summer in 1989, 1990, 1991, 1992, and 1993. Two samples with a volume of 28.5 cubic centimeters were removed from the site of spike application, which had a total volume of 1815 cubic centimeters to 10-cm depth. Each sample contained a small percents of the total soil volume, about 1.5 percent. All samples were air-dried, sieved through a 2 mm sieve, and shipped to Duke University. Ground soil samples were analyzed for 15N.
Field collections were made every even-numbered month as close to the 15th as possible.
The samples taken in 1989-1993 and 1999 were analyzed by Larry Giles on a mass spectrometer at the Duke University
Vegetation throughout the southwestern United States has changed from perennial grassland to woody shrubland over the past century. Previous studies on the development of 'islands of fertility' focused primarily on only the most limiting, plant-essential element, soil nitrogen (N). The research presented here addressed the question of whether other plant-essential elements, namely phosphorus (P) and potassium (K), showed similar concentration gradients under the desert shrub Larrea tridentata (creosotebush). It also examined whether the spatial distribution of N, P, and K differed from that of essential, but non-limiting nutrients, namely calcium (Ca), magnesium (Mg), and sulfur (S), and non-essential elements, namely sodium (Na), chloride (Cl), and fluoride (F). Within adjacent grassland and shrubland plots, surface soils were collected under and between vegetation and analyzed for a suite of soil nutrients. Soil nutrient distribution followed a uniform pattern that mirrored the spatial homogeneity of bunchgrasses in the grassland, but followed a patchy distribution that mirrored the spatial heterogeneity of individual shrubs in the shrubland. The main differences were that in the grassland, all elements were uniformly distributed, but in the shrubland the plant-essential elements, nitrogen, phosphorus, and potassium, were concentrated under the shrub canopy, and the non-limiting and non-essential elements were either concentrated in the intershrub spaces or were equally concentrated under shrubs and in the interspaces. Our results show how vegetation shifts from grassland to shrubland contribute to long-term, widespread change in the structure and function of desert ecosystems.
The research presented here addressed the question of whether other plant-essential elements, namely phosphorus (P) and potassium (K), showed similar concentration gradients under the desert shrub Larrea tridentata (creosotebush). It also examined whether the spatial distribution of N, P, and K differed from that of essential, but non-limiting nutrients, namely calcium (Ca), magnesium (Mg), and sulfur (S), and non-essential elements, namely sodium (Na), chloride (Cl), and fluoride (F).
Four 10 X 10-m plots were established in grassland and shrubland sites in 1989. In the grassland, two plots were located where B. eriopoda dominates and two plots were located where B. gracilis dominates. In the shrubland, paired plots were located in two shrubland areas dominated by L. tridentata.
Aboveground plant biomass was estimated for each vegetation type at the height of the summer growing season in July 1989. Estimates of grassland biomass were based on clippings of aboveground plant material from a composite of three separate 1 m2 quadrats adjacent to each grassland plot. Estimates of shrub biomass were calculated from shrub volume measures – one height and two canopy diameter measurements – taken on all L. tridentata shrubs in each shrubland plot following Ludwig et al. (1975). Volume measurements were not taken on the few desiccated or dead individuals of the sub-shrub G. sarothrae in the plots.
Ludwig, J. A., Reynolds, J. F. & Whitson, P. D. 1975. Size-biomass relationships of several Chihuahuan Desert shrubs. Am. Midl. Nat. 94:451-461.
To characterize overall soil nutrient composition and soil properties, we collected 25 soil samples from 0–10 cm in depth using a stratified-random sampling design in each of the eight 10 X 10-m plots, noting whether the sample was taken from beneath vegetation or in the bare space between plants. This resulted in a total of 100 soil samples from the grassland and 100 samples from the shrubland habitats. Soil samples were taken at the height of summer drought and although they appeared dry to the touch, all samples were air dried and sieved through a standard 2 mm mesh sieve prior to analysis.
Soil Nutrient Analysis
We analyzed all soil samples for NO3-N, total N, K, total organic C, Ca, Mg, SO4-S, F, Cl, Na, and P. Ground soil samples were analyzed for total organic carbon and total nitrogen using a Carlo-Erba CHN Analyzer. Anions, except for phosphorus, were extracted by shaking a 6-g sample in 30 ml of deionized H2O for 30 min. The extract was filtered through a 0.45 um millipore filter, and analyzed with a Dionex 2010i ion chromatograph. Cations were extracted by shaking a 10-g soil subsample with 50 ml of NH4C2H2O2 (ammonium acetate) at pH 7.0. The extract was filtered gravimetrically through a #40 Whatman filter and analyzer with a Perkin Elmer 3100 Atomic Absorption Spectrophotometer. Phosphorus was extracted using a modified sequential Hedley fractionation (Tiessen et al. 1984; Tiessen &Moir 1993). A 2-g soil sample was placed in a 50 ml plastic centrifuge tube with 30 ml of deionized water and a 2.5 cm2 anion exchange membrane (AR- 204UZR-412 Ionics, Watertown, MA). Samples were shaken end-over-end for 16 h at 25 degrees C. The anion-exchange membrane was removed and phosphorus retained on the membrane was eluted by shaking the strip with 30 ml of 1 M HCl for 4 h (resin-extractable P). Subsequently,the remaining soil sample was extracted with 30 ml of 0.5 M NaHCO3 (pH 8.5) in the 50-ml centrifuge tube (bicarbonate-extractable P). This process was repeated with increasingly stronger reagents that remove more tightly bound, less plant-available, fractions using NaOH, HCl, and H2SO4-H2O2. Each sample was also sonicated and resuspended in NaOH to remove P that is otherwise encapsulated in Al and Fe minerals. All extracts were analyzed for orthophosphate with the Total Phosphorus procedure for the TRAACS 800 Autoanalyzer.
Tiessen, H., Stewart, J. W. B. & Cole, C. V. 1984. Pathways of phosphate transformations in soils of differing pedogenesis. Soil Sci. Soc. Am. J. 48: 853-858.
Tiessen, H. & Moir, J. O. 1993. Characterization of available P by sequential fractionation. Pp. 75-86. In: Carter, M. R. (ed.), Soil sampling and methods of analysis. Lewis Publishers, Boca Raton.
We studied the diversity of arbuscular mycorrhizal fungi (AMF) in a semiarid grassland and the effect of long-term nitrogen (N) fertilization on this fungal community. Root samples of Bouteloua gracilis were collected at the Sevilleta National Wildlife Refuge (New Mexico, USA) from control and N-amended plots that have been fertilized since 1995. Small subunit rDNA was amplified using AMF specific primers NS31 and AM1. The diversity of AMF was low in comparison with other ecosystems, only seven operational taxonomic units (OTU) were found in B. gracilis and all belong to the genus Glomus. The dominant OTU was closely related to the ubiquitous G. intraradices/G. fasciculatum group. N-amended plots showed a reduction in the abundance of the dominant OTU and an increase in AMF diversity. The greater AMF diversity in roots from N-amended plots may have been the result of displacement of the dominant OTU, which facilitated detection of uncommon AMF. The long-term implications of AMF responses to N enrichment for plant carbon allocation and plant community structure remain unclear.
Sampling Design Roots from three Bouteloua gracilis plants were collected from three control and three N-amended plots (a total of 72 plants).
Field Methods Plant samples were collected in plastic bags, kept at 4 C and processed the same or next day after collection.
Lab Procedures Whole roots from one plant from each plot (6 plants/date) were cleaned under running tap water, rinsed twice with sterile water, and dried on paper towels. Roots were determined to be alive if they did not exhibit lesions, were not obviously damaged, possessed a prominent number of root hairs and were connected to green leaves. A subset of these roots were microscopically analyzed to confirm AMF colonization, and the others were stored at -20 C until their DNA was extracted.
Microscopy The microscopy results showed very low AMF colonization, as a compromise between the need for a large sample of clones (to reliably characterize the AMF community within each plant) and a significant sample of plants from control and N-amended plots (to study the effect of nitrogen on colonization), we decided to select 3 plants from control and 3 plants from N-amended plots and sequence approximately 100 clones per plant using fungal and AMF specific primers.
DNA Extraction Three to five roots segments (approximately 3 cm long) from each plant were used for DNA extraction. DNA was extracted using a DNeasy plant Mini Kit (Qiagen, Chatsworth, CA). Primers NS31 and AM1 were used to specifically amplify AMF (Helgalson et al., 1998; Simon et al., 1992). For each of the 6 root samples, a total of 25 to 47 random clones were sequenced, 260 sequences in all. PCR was performed using the following protocol: initial denaturation at 95 C for 5 min, followed by 30 cycles of 95 C for 30 s, 53 C for 30 s, and 72 C for 45 s, with a final extension of 72 C for 7 min. DNA was amplified in 25 uL reactions with 12.5 uL Premix Taq (Takara Bio), 1.0 uL of each primer (5 uM), 3 uL of BSA 1%, 6.5 uL of milliQ water, and 1 uL of template DNA. The first PCR products were cleaned with ExoSAP-IT (USB, Cleveland, Ohio) and 1 uL of the cleaned PCR product was used as template for the second PCR. Products were cloned with TOPO-TA cloning kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Clones were amplified and sequenced using rolling circle amplification (TempliPhi, Amersham, Buckinghamshire, England) and BigDye Terminator v1.1 Sequencing Kit (Applied Biosystems, Foster City, CA), respectively. Sequencing was conducted at the Molecular Biology Facility of The University of New Mexico. Forward and reverse sequences were assembled and edited with Sequencher 4.0 (Gene Codes, Ann Arbor, MI).
Sequence Analysis The program CHIMERA CHECK 2.7 of the Ribosomal Database Project (http:// rdp.cme.msu.edu/html/analyses.html) was used to check for chimeric 18S nrDNA sequences. Sequences were BLASTed against GenBank and information from GenBank obtained using phd, bioperl scripts and a mysql database written by George Rosenberg, Molecular Biology Facility of the University of New Mexico. Glomeromycota sequences were submitted in GenBank under accession numbers EF154520 and EF154698. OTUs were determined using the DOTUR program (Schloss and Handelsman, 2005). Distance matrices generated with the F84 evolutionary model using the DNADIST program from PHYLIP (Felsenstein, 2005) were used as input files to DOTUR. A similarity level of 97% has been used as the lower boundary to define OTUs in several studies of AMF (e.g. Helgason et al., 1999). We performed our analysis using both 97 and 99% of similarity to evaluate how mycorrhizal fungi at different taxonomic levels respond to N deposition. Rarefaction curves and diversity estimators (Chao, ACE) were calculated for the pooled data (N and control plots) and for N and control treatments with DOTUR. The effect of N enrichment on the AMF community also was evaluated in a phylogenetic context using UniFrac (Lozupone and Knight, 2005). The UniFrac metric estimates differences between microbial communities inhabiting different environments based on phylogenetic distances. In our study, we used this metric to evaluate the percentage of branch length in a phylogenetic tree that leads to descendants from N- amended and control plots. A phylogenetic tree generated with PAUP 4.0b10 (Swofford, 2002) that include all the Glomeromycota sequences from this study was used as input file to calculate UniFrac significance. Trees were constructed using the neighbor-joining (NJ) algorithm and maximum parsimony (MP) in PAUP 4.0b10 (Swofford, 2002). Bootstrap values were estimated from 1000 replicates for the MP and NJ analysis. A NJ phylogenetic tree that includes only representative OTUs (defined at 99% similarity with DOTUR) was used for normalized weighted principal coordinate analysis in UniFrac (Lozupone and Knight, 2005). The weighted UniFrac accounts for the relative sequence abundance in each sample. The NJ tree and a text file that includes OTU abundances for each sample were used as input files.
PTC 200, Pertier Thermal Cycler PCR machine DNA Engine
Changes to the data: File created on 6/13/2008 by Andrea Porras-Alfaro.
Data are available from Genbank: http://www.ncbi.nlm.nih.gov/Entrez/index.html Accessions EF154520-EF154698
Associated with a project that was based upon the assumption that nitrogen may limit net primary plant production in desert grasslands, this project began measuring available inorganic soil N and potentially mineralizable N of soils at two desert grassland locations. Both available N and potentially mineralizable N were greatest following a drought period in 1989, declined during wetter periods that followed and remained relatively stable until another extended drought period. After drought in 1995-6, both forms of soil N increased, indicating the potential for greater NPP following drought and lower potential NPP during periods of normal precipitation.
This project began as part of a study to identify the interactions between soil inorganic nitrogen supply and El Nino-La Nina oscillations that bring wetter-than-average or drier-than-average (respectively) winter-spring precipitation to the central New Mexico region (El Nino-La Nina Fertilization Experiment). This project was known locally as "The Fertilizer Study" and was initiated by Dr. Sandra Turner. To test whether soil inorganic N was limiting aboveground net primary production, fertilizer was added to designated plots within a block of study plots. At first, the soil sampling was designed to confirm or establish the actual fertilizer contribution to each site. The amount added was the amount of inorganic N in soils from the fertilized plots minus that in the control plots. The amount in the controls plots was interpreted as being the amount of inorganic N that had not been utilized by the plant/microbial community and was "available" at that time for utilization in the future. Potentially mineralizable N measurements were performed to determine how the amount of readily mineralizable N was changed by the fertilizer addition; termed a priming effect when the addition of inorganic N results in even greater increases in mineralizable N. Early results from this study were reported at the 1990 Ecological Society of America annual meetings (White et al. 1990).
Following the first year's work, measurements of soil available N and potentially mineralizable N were continued. Except in 1990 when a computer hard-drive that contained most of that year's soil N data crashed and the data were not able to be recovered, the period from 1989 until the spring- summer of 1999 was covered by at least one collection per year.
Each 30m by 30m plot was given a number at the beginning of the study. Before each year's fertilizer application or collection, plots were chosen by selection of random numbers from a table or by using a phone book and using the last 2 digits of the phone numbers. Plots that were used in prior years were excluded from future use. The outer 2.5 m of each plot were left as a buffer strip, so the inside 25m by 25m plot constituted the actual sampling unit.It is not known if the fertilizer was applied to the 30 x 30 m plot or only to the 25 x 25 m plot excluding the buffer. Thus, values given below are based upon application to the 30 x 30 m plot and should be used cautiously.
In 1989, fertilizer treatments were applied as NH4NO3 in 2 treatment levels with controls (low, high, and no fertilizer). Three plots received 61.8 kg N/ha, three plots received 30.9 kg N/ha, and 3 plots were not fertilized (controls). All of these plots were selected in a random manner. It turned out that plot 7 on the west side was too disturbed to be used, so plot 51 was substituted. On the east side, plot 66 was mistakenly fertilized instead of plot 56, which had been selected, so that plot 66 became the 3rd heavily fertilized plot. Hence the plots used in their respective treatments were as follows:
Treatments East side West side
---------- ---------- ----------
Control 16, 33, 42 16, 33, 42
Low 7, 10, 25 19, 25, 51
High 1, 26, 66 1, 26, 56
The fertilizer was applied in April on the east and west sides. Fertilizer was applied with a cyclone spreader by a person walking 5 equally spaced paths across each plot while cranking the spreader. The light treatment was applied with passes in only one direction, while passes were made in both directions across the plot for the heavy treatments.
Soil samples were collected on April 18, 1989, to verify the nitrogen additions and to measure net N mineralization and nitrification potentials. Soil cores (4.2 mm in diameter) were taken to a depth of 20 cm at 4 m intervals along a diagonal from the NE to the SW corner of the plot (starting at the edge of the 2.5 m buffer zone). These cores (15 in all) were placed in the same bag and comprised one sample. The 3 plots represented the sample units (n=3). Plot 66 was not collected until July 4th, when the mistake was identified because plot 56 had no elevated levels of nitrate or ammonium.
In 1990, it was decided that the number of treatment levels would be reduced to one fertilizer application rate and that the number of replicate treatment plots be increased to 4. Fertilizer was applied in two applications, instead of the single application in 1989, in attempts to reduce "burning" from the fertilizer. The first application was on March 1, 1990, and the second was April 4, 1990. Over an inch of rain occurred on both the east and west sides between applications. For each application, fertilizer was applied with a cyclone spreader by a person walking 3 evenly spaced passes across each plot in one direction, and then repeating the process in the perpendicular direction. The setting on the cyclone spreader was 3 1/4. The plots used were:
Treatments East side West side
---------- -------------- --------------
Control 24, 34, 43, 48 24, 34, 43, 48
High 3, 22, 30, 60 3, 22, 30, 60
Within each 30m by 30m plot, 3 ea. half-meter-square (1/2 or 0.5 m^2) quadrats were randomly selected for soil sampling within the northwest quarter of the plot (the 12.5 m by 12.5 m portion excluding the 2.5 m buffer). To identify the location of the 0.5 m^2 quadrats, two tape measures were run parallel to each other, one along the top of the plot and the second along the mid-line of the NW quarter. A third tape was run between the two tapes on the edges to locate the long side of the randomly selected 0.5 m^2 quadrats. Along each diagonal, 4 soil cores were taken at equal spacing starting with the corner of the quadrat and ending in the diagonal corner. Soil cores (4.2 mm diameter) were taken to a depth of 20 cm. The 4 cores from the quadrat were placed in the same bag and comprised one sample. Thus, each plot had 3 soil samples, one from each quadrat, with each treatment having 4 plots.
Protocols for 1991 through 1994
Starting in 1991 and continuing through 1994, 4 plots were sampled with 3 quadrats in each plot; however, only three soil cores were collected (2 from the opposing corners and one from the middle of the quadrat) per quadrat. As in the other years (except 1989 where only one sample was taken per plot), soils from each quadrat were analyzed separately and later averaged to get the value for the plot (3 replicate soil analyses, one per quadrat, and these values were averaged to get a plot value for each of 4 plots; n=4).
Protocols for 1995 to present: Soil Bridges and associated soil collections
In 1995, 5 soil bridges were installed about 30 m east of the Fertilizer Plots. The primary function of the soil bridge is to measure small changes in soil microtopography; erosion or deposition. At the time of soil bridge measurements, two soil cores (same dimensions, 4.2 mm diameter to a depth of 20 cm) for initial available N and potentially mineralizable N were collected from vegetated (mostly grass, 2 cores each) and unvegetated (2 cores each) areas that appeared comparable to those areas beneath the soil bridge. The two sample-types (under plants or open soils) were analyzed separately and the "average" for the bridge was determined by weighting the soil- types by their respective coverages determined beneath the bridge. For example, if vegetation cover was 35% (0.35 of area beneath the bridge had vegetation) and bare soil 65%, the N values for the soil beneath the vegetation were multiplied by 0.35 and summed with the N values for the bare soils multiplied by 0.65. This procedure gave 5 average values for soil N at each collection.+
In the summer of 1998, a study was performed to make direct comparison of the two different collection techniques (collection of soils from fertilizer plots or soils associated with the bridges) and the potential differences the collection method could have on soil nitrogen mineralization potentials.
Methods used for comparative study
Collection on fertilizer plots (fp): Four plots were randomly selected as per previous methods; 4 each 30 X 30 m plots within a 300 X 300 m study site. Within each plot, three randomly selected 0.5 m^2 quadrats within the 15 X 15 m NW quarter of the plot were selected. Within each 0.5 m^2 quadrat, a soil core (4 cm diameter by 20 cm long) was taken from the NW and SE corners of the plot. Both cores were composited to make one sample from each quadrat. This sampling method gave 3 soil samples for each of four plots, for a total of 12 samples.
Collection from the bridge sites: The 5 bridges were equally spaced along the 300 m east border of the fp study area. At each bridge, 2 soil sample cores (4 cm diameter by 20 cm long) were taken from under vegetation cover (under) and 2 from non-vegetative soil surfaces (open). The two cores were composited to give a single soil sample from under and open at each bridge.
Analyses of N mineralization potentials: After determining the water holding capacity (WHC; White and McDonnell 1988), a portion of each sample was adjusted to 50% of determined WHC and 5 subsamples were apportioned into plastic cups. Each cup contained approximately 30 g (+/- 0.05 g) dry-weight mineral soil. One subsample of each sample was immediately extracted with 100 ml 2 N KCl for NH4+-N and NO3--N analyses. The remainder of the cups were covered with plastic wrap, sealed with a rubber band and incubated in the dark at 20 degrees C. Moisture content was monitored by mass loss and replenished as needed. At days 14, 28, 35, and 42, one subsample of each sample was removed and extracted with KCl for 18 - 24 hours. The clarified KCl was filtered through a Kimwipe and analyzed on a Technicon AutoAnalyzer.
Fp: Extractable N at each time interval was averaged for the three soil samples from the 0.5 m^2 quadrats per plot to yield a mean value for each plot. The mean values then were used for statistical analysis (n=4).
Bridges: The relative cover for under and open was obtained from the most recent bridge measurements (made within 2 weeks of soil collections). Extractable N in each soil sample was multiplied by the respective relative cover, and N conc.-times -cover values for soils collected at each bridge were summed to give a cover-weighted value for each bridge (n=5).
The extractable N values for the four fp's were compared to the five bridges over the entire extraction period to determine differences between collection techniques (Factor 1), the effect of extraction time (Factor 2), and their interaction (Factor 1 X 2) at each time interval by analysis of variance. In addition, the variance associated with each collection method was determined and analyzed by analysis of variance to determine if the techniques significantly affected the variance associated with each technique.
The two collection techniques were not significantly different for N mineralization (P = 0.47). There was no significant incubation time x collection technique interaction (P = 0.87). The bridge method did increase variance slightly, but not significantly (P = 0.114).
Net N Mineralization Potentials by different Collection Techniques:Factor d.f. S.S. M.S. F-test P-value------ ---- ---- ---- ------ -------Collection 1 0.316 0.316 0.531 0.47TechniqueExtraction/ 4 330 87.5 147 0.001Incubation timeColl. X time 4 0.719 0.18 0.302 0.87error 35 70.8 0.596 Net N. Mineralization Potentials for Soil Collected by Different Techniques (sum ammonium and nitrate; mg N / kg soil).Incubation Bridge Collections Fertilizer Plots CollectionsTime (days) Mean S.E. Mean S.E.----------- ------------------ ---------------------------- 0 1.96 0.43 2.02 0.24 14 5.55 0.804 5.28 0.45 28 7.33 0.77 7.6 0.17 35 8.29 0.74 8.62 0.63 43 9.59 1.34 10.04 1.12Soil Collection MethodsAll soil samples were taken with a 4.2 mm diameter soil corer to a depth of 20 cm and placed into an ice chest and transported on ice to the University of New Mexico, where they were sieved (2 mm), obvious live roots removed, and stored at 5 degrees C.Soil Analysis MethodsSoil Moisture and Organic MatterFor each collection, soil moisture content of each fresh sample was determined by mass loss upon heating at 105 degrees C for 24 hours. Organic matter was determined by loss-upon-ignition from oven-dried samples placed in a muffle furnace and brought to 500 degrees C for 2 hours. Soil Texture and Total N and P.At select times, texture (percent of sand, silt, and clay) was determined by the hydrometer method (Day, 1965). Total nitrogen and phosphorus were determined by Kjeldahl digestion with copper sulfate as catalyst (Schuman et al. 1973) followed by analysis of ammonium by an automated phenolate method (Technicon AutoAnalyzer Industrial Method #19-69W) and orthophosphate by an ascorbic acid method (Technicon AutoAnalyzer Industrial Method #94-70W).Water-holding capacityWater-holding capacity (WHC) was determine by saturating about a 50 g portion of the sieved soil contained in a funnel with DI water, allowing to saturate for 30 min., and then to drain by gravity for 30 min. The drained soil was transferred to pre-weighed soil tins and dried in an oven at 105 degrees C for 24 hours. The water lost upon drying was the water-holding capacity of the soil sample.Field available N and N mineralization/nitrification potentialsAfter determining water-holding capacity (WHC), a portion of each sample was adjusted to 500f determined WHC and subsamples were apportioned into plastic cups. Each cup contained approximately 30 g dry-weight mineral soil. One subsample of each sample was immediately extracted with 100 ml 2 N KCl (with 5 ppm phenyl mercuric acetate as a preservative) for NH4+-N and NO3--N analyses. The remainder of the cups were covered with plastic wrap, sealed with a rubber band, and incubated in the dark at 20 degrees C. The plastic wrap minimized water loss during incubation, yet exchange of CO2 and O2 was sufficient to keep the subsamples aerobic during incubation. Moisture content was monitored by mass loss and replenished as needed. At approximately weekly intervals, one subsample of each sample was removed, 100 ml KCl was added and shaken with the plastic wrap coveringthe top, and allowed to settle for 18-24 h. The clarified KCl was filtered through a Kimwipe and analyzed for NH4+-N and NO3--N+NO2--N on a Technicon AutoAnalyzer (Technicon, Terrytown, NY) as described in White (1986). This procedure gave up to three different measurements for inorganic ammonium and nitrate: The initial extractions (time 0, wetted but not incubated) indicated field available concentrations (their sum gave total field available N); The amount of ammonium and nitrate extracted after 35-day incubation; and The maximum sum of ammonium and nitrate in any extraction up to the 35-day incubation (some samples peaked prior to day 35).
File assembled 1/19/2001 JAC. File updated with data through 2005, 10/25/2005 Carl White.
File updated with data through 2008, 5/20/2009 Carl White.
Additional Information on the Data Collection Period
Data collected four times per year (usually January, April, July and October). Sampled one week per season, four seasons per year.
Site DescriptionThe fertilizer study was initiated at two locations on the Sevilleta NWR; one on the east side and one on the west side of the refuge. The east side study site was located within a 300m by 300m block on MacKenzie Flats. The vegetation was dominated by black grama and was thought to be a "typical" black grama desert-grassland. Within that area were 100 ea. 30m by 30m plots (10 plots along the side). The outer 2.5 m of the plot were left as a buffer strip, so the inside 25m by 25m plot constituted the actual sampling unit. When the fertilizer study was no longer active, the plots were sampled to continue the long-term soil record. In 1995, 5 locations along the east 300 m border of the fertilizer study area were established for future soil monitoring. Each of these new sites had soil erosion bridges installed to measure changes in soil microtopography (erosion/deposition). In the methods section are the results of a study that verified that sampling at the 5 new locations gave results comparable to those from the 4 random plots within the fertilizer study area. In 2003, the 5 new locations of 1995 were burned by prescribed fire on 19 June 2003. Additional bridge and soil sites were established within the original fertilizer study area (established in control or untreated plots) and sampled before the prescribed fire to establish similarities to continue the control or untreated soil collections at that area. These new bridge sites are numbered 7196, 7197, 7198, 7199, and 7200.The original study also had a grassland site on the west side of the Sevilleta NWR. This site was west of the Field Station, accessed by entrance at the north gate near Bernardo and following the roadbed of the old highway alignment. This site was dominated by C-3 grasses with C-4 grasses a minor component. Shrubs were primarily 4-wing saltbush. The west side only had room for 61 each 30m by 30m plots. When the fertilizer study was ceased, no further soil collections were made on the west side for long-term study.
Method ReferencesDay, Paul R. 1965. Particle Fractionation and Particle-size Analysis. IN: C.A. Black (ed). Methods of Soil Analysis. Part 1. American Society of Agronomy, Inc., Madison, USA.
Schuman, G.E., Stanley, M.A., and Knudsen, D. 1973. Automated total nitrogen analysis of soil and plant samples. Soil Sci. Soc. Am. Proc. 37:480-481.
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