# All Methods

# Methods for Experiment 142 -

## Field Operations: Burning

A prescribed burning program was initiated at CCESR in 1964 to restore and maintain oak savanna and woodland vegetation and to test the effectiveness of different prescribed burning treatments. An area of about 210 hectares was divided into 14 management units of 2.4 to 30 hectares each. Field D is in burn unit 106. Each unit was assigned to one of seven burn frequency treatments, ranging from annual burns to complete fire exclusion. E142 is burned approximately two out of every three years. The entire field is burned which includes all plots and points. Dates for field D burnings are show in the table below.

Year | Month | Day | Burned (1=yes, 0=no) | ||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

1966 | April | 25 | 1 | ||||||||||||||||||

1967 | July | 31 | 1 | ||||||||||||||||||

1968 | 0 | ||||||||||||||||||||

1969 | April | 23 | 1 | ||||||||||||||||||

1970 | May | 19 | 1 | ||||||||||||||||||

1971 | 0 | ||||||||||||||||||||

1972 | April | 17 | 1 | ||||||||||||||||||

1973 | May | 15 | 1 | ||||||||||||||||||

1974 | 0 | ||||||||||||||||||||

1975 | May | 12 | 1 | ||||||||||||||||||

1976 | April | 29 | 1 | ||||||||||||||||||

1977 | 0 | ||||||||||||||||||||

1978 | May | 1 | 1 | ||||||||||||||||||

1979 | May | 15 | 1 | ||||||||||||||||||

1980 | 0 | ||||||||||||||||||||

1981 | May | 5 | 1 | ||||||||||||||||||

1982 | May | 24 | 1 | ||||||||||||||||||

1983 | 0 | ||||||||||||||||||||

1984 | April | 17 | 1 | ||||||||||||||||||

1985 | April | 11 | 1 | ||||||||||||||||||

1986 | 0 | ||||||||||||||||||||

1987 | 0 | ||||||||||||||||||||

1988 | May | 18 | 1 | ||||||||||||||||||

1989 | 0 | ||||||||||||||||||||

1990 | May | 3 | 1 | ||||||||||||||||||

1991 | May | 14 | 1 | ||||||||||||||||||

1992 | May | 14 | 1 | ||||||||||||||||||

1993 | May | 5 | 1 | ||||||||||||||||||

1994 | April | 20 | 1 | ||||||||||||||||||

1995 | 0 | ||||||||||||||||||||

1996 | April | 29 | 1 | ||||||||||||||||||

1997 | April | 21 | 1 | ||||||||||||||||||

1998 | 0 | ||||||||||||||||||||

1999 | April | 24 | 1 | ||||||||||||||||||

2000 | April | 28 | 1 | ||||||||||||||||||

2001 | 0 | ||||||||||||||||||||

2002 | April | 26 | 1 | ||||||||||||||||||

2003 | April | 8 | 1 | ||||||||||||||||||

2004 | 0 | ||||||||||||||||||||

2005 | April | 21 | 1 | ||||||||||||||||||

2006 | April | 19 | 1 | ||||||||||||||||||

2007 | 0 | ||||||||||||||||||||

2008 | May | 1 | 1 | ||||||||||||||||||

2009 | April | 22 | 1 | ||||||||||||||||||

2010 | 0 | ||||||||||||||||||||

2011 | April | 25 | 1 | ||||||||||||||||||

2012 | March | 28 | 1 | ||||||||||||||||||

2013 | 0 | ||||||||||||||||||||

2014 | April | 22 | 1 | ||||||||||||||||||

## Field Operations: Fertilization

This long-term fertilization experiment began in 1982. Nine plots 20 m wide and 50 m long were placed across the savanna in Field D and randomly assigned to one of two fertilizer treatments or unfertilized control. Unfertilized boundary areas separate adjacent plots.
The fertilizer treatments include two levels of nitrogen: 5.4 and 17.0 g N g/m2/year added as NH_{4}NO_{3} (commercial 34-0-0) slow release pellets. To offset indirect effects of N addition, each fertilization treatment also receives equal background levels of P, K, Ca, Mg, S and citrate-chelated trace metals applied in the following forms and rates: P2O5, 20.0 g/m2/year (as commercial 0-46-0 fertilizer); K2O, 20.0 g/m2/year (commercial 0-0-61); CaCO3, 40.0 g/m2/year (as fine-ground commercial lime); MgSO4, 30.0 g/m2/year (US Pure Epsom salts); CuSO4, 18.0 ??g/m2/year; ZnSO4, 37.7 ??g/m2/year; CoCO_{2}, 15.3 ??g/m2/year; MnCl2,322.0 ??g/m2/year; NaMoO4, 15.1 ??g/m2/year; and H3BO3, 12.0 ??g/m2/year. Control plots remained unfertilized, no nitrogen or micronutrients are added. Fertilization occurs twice annually in early May and in late June.

Experiment | Plot | Point | Microplot Fertilizer Treatment | Annual Nitrgen fertilizer addition (g/m2/year) | Annual percent Nitrogen addition (g/m2/year) | 1996 Canopy cover | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

142 | 1 | 1 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 2 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 3 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 4 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 5 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 6 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 7 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 8 | I | 0 | 0 | in | |||||||||||||||

142 | 1 | 9 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 10 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 11 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 12 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 13 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 14 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 15 | I | 0 | 0 | out | |||||||||||||||

142 | 1 | 16 | I | 0 | 0 | out | |||||||||||||||

142 | 2 | 1 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 2 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 3 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 4 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 5 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 6 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 7 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 8 | G | 50 | 17 | in | |||||||||||||||

142 | 2 | 9 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 10 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 11 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 12 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 13 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 14 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 15 | G | 50 | 17 | out | |||||||||||||||

142 | 2 | 16 | G | 50 | 17 | out | |||||||||||||||

142 | 3 | 1 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 2 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 3 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 4 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 5 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 6 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 7 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 8 | E | 16 | 5.44 | in | |||||||||||||||

142 | 3 | 9 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 10 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 11 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 12 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 13 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 14 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 15 | E | 16 | 5.44 | out | |||||||||||||||

142 | 3 | 16 | E | 16 | 5.44 | out | |||||||||||||||

142 | 4 | 1 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 2 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 3 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 4 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 5 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 6 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 7 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 8 | I | 0 | 0 | in | |||||||||||||||

142 | 4 | 9 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 10 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 11 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 12 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 13 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 14 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 15 | I | 0 | 0 | out | |||||||||||||||

142 | 4 | 16 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 1 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 2 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 3 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 4 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 5 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 6 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 7 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 8 | I | 0 | 0 | in | |||||||||||||||

142 | 5 | 9 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 10 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 11 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 12 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 13 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 14 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 15 | I | 0 | 0 | out | |||||||||||||||

142 | 5 | 16 | I | 0 | 0 | out | |||||||||||||||

142 | 6 | 1 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 2 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 3 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 4 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 5 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 6 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 7 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 8 | E | 16 | 5.44 | in | |||||||||||||||

142 | 6 | 9 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 10 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 11 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 12 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 13 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 14 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 15 | E | 16 | 5.44 | out | |||||||||||||||

142 | 6 | 16 | E | 16 | 5.44 | out | |||||||||||||||

142 | 7 | 1 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 2 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 3 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 4 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 5 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 6 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 7 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 8 | G | 50 | 17 | in | |||||||||||||||

142 | 7 | 9 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 10 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 11 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 12 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 13 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 14 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 15 | G | 50 | 17 | out | |||||||||||||||

142 | 7 | 16 | G | 50 | 17 | out | |||||||||||||||

142 | 8 | 1 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 2 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 3 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 4 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 5 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 6 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 7 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 8 | E | 16 | 5.44 | in | |||||||||||||||

142 | 8 | 9 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 10 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 11 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 12 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 13 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 14 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 15 | E | 16 | 5.44 | out | |||||||||||||||

142 | 8 | 16 | E | 16 | 5.44 | out | |||||||||||||||

142 | 9 | 1 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 2 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 3 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 4 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 5 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 6 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 7 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 8 | G | 50 | 17 | in | |||||||||||||||

142 | 9 | 9 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 10 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 11 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 12 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 13 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 14 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 15 | G | 50 | 17 | out | |||||||||||||||

142 | 9 | 16 | G | 50 | 17 | out | |||||||||||||||

## Layout of plots and point selection

Plots for this experiment are 20 x 50 meters. In 1996, within each of the 9 macroplots, 8 sampling points were randomly placed in areas with oak overstory canopy present and 8 were placed in areas with no overstory canopy cover. Random coordinates were drawn until the 16 points had been located within the proper category of canopy condition. 'IN' plots were underneath the tree canopy cover when selected in 1996, 'OUT' plots were outside the tree canopy cover.

# adie142 - Tree survey

## Tree survey

Every five years the trees in E133 and E142 are surveyed. We survey each tree within each plot. Each plot is divided into 6 sections so that finding and mapping trees is easier. Once we located and identified the tree number we measured the diameter breast height (DBH) of the tree. The DBH was measured at a point 1.35 m from the ground unless the notes specified otherwise. We measured 1.35 m from the ground on the side of the tree where the tag was located. There could be topography changes around the tree and this gives a consistent measuring point from year to year. Some trees split below 1.35 m. These trees were measured below the crotch. We noted any changes in where the DBH was measured (if there are any).

We also recorded the status of the tagged tree. The tree was classified as alive (L), dead (D), snapped (S), or fallen (X). If a tree had even one leaf left we called it alive. Dead trees were those which were still standing, but not alive. Snapped trees included those whose trunk was snapped and broken off above the tree tag. Some were still attached, while others were on the ground. This was a tall stump. Fallen trees had trunks which had been broken and snapped off below the tag; they are fallen dead trees. We measured all trees in this way. If we were unable to find a tree we made a note of it.

If a tree had not been previously tagged and it was more than 5 cm in diameter we tagged this tree as a new recruit. We gave it a new number and tagged it with that number. See the tree maintenance protocol on where to tag new trees. We measured the DBH, recorded the status, and identified the species of the new trees. We also added any new recruits to the maps we were using.

When entering this data into the computer we double checked all data for entry errors. We re-checked any diameters that were smaller than the last survey. If a tree was still smaller than the last survey we noted that the tree was checked twice. We also re-checked any trees that had a greater than 10 cm DBH growth spurt.

# adke142 - Shrub survey

## Shrub survey

Shrubs are surveyed in E133, E142, and E133resto in order to examine how plant compositions within plots are changing across time and how shrub compositions differ across a burn or fertility gradient.

E142 plots are sampled approximately once every five years. All points within each plot are sampled. The sampling area is a circular quadrant with a 1-meter radius around each of the sample points in the plot. For each woody species present within the quadrant all stems (dead and alive) of each individual are counted and recorded by plot and point.

Anything that is less than 1.5m tall, no matter how small, of all woody species except Parthenocissus vitacea and Rhus radicans is counted as a shrub. The only exceptions to the 1.5m cutoff are Corylus americana and Rhus glabra because they are always shrubs and never trees.

Past Notes: We noted if a species was a 'grub'. A grub is defined as any well-established (most likely re-sprouting) individual that is bigger than a seedling, but not as large as a sapling, which is defined as being taller than 1.5 m.

# adle142 - Plant aboveground biomass

## Plant aboveground biomass

Once a year aboveground biomass in E142 was harvested. The harvest area was changed yearly and is away from the permanent vegetation survey. An area of 0.5 m x 0.5 m was clipped using hedge trimmers and the vegetation was sorted to herbs and shrubs. Everything rooted in the frame is clipped, except large trees that cannot be clipped using clippers, a centimeter above the soil. The biomass is sorted into herbs and shrubs and put into two separate bags while in the savanna. The vegetation was then dried at approximately 40 degrees Celsius and weighed.

# adme142 - Plant belowground biomass

## Plant belowground biomass

We harvest the belowground biomass in the E133 and E142 experiments to examine the effects of fire frequency (E133) and nitrogen addition (E142) on the belowground biomass of the plots.

Belowground harvest takes place during the week directly following the aboveground harvest. One core is taken in the center of the aboveground clipping square. When there is no aboveground harvest, cores are taken within 2 meters of the point marker. Before the cores are taken, litter or other debris on the soil surface is wiped away by hand. A 2 inch diameter schedule 40 PVC pipe is used to take the cores. Cores can be taken at 1 or 2 different depths, 0 to 20cm and 20 to 40cm. The soil from each single depth are placed in a T-shirt bag. The bags are then taken to the root washing shed. The soil cores are washed and the roots sorted according to fine roots, coarse roots, and crowns. Roots are dried at approximately 40 degrees Celsius and weighed.

# adne142 - Herbaceous Vegetation Survey

## Herbaceous Vegetation Survey

Once every five years the herbaceous vegetation of E133, E133resto and E142 are surveyed. In E142 all 16 points in each plot (1-9) are sampled. An area of 0.5m x 1.0m is surveyed at each point. Percent cover is estimated for all herb species based on cover classes. The cover classes are based on a modified Domin scale (1 = 1%, 2 = 2-5%, 3 = 6-25%, 4 = 26-50%, 5 = 51-75%, and 6 = 76-100%). Percent cover estimates were based on vertical projection of plant parts onto the area of the frame. Plants lower than breast height (1.37m) were considered in the percent cover estimates. Plants did not have to be rooted within the area of the frame to be recorded. We imposed no upper limit on total cover, unlike some other studies within Cedar Creek Ecosystem Science Reserve, so a given point could have over 100% cover if there was significant overlap of leaves. Basically each species was considered independent of all others.

Interns completing herbaceous surveys must calibrate first with one another. The interns estimate the percent cover for the same plots and compare values. This continues until they are within a reasonable percentage (2-4%) of each other for the plots. Once the interns are calibrated to each other they start measuring percent cover on separate plots. Percent cover will be estimated for all herb and shrub species, along with bareground and litter values.

# adoe142 - Plant aboveground biomass carbon and nitrogen

## Analysis equipment

Samples were analyzed for Carbon Nitrogen with C-N Analyzers (NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc., Valencia, CA, USA). Analyses were done at The Ecosystems Analysis Laboratory at the University of Nebraska, Lincoln.

## Sample processing for analysis

Once a year aboveground biomass in E142 plots are harvested. The harvested area changes yearly. The biomass collected is sorted to herbs and shrubs and then bagged, dried and weighed. The dried and weighed biomass is then ground and put into individual vials. The ground biomass is then packed and sent to the Ecosystems Analysis Laboratory.

Aggregating samples: Combine all aboveground biomass samples for each plot-canopy combination for each herb and shrub sample. There are a total of 9 plots, each with 8 sampled points (only half of the points in each plot-canopy combination are sampled). Points numbered anywhere from 1 to 8 comprise the IN canopy treatment; and points numbered between 9 to 16 comprise the OUT canopy treatment.

Grinding: Organize and sort samples according to nitrogen treatment and plot number. Grind all the ambient plots (treatment I = plots 1, 4, 5), then low (treatment E = plots 3, 6, 8), and high (treatment G = plots 2, 7, 9) nitrogen plots, wiping the grinder(s) down with ethanol between different nitrogen treatments. We do this to minimize N contamination.

# adpe142 - Plant belowground biomass carbon and nitrogen

## Analysis equipment

Samples were analyzed for Carbon Nitrogen with C-N Analyzers (NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc., Valencia, CA, USA). Analyses were done at The Ecosystems Analysis Laboratory at the University of Nebraska, Lincoln.

## Sample processing for analysis

Once a year belowground biomass in E142 plots are harvested. The biomass collected is washed and separated into 0-20 cm roots and 20-40 cm roots, which are then bagged, dried and weighed. The dried and weighed biomass is then ground and put into individual vials. The ground biomass is then packed and sent to the Ecosystems Analysis Laboratory at the University of Nebraska, Lincoln where the samples are analyzed for their CN ratio.

Combine all root samples for each plot-canopy combination for each depth. There are a total of 9 plots, each with 8 sample points. Points numbered anywhere from 1 to 8 comprise the IN canopy treatment; and points numbered between 9 to 16 comprise the OUT canopy treatment. Grinding: Organize and sort samples according to nitrogen treatment and plot number. Grind all the ambient plots (treatment I = plots 1, 4, 5), then low (treatment E = plots 3, 6, 8), and high (treatment G = plots 2, 7, 9) nitrogen plots, wiping the grinder(s) down with ethanol between different nitrogen treatments. We do this to minimize N contamination.

# adqe142 - Canopy litter biomass carbon and nitrogen

## Analysis equipment

Samples were analyzed for Carbon Nitrogen with C-N Analyzers (NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc., Valencia, CA, USA). Analyses were done at The Ecosystems Analysis Laboratory at the University of Nebraska, Lincoln.

## Sample processing for analysis

Once a year litter biomass in the E142 plots was collected. The biomass collected is separated into Oak leaves, Acorns, and Miscellaneous litter; which are then bagged, dried and weighed. The dried and weighed Oak leaf litter biomass is then ground and put into individual vials. The ground biomass is then packed and sent to the Ecosystems Analysis Laboratory and the samples are analyzed for their CN ratio.

Combine all Oak leaf litter samples for each plot-canopy combination. There are 9 plots (N1-N9) and two treatments within each plot (points 1-8 = IN the canopy; points 9-16 = OUT of the canopy). You should have 18 composite samples after grinding. Grind samples according to nitrogen treatment and plot number. Grind all the ambient plots (treatment I = plots 1, 4, 5), then low (treatment E = plots 3, 6, 8), and high (treatment G = plots 2, 7, 9) nitrogen plots, wiping the grinder(s) down with ethanol between different nitrogen treatments. We do this to minimize N contamination.

# adre142 - Soil carbon flux

## Soil CO2 flux

Soil CO_{2} flux was measured once a month starting in May and continuing through October. All of these samplings were done using a LiCor 6200 on the SCF collar, which is near every intensively sampled savanna point. During the above- and belowground harvests in mid-July, a SCF collar was placed in the center of the freshly clipped area and the flux data was taken there.