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Methods for Experiment 120 -

E120 Construction

Photos from the construction of Experiment 120

Goals and Additional Design Details for E120

Goals To control plant species diversity in a well-replicated, long-term field experiment so as to determine the potential effects of plant species richness and plant functional-group richness on (1) stability of primary productivity in response to natural and experimentally induced climatic variation and in response to herbivores, pathogens, seed predators, and disease; (2) the species composition, abundances, stability and diversity of herbivorous insects, seed predators, predaceous insects, and parasitoids; (3) the densities, dynamics, stability and habitat choice of small mammals; (4) the dynamics of soil C and N, including rates of accretion, leaching losses, rates of mineralization, rates of fixation, and turnover of pools; and (5) the dynamics, species composition and biodiversity of soil micro- and macro-organisms, including soil mycorrhizal fungi, nitrifying bacteria, other bacteria, other fungi, soil micro-arthropods, earthworms, and soil arthropods. Additional Design Details Biodiversity II is contained within a block of 342 plots laid out in a grid adjacent to Biodiversity I. Each plot was laid out as a 13 m x 13 m square, but only the central 9 m x 9 m is actively maintained to contain the specified species and level of plant diversity. This is the only portion sampled. Plots were planted with grassland perennial herbaceous and savanna woody species. These plant species were in either the C4 grass, C3 grass, legume, other forb, or woody functional groups. The species composition of the plots was chosen by separate random draws of the appropriate number of species (1, 2, 4, 8, or 16 plant species) from a pool of 18 species. For diversity levels 1, 2, 4, 8, and 16 species, there are 39, 35, 29, 30, and 35 replicates, respectively. To initiate the experiment, a field at Cedar Creek Natural History Area, Minnesota, was treated with herbicide and burned in August, 1993, had 6-8 cm of soil removed to reduce the seed bank, was plowed and harrowed, then divided into plots of which 168 form this experiment. Plots for Biodiversity II were manually seeded in May 1994 using seed addition rates and methods like those of Biodiversity I. All plots received, in total, 10g/m2 of pure live seed in May 1994 with seed mass divided equally among species. Due to insufficient water resources, the plots were not irrigated in 1994. Because a dry and windy spring led to some soil and seed erosion and to poor germination, plots were re-seeded in May 1995, at half the 1994 rate, and watered once or twice weekly with an irrigation system. Two species that did not germinate in 1994 were replaced with different species in the same functional groups in the 1995, with their seeding proportionate to 10g/m2 or pure live seed. Three species (Elymus canadensis, Poa pratensis, Panicum virgatum) did not establish in one of their two original monocultures, even after reseeding, and these three plots were abandoned after 1996. All plots were mown in July 1994 to help control weeds, and manually weeded in August 1994. They were manually weeded twice in 1995, four times in 1996, and three or four times from 1997 onward. Selective herbicides were also used through 1997. Plots that were designated to contain only legumes and/or other forbs were sprayed with Assure (Quizalofop P-ethyl (Ethyl(R)-2-[4-(6-chloroquinoxalin-2-yl oxy)-phenoxy]propionate)), a selective herbicide against grasses, twice each growing season from 1994 through 1997. The plots designated to contain just grasses were sprayed twice a year through 1997 with 2,4-dichlorophenoxyacetic acid, a selective herbicide against forbs. All plots in Biodiversity II were burned each spring in late April or early May before plant growth had begun. There were two problems in implementing Biodiversity II. The first concerned the legume Petalostemum villosum. It had, as a seed contaminant, a congener P. candidum, causing both species to be planted, but at approximately half the desired density for each species, in plots designated for P. villosum. Moreover, seed of the legume Amorpha canescens was inadvertently substituted the legume P. villosum in 16-species plots. Second, because virtually no Solidago rigida, a non-legume forb, germinated during 1994, we reseeded all plots planted originally to Solidago rigida with the non-legume forb Monarda fistulosa. Some Solidago rigida did germinate in 1995 and occurs in plots where originally planted. Location The experiment occupies a 10 hectare block of land in a former "brome field" on the land that formerly was part of the Peterson farm. Other Plots Within the Experimental Block Plots within the experimental zone which are not used in E120 are 76 additional plots that had functional group compositions drawn from an augmented species pool, 46 plots planted to 32 species, and 48 plots designated for other uses.

History of Big Biodiversity Field Plots

One hundred seventy plots were planted in the block of 342 big biodiversity plots which were not included in experiment e120. The assignment of species to these plots were randomly allocated in different ways than for the E120 plots. The following includes details about the plots within this group.

Numbering of the plots for E120

Plots are numbered starting from the northeast corner, incrementing left to right in odd rows and right to left in even rows. Walking paths separate every row and driving paths divide the plots into 6x6 groups in the following arrangement. There are 18 Rows with 19plots each.

Seeding rates

To assure adequate establishment, plots were seeded both in 1994 and 1995. Rates for 1995 are related to those of 1994 by an adjustment factor, with one factor for plots that retained their identities and another factor for plots in which species were changed or added. Factors for plots that were changed are marked with asterisks (*) below. Columns labelled B, C, D, etc. contain the grams of seed applied per plot for the corresponding treatment. The table describes the species planted within any of the 342 plots in the block of plots containing the plots for E120.

Species Assigned to Each Plot

Species abbreviations

Species codes of five characters are assigned from the first three characters of the genus name and the first two of the species. These abbreviations are used in other files to shorten documentation of the experiment.

Treatments applied to plots

A plot's group code shows which functional groups are present in the plot, with the left-most column representing C-3 grasses, the second column representing C-4 grasses, the third representing forbs other than legumes, the fourth representing legunes, and the fifty representing woody plants. Again, if a column is 1, the corresponding group is present in the plot. If 0, it is not. For example, Plot 3 has group code 01100, which means the plot has both C-4 grasses and forbs other than legumes, but nothing else.

aafe120 - Plant traits

Abstract

In the summers of 2007 and 2008, leaf traits were measured in the Cedar Creek biodiversity experiment [E120]. Three fully mature leaves were sampled from ten individuals of each species collected within the maintained experimental plots as well as from the unmaintained experimental plots. Each individual was identified from a randomly chosen plot to cover the range of diversity treatments. Fresh leaves were scanned on a flatbed scanner on the same day as collection with petioles and sheaths removed. Leaf area, perimeter and Feret's diameter were calculated from the scanned leaves using ImageJ software (Rasband 1997-2004). This allowed calculation of perimeter per area (P/A, cm/cm-2), which is empirically correlated with leaf hydraulic conductance across a wide range of taxa (Sack et al. 2003). Perimeter per leaf area x Feret's diameter is a unitless measure of leaf lobedness Cavender-Bares et al 2006 that influences the leaf radiation balance (Givnish 1976). After scanning, leaves were dried at 65oC for three days and weighed to calculate specific leaf area (cm2/g). Seed mass was determined by collecting seed heads for ten plants per species with fully mature seeds, air drying the seed heads, and then weighing together ten seeds (and dividing by ten) to calculate a mean seed mass per plant. For five species, seed mass was taken from online commercial databases, including the Native Seed Network, Wildflower Farm Inc., and Prairie Moon Nursury. Plant height was measured at maturity from the root collar to the apical meristem or to the top of the vegetative canopy for five to ten individuals per species. Some values were also taken from the USDA Plants Database.

Sampling

Obtain 5 samples from within the biodiversity experiment and 5 samples from the out-group of not maintained plots. Each sample should consist of 3 mature leaves of the plant. Keep these leaves cold and dry until scanning.

Scanning

Scanning of leaf samples should ideally be done on the same day that samples are obtained. If this is not possible, keep samples refrigerated. To begin scanning, cut petioles from the leaves and measure with a ruler. Number a transparency and put leaves in appropriate area on transparency. Scanning is easiest if transparency remains on the scanner with ruler. Make sure scan contains all leaves and the ruler. Make sure the scan is in true color. Save the image as: Plant AbbreviationNumber_mm_dd_yy Use a sharpie to number the leaves. After the scan, the leaves should be returned to an envelope and placed in a drier at 65??C then weighed when dried Using ImageJ: Open the image from File / Open. This should bring up the image in true color in another window. Setting scale: Click analyze / set scale. Run a line across the ruler for 8 centimeters. Enter known distance as =8.0 and Unit of Length as =cm. Click global in order to make this the default for all following images. Measuring Parameters: Click Process / Binary / Threshold (or Make Binary depending on version. Click Analyze / Set Measurements. This will bring up a window and make sure that Area Perimeter and Feret???s Diameter are the only boxes checked. Select the wand from the toolbar and select one of the leaves. A yellow outline should appear on the black object. Click analyze / measure. This will bring up a third window with the data. Save this into an excel or spss spreadsheet.

aage120 - Main Plots All Arthropod Insect Sweepnet Sampling 1996-2006

Main Plots All Arthropod Insect Sweepnet Sampling 1996-2006

This dataset contains arthropods samples from the main group of 172 plots in E120. Please see main E120 web page for details on plot treatments and other information. These main plots were sampled by sweepnet one to three times per summer from 1996-2006. Sampling dates were: 16/Aug/1996, 20/Jun/1997, 28/Jul/1997, 22/Aug/1997, 22/Jun/1998, 20/Aug/1998, 22/Jun/1999, 20/Aug/1999, 26/Jun/2000, 22/Aug/2000, 23/Jun/2001, 19/Aug/2001, 22/Jun/2002, 20/Aug/2002, 18/Aug/2003, 17/Aug/2004, 17/Aug/2005, 16/Aug/2006. August samples corresponded to approximate peak plant biomass. Problems with spoilage in June samples containing lupine (samples were difficult to dry before mold became a problem) prompted the termination of June sampling after 2002. Samples are not available from all plots on all dates, either due to spoilage or because they were not sampled. Plots were sampled regardless of any changes in plot treatment. In each month, all plots were swept in one day. A 38cm diameter muslin sweep net was used to make 25 sweeps while walking a line about 10m long through the interior of each plot about 2-3m from the plot edge. A ?sweep? consisted of a quick, approximately 2-meter-long horizontal swing of the net. Each sweep sample was transferred to a 1-gallon plastic bag with label identifying plot number. Bags were placed in a chest freezer, and later opened, thawed, and sorted. Extraneous plant material was shaken and discarded. The remaining sample was viewed under a dissecting scope, and species and the number of each encountered was tabulated. Identification was generally to species or genus, but occasionally a morphological descriptive is used when identification was uncertain. Many specimens were mounted and labeled, to serve as reference material and to more closely examine and determine problematic species. As of 2012, reference specimens have primarily been transferred to the University of Minnesota Insect collection on the Saint Paul Campus. Photos of some morphospecies are posted on the Insects of Cedar Creek website. *Be Cautious comparing across studies, there is no guarantee that morphospecies descriptions within this study and others match up. Plots which were designated part of the main e120 experiment and which are included in this data set are as follows: 2, 3, 5, 6, 9, 11, 12, 14, 15, 16, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 44, 45,46, 48, 50, 53, 56, 57, 58, 62, 67, 68, 69, 70,73, 74,75, 81, 82, 83, 87, 89, 92, 93, 94, 98, 104, 107, 108, 109, 110, 111, 115, 117, 118, 125, 126, 127, 129, 130, 133, 135, 136, 137, 138, 139, 142, 146, 149, 151, 153, 156, 157, 160, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171,174,175, 176, 177, 178, 185, 186, 189, 190, 193, 197, 199, 201, 202, 205, 206, 208, 210, 211, 213, 220, 221, 223, 224, 225, 227, 229, 230, 232, 233, 234, 235, 236, 237, 239, 242, 244, 248, 253, 255, 256, 257, 259, 265, 266, 267, 268, 272, 273, 278, 280, 282, 283, 286, 287, 290, 291, 292, 293, 296, 299, 300, 301, 302, 303, 304, 307, 308, 311, 313, 318, 322, 324, 325, 328, 329, 330, 331, 333, 334, 335, 336, 338, 339, 342.

Main Plots All Arthropod Insect Sweepnet Sampling 1996-2006: Data Preparation

Data was prepared primarily by Nick Haddad, John Haarstad and Stephanie Pimm Lyon. Colleen Satyshur corrected a sampling date discrepancy and re-formatted columns to match 2013 standard arthropod attributes.

acqe120 - Developing seedheads treated with fungicide and insecticide

Developing seedheads treated with fungicide and insecticide

In 2005, we selected six species based on the availability of sufficient monoculture replication. For most species in the biodiversity experiment there were only one or two monoculture plots; we used all species for which there were three monoculture plots except for one, Andropogon gerardii, which had two monoculture and a two-species plot in which its woody species competitor was almost entirely absent (which made it a de facto monoculture). For each of the six species we randomly selected three 16-species plots (hereafter polycultures) from the 35 polycultures in the larger biodiversity experiment, with the stipulation that they contained the focal species and were not already assigned to some other focal species. In total, 36 plots (18 monocultures and 18 polycultures) were used for our natural enemy removal experiment. Eight seedheads of six species were selected and randomly assigned to one of four treatments, namely, insecticide, fungicide, insecticide and fungicide, or water (controls). Plots contained two replicate seedheads per treatment. After flowering, between mid-July and mid-August, seedheads were saturated weekly with the respective treatment using spray bottles. We used a 3 % concentration of Mavrik Aquaflow (Wellmark International, Schaumburg, IL), a pyrethroid insecticide with the active ingredient taufluvalinate (22.3 % by volume) and a 1 % concentration of Captan (Bonide Products, Inc., Oriskany, NY), a phthalimide fungicide (50 % by volume). As seeds ripened from August to September, we visited plots three times a week and harvested seedheads, along with >10 cm of stem, once they were fully mature but before seeds dispersed. Collected seedheads were stored dry at room temperature and ambient humidity for several weeks to after-ripen. We separated seeds from stems and vegetative materials and hand-counted the apparent number of seeds per seedhead. Next, to assess differences in seed viability due to natural enemy treatments, we tested germination on 30 apparent seeds per seedhead wherever possible. Apparent seeds that contained insects or seeds consumed by fungi would not germinate, providing a means to indirectly measure seeds consumed by predators. Further details on this dataset can be found in the publication: Beckman, Noelle G.; Dybzinski, Ray; Tilman, G. David; Neighborhoods have little effect on fungal attack or insect predation of developing seeds in a grassland biodiversity experiment; Oecologia, 2014; 174(2): 521 - 532. 2014

acve120 - Local plant diversity and soybean biological control 2011 Aphid and Enemy Surveys

Local plant diversity and soybean biological control Methods

Establishment In May 2011, we dug six 76.2 cm holes using a 20.3 cm auger, immediately adjacent to each of these 33 plots;eleven 1-species plots, eleven 4-species plots, and eleven 16-species plots, chosen randomly from the pool of maintained plots that were not heavily used for other experiments. To limit root competition of soybean plants with prairie vegetation, we sunk 20.3 cm diameter HVAC ducting into each hole so that it protruded approximately 5 cm above ground level. We mixed soil removed from the holes with purchased garden soil at a 1:1 ratio and filled the holes with the mixture. Soybeans and Soybean Aphids We conducted similar experiments in 2011 and 2012. On May 20, 2011 and May 8, 2012, soybeans (Syngenta NK S19-R5) were dipped in soybean inoculant slurry and planted, three beans within each HVAC pot. When the plants germinated, 91.4 cm tall cylindrical cages of hardware cloth were positioned around the plants to prevent small-mammal herbivory; the Biodiversity II field is surrounded by a deer fence, and we observed no mammal herbivory in either year. Three weeks after planting, pots without germination received a transplant from pots with multiple plants germinating; a week later, all pots were thinned to one plant. Soybean plants were watered, as needed, throughout each summer to prevent severe drought stress. Each plant received 0.95 liters of water on watering occasions. On June 24, 2011, approximately 40 lab-cultured soybean aphids were added to each soybean plant. The plants had an average of 2.5 trifoliates. On June 12, 2012, approximately 50 aphids were added to five of the six plants at each plot; one plant at each plot was left as an aphid-free control. The plants had an average of 2 trifoliates. For ten weeks each summer, we surveyed each plant once per week, recording the plant size (in trifoliates) and the number of aphids. In 2012, we removed immigrant (winged) aphids from the aphid-free control plants during these surveys. We also surveyed each plant once (2011) per week, recording the number and identity of all insects found on the plants that were not soybean aphids. In 2011, these insect surveys were done in conjunction with the aphid surveys; the plant was first assessed for mobile insects, and then other insects were noted as aphids were counted. We stopped surveys when plants began to senescence. Natural Enemy Exclusion On June 24 2011, we conducted a natural enemy exclusion experiment. We covered three (2011) plants at each plot with no-see-um mesh by wrapping the mesh around the hardware cloth cage, twisting and clipping mesh edges together, and burying the bottom edges of the mesh. Unclipping mesh edges allowed for access to the plants to conduct surveys during the natural enemy exclusion. Natural enemies were manually removed from the plants immediately prior to mesh application and during surveys. Mesh was left on for two weeks and was removed on July 6, 2011. To test for microclimate effects of the mesh, we took temperature and humidity readings from two adjacent plants,one with mesh and one without, at each plot twice: once in the morning when the temperature was relatively low and once mid-afternoon when the temperature was approximately at its peak. We also counted winged aphids (alates) on all plants during the aphid surveys while mesh was on and immediately following mesh removal.

acwe120 - Local plant diversity and soybean biological control 2012 Aphid Surveys

Local plant diversity and soybean biological control

Establishment In May 2011, we dug six 76.2 cm holes using a 20.3 cm auger, immediately adjacent to each of these 33 plots;eleven 1-species plots, eleven 4-species plots, and eleven 16-species plots, chosen randomly from the pool of maintained plots that were not heavily used for other experiments. To limit root competition of soybean plants with prairie vegetation, we sunk 20.3 cm diameter HVAC ducting into each hole so that it protruded approximately 5 cm above ground level. We mixed soil removed from the holes with purchased garden soil at a 1:1 ratio and filled the holes with the mixture. Soybeans and Soybean Aphids We conducted similar experiments in 2011 and 2012. On May 20, 2011 and May 8, 2012, soybeans (Syngenta NK S19-R5) were dipped in soybean inoculant slurry and planted, three beans within each HVAC `pot`. When the plants germinated, 91.4 cm tall cylindrical cages of hardware cloth were positioned around the plants to prevent small-mammal herbivory; the Biodiversity II field is surrounded by a deer fence, and we observed no mammal herbivory in either year. Three weeks after planting, pots without germination received a transplant from pots with multiple plants germinating; a week later, all pots were thinned to one plant. Soybean plants were watered, as needed, throughout each summer to prevent severe drought stress. Each plant received 0.95 liters of water on watering occasions. On June 24, 2011, approximately 40 lab-cultured soybean aphids were added to each soybean plant. The plants had an average of 2.5 trifoliates. On June 12, 2012, approximately 50 aphids were added to five of the six plants at each plot; one plant at each plot was left as an aphid-free control. The plants had an average of 2 trifoliates. For ten weeks each summer, we surveyed each plant once per week, recording the plant size (in trifoliates) and the number of aphids. In 2012, we removed immigrant (winged) aphids from the aphid-free control plants during these surveys. We also surveyed each plant twice per week, recording the number and identity of all insects found on the plants that were not soybean aphids. In 2011, these insect surveys were done in conjunction with the aphid surveys; the plant was first assessed for mobile insects, and then other insects were noted as aphids were counted. In 2012, insect surveys were done separately from aphid surveys; every plant was searched for the same length of time each survey, from 30 seconds each when the plants were small to 120 seconds each when the plants were large. We stopped surveys when plants began to senescence. Natural Enemy Exclusion On June 22, 2012, we conducted a natural enemy exclusion experiment. We covered three (2011) or two (2012) plants at each plot with no-see-um mesh by wrapping the mesh around the hardware cloth cage, twisting and clipping mesh edges together, and burying the bottom edges of the mesh. Unclipping mesh edges allowed for access to the plants to conduct surveys during the natural enemy exclusion. Natural enemies were manually removed from the plants immediately prior to mesh application and during surveys. Mesh was left on for two weeks and was removed on July 6, 2011, and July 6, 2012. To test for microclimate effects of the mesh, we took temperature and humidity readings from two adjacent plants, one with mesh and one without , at each plot twice: once in the morning when the temperature was relatively low and once mid-afternoon when the temperature was approximately at its peak. We also counted winged aphids (alates) on all plants during the aphid surveys while mesh was on and immediately following mesh removal. Ant Exclusion In 2012, we attempted to reduce the presence of aphid-tending ants on the soybean plants. We painted Tangle-Trap coating (Contech Inc.) around the lip of all HVAC pots on June 8, but stickiness was quickly lost after soil splattered on the coating during rain showers. We also used ant-bait traps (Terro Liquid Ant Baits T300) within each HVAC pot, which were placed on June 15 and July 20. During surveys, ants found on the plants were manually removed.

acxe120 - Local plant diversity and soybean biological control 2011 Harvest Measures

Local plant diversity and soybean biological control methods

Establishment In May 2011, we dug six 76.2 cm holes using a 20.3 cm auger, immediately adjacent to each of these 33 plots;eleven 1-species plots, eleven 4-species plots, and eleven 16-species plots, chosen randomly from the pool of maintained plots that were not heavily used for other experiments. To limit root competition of soybean plants with prairie vegetation, we sunk 20.3 cm diameter HVAC ducting into each hole so that it protruded approximately 5 cm above ground level. We mixed soil removed from the holes with purchased garden soil at a 1:1 ratio and filled the holes with the mixture. Soybeans and Soybean Aphids We conducted similar experiments in 2011 and 2012. On May 20, 2011 and May 8, 2012, soybeans (Syngenta NK S19-R5) were dipped in soybean inoculant slurry and planted, three beans within each HVAC `pot`. When the plants germinated, 91.4 cm tall cylindrical cages of hardware cloth were positioned around the plants to prevent small-mammal herbivory; the Biodiversity II field is surrounded by a deer fence, and we observed no mammal herbivory in either year. Three weeks after planting, pots without germination received a transplant from pots with multiple plants germinating; a week later, all pots were thinned to one plant. Soybean plants were watered, as needed, throughout each summer to prevent severe drought stress. Each plant received 0.95 liters of water on watering occasions. On June 24, 2011, approximately 40 lab-cultured soybean aphids were added to each soybean plant. The plants had an average of 2.5 trifoliates. On June 12, 2012, approximately 50 aphids were added to five of the six plants at each plot; one plant at each plot was left as an aphid-free control. The plants had an average of 2 trifoliates. In 2011, we harvested two plants from each plot as soon as the start of senescence was detected to determine aboveground biomass. The plants were dried and weighed. After remaining plants had fully senesced, but before dropping bean pods, we harvested bean pods from remaining plants (4 in 2011). Beans were removed from pods and were counted and weighed. Beans were then dried and reweighted. Natural Enemy Exclusion On June 24 2011, we conducted a natural enemy exclusion experiment. We covered three (2011) plants at each plot with no-see-um mesh by wrapping the mesh around the hardware cloth cage, twisting and clipping mesh edges together, and burying the bottom edges of the mesh. Unclipping mesh edges allowed for access to the plants to conduct surveys during the natural enemy exclusion. Natural enemies were manually removed from the plants immediately prior to mesh application and during surveys. Mesh was left on for two weeks and was removed on July 6, 2011. To test for microclimate effects of the mesh, we took temperature and humidity readings from two adjacent plants,one with mesh and one without, at each plot twice: once in the morning when the temperature was relatively low and once mid-afternoon when the temperature was approximately at its peak.

adce120 - Local plant diversity and soybean biological control 2012 Enemy Surveys

Local plant diversity and soybean biological control methods

Establishment In May 2011, we dug six 76.2 cm holes using a 20.3 cm auger, immediately adjacent to each of these 33 plots;eleven 1-species plots, eleven 4-species plots, and eleven 16-species plots, chosen randomly from the pool of maintained plots that were not heavily used for other experiments. To limit root competition of soybean plants with prairie vegetation, we sunk 20.3 cm diameter HVAC ducting into each hole so that it protruded approximately 5 cm above ground level. We mixed soil removed from the holes with purchased garden soil at a 1:1 ratio and filled the holes with the mixture. Soybeans and Soybean Aphids We conducted similar experiments in 2011 and 2012. On May 20, 2011 and May 8, 2012, soybeans (Syngenta NK S19-R5) were dipped in soybean inoculant slurry and planted, three beans within each HVAC `pot`. When the plants germinated, 91.4 cm tall cylindrical cages of hardware cloth were positioned around the plants to prevent small-mammal herbivory; the Biodiversity II field is surrounded by a deer fence, and we observed no mammal herbivory in either year. Three weeks after planting, pots without germination received a transplant from pots with multiple plants germinating; a week later, all pots were thinned to one plant. Soybean plants were watered, as needed, throughout each summer to prevent severe drought stress. Each plant received 0.95 liters of water on watering occasions. On June 24, 2011, approximately 40 lab-cultured soybean aphids were added to each soybean plant. The plants had an average of 2.5 trifoliates. On June 12, 2012, approximately 50 aphids were added to five of the six plants at each plot; one plant at each plot was left as an aphid-free control. The plants had an average of 2 trifoliates. For ten weeks each summer, we surveyed each plant once per week, recording the plant size (in trifoliates) and the number of aphids. In 2012, we removed immigrant (winged) aphids from the aphid-free control plants during these surveys. We also surveyed each plant twice per week, recording the number and identity of all insects found on the plants that were not soybean aphids. In 2011, these insect surveys were done in conjunction with the aphid surveys; the plant was first assessed for mobile insects, and then other insects were noted as aphids were counted. In 2012, insect surveys were done separately from aphid surveys; every plant was searched for the same length of time each survey, from 30 seconds each when the plants were small to 120 seconds each when the plants were large. We stopped surveys when plants began to senescence. Natural Enemy Exclusion On June 22, 2012, we conducted a natural enemy exclusion experiment. We covered three (2011) or two (2012) plants at each plot with no-see-um mesh by wrapping the mesh around the hardware cloth cage, twisting and clipping mesh edges together, and burying the bottom edges of the mesh. Unclipping mesh edges allowed for access to the plants to conduct surveys during the natural enemy exclusion. Natural enemies were manually removed from the plants immediately prior to mesh application and during surveys. Mesh was left on for two weeks and was removed on July 6, 2011, and July 6, 2012. To test for microclimate effects of the mesh, we took temperature and humidity readings from two adjacent plants, one with mesh and one without , at each plot twice: once in the morning when the temperature was relatively low and once mid-afternoon when the temperature was approximately at its peak. We also counted winged aphids (alates) on all plants during the aphid surveys while mesh was on and immediately following mesh removal. Ant Exclusion In 2012, we attempted to reduce the presence of aphid-tending ants on the soybean plants. We painted Tangle-Trap coating (Contech Inc.) around the lip of all HVAC pots on June 8, but stickiness was quickly lost after soil splattered on the coating during rain showers. We also used ant-bait traps (Terro Liquid Ant Baits T300) within each HVAC pot, which were placed on June 15 and July 20. During surveys, ants found on the plants were manually removed.

agfe120 - Fire Severity

Calipers

Calipers and meter sticks were used to measure stem diameters and plant heights, respectively

agge120 - Fire Fuel Load

Biomass Clipping

To assess how well summer (late July) biomass represented fuel loads the following spring, we clipped biomass (not separating previous-season biomass from older litter) immediately before burning in spring (March) 2015 in strips adjacent to those clipped the previous summer in 30 plots ranging from grass-dominated to forb-dominated. Strips were 0.1 m wide and 6 m long, 2 per plot. Biomass was dried to constant mass and weighed.

Clippers

Electric clippers were used.

agie120 - Fire Temperatures

Temperature Measurements

Fire temperature was estimated using metal tags ??? pyrometers ??? with Omega Laq paints (Omega Engineering, Stamford, Connecticut, USA) of varying melting points, placed in each plot before burning. In 2000, 9 paints with melting points spanning 70-650 ??C were spotted on aluminum tags wrapped in aluminum foil and clipped 10 cm above the ground to reinforcing bars at each corner of a 5 x 5 m square centered in each of 176 plots. In 2011, 13 paints with melting points spanning 79-788 ??C were spotted on copper plant tags (National Band and Metal, Newport, Kentucky, USA), covered by a second tag, and placed at ground level and on stakes 50 cm above ground at three corners of the same central 5 x 5 m square in each of the 154 core plots that were still maintained at that date. After the burn, we estimated the proportion of a circle with 20 cm radius around each pyrometer that carried fire. We used identical tags in a subset of 100 plots in 2010 and 90 plots in 2014. We verified the paints??? rated melting points by placing sample tags in a calibrated muffle furnace for 1 min at increasing temperatures, and scored whether paints had melted using a dissecting microscope and reference images. Tags were assigned the melting temperature of the highest melted paint; when no paints were melted, they were assigned 20 ??C to approximate ambient temperature. Work was co-ordinated in 2000 by Troy Mielke

Temperature Paints

Omega Laq paints (Omega Engineering, Stamford, Connecticut, USA) of varying melting points; copper plant tags (National Band and Metal, Newport, Kentucky, USA)

aime120 - Soil phosphorus

Instrumentation - lab

Brinkmann PC 900 probe colorimeter (Thermo Fisher Scientific, Waltham, MA, USA).

Sampling and lab analysis

Extractable Bray P was measured from archived soil samples collected from each plot before planting in 1994 at 0-20 cm and soil samples collected after 23 years of growth in 2017 from depth increments of 0.20 cm, 20.40 cm and 40.60 cm. The University of Minnesota Research Analytical Laboratory (Saint Paul, MN, USA) analyzed these soil samples for extractable P using a standard Bray-1 extract (0.025 M HCl and 0.03 M NH₄F) and analyzed colorimetrically. Lab methods from; R. Eliason, R. J. Goos, B. Hoskins, Recommended chemical soil test procedures for the North Central Region, Rev. ed.. (Missouri Agricultural Experiment Station, University of Missouri, 2015).

invne120 - Invasion strip soil nitrogen

Soil nitrogen sampling and analysis

The 13 x 13 m plots planted in 1994 with 1, 2, 4, 8 or 16 randomly selected prairie-savannah species from a pool of 18 possible species were weeded to maintain the planted species composition. In 2000, weeded plots areas were shrunk to 9 x 9 m plus an unweeded 1 x 9 m strip along one edge of the plots where invaders were allowed to colonize. The invasion strip subplots were sampled for soil nitrogen, on 7 August 2002. 10-g subsamples from four pooled 2-cm diameter cores were extracted for nitrate using 0.01 M KCl, shaken for 30 min, and allowed to settle overnight at 4 C. The supernatant was removed the following day, and was frozen until analysed for nitrate on an Alpkem analyzer (Pulse Instruments Ltd, Saskatoon, Canada).

invre120 - Invasion strip root biomass

Root biomass sampling

The 13 x 13 m plots planted in 1994 with 1, 2, 4, 8 or 16 randomly selected prairie-savannah species from a pool of 18 possible species were weeded to maintain the planted species composition. In 2000, weeded plots areas were shrunk to 9 x 9 m plus an unweeded 1 x 9 m strip along one edge of the plots where invaders were allowed to colonize. In 2002 six soil cores, 5.08 cm diameter, 0-30cm depth were taken from below the clipped aboveground biomass and aggregated. Roots were washed free of soil, sorted from other organic material, dried and weighed.

nbe120 - Plant aboveground biomass carbon and nitrogen

Instrumentation-Plant aboveground biomass carbon and nitrogen

Samples were analyzed using C-N Analyzers, NA1500, Carlo-Erba Instruments or ECS 4010, COSTECH Analytical Technologies Inc., Valencia, CA, USA Lab analysis were done at University of MN or at the Ecosystems Analysis Lab, University of Nebraska, Lincoln

Plant aboveground biomass carbon and nitrogen

Clip strip harvests were 10cm wide by 6 meters in length. Four strips per plot were harvested, typically in late July-early August. Clip strip locations in the plots were rotated each year to minimize sampling effect. For this dataset, unsorted biomass from two clip-strips was air dried at 40 degrees C. After drying, biomass samples were ground with a standard Thomas Wiley? Mill. The resulting ground sample was stirred to homogenize, then a sub-sample was re-ground in a Wiley? Mini-Milll using a 20 mesh screen. Final ground samples were placed in labelled glass scintillation vials and re-dried prior to lab analysis for percent carbon and nitrogen.

pce120 - Plant species percent cover data

Vegetation Sampling

Percent cover of species within 4 permanently marked quadrats within each plot has been recorded annually in July from 1996 to 2000.

ple120 - Plant aboveground biomass data

Vegetation Sampling

Aboveground biomass from each plot has been sampled by clipping narrow strips. The biomass has been sorted, dried, and weighed separately for each strip. Different areas of the plot have been sampled each year and the size of the clipped strips have periodically changed. See aboveground vegetation sampling for details on equipment and other field methods.

rbe120 - Root biomass data

Sampling

Beginning in 1997, root biomass is sampled after clipping by collecting three 5 cm diameter x 30 cm deep cores per clipped strip. Roots are washed free of soil, sorted from other organic material, dried and weighed.