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Methods for Experiment 271 -

Experiment Design

The FAB 1 High density diversity experiment consists of 8,960 trees of 12 native species. Four of these species are gymnosperms: eastern red cedar (Juniperus virginiana) and white (Pinus strobus), red (P. resinosa), and jack (P. banksiana) pine. The eight angiosperm species include red (Quercus rubra), pin (Q. ellipsoidalis), white (Q. alba), and bur (Q. macrocarpa) oak; red maple (Acer rubrum) and box elder (A. negundo); paper birch (Betula papyrifera); and basswood (Tilia americana). Each of FAB`s three blocks(spaced 4.5 m apart) consists of either 46 or 47 square plots, each 3.5 m on the edge; plots are planted with one, two, five, or 12 species, with two-species plots additionally designed to tease apart functional and phylogenetic diversity. Each plot contains 64 trees, planted at 0.5 m intervals. Within a block, all trees are planted on a contiguous grid, without extra space in between plots. Each block contains 12 monocultural plots and 28 bicultural (two-species) plots; each of these plot types (or compositions) is therefore replicated three times across the experiment. Each block also contains either three or four random-draw five-species poly-cultures, the compositions of which are not replicated in the experiment, giving replication of the five-species level of richness but not of each five-species polyculture's composition. Each block also contains three or four 12-species polycultures, such that this composition is replicated 10 times across the experiment. Half of the 28 bicultural plot compositions were chosen by random draw. The other remaining bicultures were chosen using a stratified random approach designed to provide plots both low and high in PD and FD.

Planting and establishment

The experimental site was burned, then mulched with wood chips (from non-native western red cedar [Thuja plicata]) to prevent regrowth of herbaceous species. The experiment was planted over one week in late May 2013 with regionally sourced bare root seed-lings of unknown genetic relatedness that ranged from 1 to 2 years in age. Prior to planting, seedling roots were coated with commercial ectomycorrhizal and endomycorrhizal inoculum including species known to associate with all genera included in the experiment (Bio Organics, New Hope, PA). We used sprinkler irrigation to water newly planted seedlings ad libitum through June and July 2013. We replanted seedlings as needed in May/June 2014 to 2015; mortality was roughly 7 to 10 percent following replanting.

aepe271 - Sapling Census

FAB Sapling Census Protocol

FAB Sapling Census Protocol Height Height Use a folding ruler to measure the height of each tree. Measure from the base of the soil to the tip of the sapling's leading stem. Measure from the soil surface, not from the mulch surface. Insert ruler into mulch if necessary. The leader is the tallest live, vegetative stem. Measure to the base of the leader's apical bud. Do not include bud scales or, for conifers, new needles. If there are multiple stems, pick the tallest living stem. Make sure that the ruler follows the contours of the stem, especially for curved stems. Report height in centimeters to the nearest 0.5 cm. (All heights should end in '.0' or '.5') Diameter Use calipers to measure the diameter of the sapling at 5.0 cm from the soil surface. Before measuring and repeatedly throughout the day, check the caliper to make sure that it reads 0.0 cm when closed. If the tree has already been measured, find the orange or pink paint pen mark indicating where it was measured previously. Use calipers to record the measurement at that point. If the tree has not been measured previously or a paint pen mark is not apparent, measure 5.0 cm from the soil surface (see Height) using a ruler and mark the tree with a ring there. Measure from that point. If the stem forks before 5 cm or is otherwise not amenable to measurement at 5 cm, mark and measure at a different height and note this on the data sheet. If the stem forks and both resultant stems seem vigorous, measure below the fork. Report Diameter in centimeters to the nearest 0.5 mm. (All diameters should end in '.0' or '.5') DBH If the tree height is above 1.37 meters, use calipers to measure the diameter of the sapling at this level. (All diameters should end in '.0' or '.5') Reproducing Observe if the sapling is producing any reproductive structures (e.g. flowers, cones, seeds). If yes, mark 'Y'. Leave blank if none present.

aere271 - Initial soil pH

Initial soil pH -instrumentation

Orion 420A pH meter

Intital Soil pH protocol

Soils were collected at depths of 0-15cm, 15-30cm, and 30-60cm, sieved through a 2mm sieve, air dried, and 10g soil (plus 20ml DI water) weighed for pH analysis.

afee271 - Soil bulk density

Soil bulk density protocol

Soil cores were taken from 0-15cm, 15-30cm, and 30-60cm in randomly chosen FAB1 plots. The soil samples were dried and weighed. Dry weights were divided by the volume of the soil core (diameter=5cm).

agke271 - FAB Leaf Herbivory

Methods FAB Leaf Herbivory

Leaf herbivory was measured in a subset of angiosperm species (2014: ACNE, ACRU, BEPA, QUEL, QURU, and TIAM; 2015,2016: all of these plus QUAL and QUMA) across a range of plots varying in functional and phylogenetic diversity. All measurements were taken by Jacob Grossman. For each plot surveyed, three trees per species per plot were measured. The newest five fully expanded leaves on the leading stem were measured. 1 is the first (youngest) fully expanded leaf on a leader and 5 is the fifth youngest. If there weren't 5 leaves on the tallest leader's branch, another branch was selected for additional sampling. Grossman used a laminated, translucent 1 cm2 grid to estimate leaf area and leaf area removed. Necrotic or chlorotic tissue was not counted as removed. Some plots were inaccessible due to poison ivy. For leaf area measurements, he rounded to the nearest 0.5 cm2 for all leaf area and leaf area removed. However, he used a value of 0.1 to include all herbivory smaller than 0.5 cm2. Galls were only counted on oaks and were counted in all years. Leaf miners were only counted on oaks and paper birch and were only surveyed in 2015 and 2016. "Spots" (anthracnose per B. Blanchette) were assessed on only red maple and only in 2015 and 2016. PLot Ordination Details FAB 1 Plot Rows: 1 is the northern row, 8 is the southern row FAB 1 Plot Columns: A is the western column, H is the eastern column Results from this dataset published in: Grossman, J. J., Cavender - Bares, J., Reich, P. B., Montgomery, R. A., & Hobbie, S. E. (2018). Neighborhood diversity simultaneously increased and decreased susceptibility to contrasting herbivores in an early stage forest diversity experiment. Journal of Ecology, 107(3), 1492-1505. doi:10.1111/1365-2745.13097

ahe271 - Soil lipid (P/NLFA) and AMF (spore and sequence) data from selected plots

Arbuscular Mycorrhizal Fungi (AMF) spore extraction methods

AMF spores were extracted from an additional subset of the June soil samples for each plot using the International Collection of Vesicular and Arbuscular Mycorrhizal Fungi (INVAM) method (; Daniels and Skipper 1982). Briefly, soil samples were homogenized by blending and AMF spores were collected on a 38 micrometer sieve and separated from other soil matter using sucrose gradient centrifugation. Spore counts per gram of soil as well as spore morphotype richness, a proxy of AMF diversity, per plot were assessed using light microscopy.

Lipid analysis methods

Lipid analysis was carried out in the Gutknecht Lab at the University of Minnesota. Samples designated for lipid analysis were originally stored at minus 20 degrees celsius following collection, then thawed and freeze dried. Though the original freeze and thaw step may have altered samples lipid content, samples from all plots experienced the same treatment; we expect changes to be, therefore, independent with respect to the diversity of samples plots of origin. Following freeze drying, we extracted total soil lipids from a 10 g subsample from each plot and quantified both neutral lipid (NLFA) and phospholipid fatty acids (PLFA; Schmidt et al. 2017). Dissolved fatty acids were extracted from 2g of freeze dried soil through three extractions with a 1:1:0.9 chloroform to methanol to citrate buffer. Fatty acids were then converted to methyl esters through acid methylation and analyzed on a GC - MS (Agilent, HP DB5 column) spectrometer. Using an internal standard (13:0 tridecanoic methyl ester) for quantification, we converted peak areas to nmol g soil-1. We quantified abundance of 24 microbial lipids 12:0, 13:0, 14:0, 15:0, i15:0, a15:0, 16:0, Me16:0, 16:1w5c, 16:1w7c, 16:1w9c, 17:0, a17:0, i17:0, cy17:0, 18:0, Me1018:0, 18:1w7c, 18:1w9c, 18:1w9t, 18:2w6,9c, 19:0, cy19:0, and 20:0) and for each of these, calculated its mol percent, or relative abundance, of a sample`s total lipid mass. Reference cited in this method: Schmidt, J., T. Fester, E. Schulz, B. Michalzik, F. Buscot, and J. Gutknecht. 2017. Effects of plant-symbiotic relationships on the living soil microbial community and microbial necromass in a long-term agro-ecosystem. Science of the Total Environment 581 - 582:756 - 765.

Sequencing methods

Sequencing was carried out in the Kennedy Lab at the University of Minnesota. The same soil samples collected in August 2016 for PFLA analysis were also used to assess AMF community richness and composition. We thawed subsamples from all plots and extracted DNA from 250 mg of each pooled soil sample using PowerPlant Pro DNA isolation kit (MoBio Laboratories, Inc. Solana Beach, CA). We then used a two - step PCR protocol to generate amplicon libraries (Lekberg Lab, MPG Ranch, Florence, MT; appendix S1 of Lekberg et al. 2018). The first PCR step entailed amplification using the universal eukaryotic primer WANDA (SI) and an AMF - specific primer, AML2. The second PCR step allowed us to add barcode adaptors and Illumina flowcell adaptors (P5 and P7, Illumina Inc., San Diego, CA, USA). PCR products were purified and normalized using a ``Just-a-Plate`` kit (Charm Biotech, San Diego, CA) and pooled to equimolar concentrations, and then sequenced at the University of Idaho`s Institute for Bioinformatics and Evolutionary Studies (iBEST) genomics resources core (; Moscow, ID) on an Illumina MiSeq sequencing platform (Illumina Inc., San Diego, CA) using v2 (2 x 250 bp) chemistry. Raw single-end Illumina sequence files were denoised and dereplicated using ``dada2`` and trimmed reads to 200 bp. Sequences were then clustered using the MaarjAM database (Opik et al. 2010), removing all sequences that did not match at least 90 percent identity and 90 percent coverage to sequences within MaarjAM. Taxonomy was assigned using BLAST and a 97 percent sequence similarity threshold, resulting in assignments that were at least 97 percent identical over 90 percent of their sequence to those in MaarjAM. Any sequence reads present in negative controls were subtracted from sample read counts. Refernces cited in this method: Lekberg, Y., M. Vasar, L. S. Bullington, S.-K. Sepp, P. M. Antunes, R. Bunn, B. G. Larkin, and M. Opik. 2018. More bang for the buck? Can arbuscular mycorrhizal fungal communities be characterized adequately alongside other fungi using general fungal primers? New Phytologist:971 - 976. Opik, M., A. Vanatoa, E. Vanatoa, M. Moora, J. Davison, J. M. Kalwij, U. Reier, and M. Zobel. 2010. The online database MaarjAM reveals global and ecosystemic distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota). New Phytologist 188:223 - 241. doi: 10.1111/j.1469-8137.2010.03334.x.

Soil moisture instrumentation

Time-Domain Reflectometer used, Fieldscout TDR 300, Spectrum Technologies, Inc.

Soil moisture methods

Soil moisture of all plots was measured in the field using a time-domain reflectometer probe using 7 inch rods. Samples collected on that date for environmental characterization and spore abundance and diversity were processed upon collection. One subset of fresh soil samples from all plots was oven-dried for 24 hours at 60 degrees celsius and ashed at 550 dedrees celsius. Its phosphorus content was measured using the sulfuric acid digestion with absorbic acid (APHA 1999). A second subset of fresh subsamples was diluted with DDI water (20 mL to 10 g soil) prior to pH measurements of resulting soil slurries.

Soil sampling methods

We collected soil samples twice, on 23-24 June and 24 August 2016. Recent rainfall prior to both events was similar, so the soil was moist, but not waterlogged when sampled. June soil samples were used to assess environmental variables (soil moisture, pH, and phosphorus) and for microscopic counting and identification of spores; August samples were collected for sequencing and lipid analysis. In both cases, surface vegetation and mulch were pushed aside and three 2.5 x 10 cm deep cores were taken at points in the interior of each plot (> 1.0 m from plot edges). Cores were pooled and stored in plastic bags, and sampling equipment was sterilized with ethanol between plots to prevent cross-contamination. In August, we also collected several samples from outside of the FAB experiment to serve as controls; these included samples from adjacent disturbed grasslands and unvegetated roadsides adjacent to FAB. Samples were kept in a dark cooler and pooled at the plot level following collection.

Tree biomass methods

Tree biomass was calculated allometrically based on basal diameter (calipers) and height (yard sticks). Species-specific allometric equations are given in Grossman et al. (2017). Grossman, J. J., J. Cavender-Bares, S. E. Hobbie, P. B. Reich, and R. A. Montgomery. 2017. Species richness and traits predict overyielding in stem growth in an early-successional tree diversity experiment. Ecology 98:2601-2614. DOI: 10.1002/ecy.1958 ecy1958-sup-0001-AppendixS1

ahfe271 - Litterbag mass and chemistry

Lab analysis

Carbon fraction analysis was carried out to separate soluble cell contents, hemicellulose and bound proteins, cellulose, and acid unhydrolyzable residues (including lignin and hereafter referred to as AUR) of dried, ground leaves. All mass and carbon fraction data are reported here without having ash factored out and ash weight (or recalcitrant weight) is presented as a percent of total mass at collection. Thus, ash-free weight can be calculated, as in the accompanying manuscript. Three bags were not recovered, giving a final sample size of 597 bags across 150 strings. Carbon fraction data is also incomplete for some bags due to loss of ground litter during analysis.

Lab analysis instrumentation

We used an ANKOM 200 fiber analyzer (Macedon, NY, USA) to measure the concentration by mass of four operational `carbon fractions` in ground litter.

Litterbag methods

Litterbags were constructed with either 1 species of litter or mixtures of 2, 5, or 12 species. In October 2014, we collected freshly senesced litter from 12 species of adult trees of native provenance on private property in Hudson, WI, USA (Juniperus virginiana; eastern red cedar) and at Cedar Creek Ecosystem Science Reserve (CCESR; all other species). Litter was air dried and stored at room temperature in darkness. In spring 2015, litter was used to fill 20 cm by 20 cm square bags constructed of 1 mm fiberglass mesh. Bags were filled with 2.5 g of air-dried litter and heat-sealed. All weights were adjusted to reflect oven-dried (> 24 hours at 60 degrees Celsius) weight and loss-on-handling as estimated from one-species litterbags that had been assembled, deployed in the field, and immediately returned and weighed. All litterbags were deployed in a common ``garden`` at CCESR on 12 June, 2015. The common garden was located in a secondary, unmanaged stand of trees, consisting primarily of Populus grandidentata (bigtooth aspen) and Pinus strobus (white pine) interspersed with Acer spp. (maples). Understory growth was minimal and largely consisted of the seasonally abundant legume Amphicarpaea bracteata (hog peanut). A duff layer of roughly 0.25 cm in depth covered the mineral soil horizon in the common garden and was left intact. Four replicate litterbags with the same composition were tied together to form 150 strings. Each string of four litterbags was stretched to its full length so that bags were not touching and staked in place so that the entire bottom surface of each bag was in contact with the existing litter layer. Bags were not covered when deployed but became covered with a layer of freshly fallen litter from four months post-deployment onward. Because bags were deployed over an area large enough to vary in microtopography, overstory vegetation, exposure to deer trampling, etc., we divided strings into three blocks, with 50 strings arranged randomly within each garden block. Strings were assigned to blocks so that each bag composition was represented across all three blocks. One litterbag from each of the 150 strings was collected at 62 days (two months), 124 days (four months), 363 days (one year), and 731 days (two years) following deployment. On collection, each bag was cleaned manually of mineral soil, allochthonous litter, ingrown plant material, and soil animals (including small earthworms). Litter was removed from each bag, cleaned further, oven dried at 60 degrees for > 24 hours, and weighed. Dried litter was then ground in a Wiley mill and carbon fractions were quantified as described in lab analysis. Post-decomposition litter was ashed at 550 degrees Celsius for four hours.

ahoe271 - Chlorophyll fluorescence


Chlorophyll fluorescence parameters were measured with a FMS2 Pulse-Modulated Fluorometer with dark-acclimation leaf clips from Hansatech Instruments.

Measurement protocol

For each of the eight broadleaf species in FAB 1, we selected three plots from each of three treatments (monoculture, twelve-species, and shaded biculture i.e. with conifers). From each plot, we measured chlorophyll fluorescence parameters from top fully expanded leaves of two trees. We measured dark Fv/Fm in the early morning after dark-acclimating leaves with specialized shading clips. On the same leaves, we measured light acclimated parameters at midday following 15 second exposure to 1000 umol/m2/s actinic light.

ahpe271 - Tree light availability


We used an Accupar LP80 light meter.

Measurement protocol

We measured relative light availability, the ratio of ambient light reaching the top leaves of a given tree, above 138 trees in the FAB experiment. For each tree, we took four measurements in the open and 2-4 above the given tree. The trees were chosen to correspond with those selected for gas exchange, chlorophyll fluorescence, and spectral measurements.

ahqe271 - Tilia americana leaf senescence phenology

Survey protocol

We labeled leaves from 120 T. americana trees, half in monoculture, half in shaded biculture. We did an initial survey of half of those trees in early August where we counted how many of the top five leaves still remained. We tracked the senescence of the remaining marked leaves on a presence/absence basis until all were senesced.

Survey protocol

The initial survey was only done on half of the trees included. We assumed that all trees in a plot would have, on average, the same proportion of leaves in the initial survey as those actually surveyed.

ahre271 - Photosynthetic light-response curves


Photosynthetic light-response curve measurements were taken using a LICOR 6400 and 6400 XT with fluorescence chambers.

Measurement protocol

For four focal species (T. americana, B. papyrifera, Q. ellipsoidalis, and A. rubrum), we selected three plots from each of three treatments (monoculture, twelve-species, and shaded biculture i.e. with conifers). From each plot, we measured photosynthetic light-response curves from top fully expanded leaves of two trees under favorable chamber conditions. We used a fluorescence head to take simultaneous chlorophyll fluorescence measurements. Only two shaded bicultures were available for B. papyrifera, so we picked three individuals from each one. For 12_ACRU_2D, there was condensation inside the chamber, throwing off Ci and conductance, which we removed, but photosynthesis looks fine. A similar problem occurred for 96_ACRU_7E.

ahse271 - SSU amplicons of arbuscular mycorrhizal fungal communities in soils

SSU amplicons of arbuscular mycorrhizal fungal communities in soils

Arbuscular mycorrhizal fungal communities in soils of FAB experiment at Cedar Creek Ecosystem Science Reserve, Minnesota, USA While the relationship between plant and microbial diversity has been well studied in grasslands, less is known about similar relationships in forests, especially for obligately symbiotic arbuscular mycorrhizal (AM) fungi. To assess the effect of varying tree diversity on microbial alpha- and beta-diversity, we sampled soil from plots in a high-density tree diversity experiment in Minnesota, USA three years after establishment. Three of 12 tree species are AM hosts; the other 9 primarily associate with ectomycorrhizal fungi. We used phospho- and neutral lipid fatty acid analysis to characterize the biomass and functional identity of the whole soil bacterial and fungal community and high throughput sequencing to identify the species-level richness and composition of the AM fungal community. Results are published in: Grossman, J. J., Butterfield, A. J., Cavender-Bares, J., Hobbie, S. E., Reich, P. B., Gutknecht, J., & Kennedy, P. G. (2019). Non-symbiotic soil microbes are more strongly influenced by altered tree biodiversity than arbuscular mycorrhizal fungi during initial forest establishment. FEMS microbiology ecology, 95(10). doi:10.1093/femsec/fiz134