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Methods for Experiment 290 -

Summary of Cedar Creek experiments used for e290 - Dimensions of Biodiversity

e120 - Biodiversity II: Effects of Plant Biodiversity on Population and Ecosystem Processes This experiment (often called the "Big" Biodiversity Experiment or Big Bio) determines effects of plant species numbers and functional traits on community and ecosystem dynamics and functioning. Planted in 1994, it manipulates the number of plant species in 168 plots, each 9 m x 9 m, by imposing plant species numbers of 1, 2, 4, 8, or 16 perennial grassland species. The species planted in a plot were randomly chosen from a pool of 18 species (4 species, each, of C4 grasses, C3 grasses, legumes, non-legume forbs; 2 species of woody plants). Its high replication (about 35 plots at each level of diversity) and large plots allow observation of responses of herbivorous, parasitoid and predator insects and allow additional treatments to be nested within plots. e271 - FAB 1 : Forests and Biodiversity Experiment - High density diversity experiment A forest biodiversity experiment (FAB) focused on trees of our region investigates the consequences of multiple dimensions of tree diversity for soil, food webs, plant communities and ecosystems. FAB is designed to unravel effects of three forms of biological diversity: species richness (SR), functional diversity (FD), and phylogenetic diversity (PD). The 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 FABs 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. e277 Consequences of intraspecific and interspecific biodiversity in willows and poplars for community processes and ecosystem function (BiWaP) (est. 2014): trees of three species are planted at 0.5m in 27-tree plots that vary in species richness and genetic diversity . Plants were produced clonally, so each species is represented by three genotypes in the experiment and the exact genetic identity of each tree is known. This will allow for exploration of the joint effects of diversity within and among species. The specific aims of this project are to i) create an ecologically realistic, artificial plant community in which to investigate the relationship between biodiversity and ecological function over the next 5-15 years, ii) assess the unique and interacting effects of intraspecific tree diversity and interspecific tree diversity as drivers of ecological function in the community, and iii) provide a platform for collaboration with colleagues at the University of Minnesota and elsewhere who could take advantage of the study system to ask related questions. We hypothesize that: i) focal plant and whole ecosystem metrics of ecological function will be higher in the presence of higher intraspecific and interspecific tree diversity, ii) we will observe an additional synergistic increase in function as both types of diversity are experimentally increased, and iii) ecological functions will respond to increasing diversity in unique ways. The primary motivation for this project is to orthogonally assess the roles of intraspecific and interspecific diversity in driving ecological function.

afse290 - Soil microbial biomass carbon

Instrumentation soil microbial biomass carbon

Analyzed with Shimadzu TOC-V, Shimadzu Corporation, Kyoto, Japan

Protocol soil microbial biomass carbon

Soil from e120 plots was sampled to a depth of 10 cm (2 cm diameter). Five soil cores were sampled from each plot and composited into a single sample. Fresh, 2 mm sieved soil was extracted with 0.05 M K2SO4 and filtered (Whatman No. 42; 2.5 mm pore size). A replicated soil sample was fumigated with chloroform in a vacuum for 72 h, extracted with 0.05 M K2SO4 and filtered. Filtered extracts were analyzed for total dissolved organic C and total dissolved N. Results from this data are published in: Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2017). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology, DOI: 10.1002/ecy.2075

afte290 - Root carbon fraction chemistry

Instrumentation - root carbon fraction chemistry

Root carbon fractions were analyzed using an ANKOM forage analyzer (Macedon, NY).

Root carbon fraction chemistry sampling and processing

Soil sampling: Soil was sampled to a depth of 10 cm (2 cm diameter). Five soil cores were sampled from each e120 plot and composited into a single sample. Processing: Roots were removed from soil samples using a 2 x 2 mm sieve and dried at 60 degrees C for 48 hours then analyzed for root carbon fractions (cell solubles, cellulose, hemicellulose+bound proteins, nonhydrolyzable). Results from this data are published in: Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2017). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology, DOI: 10.1002/ecy.2075

afue290 - Enzyme activity of soil communities

Sampling and lab analysis

Soil sampling: Soil was sampled to a depth of 10 cm (2 cm diameter) from 35 e120 plots in July 2014. Five soil cores were sampled from each plot and composited into a single sample. Lab: We estimated the hydrolytic, chitinolytic, and lignolytic enzyme activity of soil communities using extracellular enzyme assays. Specifically, we measured the activity of alpha-glucosidase (AG; EC 3.2.1.20), beta-1,4-glucosidase (BG; EC 3.2.1.21), cellobiohydrolase (CBH; EC 3.2.1.91), beta-1,4-xylosidase (BX; EC 3.2.1.37), and N-acetyl-N-glucosaminidase (NAG; EC 3.1.6.1), as well as phenol oxidase (PO; EC 1.10.3.2) and peroxidase (PX; EC 1.11.1.7) activity. One gram of soil mixed and homogenized in 125 ml of 25 mM maleate buffer (adjusted to pH 6) for 1 minute. In 96-well plates, MUB-linked enzyme assays included 100 uL soil-buffer solution, in addition to 50 uL of substrate. Controls included soil plus buffer, buffer plus substrate, and, for methylumbellyferyl-linked substrates (MUB), soil and MUB. Cellobiohydrolase, beta-1,4-glucosidase , beta-1,4-xylosidase, and N-acetyl-glucosaminidase assays were incubated in the dark at 20 degrees C for 2 h. Afterwards, 10 uL of 1 M NaOH was added to each well to stop the reaction and increase fluorescence. Enzyme activity was measured at 365 nm excitation wavelength and 450 nm emission wavelength (8 replicates per sample). Soil samples were incubated with substrates in 2 ml micro-centrifuge tubes for 24 hours, followed by centrifugation at 3600 rpm for five minutes. Supernatant was pipetted into 96-well places and read spectrophotometrically at 460 nm absorbance (4 replicates per sample; Madritch et al. 2007). A 25-mM L-dihydroxy-phenylalanine substrate was used to assay phenol oxidase and peroxidase activity. Publications cited in these methods: Madritch, M. D., J. R. Donaldson, and R. L. Lindroth. 2007. Canopy herbivory can mediate the influence of plant genotype on soil processes through frass deposition. Soil Biology & Biochemistry 39:1192?1201. Results from this data are published in: Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2017). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology. DOI: 10.1002/ecy.2075

afve290 - Net N mineralization

Net nitrogen mineralization

In situ net N mineralization rates were estimated using 1.9 cm diameter plastic tubes (PVC) sunk to a depth of 10 cm at two different locations within each plot and covered with caps to prevent leaching losses. NH₄+ and NO₃- was measured initially in soil subsamples, as well as following 30 days of soil incubation within each PVC. We calculated net N mineralization rates (ug N g-1 soil d-1) as the difference between initial and final extractable concentrations of NH₄+ and NO₃- (Doane and Horwath 2003) using a BioTek Synergy H1 microplate reader (Winooski, VT). Soil samples were collected to 10 cm soil depth for laboratory incubation, sieved using a 2 x 2 mm sieve, extracted with 1 M KCl, shaken for 0.5 h, settled overnight at 4 degrees C and analyzed for NH₄+ and NO₃-. Adjusted to 70 percent field capacity, incubations were stored at 22 degrees C in a dark room, and NH₄+ and NO₃- concentrations in soil were extracted and measured after 30 days. Publications cited in these methods: Doane, T. A, and W. R. Horwath. 2003. Spectrophotometric determination of nitrate with a single reagent. Analytical Letters 36:2713 - 2722. Results from this data are published in: Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2017). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology. DOI: 10.1002/ecy.2075

afwe290 - Root biomass

Root biomass sampling

Soil was sampled to a depth of 10 cm (2 cm diameter) from 35 plots in July 2014. Five soil cores were sampled from each plot and composited into a single sample. Roots were removed from soil samples using a 2 x 2 mm sieve and dried at 60 degrees Celsius for 48 hours. Any plant material above soil core was also clipped and collected

afxe290 - Soil percent carbon and nitrogen

Soil percent carbon analysis

Soil Carbon and Nitrogen were analyzed with a Costech ECS4010 elemental analyzer.

Soil percent carbon analysis

Soil was sampled to a depth of 10 cm (2 cm diameter). Five soil cores were sampled from each plot and composited into a single sample. Soil C and N content was measured by combustion using an Elemental Analyzer.

afye290 - Soil pH

Protocol soil pH

Soil was sampled to a depth of 10 cm (2 cm diameter). Five soil cores were sampled from each plot and composited into a single sample. Roots were removed from soil samples using a 2 x 2 mm sieve. We measured soil pH using a 1:1 (v/v) soil: water mixture.

afze290 - Soil microbial respiration rate

Methods - soil microbial respiration

Soil was sampled to a depth of 10 cm (2 cm diameter) from 35 plots in July 2014. Five soil cores were sampled from each plot and composited into a single sample. Microbial respiration rate (mg CO₂-C g-1 soil day-1) was quantified from a 50 g subsample of fresh, sieved soil by measuring the accumulation of CO₂ in airtight 1 L Mason jars during 24 - 48 h intervals on days 5, 7, 9, 12, 19, 26, 54, 84, 117, 178, 207, 237, 269, 301, 325, 355 of the incubation. We measured CO₂ concentration inside the jars at the start and end of each interval by analyzing air samples collected via syringe with an infrared gas analyzer (LICOR 7000). When not being measured, soil samples were covered with gas-permeable, low-density poly-ethylene film and kept at 20 degrees Celcius in the dark. Soil samples were maintained at 70 percent field capacity throughout the incubation. Results from this data are published in: Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2017). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology. DOI: 10.1002/ecy.2075

agve290 - Molecular characterization of fungi in soil

Abstract

LinkURL will direct to 124 PCR amplicon soil metagenome raw sequences archived at National Center for Biotechnology Information (NCBI) under Sequence Read Archive SRP108802. Samples were taken from the e120 - Biodiversity II experiment as part of e290.

Molecular characterization of fungi in soil protocols

In July 2015, soil from 125 plots in the e120 - Biodiversity II: Effects of Plant Biodiversity on Population and Ecosystem Processes experiment were sampled to a depth of 10 cm (2 cm diameter) for fungal molecular characterization. Five soil cores were sampled from each plot and composited into a single sample. Sieved 2015 soils were transported on ice and stored at minus 80 degrees C until fungal molecular characterization was initiated. DNA was extracted from 0.5 g soil using the FastDNA SPIN Kit (MP Biomedical, Solon, Ohio, USA). PCR amplification of the ITS1 gene region was conducted using primers ITS1F and ITS2 (Smith and Peay 2014). See Appendix S1 (Cline 2018) for details of PCR conditions, amplicon clean up, and normalization. Sequencing was performed on the MiSeq platform (Illumina, San Diego, California, USA) with 250 paired - end reads at West Virginia University's Genomic Core Facility. Low quality reads were removed bioinformatically using the FAST pipeline (https://github.com/ZeweiSong/FAST; see Cline et al 2018 Appendix S1). Taxonomy was assigned using the BLAST algorithm (Altschul et al. 1990) against the UNITE database (v.7.0.; Koljalg et al. 2013), and functional assignments (i.e., trophic mode and guild) were made in FunGuild using taxonomic assignments (Nguyen et al. 2016). Results from theis data are published in: Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2018). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology 99(1): 204-216. https://doi.org/10.1002/ecy.2075 Methods cited: Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J.. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403 - 410. Cline, L. C., S. E. Hobbie, M. Madritch, C. R. Buyarski, D. Tilman and J. M. Cavender-Bares (2018). "Resource availability underlies the plant-fungal diversity relationship in a grassland ecosystem." Ecology 99(1): 204-216. https://doi.org/10.1002/ecy.2075 Koljalg, U., et al. 2013. Towards a unified paradigm for sequence‐based identification of fungi. Molecular Ecology 22: 5271? 5277. Nguyen, N. H., Song, Z., Bates, S. T., Branco, S., Tedersoo, L., Menke, J., Schilling, J. S., and Kennedy, P. G.. 2016. FUNGuild: an open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecology 20: 241? 248. Smith, D. P., and Peay, K. G.. 2014. Sequence depth, Not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS ONE 9: e90234.