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Methods for Experiment 263 -

Experiment design

Experimental gardens: We propose to plant twenty common garden pairs containing fifteen willow and poplar species (Table 1) in paired wetland and upland habitats at Cedar Creek (40 gardens total; 5m x 17m). In each of these gardens, we will plant two individuals per species, one of which will be contained in an herbivore exclosure. The other will have an open-sided dummy exclosure that will have the same effect on light attenuation but not exclude insects. Cuttings, cultivated in a greenhouse, will be planted 1.5 m apart. Plants and exclosure treatments within each garden will be randomized spatially. At most sites, the plots will be grouped in sets of four (2 wetland (W) plots and 2 upland plots (D)). These groupings (86.7m2 total) will be surrounded by deer fencing (2.5m tall). In sites where it is not possible to group four sites together, two pairs of plots will be fenced separately.

Experiment species

Table 1 Species ordered based on their distributions across a water availability gradient from wet to dry habitats

afae263 - Effects of Salicaceae species litter type and moisture at 10 paired wetland by upland plots

Instrumentation litter bag carbon and nitrogen analysis

COSTECH ESC 4010 Element Analyzer, University of Nebraska, Lincoln

Methods for litter decomposition bags

To make leaf litter decomposition bags, freshly fallen leaf litter was collected during September and October 2010 from natural populations of 12 closely related species in the willow family. We collected from populations at Cedar Creek, as well as nearby Carlos Avery State Wildlife Management Area (Forest Lake, MN, 45 degree 19 degree N, 93 degree 05 degree W), that were located in habitats representative of the species water regime affinities. After air-drying the leaf litter, a subsample was ground in a Wiley Mill (.425 mm). Initial leaf litter chemistry of the ground subsample was analyzed for: concentrations of carbon (C) and nitrogen (N). Leaf litter decomposition bags (15 cm x 15 cm size) were constructed out of 1-mm fiberglass mesh. Each bag was filled with 3 g air-dried leaf litter. Additional leaf litter was also dried for >48 hours at 65 degrees Celsius to calculate an air-dried:oven-dried weight conversion factor for each species. Replicate bags were constructed to allow for six harvests of each species in each plot (wetland or upland). Litter bags were deployed at the start of winter in 2010 (27-Nov-2010). Bags were deployed at a minimum of six (of ten) randomly selected sites. Where there was sufficient leaf litter material, bags were deployed at all ten sites. Leaf litter decomposition bags were harvested from each species-plot-site combination six times over the course of three years. There were four harvests in 2011 (29-Apr, 26-May, 1-Aug, 17-Sep), one in 2012 (30-Sep), and one in 2013 (20-Sep). At each harvest, litter was removed from the bags and cleaned of debris, including in-grown roots. Clean leaf litter was dried at 65 degrees Celsius for >48 hours in a forced-air drying oven and weighed. We calculated mass loss (% initial mass) and analyzed % C and % N of the remaining litter sample. Results from these data are published in: Riggs, C. E., S. E. Hobbie, J. Cavender-Bares, J. A. Savage, and X. Wei. 2015. Contrasting effects of plant species traits and moisture on the decomposition of multiple litter fractions. Oecologia DOI 10.1007/s00442-015-3352-0 2015

afbe263 - Initial Salicaceae species litter chemistry

Instrumentation Salicaceae species litter chemistry

COSTECH ESC 4010 Element Analyzer, University of Nebraska, Lincoln) (Ankom Fiber Analyzer, Ankom Technology, Macedon, New York, USA)

Lab methods Initial Salicaceae species litter chemistry

Initial leaf litter chemistry of the ground subsample was analyzed for: concentrations of carbon (C) and nitrogen (N), phosphorous (P) by digestion with 10 N sulfuric acid after ashing samples at 300 degrees C for 30 min. followed by 500 degrees C for 2 hours (DeMott et al. 1998), C fractions (soluble cell contents (SCC), cellulose (CELL), hemicellulose plus bound proteins (HBP), and lignin plus other recalcitrant compounds (LR); and condensed tannins (CT) by acid butanol assay with quebrancho extract standard (Porter et al. 1986). References cited: DeMott WR, Gulati RD, Siewertsen K (1998) Effects of phosphorusdeficient diets on the carbon and phosphorus balance of Daphnia magna. Limnol Oceanogr 43:1147 to 1161 Porter LJ, Hrstich LN, Chan BG (1986) The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25:223 to 230

ahge263 - 2012 growing season water table depth in common gardens

Water table depth in common gardens

During 2012 growing season, we measured water table depth from the water wells installed in each common garden biweekly using a metal tape measure. A positive number represent a standing water table, a negative number represents a water table that was below ground level. Results from this data are published in: Wei, X., Savage, J. A., Riggs, C. E., & Cavender-Bares, J. (2017). An experimental test of fitness variation across a hydrologic gradient predicts willow and poplar species distributions. Ecology, 98(5), 1311-1323. doi:10.1002/ecy.1784

ahhe263 - Percent carbon and nitrogen in leaf tissue

Percent nitrogen and carbon in leaf tissue

In August,2012, one uppermost, fully-expanded leave per plant was collected, oven-dried at 60 Celsius degree, and analyzed using dry combustion gas chromatography for percent of carbon and nitrogen. The analysis was performed by the Ecosystem Analysis Laboratory, University of Nebraska, Lincoln.

Percent nitrogen and carbon in leaf tissue

Leaf tissue was analyzed for tissue nitrogen concentration with a C-N Analyzer (ECS 4010, COSTECH Analytical Technologies Inc.).