These methods, instrumentation and/or protocols apply to all data in this dataset:| Methods and protocols used in the collection of this data package |
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| Description: |
Abiotic Measurements:
Hourly abiotic conditions were recorded at the plot-level at each site using HOBO products (Onset Computer Corporation, Bourne, MA). Three ambient and three warmed plots were equipped with four-channel external U12-008 data loggers that recorded air temperature at 10cm and soil temperature at 5cm belowground. These plots also contained microstation H21-002 data loggers that recorded air temperature at 1m and soil moisture at 5cm belowground. Plastic dish solar shields were installed above each air temperature sensor to mitigate the impact of solar radiation on air temperature readings. The timezone is EST.
| | Instrument(s): | HOBO products (Onset Computer Corporation, Bourne, MA), four-channel external U12-008 data loggers and microstation H21-002 data loggers. |
| | Description: |
Abiotic Measurements (Temperature pendants): Onset HOBO temperature pendants were installed on wooden stakes at the perimeter of each 1m2 plot (excluding the plots with HOBO microstations). Each pendant was covered by a dish solar shield to mitigate solar radiation impacts on accurate temperature measurements. These pendants measured temperature (C) and light levels (Lux). Occasionally, HOBO pendants malfunctioned and required replacement. Pendants logged data once every hour. The timezone is EST.
| | Instrument(s): | Onset HOBO temperature pendants. Model #: early model = UA-002-64; later blue-tooth model= MX2202. Later model was phased in starting in 2020. |
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Leaf Herbivory:
Leaf herbivory was measured once per season at peak biomass prior to senescence, typically July-August. We haphazardly selected four leaves vertically distributed across the stem of three individuals of each species in each plot. We then visually estimated percent of the leaf eaten and percent of the leaf that was damaged (due to senescence, fungus, sucking insects, disease, or other). The analyses for this paper focus on percent of leaf eaten for leaf herbivory, and not leaf damage, as we wanted to focus on the amount eaten by herbivores rather than other causes of leaf damage.
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Plant Phenology:
Phenology of all plant species within all plots was monitored every 3-4 days. The beginning of data collection at each site was determined by the last snow melt of the year in the spring. Phenology consisted of green-up, flowering, and seed set. Green-up was recorded as the percent cover of each species in each plot from the time of the first occurrence of each species until all species had emerged. We used percent cover measurements to record green-up in order to record the date at which a plot reached half of its maximum percent cover. A species was recorded as flowering during the period between flower bud break (anthers exposed) and flower senescence. Seed set was determined when an individual exhibited a mature seed that was ready to be dispersed (pappus/achene, florets dehiscent, etc.). For both flowering and seed set, we recorded the date when at least one individual for each species in each plot exhibited the phenological stage.
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Plant Composition:
Percent cover was visually estimated within all 1m2 plots as the percentage of the total plot occupied by each species (0-100%). This measurement was taken every 3-4 days through green-up and once a month post green-up.
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Leaf Traits (C, N, SLA):
Prior to senescence, green leaves were harvested for measurements of foliar C and N content and specific leaf area (SLA). The species selected for these measurements were commonly found across all plots at each respective site (Appendix S1: Table S2). We chose 3-5 plants of the same species in each plot and harvested 4-5 green, mature leaves with little to no obvious insect damage or disease. The selected leaves were haphazardly selected from the top to the bottom of the plant to be representative of the whole plant. The youngest fully expanded leaf from each individual plant sample was chosen for SLA, while the remaining leaves were stored separately for C and N analysis. SLA leaves were scanned fresh with a LI-COR LI-3000A Portable Leaf Area Meter with conveyor belt LI-3050A at KBS and a LI-COR LI-3100c at UMBS. After SLA leaves were scanned, all leaf samples, including those harvested for C and N analysis, were placed in a drying oven at 60°C for 36-48 hours and subsequently weighed.
For C and N combustion analysis, dried leaves were ground to a powder using two different instruments: for forb species, we used a Thermo Savant FastPrep 120 with 2.38mm metal beads (Qiagen), and for graminoid species, we used a Shatterbox 8530. Tissue was typically ground for 1-3 minutes per sample in order to achieve a finely ground homogenous sample. For species with dominant midribs (e.g., Solidago canadensis and Centaurea stoebe), the midribs were removed prior to grinding. Once ground, plant material was weighed and packed into 8 x 5mm tin capsules (EA consumables, Elemental Microanalysis). We then performed combustion analysis (Costech Elemental Combustion System 4010) to yield % C and % N by mass.
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Plant Community Biomass:
In 2021, plant biomass was harvested in a 0.20m x 1m area within all 1m2 plots at both sites. Plant material was sorted to species, placed in individual paper bags, dried at 60°C for 3-4 days, and weighed for a final dry biomass weight per species and plot. Biomass was harvest from 09-14-2021 to 09-19-2021 at KBS, and 08-06-2021 to 08-07-2021 at UMBS.
2020 biomass was harvested in ancillary plots, outside of the main warming experiment, from 08-14-2020 to 08-27-2020.
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Insecticide Application: Beginning in 2015, insecticide was applied to reduce insect herbivores in half of the plots as follows. Insect herbivory was manipulated through insecticide applications throughout the growing season. Merit (imidacloprid 75% active ingredient by weight; EPA Reg. No. 432-1318), a systemic broad spectrum insecticide, was sprayed every 2-3 weeks from 2015-2018 at a rate of 0.031 grams per liter of water. In an attempt to be bee friendly and avoid insect resistance, we introduced a mixture of Endeavour (pymetrozine 50% active ingredient by weight; EPA Reg. No. 100-913) and Talstar (bifenthrin 7.9% by weight; EPA Reg. No. 279-3206) in the summer of 2018. This mixture has a longer residual time of 4-6 weeks (compared to 2-3 weeks for Merit), and was rotated with the Merit application subsequently for 2019-2021. The Talstar + Endeavour combination was applied at a rate of 0.78 mL per liter of water and 0.37 grams per liter of water, respectively. For non-insecticide plots, an equal amount of water was applied immediately after insecticide treatment application. Insecticide plots are termed “reduced herbivory”, and non-insecticide plots are termed “herbivory” or “background herbivory” plots.
| | Instrument(s): | Merit (imidacloprid 75% active ingredient by weight; EPA Reg. No. 432-1318). Endeavour (pymetrozine 50% active ingredient by weight; EPA Reg. No. 100-913). Talstar (bifenthrin 7.9% by weight; EPA Reg. No. 279-3206). The above pesticides were mixed and applied by USDA Pesticide certified applicator personnel, using standard hand pump sprayers. |
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Open Top Chambers (OTCs): Warming was achieved through the use of hexagonal OTCs designed for taller stature plant communities (Welshofer et al. 2018 Methods in Ecology & Evolution doi: https://doi.org/10.1111/2041-210X.12863). OTCs simulate climate warming by passively increasing air temperatures in situ while also allowing for natural levels of precipitation, gas exchange, and solar radiation. These chambers remained on the plots year-round and were constructed with clear, UV-transmitting 1/8′′ Lexan Polycarbonate (ePlastics, San Diego, CA). OTCs remained in place year-round, beginning in May/June 2015.
| | Instrument(s): | Open Top Chambers (OTCs) from Welshofer et al. 2018 Methods in Ecology & Evolution doi: https://doi.org/10.1111/2041-210X.12863. Material: pressure-treated wood, zipties, fasteners, UV-transmitting 1/8′′ Lexan Polycarbonate (ePlastics, San Diego, CA) |
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2015 insect collection: The “Blow and Vac” method of sampling was used to collect invertebrates present within the plots (Stewart and Wright 2008). Insect sampling occurred every 13-15 days after insecticide treatment prior to the next treatment. We sampled mid-day from 12-5pm for consistency as the literature suggests the presence of different invertebrates vary throughout the day. This process is only possible with at least two people as you will need someone to hold the vacuum when emptying the paint strainer and to hand the vacuum over once inside the chambered plots.
Stewart, A.J.A., and A.F. Wright. “A New Inexpensive Suction Apparatus for Sampling Arthropods in Grassland.” Ecological Entomology 20, no. 1 (February 1, 1995): 98–102. doi:10.1111/j.1365-2311.1995.tb00434.x.
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