Li 820

Positive pressure flow-through respirometry was used to measure whole animal rates of oxygen consumption ( ${\dot{V}}_{O_2}$ , ml O₂ h⁻¹) and carbon dioxide production ( ${\dot{V}}_{C{O}_2}$ , ml CO₂ h⁻¹), as a proxy for whole animal metabolism (the sum of both respiratory and cutaneous respiration). Dry, CO₂–free air passed through the metabolic chamber (50 ml, 300 ml or 500 ml glass air-tight container), at a controlled flow rate of either 30 ml min⁻¹ (frogs <10 g), 50 ml min⁻¹ (10–25 g) or 80 ml min⁻¹ (>25 g) and re-scrubbed of water vapour before passing through an infrared CO₂ gas analyser (LI-820, LI-COR^® Biosciences Inc., Lincoln, NE, USA) and an O₂ analyser (Oxzilla II; Sable Systems, Las Vegas, NE, USA). The fractional concentrations of the CO₂ and O₂ in the excurrent air (F_eco₂ and F_eo₂) were recorded in a PowerLab 4/35 interface and imported into Labchart software (ADinstruments).

We exposed grasses to five CO₂ treatments: ambient concentration (400 ± 10 ppm) or elevated concentrations (600, 800, 1000, and 1200 ± 10 ppm). In order to minimize confounding effects of environmental variation between different chambers, we randomly changed the CO₂ concentration of each growth chamber every three days, and then relocated the CO₂ treated grasses to the growth chambers with corresponding CO₂ concentrations. The experiment was arranged in a randomized complete block design with four replicates (pots) per treatment. The ambient and elevated CO₂ concentrations within the chambers were maintained through an automatic CO₂ control system connected to a CO₂ source-tank containing 100% research-grade CO₂ (Airgas, Inc.). The CO₂ concentrations inside the chambers were continuously monitored through an infrared gas analyzer (LI-820; LICOR, Inc., Lincoln, NB, USA) connected to a computer logger maintaining the CO₂ concentration within 10 ppm of the ambient and elevated target levels.

Changes in soil pH, macro and micronutrients, percent of soil moisture content, and bulk density, and soil respiration were measured before planting and at harvest in 2012 and 2013 growing seasons. Soil moisture content level in each sample was determined by weight loss after oven dry at 104°C for 24 hr. Bulk density measurements were done drying cores of soil at 104°C for 24 h (Blake and Hartge, 1986 ). Soil pH, phosphorus, potassium, calcium, magnesium, soil organic matter, nitrate-nitrogen, ammonium-nitrogen, and cation exchange capacity were determined by the MSU Soil and Plant Nutrient Laboratory using standard procedures (Huffman and Barbarick, 1981 ; Nelson, 1983 ). The rate of CO₂ emission from the soil samples was used as an indicator of relative soil respiration and of level of biological activity (Ettema et al., 1998 ; Ferris and Matute, 2003 ; Treonis et al., 2010 ). In total, 15 g of fresh soil sample was incubated in 237 ml glass jars at 22°C for 7 days at field soil moisture content during sampling (Treonis et al., 2010 ). The CO₂ concentration of a 0.5 ml headspace gas sample was withdrawn from the jar through the rubber stopper using 1 ml syringe. The concentration was determined after 7 days of incubation using an infrared gas analyzer (LI-820, LI-COR, Inc., Lincoln, NE, USA; Zibilske, 1994 ) and expressed as µg CO₂-C per gram of soil per day.