Background/Question/Methods: We measured carbon isotope discrimination (D¹³C) and tissue N content in above ground biomass over four years at the Prairie Heating and CO₂ Enrichment (PHACE) experiment in southeastern Wyoming, USA. This factorial experiment combined FACE (ambient and elevated [600 ppm] CO₂ concentration), experimental warming (1.5˚C daytime, 3˚C nighttime) and irrigation to evaluate direct, indirect and interactive effects of global change factors on semi-arid grassland dynamics and production. We sampled species representing three functional types: a C4 grass Bouteloua gracilis; a C3 taprooted forb Sphaeralcea coccinea; and three C3 graminoids Pascopyrum smithii, Carex eleocharis, and Hesperostipa comata.

Results/Conclusions: We found D¹³C was reduced similarly by about 2‰ for all species under elevated compared to ambient [CO₂], suggesting that at least for C3 species stomatal closure under elevated [CO₂] reduced CO₂ supply relative to carboxylation capacity. The ratio of leaf internal to ambient CO₂ concentration (Ci/Ca) calculated from D¹³C values in P. smithii, the dominant C3 graminoid, was negatively correlated with tissue N content in elevated [CO2], but not in ambient [CO₂].  Tissue N content on a per unit mass basis was lower in the elevated (1.4±0.07%) compared to the ambient (1.8±0.07%) [CO₂] treatment. Experimental warming by itself did not alter D¹³C for any species, but warming lessened effects of elevated [CO₂] on this integrated gas exchange trait. Irrigation had little effect on D¹³C or tissue N content. The apparent interaction between elevated [CO₂] and warming on D¹³C and tissue N content was unexpected and is potentially attributed to 1) accelerated phenological development with warming; 2) warming-induced changes in leaf structure and anatomy; or 3) indirect and interactive effects of warming on soil nitrogen supply.