Using SPIRAL (Single Pollen Isotope Ratio AnaLysis) to estimate C3- and C4-grass abundance in the paleorecord

C₃ and C₄ grasses differ greatly in their responses to environmental controls and influences on biogeochemical processes (e.g. water, carbon, and nutrient cycling). Difficulties in distinguishing between these two functional groups of grasses have hindered paleoecological studies of grass-dominated ecosystems. Stable carbon isotopic analysis of individual grains of grass pollen using a spooling-wire microcombustion device interfaced with an isotope-ratio mass spectrometer holds promise for improving C₃ and C₄ grass reconstructions. This technique, SPIRAL (Single Pollen Isotope Ratio AnaLysis), has only been evaluated using pollen of known C₃ and C₄ grasses. To test the ability of SPIRAL to reproduce the abundance of C₃ and C₄ grasses on the landscape, we measured δ¹³C values of > 1500 individual grains of grass pollen isolated from the surface sediments of ten lakes in areas that span a large gradient of C₃- and C₄-grass abundance, as determined from vegetation surveys. Results indicate a strong positive correlation between the δ¹³C-based estimates of % C₄-grass pollen and the abundance of C₄ grasses on the landscape. The % C₄-grass pollen slightly underestimates the actual abundance of C₄ grasses at sites with high proportions of C₄ grasses, which can be corrected using regression analysis. Comparison of the % C₄-grass pollen with C/N and δ¹³C measurements of bulk organic matter illustrates the distinct advantages of grass-pollen δ¹³C as a proxy for distinguishing C₃ and C₄ shifts within the grass family. Thus SPIRAL promises to advance our understanding of grassland ecology and evolution.