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.