Carbon-isotopic analysis of individual pollen grains from C3 and C4 grasses using a spooling-wire microcombustion interface

Pollen grains from grasses using the C₃ and C₄ photosynthetic pathways have distinct ranges of δ¹³C values that may be used to estimate their relative abundance in paleorecords. We evaluated a spooling-wire microcombustion device interfaced with an isotope-ratio mass spectrometer (SWiM-IRMS) for δ¹³C analysis of individual grass-pollen grains. Pollen from four C₃ and four C₄ grass species was isolated through micromanipulation and analyzed as single grains suspended in water. A carbon yield greater than the 2σ range of the carbon content of blanks containing only water was used to distinguish samples containing pollen (“pollen present”) from those not containing pollen. This criterion resulted in the exclusion of ∼45% of the 946 samples applied to the wire. The average δ¹³C values (±1σ) of the remaining samples were −26.9‰ (±6.3‰) and −11.5‰ (±9.6‰) for C₃ grasses and C₄ grasses, respectively, after blank-correcting the δ¹³C data. These results suggest that the SWiM-IRMS system can be used to distinguish C₃ from C₄ grass pollen. The high variability in measured δ¹³C values is likely caused by a combination of factors. These include natural isotopic variability among individual pollen grains; the relatively poor precision that can be obtained when determining δ¹³C values of such small samples; and the uncertainty in the magnitude, isotopic composition, and stability of the analytical blank. Nonetheless, high percentages of individual pollen grains were correctly classified as being of either C₃ or C₄ origin. On average, 90% (range = 78-100%) of pollen grains from C₃ grasses had δ¹³C values more negative than the cutoff threshold of −19.2‰; while 84% (range = 77-90%) of pollen grains from C₄ grasses had δ¹³C values more positive than −19.2‰. Compared with analysis using an elemental analyzer interfaced with an IRMS (EA-IRMS), the number of pollen grains required for δ¹³C-based evaluation of C₃/C₄ grass composition is many times lower with the SWiM-IRMS. Additionally, δ¹³C data from the SWiM-IRMS does not need to be incorporated into a mixing model to derive estimates of the abundance of C₃ and C₄ grass pollen. Carbon-isotopic analysis of individual grass-pollen grains using the SWiM-IRMS system may help improve our understanding of the evolutionary and ecological significance of grass taxa in the paleorecord.