Evolution and ecology of C4 grasses: insights from carbon isotope analysis of grass pollen grains

C4 grasses account for >25% of global primary productivity and dominate tropical, subtropical, and warm-temperate grassland ecosystems. Because of limitations of conventional morphological and geochemical proxies, identification of C4 grasses in the fossil record is difficult. Thus, testing long-standing hypothesis regarding the patterns and drivers of the origins and expansion of C4 grasses has not been possible. I addressed this limitation by utilizing Single Pollen Isotope Ratio AnaLysis (SPIRAL), a technique that reliably distinguishes C4 from C3 grasses in the fossil record via δ13C values of individual pollen grains. Before applying the technique to fossil samples, I refined the original SPIRAL method by expanding its training dataset and developing a hierarchical Bayesian model that increases precision of C4 grass estimates. I applied the refined technique to samples from the Oligocene and Miocene in Western Europe, a region of the world with no previous documented evidence of C4 grasses, yet with climatic conditions similar to regions where C4 grasses are common today. In Oligocene samples from France, I found that >70% of total grasses were C4. During the Miocene, SPIRAL results show 21-71% of total grasses from a site in Spain were C4. Thus C4 grasses were present in Western Europe as early as the earliest Oligocene, ~14 million years earlier than previous isotopic evidence of the first C4 plants. The Early Oligocene was also a period with extremely high pCO2, suggesting the precipitous drop in pCO2 by the end of the Oligocene did not trigger the appearance of C4 photosynthesis. The relatively high proportion of C4 grasses during the early Oligocene to middle Miocene was likely facilitated by warm and dry climatic conditions. This interpretation is further supported by spatial variability in C4 grass abundance in Spain during the early to middle Miocene. C4 grasses were abundant in southern Spain during the early Miocene, when aridity was high, and decreased after the early-middle Miocene as the region became cooler and precipitation patterns more seasonal. This pattern contrasts with the late-Miocene expansion of C4 grasses in many other regions of the world, highlighting spatial variability in the global expansion of C4 dominated grasslands. My results help elucidate the development of grasslands in Europe, provide information on the spatiotemporal dynamics of C4 grasses in earth's history, and offer insight into the relative drivers of C4 grass expansion