Carbon isotope analyses reveal relatively high abundance of C₄ grasses during early-middle Miocene in southwestern Europe

Before their rapid expansion during the late Miocene, C₄ plants were generally uncommon or absent from most grasslands. Recent studies have furthered our understanding of shifts in C₄ abundance prior to their late-Miocene expansion; however, the early history of C₄ grasses remains poorly constrained. Distinguishing C₄ grasses from C₃ taxa (i.e., most trees, shrubs, herbs, and about half of all grasses) using isotopic data in the paleorecord is challenging, especially when C₄ plants are in relatively low abundance. We used SPIRAL (Single Pollen Isotope Ratio AnaLysis) to measure δ¹³C values of individual grass-pollen grains in early-middle Miocene sediments from three sites in Spain where grass pollen represents 5-20% of the pollen assemblages, including 25 samples from Rubielos de Mora, (20.3-15.5Ma), seven from Gor (15.2-13.7Ma) and seven from Andalucía A1 (13.7-10.1Ma). Results indicate that C₄-grass abundance varied between 21 and 72% of total grasses during the early-middle Miocene. C₄ grasses are uncommon (10-20% of the grass flora) in this region today, and thus C₄-grass abundance likely decreased after the early-middle Miocene as the region became cooler and precipitation shifted to the winter months. This pattern contrasts with that of a late-Miocene expansion of C₄ grasses in many other regions of the world. Our results thus help elucidate the history of grasslands in Europe and the spatiotemporal dynamics of C₄ grasses worldwide.