The Eocene–Oligocene Transition (EOT) is crucial for understanding the climate evolution of the Earth but is poorly documented in the terrestrial domains. A pollen record from the early Priabonian to the late Aquitanian encompassing the EOT was produced for thirty-three locations in Southeastern France. Changes in lowland flora and vegetation were documented based on the shift from dense forests with tropical-subtropical plants to open shrublands rich in xeric plants, paving the way for the future Mediterranean landscapes. Cupressaceae, particularly Tetraclinis, and Ephedra, occupied a proeminent place in the newly structured vegetation. Palaeolimatic fluctuations were quantified using the Climatic Amplitude Method, revealing the fact that the EOT was mainly characterized by a decrease of 7–9 °C in winter temperatures and by an increase in overall temperature and rainfall seasonality. Using the climatostratigraphic approach further improved the regional chronostratigraphy. This climate change occurred during a period and in a region marked by an extensive geodynamic upheaval. This work documents the pathways of some influential marine incursions of the Mediterranean Sea earlier than is usually considered. Finally, the deposition of evaporites in lakes is interpreted as the result of a combination of different factors, including the entrance of marine waters in the terrestrial realm, a decrease in regional rainfall, a decrease in the supply of water by rivers and increased seasonality. Palaeoelevations of nearby mountains were also estimated using pollen grains transported from highlands and reconstructed palaeotemperature of the lowlands.
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