The main episode of glaciation during the Late Paleozoic began just prior to the mid-Carboniferous boundary, based on evidence from Gondwanan tillites, and sequence stratigraphic and chemostratigraphic analysis in Euramerica. While gradual drawdown of atmospheric CO₂ during the Devonian and Mississippian may have preconditioned the Earth for the most widespread glacial advance since the Neoproterozoic, an abrupt onset at ~320 Ma hints at a critical role for additional factors affecting the climate system, such as oceanic gateway closure. The relative importance of gateway closure versus CO₂ drawdown in part relates to the interpretation of carbon isotope trends in Carboniferous carbonates. Carbon isotope values in European successions show a dramatic increase to a Phanerozoic maximum of ~+6‰ across the mid-Carboniferous boundary, which has been interpreted to reflect a period of globally enhanced organic carbon burial and CO₂ drawdown associated with the widespread Pennsylvanian coal basins. However, new data from western North America do not show an equally large increase at this time and raises questions concerning the true magnitude of organic carbon burial and the origin of the European d¹³C shift.

Over 700 carbonate d¹³C analyses from a 1.3 km marine succession at Arrow Canyon, Nevada, reveal a complex curve in which a large +7‰ anomaly found in the lowermost Mississippian stage (Kinderhookian) stands far above the next highest peaks of +3-4‰ in the Meramecian, Atokan and Missourian. This micrite-based curve is in good agreement with the brachiopod calcite curve of Mii et al. (1999) from the midcontinent of North America and indicates that the large, European mid-Carboniferous shift was not global. An alternative hypothesis for the large European d¹³C shift is linked to the closing of the oceanic gateway between Euramerica and Gondwana associated with the assembly of Pangea, rather than high organic carbon burial. This gateway closure just prior to the mid-Carboniferous boundary increased the transport of heat and moisture to high southern latitudes of Gondwana, resulting in widespread glacial advance and eustatic drop. Gateway closure also had profound biological consequences, triggering widespread extinctions and leading to the development of a distinct Tethyan faunal realm.