Introduction: Amazonia is Earth’s most iconic hotspot of diversity and endemism and, arguably, the most crucial terrestrial biome for its contributions to global systems ecology. To understand the importance and future of Amazonian biodiversity to Earth's systems biology, it is essential to place the present-day Amazonian biome in its historical context. Our understanding of how Amazonian biodiversity has been generated and assembled taxonomically and ecologically remains surprisingly meager, as is knowledge about how Amazonian ecosystems have responded to historical environmental change. There are also significant uncertainties about the paleogeography, age, and extent of its immense fresh-water and terrestrial ecosystems.

Objective/Hypotheses: This study aims to further understand our understanding of the assembly and evolution of the Amazon biota. While some models posit that Amazonian ecosystems were established in the Middle to Late Miocene, others see them as Plio-Pleistocene in age.

Methods: We reconstructed multiple time-calibrated trees of plant, vertebrate, and invertebrate taxa and combined this information with new geological and environmental data to test the current hypotheses on the age of Amazonian taxa and ecosystems.

Results: The Amazon biota has ancient roots, going back to the Paleocene or even to the Late Cretaceous, with high diversification rates after the K-Pg extinction. Many of the living Amazon vertebrate, invertebrate, and plant species diversity originated during the Plio-Pleistocene, with some lineages going back to the Late Miocene. The origin of several components of the Amazonian biota appears to relate to the establishment of the Amazon River drainage.

Implications: Resolving these historical uncertainties, as well as addressing how biotas within Amazonia have responded to past paleogeographic and climatic events, have significant implications for understanding past and present environmental change, for predicting the future of ecosystem structure and function at different spatiotemporal scales, and for understanding the generation and maintenance of biodiversity.