Quaternary Ice Ages Shaped Protists Phylogeography: The Case of Arcellinida in the Iberian Peninsula

The Quaternary glaciations profoundly shaped the biogeography of plants and animals, yet their impact on microbial eukaryotes remains largely unexplored. We tested the "genetic legacy of the Quaternary" (GLQ) paradigm in terrestrial protists using Arcellinida testate amoebae diversity distribution across the Iberian Peninsula, a well-established glacial refugium. To do so, we compiled the most extensive Arcellinida metabarcoding dataset to date (ArKOI), including 615 samples from multiple continents and ecosystems. Our results showed that hotspots of intra-OTU genetic diversity align with known Iberian refugia, supporting the concept of "refugia within refugia," and displaying clear ecoregion-specific climatic niches. Patterns of spatial clustering, niche breadth, and historical demographic reconstruction revealed repeated range contractions and expansions during the Pleistocene, mirroring those observed in macro-organisms. These findings extend the GLQ paradigm to protists, highlighting shared historical and ecological processes across the eukaryotic tree of life and contributing to a unified theory of biogeographic responses to climatic change.