Amoebozoan testate amoebae illuminate the diversity of heterotrophs and the complexity of ecosystems throughout geological time.

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

The Phanerozoic diversification of silica-cycling testate amoebae and its possible links to changes in terrestrial ecosystems.

The terrestrial cycling of Si is thought to have a large influence on the terrestrial and marine primary production, as well as the coupled biogeochemical cycles of Si and C. Biomineralization of silica is widespread among terrestrial eukaryotes such as plants, soil diatoms, freshwater sponges, silicifying flagellates and testate amoebae. Two major groups of testate (shelled) amoebae, arcellinids and euglyphids, produce their own silica particles to construct shells. The two are unrelated phylogenetically and acquired biomineralizing capabilities independently. Hyalosphenids, a group within arcellinids, are predators of euglyphids. We demonstrate that hyalosphenids can construct shells using silica scales mineralized by the euglyphids. Parsimony analyses of the current hyalosphenid phylogeny indicate that the ability to "steal" euglyphid scales is most likely ancestral in hyalosphenids, implying that euglyphids should be older than hyalosphenids. However, exactly when euglyphids arose is uncertain. Current fossil record contains unambiguous euglyphid fossils that are as old as 50 million years, but older fossils are scarce and difficult to interpret. Poor taxon sampling of euglyphids has also prevented the development of molecular clocks. Here, we present a novel molecular clock reconstruction for arcellinids and consider the uncertainties due to various previously used calibration points. The new molecular clock puts the origin of hyalosphenids in the early Carboniferous (∼370 mya). Notably, this estimate coincides with the widespread colonization of land by Si-accumulating plants, suggesting possible links between the evolution of Arcellinid testate amoebae and the expansion of terrestrial habitats rich in organic matter and bioavailable Si.