Eukaryotes radiated from their last common ancestor, diversifying into several supergroups with unresolved deep evolutionary connections. Heterotrophic flagellates, often branching deeply in phylogenetic trees, are arguably the most diverse eukaryotes. However, many of them remain undersampled and/or incertae sedis. Here, we conducted comprehensive phylogenomics analyses with an expanded taxon sampling of early-branching protists including 22 newly sequenced transcriptomes (apusomonads, ancyromonads, Meteora). They support the monophyly of Opimoda, one of the largest eukaryotic supergroups, with CRuMs being sister to the Amorphea (amoebozoans, breviates, apusomonads, and opisthokonts -including animals and fungi-), and the ancyromonads+malawimonads clade. By mapping traits onto this phylogenetic framework, we infer a biflagellate opimodan ancestor with an excavate-like feeding groove. Breviates and apusomonads retained the ancestral biflagellate state. Other Amorphea lost one or both flagella, enabling the evolution of amoeboid shapes, novel feeding modes, and palintomic cell division resulting in multinucleated cells, which likely facilitated the subsequent evolution of fungal and metazoan multicellularity.

Phylogenomics of neglected flagellated protists supports a revised eukaryotic tree of life