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

Eukaryotes evolved from prokaryotic predecessors in the early Proterozoic^1,2 and radiated from their already complex last common ancestor,³ diversifying into several supergroups with unresolved deep evolutionary connections.⁴ They evolved extremely diverse lifestyles, playing crucial roles in the carbon cycle.^5,6 Heterotrophic flagellates are arguably the most diverse eukaryotes^4,7,8,9 and often occupy basal positions in phylogenetic trees. However, many of them remain undersampled^4,10 and/or incertae sedis.^{4,11,12,13,14,15,16,17,18} Progressive improvement of phylogenomic methods and a wider protist sampling have reshaped and consolidated major clades in the eukaryotic tree.^{13,14,15,16,17,18,19} This is illustrated by the Opimoda,¹⁴ one of the largest eukaryotic supergroups (Amoebozoa, Ancyromonadida, Apusomonadida, Breviatea, CRuMs [Collodictyon-Rigifila-Mantamonas], Malawimonadida, and Opisthokonta-including animals and fungi).^{4,14,19,20,21,22} However, their deepest evolutionary relationships still remain uncertain. Here, we sequenced transcriptomes of poorly studied flagellates^23,24 (14 apusomonads,^25,26 7 ancyromonads,²⁷ and 1 cultured Mediterranean strain of Meteora sporadica¹⁷) and conducted comprehensive phylogenomics analyses with an expanded taxon sampling of early-branching protists. Our findings support the monophyly of Opimoda, with CRuMs being sister to the Amorphea (amoebozoans, breviates, apusomonads, and opisthokonts) and ancyromonads and malawimonads forming a moderately supported clade. By mapping key complex phenotypic traits onto this phylogenetic framework, we infer an opimodan biflagellate ancestor with an excavate-like feeding groove, which ancyromonads subsequently lost. Although breviates and apusomonads retained the ancestral biflagellate state, some early-diverging Amorphea lost one or both flagella, facilitating the evolution of amoeboid morphologies, novel feeding modes, and palintomic cell division resulting in multinucleated cells. These innovations likely facilitated the subsequent evolution of fungal and metazoan multicellularity.