Phylogenomics of neglected flagellated protists supports a revised eukaryotic tree of life Torruella, Guifré Galindo González, Luis Javier Moreira, David López-García, Purificación Ancyromonad Apusomonad Free-living flagellate Meteora Phylogenomics This work was supported by the European Research Council (ERC) Advanced Grants “Protistworld” and “Plast-Evol” (322669 and 787904, respectively) and the Horizon 2020 research and innovation program under the Marie Skłodowska-Curie ITN project SINGEK (http://www.singek.eu/; grant agreement no. H2020-MSCA-ITN-2015-675752). G.T. was supported by 2019 BP 00208, Beatriu de Pinós-3 Postdoctoral Programme (BP3), grant agreement ID: 801370. L.J.G. was funded by the Ramón y Cajal Programme (grant RYC2022-035282-I, supported by the MCIU/AEI/10.13039/501100011033 and FSE+). Eukaryotes evolved from prokaryotic predecessors in the early Proterozoic1,2 and radiated from their already complex last common ancestor3, diversifying into several supergroups with unresolved deep evolutionary connections4. They evolved extremely diverse lifestyles, playing crucial roles in the carbon cycle5,6. Heterotrophic flagellates are arguably the most diverse eukaryotes4,7-9 and often occupy basal positions in phylogenetic trees. However, many of them remain undersampled4,10 and/or incertae sedis4,11-18. Progressive improvement of phylogenomic methods and a wider protist sampling have reshaped and consolidated major clades in the eukaryotic tree13-19. This is illustrated by the Opimoda14, one of the largest eukaryotic supergroups (Amoebozoa, Ancyromonadida, Apusomonadida, Breviatea, CRuMs, Malawimonadida, and Opisthokonta –including animals and fungi–)4,14,19-22. However, their deepest evolutionary relationships still remain uncertain. Here, we sequenced transcriptomes of poorly studied flagellates23,24 (fourteen apusomonads25,26, seven ancyromonads27 and one 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. While 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. 2025-02-14T13:05:07Z 2025-02-14T13:05:07Z 2024-08-27 journal article Published version: Torruella, Guifré et al. Current Biology Volume 35, Issue 1, 6 January 2025, Pages 198-207.e4. https://doi.org/10.1016/j.cub.2024.10.075 https://hdl.handle.net/10481/102343 10.1016/j.cub.2024.10.075 eng info:eu-repo/grantAgreement/EC/H2020/675752 http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Cold Spring Harbor Laboratory