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A robustly rooted tree of eukaryotes reveals their excavate ancestry

Nature volume 640, pages 974–981 (2025)Cite this article

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Abstract

The eukaryote Tree of Life (eToL) depicts the relationships among all eukaryotic organisms; its root represents the Last Eukaryotic Common Ancestor (LECA) from which all extant complex lifeforms are descended1. Locating this root is crucial for reconstructing the features of LECA, both as the endpoint of eukaryogenesis and the start point for the evolution of the myriad complex traits underpinning the diversification of living eukaryotes. However, the position of the root remains contentious due to pervasive phylogenetic artefacts stemming from inadequate evolutionary models, poor taxon sampling and limited phylogenetic signal1. Here we estimate the root of the eToL with unprecedented resolution on the basis of a new, much larger, dataset of mitochondrial proteins that includes all known eukaryotic supergroups. Our analyses of a 100 taxon × 93 protein dataset with state-of-the-art phylogenetic models and an extensive evaluation of alternative hypotheses show that the eukaryotic root lies between two multi-supergroup assemblages: ‘Opimoda+’ and ‘Diphoda+’. This position is consistently supported across different models and robustness analyses. Notably, groups containing ‘typical excavates’ are placed on both sides of the root, suggesting the complex features of the ‘excavate’ cell architecture trace back to LECA. This study sheds light on the ancestral cells from which extant eukaryotes arose and provides a crucial framework for investigating the origin and evolution of canonical eukaryotic features.

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Fig. 1: Rooted phylogenies of eukaryotes estimated from mitochondrial proteins of alphaproteobacterial origin.
Fig. 2: Likelihood estimation of candidate eukaryote roots across complex models.
Fig. 3: Evaluating potential for LBA bias on simulated data.
Fig. 4: The recovered root suggests the morphological features of ‘typical excavates’ are ancestral traits of LECA.

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Data availability

All sequence alignments and phylogenetic trees are available at Figshare (https://figshare.com/s/59b28ecc0056dc8d0d03)34 and the accession codes for publicly available data used in these analyses are given in Supplementary Table 7. The data included in the accession codes can be found in the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov), the MMETSP database on the iMicrobe website https://www.imicrobe.us/#/projects/104 or the PhyloFisher v.1 dataset available at Figshare (https://doi.org/10.6084/m9.figshare.15141900.v1)72 as indicated in Supplementary Table 7.

Code availability

Custom scripts have been deposited at Figshare (https://figshare.com/s/59b28ecc0056dc8d0d03)34.

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Acknowledgements

We thank N. Ly-Trong for assistance with model implementation in IQ-TREE and J. Jerlström-Hultqvist for assistance with data acquisition and processing. We also thank J. Ross for assistance with figure design. This work was supported by the Moore-Simons Project on the Origin of the Eukaryotic Cell, Simons Foundation grant no. 735923LPI (https://doi.org/10.46714/735923LPI) awarded to A.J.R., E.S. and L.E. and by NSERC Discovery grants awarded to A.J.R. (grant no. RGPIN-2022-05430), A.G.B.S. (grant no. 298366-2019) and E.S. K.W. was supported by a graduate scholarship from the Killam Foundation.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology and Institute for Comparative Genomics, Dalhousie University, Halifax, Nova Scotia, Canada

    Kelsey Williamson, Laura Eme, Hector Baños, Charley G. P. McCarthy, Sergio A. Muñoz-Gómez & Andrew J. Roger

  2. Unité d’Ecologie, Systématique et Evolution Université Paris-Saclay, Gif-sur-Yvette, France

    Laura Eme

  3. Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI, USA

    Laura Eme

  4. Department of Mathematics and Statistics and Institute for Comparative Genomics, Dalhousie University, Halifax, Nova Scotia, Canada

    Hector Baños & Edward Susko

  5. Department of Mathematics, California State University San Bernardino, San Bernardino, CA, USA

    Hector Baños

  6. Graduate School of Agriculture, Kyoto University, Kyoto, Japan

    Ryoma Kamikawa

  7. Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo, Norway

    Russell J. S. Orr

  8. Total Defence Division, Norwegian Defence Research Establishment FFI, Kjeller, Norway

    Russell J. S. Orr

  9. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Sergio A. Muñoz-Gómez

  10. School of Computing, Australian National University, Canberra, Australian Capital Territory, Australia

    Bui Quang Minh

  11. Department of Biology and Institute for Comparative Genomics, Dalhousie University, Halifax, Nova Scotia, Canada

    Alastair G. B. Simpson

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Contributions

Ancestral sequence reconstruction analyses, expansion and curation of the final datasets, and all phylogenomic analyses were performed by K.W. in consultation with A.J.R., A.G.B.S. and L.E. K.W. and H.B. performed simulation analyses. H.B. developed and implemented the MEOW model and performed cross-validation testing. C.G.P.M. and E.S. performed GFmix analyses. E.S. performed topology testing. B.Q.M. implemented the FunDi model in IQ-TREE2. S.A.M.-G., R.K. and R.J.S.O. provided molecular data. K.W., A.J.R., A.G.B.S., H.B., C.G.P.M. and E.S. wrote, and all authors edited and approved, the manuscript. A.J.R. and L.E. initially conceived the study.

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Correspondence to Kelsey Williamson or Andrew J. Roger.

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Extended data figures and tables

Extended Data Fig. 1 Entropy and posterior probability of the predicted ancestral sequence at the root of Alphaproteobacteria as ingroup taxa are removed.

A. The mean, median, and standard deviation of the posterior probability as taxa are removed. B. The mean, median, and standard deviation of the entropy as taxa are removed.

Extended Data Fig. 2 Bayesian consensus tree estimated from Anae+ dataset.

Consensus phylogeny estimated under the CAT + GTR model in PhyloBayes from 4 chains after 29,000 cycles, with a burn-in of 1000. Posterior probabilities for each bipartition are indicated. Note that these 4 chains included one in which Hemimastigophora branched on the opposite side of the root, separate from its previously inferred close relative Meteora, with this non-convergence resulting in posterior probabilities ~0.75 (0.74) for several deep nodes in the Diphoda+ side of the tree.

Extended Data Fig. 3 Phylogeny estimated from Anae+ dataset with CAT-PMSF model.

Amino acid exchangeability matrix and site frequency profiles were estimated on the alignment and a guide tree in PhyloBayes. Rate variation was modelled with the Free Rate model with four classes. Support values indicated are from 100 replicates of non-parametric bootstrapping.

Extended Data Fig. 4 Bayesian consensus tree estimated from Anae- dataset.

Consensus phylogeny estimated under the CAT + GTR model in PhyloBayes from 4 chains after 16,500 cycles, with a burn-in of 1000. Posterior probabilities for each bipartition are indicated.

Extended Data Fig. 5 Phylogeny estimated from Anae- dataset with CAT-PMSF model.

Amino acid exchangeability matrix and site frequency profiles were estimated on the alignment and a guide tree in PhyloBayes. Rate variation was modelled with the Free Rate model with four classes. Support values indicated are from 100 replicates of non-parametric bootstrapping.

Extended Data Fig. 6 Phylogeny estimated from Anae+ dataset with only nuclear-encoded proteins.

Phylogeny was estimated in IQ-TREE2 under the LG + MEOW80 + G4 model with 1000 replicates each of SH-aLRT and UFBOOT2. Support values are displayed on branches as SH-aLRT/UFBOOT2.

Extended Data Fig. 7 Phylogeny estimated from Anae- dataset with only nuclear-encoded proteins.

Phylogeny was estimated in IQ-TREE2 under the LG + MEOW80 + G4 model with 1000 replicates each of SH-aLRT and UFBOOT2. Support values are displayed on branches as SH-aLRT/UFBOOT2.

Extended Data Fig. 8 Evaluating change in support for the root position as fastest-evolving sites are removed.

A. Phylogenies were estimated in IQ-TREE2 under the LG + MEOW80 + G4 model with 1000 replicates each of SH-aLRT and UFBOOT2. Support values are indicated for the bipartitions relevant to the position of the eukaryote root.

Extended Data Fig. 9 Phylogeny estimated from Anae- dataset after removal of fastest evolving taxa.

Phylogeny was estimated in IQ-TREE2 under the LG + MEOW80 + G4 model with 1000 replicates each of SH-aLRT and UFBOOT2. Support values are displayed on branches as SH-aLRT/UFBOOT2.

Extended Data Fig. 10 Phylogeny estimated from Anae- dataset after removal of most divergent genes.

Alignment after gene removal consisted of 19324 sites from 80 concatenated genes. The phylogeny was estimated in IQ-TREE2 under the LG + MEOW80 + G4 model with 1000 replicates each of SH-aLRT and UFBOOT2. Support values are displayed on branches as SH-aLRT/UFBOOT2.

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Williamson, K., Eme, L., Baños, H. et al. A robustly rooted tree of eukaryotes reveals their excavate ancestry. Nature 640, 974–981 (2025). https://doi.org/10.1038/s41586-025-08709-5

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