Genome-scale phylogenetics has produced a broadly supported tree for the Opisthokonta, the eukaryotic supergroup containing animals, fungi, and their unicellular relatives, yet its deepest divergences, an ancient and probably rapid radiation roughly a billion years ago, remain unstable. The most taxon-rich analysis to date (Liu and colleagues, 2024; 348 species, three independently constructed gene matrices) left five nodes dependent on the choice of gene matrix, substitution model, and taxon sampling: the relationships among unicellular Holozoa (Pluriformea-sister versus Teretosporea-sister, with an Ichthyosporea-sister topology recurring under sparse taxon sampling); the placement of the rogue predator Tunicaraptor unikontum; the branching order of the flagellated zoosporic fungal phyla Chytridiomycota and Blastocladiomycota; the position of the endoparasitic fungus Olpidium, which determines whether the fungal flagellum was lost once or repeatedly; and the position of Placozoa within Metazoa. The problem is to resolve these five nodes robustly, meaning stable across gene-matrix constructions and substitution-model classes and consistent with gene- and site-level concordance, thereby completing the opisthokont backbone that anchors inference of the unicellular ancestors of the animal and fungal kingdoms.

Background

Opisthokonta is the eukaryotic supergroup that contains animals (Metazoa), fungi (Fungi), and their unicellular relatives, and it comprises two main lineages: Holozoa (Metazoa plus its unicellular relatives: Choanoflagellatea, collar-flagellate bacterivores; Filasterea, unicellular amoeboid or flagellated predators such as Capsaspora and Ministeria; Ichthyosporea, mostly endoparasites of fish and invertebrates; and Pluriformea, also called Corallochytrea, which includes Corallochytrium and Syssomonas) and Holomycota (Fungi plus unicellular relatives such as nucleariids and aphelids). Filozoa denotes the clade Filasterea + Choanoflagellatea + Metazoa. Because this backbone fixes what the unicellular ancestors of animals and fungi were like, its deep branches have been attacked with phylogenomics, the reconstruction of species trees from matrices of hundreds of conserved genes, but the deepest divergences occurred close together roughly a billion years ago (Liu and colleagues dated the opisthokont crown to the Mesoproterozoic, approximately 1.08 billion years ago, under a 1.5-billion-year root-age constraint) and have resisted stable resolution.

Liu et al. (2024) assembled the most taxon-rich opisthokont phylogeny to date (348 species) from three independently built gene matrices (a BUSCO-based matrix, an OrthoFinder-based matrix, and the curated matrix of Tikhonenkov et al. 2020; BUSCO and OrthoFinder are two standard but methodologically different orthology-inference pipelines, and different gene sets can yield different trees). Each matrix was analyzed under site-homogeneous substitution models (a single amino-acid exchange process for all sites, e.g., LG+I+G4) and site-heterogeneous mixture models (site-specific amino-acid profile mixtures such as CAT-GTR or the C10-C60 family, which better control systematic error), and the authors dissected known sources of phylogenomic error: taxon-sampling density, compositional heterogeneity (unequal amino-acid frequencies across lineages), long-branch attraction (the artifactual grouping of fast-evolving lineages), and rogue taxa (species whose position varies across bootstrap replicates). A key diagnostic was concordance factors, the fraction of gene trees (gCF) or alignment sites (sCF) supporting a given split. The crux they exposed is that the sequence signal at the deepest nodes is genuinely weak: genes split almost evenly between competing topologies, and one topology with 98% ultrafast-bootstrap support was supported by only 0.7% of individual gene trees, so apparent resolution can reflect systematic error rather than history.

The prior literature offers competing answers at each unstable node. Analyses that added new protist-opisthokont genomes recovered Teretosporea, the clade of Ichthyosporea + Pluriformea, as the earliest-branching holozoan lineage (Ocaña-Pallarès et al. 2022), whereas Tikhonenkov et al. (2020), who described the flagellated microbial predator Tunicaraptor unikontum, recovered Ichthyosporea as sister to the rest of Holozoa, and the main analyses of Liu et al. (2024) favor Pluriformea as sister to the rest of Holozoa. Within Holomycota, Chang et al. (2021) placed Olpidium, an obligate endoparasitic fungus whose motile flagellated spores (zoospores) infect plant roots, as the closest living zoosporic relative of the non-flagellated terrestrial fungi, implying a single loss of the fungal flagellum, and site-heterogeneous analyses (Galindo et al. 2021; Strassert and Monaghan 2022) support Chytridiomycota (zoosporic fungi with rhizoids) diverging before Blastocladiomycota (zoosporic fungi with true hyphae and spore-bearing structures). Within Metazoa, Laumer et al. (2018) supported Placozoa, the Trichoplax animals, which are the simplest known free-living diploblastic animals, as sister to Cnidaria using genes with minimal compositional bias, whereas other matrices place it sister to the clade Cnidaria + Bilateria. Beyond sequence substitution, rare genomic changes, shared derived chromosome-scale rearrangements detectable as fusions of ancestral linkage groups (macrosynteny; ancestral linkage groups are inferred ancestral chromosomes reconstructed from conserved gene adjacency across chromosome-scale genomes), have been proposed as phylogenetic evidence largely immune to substitution-model error, most prominently supporting ctenophores as sister to the remaining animals (Schultz et al. 2023); however, the statistical procedures used to judge such shared linkages significant are themselves contested (Copley 2025).

Problem Statement

Determine, robustly, the branching order at the five deep opisthokont nodes that genome-scale analyses have so far left unstable, so that the opisthokont tree of life is resolved at these ancient divergences:

  1. Relationships among unicellular Holozoa: is Pluriformea the sister lineage to Ichthyosporea + Filozoa (the Pluriformea-sister hypothesis), do Pluriformea and Ichthyosporea together form the clade Teretosporea that is sister to Filozoa (the Teretosporea-sister hypothesis), or is Ichthyosporea the sister lineage to the rest of Holozoa (the Ichthyosporea-sister hypothesis)?

  2. The phylogenetic placement of Tunicaraptor unikontum among the unicellular holozoans, including which lineages are its closest relatives. Whether expanded sampling of those relatives would stabilize the neighboring deep nodes, in particular the root of unicellular Holozoa (deliverable 1), is an exploratory question rather than a completion requirement.

  3. The branching order of the flagellated zoosporic fungal phyla Chytridiomycota and Blastocladiomycota relative to the remaining fungi, that is, which of the two represents the earliest surviving fungal lineage.

  4. The position of the zoosporic endoparasite Olpidium: is it the sister lineage of the non-flagellated terrestrial fungi, implying a single loss of the fungal flagellum, or nested within them, implying multiple losses?

  5. The position of Placozoa within Metazoa: sister to Cnidaria alone, or sister to the clade Cnidaria + Bilateria?

These five deliverables serve one overarching objective: a resolved opisthokont backbone, the framework that fixes the nature of the unicellular ancestors of both animals and fungi. The problem is complete when each named node is resolved robustly. Resolved robustly means that the supported topology satisfies both of the following: (i) it is stable across the independent gene-matrix constructions and across the site-homogeneous and site-heterogeneous substitution-model classes, or is otherwise demonstrated to survive the identified sources of systematic error (compositional heterogeneity, long-branch attraction, orthology-method differences, and taxon-sampling density); and (ii) it is consistent with gene- and site-level concordance, in the sense that its gene-concordance factor (gCF) and its site-concordance factor (sCF) are each higher than those of every competing topology at that node. A conclusion that a node is not robustly resolvable by this standard, however well supported, reports the current state of the evidence and does not by itself complete that node's deliverable.

References

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