A 2022 phylogenomic study of the fungal tree of life (Strassert and Monaghan, Current Biology 32:3628-3635.e3) resolved several long-contested nodes of the early fungal backbone but left a small set of deep relationships ambiguous, because its answers depended on the substitution model and inference method used, and because key lineages were represented by a single genome. Work published since then shows that one flagged item - whether Microsporidia and Rozellomycota should be classified as a single phylum, which the study said would require data from additional representatives of Rozellomycota - has been substantially resolved: single-cell genomes of three new rozellid lineages form a monophyletic Rozellomycota sister to Microsporidia (Thomé et al. 2023). The remaining open core is precise and actively contested: (i) the position of the arbuscular-mycorrhizal phylum Glomeromycota, recovered as sister to Dikarya under site-heterogeneous Bayesian models but as sister to Mucoromycota + Mortierellomycota in the C60 maximum-likelihood and Bayesian analyses and in the coalescent summary tree of the same study, and in subsequent balanced-sampling analyses; and (ii) the placement of the early-branching microsporidian lineages Mitosporidium daphniae and Paramicrosporidium saccamoebae at the base of Microsporidia, which that study recovered differently under different models. The source reported the rest of the phylum-level branching order as 'consistent and well supported across multiple analyses', with the Glomeromycota 'the sole exception (on the phylum level)': it treated Olpidiomycota as sister to the non-flagellated fungi, found the Caulochytrium clade well supported in all analyses and suggested assigning it to the Chytridiomycota, mentioned the Zoopagomycota only as a rank question, and placed the aphelid representative as sister to all non-Opisthosporidia fungi in all analyses. The later-literature controversies about those items, and about the boundary of the kingdom Fungi, are therefore context rather than part of the source's open question.

Background

Fungi are a major kingdom of eukaryotes (an estimated 2-3 million species) whose deep evolutionary relationships are still being revised as genome-scale data accumulate. Current schemes recognize around 19-21 phylum-level lineages: the familiar Dikarya (the subkingdom uniting Ascomycota and Basidiomycota - yeasts, molds, and mushroom-forming fungi), a grade of aquatic flagellated lineages (Chytridiomycota and relatives, collectively Chytridiomyceta, plus Blastocladiomycota), the terrestrial 'zygomycetous' phyla Mucoromycota, Mortierellomycota and Zoopagomycota, the arbuscular-mycorrhizal Glomeromycota, and the Microsporidia - obligate intracellular parasites with highly reduced genomes that infect animals and other eukaryotes. Three further lineages of eukaryote parasites are intimately involved in the deepest divergences: rozellids (Rozellomycota, mostly known from environmental ribosomal DNA and the model hyperparasite Rozella allomycis), aphelids (Aphelidiomycota, phagotrophic parasites of algae), and the amoeba parasite Paramicrosporidium saccamoebae, whose assignment to Microsporidia or Rozellomycota has been debated since its genome was sequenced. Together, aphelids, rozellids and microsporidians were once proposed as the superphylum Opisthosporidia.

Because these lineages separated from one another roughly a billion years ago, their branching order is difficult to recover: the internal branches are short, and microsporidians and rozellids have extremely substituted sequences and reduced genomes, producing long branches that can be attracted toward other fast-evolving lineages (long-branch attraction). Deep fungal phylogenetics therefore relies on site-heterogeneous substitution models - the CAT+GTR Bayesian model, which lets amino-acid exchange preferences vary across alignment sites, and C60-style profile-mixture maximum-likelihood models - together with coalescent summary methods that combine gene trees. In the source study these frameworks disagreed exactly at the nodes that remained ambiguous, and the alternatives were weighed with Approximately Unbiased topology tests, posterior-predictive model comparison, removal of 25% and 50% of the most compositionally heterogeneous sites, progressive removal of the fastest-evolving sites, and long-branch-attraction tests in which the fastest-evolving taxa were excluded; the independent CAT+GTR Bayesian chains did not reach global convergence (maxdiff = 1).

In this setting, Strassert and Monaghan (2022) analyzed a 299-protein matrix spanning the recognized fungal diversity and resolved several contested nodes: Chytridiomyceta was sister to all other non-Opisthosporidia fungi (with Chytridiomycota sister to Monoblepharomycota + Neocallimastigomycota), Blastocladiomycota + Sanchytriomycota branched next, and the aphelid representative was sister to all non-Opisthosporidia fungi in all analyses - but Glomeromycota was recovered as sister to Dikarya only in the better-fitting CAT+GTR analyses, while their C60 maximum-likelihood and Bayesian analyses and their coalescent summary tree instead placed Glomeromycota with Mucoromycota/Mortierellomycota. The same study found Mitosporidium daphniae either sister only to Paramicrosporidium saccamoebae (coalescent and C60 analyses) or sister to all other Microsporidia including P. saccamoebae (CAT+GTR), and described the exact placement of P. saccamoebae as questionable; it recovered Rozella allomycis - then the only rozellid with genomic data - as sister to all Microsporidia and P. saccamoebae in all analyses, and left open whether Microsporidia and Rozellomycota should be classified as a single phylum pending data from additional rozellid representatives. It also reported that its CAT+GTR chains did not converge (maxdiff = 1), treats its backbone as a hypothesis that will see 'several clades ... reshuffled' as genomic and transcriptomic data accumulate, and remarks only that a comprehensive revision of fungal taxonomic classification 'is desirable'. Since then, Thomé et al. (2023) added three rozellid single-cell genomes and showed rozellids are monophyletic and sister to Microsporidia, and Wijayawardene et al. (2024) produced an updated kingdom-wide classification (19 phyla) while still flagging the arrangement of Glomeromycota, Mucoromycota, Mortierellomycota and Dikarya as ambiguous. The question below is the surviving core of the source study's flagged uncertainties.

Problem Statement

Which topology describes the earliest divergences of the fungal tree of life? Specifically, resolve with genome-scale protein data the deep nodes that Strassert and Monaghan (2022) left ambiguous, and determine whether the conflict between inference frameworks at those nodes reflects model inadequacy and systematic error - compositional heterogeneity, homoplasy at fast-evolving sites, long-branch attraction - or genuine phylogenetic signal:

(1) the position of Glomeromycota - sister to the Dikarya (Ascomycota + Basidiomycota, and possibly Entorrhizomyceta), as recovered in the site-heterogeneous CAT+GTR Bayesian analyses, a placement retained after removal of 25% and 50% of the most compositionally heterogeneous sites and also gaining support under C60 maximum likelihood once the fastest-evolving sites were removed, or sister to the Mucoromycota + Mortierellomycota clade, as recovered in the C60 maximum-likelihood and Bayesian analyses, in the multi-species-coalescent summary tree, and in later balanced-sampling analyses;

(2) the placement of the early-branching microsporidian lineages Mitosporidium daphniae and Paramicrosporidium saccamoebae at the base of Microsporidia - Mitosporidium sister only to P. saccamoebae, as in the coalescent and C60 maximum-likelihood and Bayesian analyses, or Mitosporidium sister to all other Microsporidia including P. saccamoebae, with P. saccamoebae nested between Mitosporidium and Amphiamblys sp., as under CAT+GTR, an arrangement that the fast-site analysis supported only once at least about 85% of the fastest-evolving sites had been removed.

Whether Microsporidia and Rozellomycota should be classified as one phylum - the source study's other flagged item, which it said would require data from additional representatives of Rozellomycota - is context rather than target here: Thomé et al. (2023) supplied those genomes and recovered a monophyletic Rozellomycota sister to Microsporidia. The nodes the source reported as consistent and well supported (Olpidiomycota, Caulochytrium, the Zoopagomycota, the aphelid position), the boundary of the kingdom Fungi, and the revision of the higher-level classification that the source called 'desirable' are likewise context rather than target.

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