The six-gene AFTOL (Assembling the Fungal Tree of Life) phylogeny of James et al. (2006) established the modern backbone of the fungal tree of life and concluded that the spore flagellum was lost at least four times, that the microsporidia nest on the earliest branch with the endoparasitic Rozella, and that the nutritional mode and habitat of the fungal ancestor remained equivocal. Two decades of genome-scale analyses have settled parts of this program: microsporidia are confirmed as close relatives of Rozella (Rozellomycota), classical Zygomycota is resolved into the phyla Mucoromycota and Zoopagomycota, and the zoosporic endoparasite Olpidium is repeatedly recovered as the closest living flagellated relative of the non-flagellated terrestrial fungi. But the deepest internodes - the arrangement of the rozellid-microsporidian and aphelid lineages relative to the crown (all living fungi and their last common ancestor), the positions of the zoosporic phyla, and above all the branching order among Olpidium, Zoopagomycota, Mucoromycota, and Dikarya - remain unstable across phylogenomic studies and may constitute a near-hard polytomy (an effectively simultaneous branching) from a Precambrian rapid radiation. Consequently, the definitive number and phylogenetic positions of the independent flagellum losses and the ecological portrait (parasitic versus saprotrophic; marine versus freshwater/soil) of the fungal ancestor are still open. The problem is to resolve these deep relationships - or rigorously establish their irresolvability - through dense sampling and characterization of the basal lineages and genome-scale or rare-genomic-change evidence, and on that basis to fix the count and timing of the flagellum losses and the ancestral nutritional mode and habitat of the first fungi.

Background

The kingdom Fungi comprises the familiar mushrooms, molds, and yeasts together with a diverse array of mostly microscopic aquatic lineages. Fungi and animals together form Opisthokonta, one of the major branches of the eukaryote tree; within the broader fungal side of that clade (Holomycota), the closest unicellular relatives of Fungi are the nucleariid amoebae and several groups of endoparasitic protists. Most described fungal species belong to the subkingdom Dikarya (the phyla Ascomycota and Basidiomycota), whose members produce non-motile aerial spores and entirely lack flagella. The early-diverging (non-Dikarya) fungi instead include the zoosporic lineages, which disperse by flagellated swimming spores (zoospores) in water and moist soils: the chytrids (Chytridiomycota, in the broad sense that also covers Monoblepharomycota), Blastocladiomycota, the anaerobic rumen fungi (Neocallimastigomycota), and the recently discovered Sanchytriomycota. Nesting among these are obligate endoparasites with reduced cells and genomes: the microsporidia (intracellular parasites of animals, once so atypical that they were treated as a separate protist phylum), their close relatives Rozella and the rozellids (Rozellomycota, also called Cryptomycota), and the aphelids (phagotrophic parasites of algae, which feed by engulfing their prey). A pivotal single genus is Olpidium, a zoosporic endoparasite of plant roots. The classical phylum Zygomycota (bread molds and relatives) is now known to be an unnatural grouping. Because these organisms offer few morphological characters, deep fungal relationships must be reconstructed from molecular data. The landmark study of James et al. (2006), produced by the AFTOL (Assembling the Fungal Tree of Life) consortium, combined six gene regions - the nuclear ribosomal RNA genes (18S, 5.8S, and 28S rDNA), the protein-coding gene elongation factor 1-alpha, and the two largest subunits of RNA polymerase II (RPB1 and RPB2) - for nearly 200 species. It established that Chytridiomycota is not a monophyletic clade but a grade of early-diverging lineages; that the microsporidia group with the endoparasitic chytrid Rozella allomycis on the earliest diverging branch; that Olpidium groups with the zygomycetes; and that the flagellum was lost at least four times independently rather than once. Mapping characters onto 58,611 Bayesian credible trees (Figure 1 of that paper), Dollo parsimony (a reconstruction method that lets a complex trait arise once but be lost repeatedly) and maximum-likelihood ancestral-state reconstruction left many deep nodes equivocal: the last common ancestor of Fungi was reconstructed as flagellated and probably parasitic, but whether it was marine or freshwater/soil-associated, and where transitions between parasitism and saprotrophy (growth on dead organic matter) occurred, could not be decided. The authors' stated program - denser sampling of basal taxa and additional independent loci to resolve the residual topological uncertainty, pin down the number and timing of flagellum losses, and make ancestral-state reconstructions for nutritional mode and habitat definitive - frames the problem addressed here. Resolving these deepest nodes is technically hard for known reasons. The pivotal internodes are short, consistent with a Precambrian rapid radiation, so the phylogenetic signal distinguishing alternative branching orders is weak and has partly decayed over roughly 0.9-1.4 billion years; a sufficiently rapid radiation can approach a genuine hard polytomy (an effectively simultaneous bush, for example under incomplete lineage sorting - ancestral genetic variation that fails to sort cleanly into the descendant lineages when splits follow one another in rapid succession - across successively short internodes). Fast-evolving endoparasitic lineages (microsporidia, rozellids, sanchytrids, Olpidium) induce long-branch attraction, the artifact that pulls rapidly evolving taxa together artificially; several genome-scale studies have had to exclude microsporidia for this reason. Most basal lineages are unculturable obligate parasites, so genome data must be extracted from single cells or metagenomes, with attendant missing data and host contamination. Finally, ancestral ecological states can only be coded for lineages whose way of life is known, while a growing fraction of basal fungal diversity is known only from environmental rDNA sequences ('dark fungi').

Problem Statement

Determine the deep phylogeny of the early-diverging fungi, and on that basis make the trait inferences that the 2006 six-gene study could only make conditionally. Concretely: (1) resolve the branching order of the deepest lineages of kingdom Fungi - the arrangement of the rozellid-microsporidian lineages (Rozellomycota) and the aphelids relative to the crown fungi; the positions of the zoosporic phyla (Chytridiomycota, Monoblepharomycota, Neocallimastigomycota, Blastocladiomycota, Sanchytriomycota); and above all the branching order among Olpidium, Zoopagomycota, Mucoromycota, and Dikarya, which current genome-scale analyses recover either as unstable across data matrices or as a near-hard polytomy - to the standard that the competing placements found in the literature are statistically rejected, or the radiation is rigorously demonstrated to be effectively simultaneous (unresolvable with available signal); (2) on that topology, determine the exact number, phylogenetic positions, and timing of the independent losses of the flagellum during fungal evolution, replacing the current topology-contingent estimates, which range from a single loss (under the Chang et al. 2021 topology) through three losses (from flagellum-protein distributions) to the four to six losses of the 2006 six-gene analysis; and (3) determine the nutritional mode (parasitic versus saprotrophic, and the earlier transition from phagotrophy) and the aquatic habitat (marine versus freshwater or soil-associated) of the fungal common ancestor, together with the deep-node transitions among these states. These deliverables share one scientific objective: an evolutionary and ecological portrait of the first fungi that no longer depends on which unresolved basal topology is assumed. Establishing that the basal radiation is effectively simultaneous does not by itself complete the task: deliverables (2) and (3) must still be delivered, and where the relevant internodes are unresolvable they are to be stated as the bounds and ranges that hold across every topology the data cannot reject. Expected routes include dense taxon and genome sampling of the basal lineages (including uncultured endoparasites), improved orthology inference (correct assignment of genes across species to their single common ancestral copies) and model treatment of long-branch attraction, and independent evidence classes such as rare genome-structure changes or gene content.

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