Phylogenomics of a new fungal phylum reveals multiple waves of reductive evolution across Holomycota

Compared to well-known multicellular fungi and unicellular yeast, unicellular fungi with zoosporic, free-living flagellated stages remain poorly known and their phylogenetic position is often unresolved. Recently, 18S+28S rRNA gene molecular phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and record-long simplified kinetosomes, Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a monophyletic group without affinity with any known fungal clade. To assess their phylogenetic position and unique trait evolution, we sequenced single-cell genomes for both species. Phylogenomic analyses using 264 protein markers and a comprehensive taxon sampling retrieved and almost fully-resolved fungal tree with these species forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Chytridiomycota branched as sister to all other fungi, and the zoosporic fungus Olpidium bornovanus as sister to non-flagellated fungi. Comparative genomic analyses across Holomycota revealed an atypically reduced metabolic repertoire for sanchytrids given their placement in the tree. We infer four independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. The highly reduced sanchytrid flagellar machinery, notably their long kinetosome, might have been retained to support a putative light-sensing lipid organelle. Together with their phylogenetic position, these unique traits justify the erection of the novel phylum Sanchytriomycota. Our results also show that most of the hyphal morphogenesis gene repertoire of multicellular Fungi had already evolved in early holomycotan lineages.