Aphelids are little-known phagotrophic parasites of algae whose life cycle and morphology resemble those of the parasitic rozellids (Cryptomycota, Rozellomycota). In previous phylogenetic analyses of RNA polymerase and rRNA genes, aphelids, rozellids and Microsporidia (parasites of animals) formed a clade, named Opisthosporidia, which appeared as the sister group to Fungi. However, the statistical support for the Opisthosporidia was always moderate. Here, we generated full life-cycle transcriptome data for the aphelid species Paraphelidium tribonemae. In-depth multi-gene phylogenomic analyses using several protein datasets place this aphelid as the closest relative of fungi to the exclusion of rozellids and Microsporidia. In contrast with the comparatively reduced Rozella allomycis genome, we infer a rich, free-living-like aphelid proteome, with a metabolism similar to fungi, including cellulases likely involved in algal cell-wall penetration and enzymes involved in chitin biosynthesis. Our results suggest that fungi evolved from complex aphelid-like ancestors that lost phagotrophy and became osmotrophic.

Nucleariid amoebae (Opisthokonta) have been known since the nineteenth century but their diversity and evolutionary history remain poorly understood. To overcome this limitation, we have obtained genomic and transcriptomic data from three Nuclearia , two Pompholyxophrys and one Lithocolla species using traditional culturing and single-cell genome (SCG) and single-cell transcriptome amplification methods. The phylogeny of the complete 18S rRNA sequences of Pompholyxophrys and Lithocolla confirmed their suggested evolutionary relatedness to nucleariid amoebae, although with moderate support for internal splits. SCG amplification techniques also led to the identification of probable bacterial endosymbionts belonging to Chlamydiales and Rickettsiales in Pompholyxophrys . To improve the phylogenetic framework of nucleariids, we carried out phylogenomic analyses based on two datasets of, respectively, 264 conserved proteins and 74 single-copy protein domains. We obtained full support for the monophyly of the nucleariid amoebae, which comprise two major clades: (i) Parvularia–Fonticula and (ii) Nuclearia with the scaled genera Pompholyxophrys and Lithocolla . Based on these findings, the evolution of some traits of the earliest-diverging lineage of Holomycota can be inferred. Our results suggest that the last common ancestor of nucleariids was a freshwater, bacterivorous, non-flagellated filose and mucilaginous amoeba. From the ancestor, two groups evolved to reach smaller ( Parvularia–Fonticula ) and larger ( Nuclearia and related scaled genera) cell sizes, leading to different ecological specialization. The Lithocolla + Pompholyxophrys clade developed exogenous or endogenous cell coverings from a Nuclearia -like ancestor. This article is part of a discussion meeting issue ‘Single cell ecology’.

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The genus Diaphoropodon, Archer 1869, comprises filose amoebae with agglutinated tests made of quartz grains, diatom frustules and other particulate materials. The key trait of the genus is a hyaline theca covered with numerous 5- to 10-μm-long, hairlike rods. Based on SSU rDNA phylogeny, we show that Diaphoropodon groups closely to Lecythium, a testate amoeba genus with a flexible but naked theca. Electron microscopic images reveal that the rods of Diaphoropodon are not perforating the test but lie randomly distributed on the surface of the amoeba. Comparing fairly naked cells from our cultures with cells from the environment leads to the conclusion that these rods play a role in agglutinating the material on the test.

Aphelids are poorly known phagotrophic parasites of algae whose life cycle and morphology resemble those of the widely diverse parasitic rozellids (Cryptomycota, Rozellomycota). In previous phylogenetic analyses of RNA polymerase and rRNA genes, aphelids and rozellids formed a monophyletic group together with the extremely reduced parasitic Microsporidia, named Opisthosporidia, which was sister to Fungi. However, the statistical support for that group was always moderate. We generated the first transcriptome data for one aphelid species, Paraphelidium tribonemae. In-depth multi-gene phylogenomic analyses using various protein datasets place aphelids as the closest relatives of Fungi to the exclusion of rozellids and Microsporidia. In contrast with the comparatively reduced Rozella allomycis genome, we infer a rich, free-living-like aphelid proteome, including cellulases likely involved in algal cell-wall penetration, enzymes involved in chitin biosynthesis and several metabolic pathways. Our results suggest that Fungi evolved from a complex aphelid-like ancestor that lost phagotrophy and became osmotrophic.