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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’.

Metchnikovellids are highly specialized hyperparasites, which infect and reproduce inside gregarines (Apicomplexa) inhabiting marine invertebrates. Their phylogenetic affiliation was under constant discussion until recently, when analysis of the first near-complete metchnikovellid genome, that of Amphiamblys sp., placed it in a basal position with respect to most Microsporidia. Microsporidia are a highly diversified lineage of extremely reduced parasites related to Rozellida (Rozellosporidia = Rozellomycota = Cryptomycota) within the Holomycota clade of Opisthokonta. By sequencing DNA from a single-isolated infected gregarine cell we obtained an almost complete genome of a second metchnikovellid species, and the first one of a taxonomically described and well-documented species, Metchnikovella incurvata. Our phylogenomic analyses show that, despite being considerably divergent from each other, M. incurvata forms a monophyletic group with Amphiamplys sp., and confirm that metchnikovellids are one of the deep branches of Microsporidia. Comparative genomic analysis demonstrates that, like most Microsporidia, metchnikovellids lack mitochondrial genes involved in energy transduction and are thus incapable of synthesizing their own ATP via mitochondrial oxidative phosphorylation. They also lack the horizontally acquired ATP transporters widespread in most Microsporidia. We hypothesize that a family of mitochondrial carrier proteins evolved to transport ATP from the host into the metchnikovellid cell. We observe the progressive reduction of genes involved in DNA repair pathways along the evolutionary path of Microsporidia, which might explain, at least partly, the extremely high evolutionary rate of the most derived species. Our data also suggest that genome reduction and acquisition of novel genes co-occurred during the adaptation of Microsporidia to their hosts.

Extracting DNA from gastropods presents particular difficulties due to the capacity of the living animal to retract into the shell, resulting in poor penetration of the ethanol into the tissues. Because the shell is essential to establish the link between sequences and traditional taxonomic identity, cracking the shell to facilitate fixation is not ideal. Several methods are currently in routine use to overcome this difficulty, including chemical relaxation, drilling the shell and boiling. Most of these methods are time-consuming, may be safety hazards and constitute a bottleneck in the preparation of large numbers of specimens in the field. We have experimented with a method traditionally used to clean shells that involves placing the living gastropods in a microwave (MW) oven; the electromagnetic radiation very quickly heats both the animal and the water trapped inside the shell, resulting in separation of the muscles that anchor the animal to the shell. Done properly, the body can be removed intact from the shell and the shell voucher is preserved undamaged. To test the method, the bodies of live-collected specimens from two gastropod species were separated from their shell by microwaving and by anesthetizing/drilling. After identical extraction and PCR procedures, the gels showed no difference in DNA quantity or quality, and the resulting sequences are identical within species. The method was then implemented on a large scale during expeditions, resulting in higher percentage of DNA extraction success. The MWs are also effective for quickly and easily removing other molluscs from their shells, that is, bivalves and scaphopods. Workflows implementing the MW technique show a three- to fivefold increase in productivity compared with other methods.

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.

Compared to multicellular fungi and unicellular yeasts, unicellular fungi with free-living flagellated stages (zoospores) remain poorly known and their phylogenetic position is often unresolved. Recently, rRNA gene phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a monophyletic group without close affinity with known fungal clades. Here, we sequence single-cell genomes for both species to assess their phylogenetic position and evolution. Phylogenomic analyses using different protein datasets and a comprehensive taxon sampling result in an almost fully-resolved fungal tree, with Chytridiomycota as sister to all other fungi, and sanchytrids forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Comparative genomic analyses across fungi and their allies (Holomycota) reveal an atypically reduced metabolic repertoire for sanchytrids. We infer three main independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. Based on sanchytrids’ phylogenetic position and unique traits, we propose the designation of a novel phylum, Sanchytriomycota. In addition, our results indicate that most of the hyphal morphogenesis gene repertoire of multicellular fungi had already evolved in early holomycotan lineages.

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