A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.

Animals and fungi independently evolved from the protozoan phylum Choanozoa, these three groups constituting a major branch of the eukaryotic evolutionary tree known as opisthokonts. Opisthokonts and the protozoan phylum Amoebozoa (amoebae plus slime moulds) were previously argued to have evolved independently from the little-studied, largely flagellate, protozoan phylum, Sulcozoa. Sulcozoa are a likely evolutionary link between opisthokonts and the more primitive excavate flagellates that have ventral feeding grooves and the most primitive known mitochondria. To extend earlier sparse evidence for the ancestral (paraphyletic) nature of Sulcozoa, we sequenced transcriptomes from six gliding flagellates (two apusomonads; three planomonads; Mantamonas). Phylogenetic analyses of 173-192 genes and 73-122 eukaryote-wide taxa show Sulcozoa as deeply paraphyletic, confirming that opisthokonts and Amoebozoa independently evolved from sulcozoans by losing their ancestral ventral groove and dorsal pellicle: Apusozoa (apusomonads plus anaerobic breviate amoebae) are robustly sisters to opisthokonts and probably paraphyletic, breviates diverging before apusomonads; Varisulca (planomonads, Mantamonas, and non-gliding flagellate Collodictyon) are sisters to opisthokonts plus Apusozoa and Amoebozoa, and possibly holophyletic; Glissodiscea (planomonads, Mantamonas) may be holophyletic, but Mantamonas sometimes groups with Collodictyon instead. Taxon and gene sampling slightly affects tree topology; for the closest branches in Sulcozoa and opisthokonts, proportionally reducing missing data eliminates conflicts between homogeneous-model maximum-likelihood trees and evolutionarily more realistic site-heterogeneous trees. Sulcozoa, opisthokonts, and Amoebozoa constitute an often-pseudopodial 'podiate' clade, one of only three eukaryotic 'supergroups'. Our trees indicate that evolution of sulcozoan dorsal pellicle, ventral pseudopodia, and ciliary gliding (probably simultaneously) generated podiate eukaryotes from Malawimonas-like excavate flagellates.

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost-effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new-generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%-100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.

Protists are the most diverse eukaryotes. These microbes are keystone organisms of soil ecosystems and regulate essential processes of soil fertility such as nutrient cycling and plant growth. Despite this, protists have received little scientific attention, especially compared to bacteria, fungi and nematodes in soil studies. Recent methodological advances, particularly in molecular biology techniques, have made the study of soil protists more accessible, and have created a resurgence of interest in soil protistology. This ongoing revolution now enables comprehensive investigations of the structure and functioning of soil protist communities, paving the way to a new era in soil biology. Instead of providing an exhaustive review, we provide a synthesis of research gaps that should be prioritized in future studies of soil protistology to guide this rapidly developing research area. Based on a synthesis of expert opinion we propose 30 key questions covering a broad range of topics including evolution, phylogenetics, functional ecology, macroecology, paleoecology, and methodologies. These questions highlight a diversity of topics that will establish soil protistology as a hub discipline connecting different fundamental and applied fields such as ecology, biogeography, evolution, plant-microbe interactions, agronomy, and conservation biology. We are convinced that soil protistology has the potential to be one of the most exciting frontiers in biology

Land-use intensification can increase provisioning ecosystem services, such as food and timber production, but it also drives changes in ecosystem functioning and biodiversity loss, which may ultimately compromise human wellbeing. To understand how changes in land-use intensity affect the relationships between biodiversity, ecosystem functions, and services, we built networks from correlations between the species richness of 16 trophic groups, 10 ecosystem functions, and 15 ecosystem services. We evaluated how the properties of these networks varied across land-use intensity gradients for 150 forests and 150 grasslands. Land-use intensity significantly affected network structure in both habitats. Changes in connectance were larger in forests, while changes in modularity and evenness were more evident in grasslands. Our results show that increasing land-use intensity leads to more homogeneous networks with less integration within modules in both habitats, driven by the belowground compartment in grasslands, while forest responses to land management were more complex. Land-use intensity strongly altered hub identity and module composition in both habitats, showing that the positive correlations of provisioning services with biodiversity and ecosystem functions found at low land-use intensity levels, decline at higher intensity levels. Our approach provides a comprehensive view of the relationships between multiple components of biodiversity, ecosystem functions, and ecosystem services and how they respond to land use. This can be used to identify overall changes in the ecosystem, to derive mechanistic hypotheses, and it can be readily applied to further global change drivers.

Soil protists are increasingly appreciated as essential components of soil foodwebs; however, there is a dearth of information on the factors structuring their communities. Here we investigate the importance of different biotic and abiotic factors as key drivers of spatial and seasonal distribution of protistan communities. We conducted an intensive survey of a 10 m 2 grassland plot in Germany, focusing on a major group of protists, the Cercozoa. From 177 soil samples, collected from April to November, we obtained 694 Operational Taxonomy Units representing >6 million Illumina reads. All major cercozoan taxonomic and functional groups were present, dominated by the small flagellates of the Glissomonadida. We found evidence of environmental selection structuring the cercozoan communities both spatially and seasonally. Spatial analyses indicated that communities were correlated within a range of 3.5 m. Seasonal variations in the abundance of bacterivores and bacteria, followed by that of omnivores suggested a dynamic prey-predator succession. The most influential edaphic properties were moisture and clay content, which differentially affected each functional group. Our study is based on an intense sampling of protists at a small scale, thus providing a detailed description of the biodiversity of different taxa/functional groups and the ecological processes involved in shaping their distribution.

Amoebozoa is a key phylum for eukaryote phylogeny and evolutionary history, but its phylogenetic validity has been questioned since included species are very diverse: amoebo-flagellate slime-moulds, naked and testate amoebae, and some flagellates. 18S rRNA gene trees have not firmly established its internal topology. To rectify this we sequenced cDNA libraries for seven diverse Amoebozoa and conducted phylogenetic analyses for 109 eukaryotes (17-18 Amoebozoa) using 60-188 genes. We conducted Bayesian inferences with the evolutionarily most realistic site-heterogeneous CAT-GTR-Γ model and maximum likelihood analyses. These unequivocally establish the monophyly of Amoebozoa, showing a primary dichotomy between the previously contested subphyla Lobosa and Conosa. Lobosa, the entirely non-flagellate lobose amoebae, are robustly partitioned into the monophyletic classes Tubulinea, with predominantly tube-shaped pseudopodia, and Discosea with flattened cells and different locomotion. Within Conosa 60/70-gene trees with very little missing data show a primary dichotomy between the aerobic infraphylum Semiconosia (Mycetozoa and Variosea) and secondarily anaerobic Archamoebae. These phylogenetic features are entirely congruent with the most recent major amoebozoan classification emphasising locomotion modes, pseudopodial morphology, and ultrastructure. However, 188-gene trees where proportionally more taxa have sparser gene-representation weakly place Archamoebae as sister to Macromycetozoa instead, possibly a tree reconstruction artefact of differentially missing data.

We have compiled a database of functional traits for two widespread and ecologically important groups of protists, Cercozoa and Endomyxa (Rhizaria). The functional traits of microorganisms are crucially important for interpreting results from environmental sequencing surveys. Linking morphological and ecological traits to environmental factors is common practice in studies involving micro‐ and macroorganisms, but is rarely applied to protists. Our database provides functional and ecologically significant traits linked to morphology, nutrition, locomotion and habitats. We discuss how the use of functional traits may help to unveil underlying ecosystem processes. This database is intended as a common reference for the molecular ecology community and will boost the understanding of ecosystem functions, especially those driven by biological interactions.