How good are global DNA-based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation.

Metabarcoding approaches targeting microeukaryotes have deeply changed our vision of protist environmental diversity. The public repository EukBank consists of 18S v4 metabarcodes from 12,672 samples worldwide. To estimate how far this database provides a reasonable overview of all eukaryotic diversity, we used Arcellinida (lobose testate amoebae) as a case study. We hypothesised that (1) this approach would allow the discovery of unexpected diversity, but also that (2) some groups would be underrepresented because of primer/sequencing biases. Most of the Arcellinida sequences appeared in freshwater and soil, but their abundance and diversity appeared underrepresented. Moreover, 84% of ASVs belonged to the suborder Phryganellina, a supposedly species-poor clade, whereas the best-documented suborder (Glutinoconcha, 600 described species) was only marginally represented. We explored some possible causes of these biases. Mismatches in the primer-binding site seem to play a minor role. Excessive length of the target region could explain some of these biases, but not all. There must be some other unknown factors involved. Altogether, while metabarcoding based on ribosomal genes remains a good first approach to document microbial eukaryotic clades, alternative approaches based on other genes or sequencing techniques must be considered for an unbiased picture of the diversity of some groups.

Discrepancies between prokaryotes and eukaryotes need to be considered in soil DNA-based studies.

Metabarcoding approaches are exponentially increasing our understanding of soil biodiversity, with a major focus on the bacterial part of the microbiome. Part of the soil diversity are also eukaryotes that include fungi, algae, protists and Metazoa. Nowadays, soil eukaryotes are targeted with the same approaches developed for bacteria and archaea (prokaryotes). However, fundamental differences exist between domains. After providing a short historical overview of the developments of metabarcoding applied to environmental microbiology, we compile the most important differences between domains that prevent direct method transfers between prokaryotic and eukaryotic soil metabarcoding approaches, currently dominated by short-read sequencing. These include the existence of divergent diversity concepts and the variations in eukaryotic morphology that affect sampling and DNA extraction. Furthermore, eukaryotes experienced much more variable evolutionary rates than prokaryotes, which prevent capturing the entire eukaryotic diversity in a soil with a single amplification protocol fit for short-read sequencing. In the final part we focus on future potentials for optimization of eukaryotic metabarcoding that include superior possibility of functionally characterizing eukaryotes and to extend the current information obtained, such as by adding a real quantitative component. This review should optimize future metabarcoding approaches targeting soil eukaryotes and kickstart this promising research direction.

Freshwater protists: unveiling the unexplored in a large floodplain system.

Protists play a fundamental role in all ecosystems, but we are still far from estimating the total diversity of many lineages, in particular in highly diverse environments, such as freshwater. Here, we survey the protist diversity of the Paraná River using metabarcoding, and we applied an approach that includes sequence similarity and phylogeny to evaluate the degree of genetic novelty of the protists' communities against the sequences described in the reference database PR2 . We observed that ~28% of the amplicon sequence variants were classified as novel according to their similarity with sequences from the reference database; most of them were related to heterotrophic groups traditionally overlooked in freshwater systems. This lack of knowledge extended to those groups within the green algae (Archaeplastida) that are well documented such as Mamiellophyceae, and also to the less studied Pedinophyceae, for which we found sequences representing novel deep-branching clusters. Among the groups with potential novel protists, Bicosoecida (Stramenopiles) were the best represented, followed by Codosiga (Opisthokonta), and the Perkinsea (Alveolata). This illustrates the lack of knowledge on freshwater planktonic protists and also the need for isolation and/or cultivation of new organisms to better understand their role in ecosystem functioning.

Soil protist function varies with elevation in the Swiss Alps.

Protists are abundant and play key trophic functions in soil. Documenting how their trophic contributions vary across large environmental gradients is essential to understand and predict how biogeochemical cycles will be impacted by global changes. Here, using amplicon sequencing of environmental DNA in open habitat soil from 161 locations spanning 2600 m of elevation in the Swiss Alps (from 400 to 3000 m), we found that, over the whole study area, soils are dominated by consumers, followed by parasites and phototrophs. In contrast, the proportion of these groups in local communities shows large variations in relation to elevation. While there is, on average, three times more consumers than parasites at low elevation (400-1000 m), this ratio increases to 12 at high elevation (2000-3000 m). This suggests that the decrease in protist host biomass and diversity toward mountains tops impact protist functional composition. Furthermore, the taxonomic composition of protists that infect animals was related to elevation while that of protists that infect plants or of protist consumers was related to soil pH. This study provides a first step to document and understand how soil protist functions vary along the elevational gradient.

Contribution of soil algae to the global carbon cycle.

Soil photoautotrophic prokaryotes and micro-eukaryotes - known as soil algae - are, together with heterotrophic microorganisms, a constitutive part of the microbiome in surface soils. Similar to plants, they fix atmospheric carbon (C) through photosynthesis for their own growth, yet their contribution to global and regional biogeochemical C cycling still remains quantitatively elusive. Here, we compiled an extensive dataset on soil algae to generate a better understanding of their distribution across biomes and predict their productivity at a global scale by means of machine learning modelling. We found that, on average, (5.5 ± 3.4) × 106 algae inhabit each gram of surface soil. Soil algal abundance especially peaked in acidic, moist and vegetated soils. We estimate that, globally, soil algae take up around 3.6 Pg C per year, which corresponds to c. 6% of the net primary production of terrestrial vegetation. We demonstrate that the C fixed by soil algae is crucial to the global C cycle and should be integrated into land-based efforts to mitigate C emissions.

Cyphoderia ampulla (Cyphoderiidae: Rhizaria), a tale of freshwater sailors: The causes and consequences of ecological transitions through the salinity barrier in a family of benthic protists.

The salinity barrier that separates marine and freshwater biomes is probably the most important division in biodiversity on Earth. Those organisms that successfully performed this transition had access to new ecosystems while undergoing changes in selective pressure, which often led to major shifts in diversification rates. While these transitions have been extensively investigated in animals, the tempo, mode, and outcome of crossing the salinity barrier have been scarcely studied in other eukaryotes. Here, we reconstructed the evolutionary history of the species complex Cyphoderia ampulla (Euglyphida: Cercozoa: Rhizaria) based on DNA sequences from the nuclear SSU rRNA gene and the mitochondrial cytochrome oxidase subunit I gene, obtained from publicly available environmental DNA data (GeneBank, EukBank) and isolated organisms. A tree calibrated with euglyphid fossils showed that four independent transitions towards freshwater systems occurred from the mid-Miocene onwards, coincident with important fluctuations in sea level. Ancestral trait reconstructions indicated that the whole family Cyphoderiidae had a marine origin and suggest that ancestors of the freshwater forms were euryhaline and lived in environments with fluctuating salinity. Diversification rates did not show any obvious increase concomitant with ecological transitions, but morphometric analyses indicated that species increased in size and homogenized their morphology after colonizing the new environments. This suggests adaptation to changes in selective pressure exerted by life in freshwater sediments.

Deconstructing Difflugia: The tangled evolution of lobose testate amoebae shells (Amoebozoa: Arcellinida) illustrates the importance of convergent evolution in protist phylogeny.

Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.

Niche Conservatism Drives the Elevational Diversity Gradient in Major Groups of Free-Living Soil Unicellular Eukaryotes.

Ancestral adaptations to tropical-like climates drive most multicellular biogeography and macroecology. Observational studies suggest that this niche conservatism could also be shaping unicellular biogeography and macroecology, although evidence is limited to Acidobacteria and testate amoebae. We tracked the phylogenetic signal of this niche conservatism in far related and functionally contrasted groups of common soil protists (Bacillariophyta, Cercomonadida, Ciliophora, Euglyphida and Kinetoplastida) along a humid but increasingly cold elevational gradient in Switzerland. Protist diversity decreased, and the size of the geographic ranges of taxa increased with elevation and associated decreasing temperature (climate), which is consistent with a macroecological pattern known as the Rapoport effect. Bacillariophyta exhibited phylogenetically overdispersed communities assembled by competitive exclusion of closely related taxa with shared (conserved) niches. By contrast, Cercomonadida, Ciliophora, Euglyphida and Kinetoplastida exhibited phylogenetically clustered communities assembled by habitat filtering, revealing the coexistence of closely related taxa with shared (conserved) adaptations to cope with the humid but temperate to cold climate of the study site. Phylobetadiversity revealed that soil protists exhibit a strong phylogenetic turnover among elevational sites, suggesting that most taxa have evolutionary constraints that prevent them from colonizing the colder and higher sites of the elevation gradient. Our results suggest that evolutionary constraints determine how soil protists colonize climates departing from warm and humid conditions. We posit that these evolutionary constraints are linked to an ancestral adaptation to tropical-like climates, which limits their survival in exceedingly cold sites. This niche conservatism possibly drives their biogeography and macroecology along latitudinal and altitudinal climatic gradients.

Description of Phaeobola aeris gen. nov., sp. nov (Rhizaria, Cercozoa, Euglyphida) Sheds Light on Euglyphida's Dark Matter.

The majority of Euglyphida species are characterised by shells with imbricated silica scales. Environmental surveys indicate a large unexplored diversity and recent efforts hinted at a certain diversity of yet undescribed, inconspicuous, scale-lacking Euglyphida. Here we describe Phaeobola aeris gen. nov., sp. nov. that shows a variety of morphological characters typical for the Euglyphida but lacks silica scales-instead, this species bears an agglutinated test. Neither its morphology nor phylogenetic placement allows its assignment to any currently described family. We erected the yet monospecific genus Phaeobola gen. nov., which with yet available data remain Euglyphida incertae sedis.

Superficially described and ignored for 92 years, rediscovered and emended: Apodera angatakere (Amoebozoa: Arcellinida: Hyalospheniformes) is a new flagship testate amoeba taxon from Aotearoa (New Zealand).

Eukaryotic microbial diversity is known to be extensive but remains largely undescribed and uncharted. While much of this unknown diversity is composed of inconspicuous flagellates and parasites, larger and morphologically distinct protists are regularly discovered, most notably from poorly studied regions. Here we report a new flagship species of hyalospheniid (Amoebozoa; Arcellinida; Hyalospheniformes) testate amoeba from New Zealand and an unusual story of overlooked description under a preoccupied name and subsequent oversight for nearly one century. Through a process involving The Maori Language Commission, we named the species Apodera angatakere, meaning "a shell with a keel." This species resembles Apodera vas but differs by the presence of a distinctive hollow keel. Cytochrome Oxidase Subunit 1 (COI) sequence data show that this species forms a distinct clade nested within genus Apodera. This conspicuous species is so far known only from New Zealand and is restricted to peatlands. It is one of the few examples of endemic microorganisms from this biodiversity hotspot and biogeographer's paradise. As over 90% of New Zealand's peatlands have been lost since European colonization and much of the remaining surfaces are threatened, Apodera angatakere could be a flagship species not only for microbial biogeography but also for island biodiversity conservation.

Perceptions of Change in the Natural Environment produced by the First Wave of the COVID-19 Pandemic across Three European countries. Results from the GreenCOVID study.

Although different studies have evaluated the positive impacts of the COVID-19 pandemic and lockdown measures on reducing noise pollution and traffic levels and improving air quality, how populations have perceived such changes in the natural environment has not been adequately evaluated. The present study provides a more in-depth exploration of human population perception of enhanced natural exposure (to animal life and nature sounds) and reduced harmful exposure (by improved air quality and reduced traffic volume) as a result of the COVID-19 pandemic lockdown. The data is drawn from 3,109 unselected adults who participated in the GreenCOVID survey from April to July 2020 in England, Ireland, and Spain. The findings suggest that the positive impacts to the natural environment as a result of the lockdown have been better received by the population in Spain and Ireland, in comparison to England. Participants who resided in urban areas had better perceived improvements in nature sounds, air quality, and traffic volume compared to those in rural areas. Older populations and those with lower smoking and alcohol consumption were found to perceive this improvement the most. Furthermore, the greater perception of improvements in environmental elements was also associated with better self-perceived health and improved wellbeing. In the binary logistic regression, living in Ireland or Spain, urban areas, female gender, older age, and good overall wellbeing were associated with a greater perception of improvements in the natural environment, while the factors most associated with a greater perception of reduced harmful exposure were living in Spain, had a good self-perceived health status and older age.

Greater topoclimatic control of above- versus below-ground communities.

Assessing the degree to which climate explains the spatial distributions of different taxonomic and functional groups is essential for anticipating the effects of climate change on ecosystems. Most effort so far has focused on above-ground organisms, which offer only a partial view on the response of biodiversity to environmental gradients. Here including both above- and below-ground organisms, we quantified the degree of topoclimatic control on the occurrence patterns of >1,500 taxa and phylotypes along a c. 3,000 m elevation gradient, by fitting species distribution models. Higher model performances for animals and plants than for soil microbes (fungi, bacteria and protists) suggest that the direct influence of topoclimate is stronger on above-ground species than on below-ground microorganisms. Accordingly, direct climate change effects are predicted to be stronger for above-ground than for below-ground taxa, whereas factors expressing local soil microclimate and geochemistry are likely more important to explain and forecast the occurrence patterns of soil microbiota. Detailed mapping and future scenarios of soil microclimate and microhabitats, together with comparative studies of interacting and ecologically dependent above- and below-ground biota, are thus needed to understand and realistically forecast the future distribution of ecosystems.

Correction to: Rain-Fed Granite Rock Basins Accumulate a High Diversity of Dormant Microbial Eukaryotes.

The original version of this article contained an erratum of omission in the Acknowledgments section.

Rain-Fed Granite Rock Basins Accumulate a High Diversity of Dormant Microbial Eukaryotes.

Rain fed granite rock basins are ancient geological landforms of worldwide distribution and structural simplicity. They support habitats that can switch quickly from terrestrial to aquatic along the year. Diversity of animals and plants, and the connexion between communities in different basins have been widely explored in these habitats, but hardly any research has been carried out on microorganisms. The aim of this study is to provide the first insights on the diversity of eukaryotic microbial communities from these environments. Due to the ephemeral nature of these aquatic environments, we predict that the granitic basins should host a high proportion of dormant microeukaryotes. Based on an environmental DNA diversity survey, we reveal diverse communities with representatives of all major eukaryotic taxonomic supergroups, mainly composed of a diverse pool of low abundance OTUs. Basin communities were very distinctive, with alpha and beta diversity patterns non-related to basin size or spatial distance respectively. Dissimilarity between basins was mainly characterised by turnover of OTUs. The strong microbial eukaryotic heterogeneity observed among the basins may be explained by a complex combination of deterministic factors (diverging environment in the basins), spatial constraints, and randomness including founder effects. Most interestingly, communities contain organisms that cannot coexist at the same time because of incompatible metabolic requirements, thus suggesting the existence of a pool of dormant organisms whose activity varies along with the changing environment. These organisms accumulate in the pools, which turns granitic rock into high biodiversity microbial islands whose conservation and study deserve further attention.

Assessing the life-cycle sustainability of algae and bacteria-based wastewater treatment systems: High-rate algae pond and sequencing batch reactor.

High Rate Algae Ponds (HRAPs) are a promising technology for the treatment of municipal wastewater in locations with sufficient space and solar radiation. Algae-based processes do not require aeration, and thus have the potential to be less energy-intensive than activated sludge processes. We used a combination of LCA and LCCA analysis to evaluate the sustainability of HRAP systems, using data from the construction and operation of two demonstration-scale systems in Almería and Cádiz, Spain. As a reference for comparison, we used data from an activated sludge-based Sequencing Batch Reactor (SBR) treatment system in operation in Leppersdorf, Germany, which has comparable removal rates for a similar inflow. We focused solely on the actual wastewater treatment aspect of these technologies, excluding sludge treatment from this analysis. Based on our analysis, the current HRAP technology is more energy-efficient than activated sludge-based SBRs and requires only 22% of its electricity consumption. In addition, HRAP is more advantageous both economically (0.18 €/m3 versus 0.26 €/m3) and environmentally, with both lower global warming and eutrophication potentials (146.27 vs. 458.27 × 10-3 kg CO2 equiv./m3; 126.14 vs. 158.01 × 10-6 kg PO4 equiv./m3). However, the Net Environmental Benefit of SBR was slightly more favorable than of HRAP because of the higher removal rate for nutrients of SBR.

Global distribution of Trebouxiophyceae diversity explored by high-throughput sequencing and phylogenetic approaches.

Trebouxiophyceae are a ubiquitous class of Chlorophyta encountered in aquatic and terrestrial environments. Most taxa are photosynthetic, and many acts as photobionts in symbiotic relationships, while others are free-living. Trebouxiophyceae have also been widely investigated for their use for biotechnological applications. In this work, we aimed at obtaining a comprehensive image of their diversity by compiling the information of 435 freshwater, soil and marine environmental DNA samples surveyed with Illumina sequencing technology in order to search for the most relevant environments for bioprospecting. Freshwater and soil were most diverse and shared more than half of all operational taxonomic units (OTUs), however, their communities were significantly distinct. Oceans hosted the highest genetic novelty, and did not share any OTUs with the other environments; also, marine samples host more diversity in warm waters. Symbiotic genera usually found in lichens such as Trebouxia, Myrmecia and Symbiochloris were also abundantly detected in the ocean, suggesting either free-living lifestyles or unknown symbiotic relationships with marine planktonic organisms. Altogether, our study opens the way to new prospection for trebouxiophycean strains, especially in understudied environments like the ocean.

Dispersal limitations and historical factors determine the biogeography of specialized terrestrial protists.

Recent studies show that soil eukaryotic diversity is immense and dominated by micro-organisms. However, it is unclear to what extent the processes that shape the distribution of diversity in plants and animals also apply to micro-organisms. Major diversification events in multicellular organisms have often been attributed to long-term climatic and geological processes, but the impact of such processes on protist diversity has received much less attention as their distribution has often been believed to be largely cosmopolitan. Here, we quantified phylogeographical patterns in Hyalosphenia papilio, a large testate amoeba restricted to Holarctic Sphagnum-dominated peatlands, to test if the current distribution of its genetic diversity can be explained by historical factors or by the current distribution of suitable habitats. Phylogenetic diversity was higher in Western North America, corresponding to the inferred geographical origin of the H. papilio complex, and was lower in Eurasia despite extensive suitable habitats. These results suggest that patterns of phylogenetic diversity and distribution can be explained by the history of Holarctic Sphagnum peatland range expansions and contractions in response to Quaternary glaciations that promoted cladogenetic range evolution, rather than the contemporary distribution of suitable habitats. Species distributions were positively correlated with climatic niche breadth, suggesting that climatic tolerance is key to dispersal ability in H. papilio. This implies that, at least for large and specialized terrestrial micro-organisms, propagule dispersal is slow enough that historical processes may contribute to their diversification and phylogeographical patterns and may partly explain their very high overall diversity.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Contrasted Micro-Eukaryotic Diversity Associated with Sphagnum Mosses in Tropical, Subtropical and Temperate Climatic Zones.

Sphagnum-dominated ecosystem plays major roles as carbon sinks at the global level. Associated microbial communities, in particular, eukaryotes, play significant roles in nutrient fixation and turnover. In order to understand better the ecological processes driven by these organisms, the first step is to characterise these associated organisms. We characterised the taxonomic diversity, and from this, inferred the functional diversity of microeukaryotes in Sphagnum mosses in tropical, subtropical and temperate climatic zones through an environmental DNA diversity metabarcoding survey of the V9 region of the gene coding for the RNA of the small subunit of the ribosomes (SSU rRNA). As microbial processes are strongly driven by temperatures, we hypothesised that saprotrophy would be highest in warm regions, whereas mixotrophy, an optimal strategy in oligotrophic environments, would peak under colder climates. Phylotype richness was higher in tropical and subtropical climatic zones than in the temperate region, mostly due to a higher diversity of animal parasites (i.e. Apicomplexa). Decomposers, and especially opportunistic yeasts and moulds, were more abundant under warmer climates, while mixotrophic organisms were more abundant under temperate climates. The dominance of decomposers, suggesting a higher heterotrophic activity under warmer climates, is coherent with the generally observed faster nutrient cycling at lower latitudes; this phenomenon is likely enhanced by higher inputs of nutrients most probably brought in the system by Metazoa, such as arthropods.

Environmental filtering and phylogenetic clustering correlate with the distribution patterns of cryptic protist species.

The community composition of any group of organisms should theoretically be determined by a combination of assembly processes including resource partitioning, competition, environmental filtering, and phylogenetic legacy. Environmental DNA studies have revealed a huge diversity of protists in all environments, raising questions about the ecological significance of such diversity and the degree to which they obey to the same rules as macroscopic organisms. The fast-growing cultivable protist species on which hypotheses are usually experimentally tested represent only a minority of the protist diversity. Addressing these questions for the lesser known majority can only be inferred through observational studies. We conducted an environmental DNA survey of the genus Nebela, a group of closely related testate (shelled) amoeba species, in different habitats within Sphagnum-dominated peatlands. Identification based on the mitochondrial cytochrome c oxidase 1 gene, allowed species-level resolution as well as phylogenetic reconstruction. Community composition varied strongly across habitats and associated environmental gradients. Species showed little overlap in their realized niche, suggesting resource partitioning, and a strong influence of environmental filtering driving community composition. Furthermore, phylogenetic clustering was observed in the most nitrogen-poor samples, supporting phylogenetic inheritance of adaptations in the group of N. guttata. This study showed that the studied free-living unicellular eukaryotes follow to community assembly rules similar to those known to determine plant and animal communities; the same may be true for much of the huge functional and taxonomic diversity of protists.