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A library of eight different cationic emitters with emission properties in solution and in solid-state (solution and solid-state emitters – SSSE) is presented. These compounds, bearing either ammonium or pyridinium groups, have been investigated regarding their photophysical properties as well as their potential application in biological imaging. Besides high quantum yields as well as a high degree of stability during the imaging process, it was additionally revealed that a broad range of biological targets can be addressed, such as different bacterial strains, human cells as well as protists. The reported SSSE approach employing the mentioned robust emitters for biological imaging, will contribute to a rapid and facile way to design and apply affordable emitters with outstanding properties. Additionally, these emitters will overcome the drawbacks of classical luminophores and agents featuring well-known aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ) properties. in press

Taxonomic assignment of OTUs is an important bioinformatics step in analyzing environmental sequencing data. Pairwise-alignment and phylogenetic-placement methods represent two alternative approaches to taxonomic assignments, but their results can differ. Here we used available colpodean ciliate OTUs from forest soils to compare the taxonomic assignments of VSEARCH (which performs pairwise alignments) and EPA-ng (which performs phylogenetic placements). We showed that when there are differences in taxonomic assignments between pairwise alignments and phylogenetic placements at the subtaxon level, there is a low pairwise similarity of the OTUs to the reference database. We then showcase how the output of EPA-ng can be further evaluated using GAPPA to assess the taxonomic assignments when there exist multiple equally likely placements of an OTU, by taking into account the sum over the likelihood weights of the OUT placements within a subtaxon, and the branch distances between equally likely placement locations. We also inferred evolutionary and ecological characteristics of the colpodean OTUs using their placements within subtaxa. This study demonstrates how to fully analyse the output of EPA-ng, by using GAPPA in conjunction with knowledge of the taxonomic diversity of the clade of interest.

Species may co-occur due to responses to similar environmental conditions, biological associations, or simply because of coincident geographical distributions. Disentangling patterns of co-occurrence and potential biotic and abiotic interactions is crucial to understand ecosystem function. Here we used DNA metabarcoding data from litter and mineral soils collected from a longitudinal transect in Amazonia to explore patterns of co-occurrence. We compared data from different Amazonian habitat types, each with a characteristic biota and environmental conditions. These included non-flooded rainforests (terra-firme), forests seasonally flooded by fertile white waters (várzeas) or by unfertile black waters (igapós), and open areas associated with white sand soil (campinas). We ran co-occurrence network analyses based on null models and Spearman correlation for all samples and for each habitat separately. We found that one third of all operational taxonomic units (OTUs) were bacteria and two thirds were eukaryotes. The resulting networks were nevertheless mostly composed of bacteria, with fewer fungi, protists, and metazoans. Considering the functional traits of the OTUs, there is a combination of metabolism modes including respiration and fermentation for bacteria, and a high frequency of saprotrophic fungi (those that feed on dead organic matter), indicating a high turnover of organic material. The organic carbon and base saturation indices were important in the co-occurrences in Amazonian networks, whereas several other soil properties were important for the co-exclusion. Different habitats had similar network properties with some variation in terms of modularity, probably associated with flooding pulse. We show that Amazonian micro-organism communities form highly interconnected co-occurrence and co-exclusion networks, which highlights the importance of complex biotic and abiotic interactions in explaining the outstanding biodiversity of the region.

13 pages, 3 figures.-- This article resulted from the symposium “Ten Years of Metabarcoding: What Have we Learned and How Do we Move Forward?” held during the VIII European Congress of Protistology-ISOP Joint Meeting, Rome, Italy, in July 2019.-- This is the pre-peer reviewed version of the following article: L.F. Santoferrara, F. Burki, S. Filker, R. Logares, M. Dunthorn, G. McManus, Perspectives from ten years of protist studies by high-throughput metabarcoding, Journal of Eukaryotic Microbiology 67(5):612-622(2020), which has been published in final form at https://doi.org/10.1111/jeu.12813. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions, During the last decade, high-throughput metabarcoding became routine for analyzing protistan diversity and distributions in nature. Amid a multitude of exciting findings, scientists have also identified and addressed technical and biological limitations, although problems still exist for inference of meaningful taxonomic and ecological knowledge based on short DNA sequences. Given the extensive use of this approach, it is critical to settle our understanding on its strengths and weaknesses and to synthesize up-to-date methodological and conceptual trends. This article summarizes key scientific and technical findings, and identifies current and future directions in protist research that uses metabarcoding, Additional support for this work includes a grant by the US National Science Foundation (OCE1924527) to GM and LS, and a fellowship from the Science for Life Laboratory (SciLifeLab) to FB, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

One of the major goals in microbial ecology is to understand whether the empirical biogeographic patterns of macroorganisms also apply to microorganisms. Here, we used morphological data from live organisms, along with molecular data, to investigate the importance of spatial factors and environmental variables in influencing ciliate composition from floodplain lakes. Our main goal was to use 2 different approaches (morphological and molecular) to compare ciliate diversity and distribution patterns as well as to compare how these methods differ in their ability to detect distribution patterns and the roles of spatial and environmental factors that shape ciliate assemblages in the 4 largest floodplains in Brazil. Planktonic water samples were gathered from 33 lakes associated with 4 different river floodplain systems in Brazil. We analyzed ciliates in vivo and sequenced surface water DNA using a metabarcoding approach with general eukaryotic primers. We showed that the diversity of operational taxonomic units was much higher than that of morphospecies. Regardless of the method of identification, we found a consistent spatial assembly pattern of ciliate assemblages across the 4 floodplain systems. We also found that environmental filters had a stronger association with the morphological than with the molecular site-by-site dissimilarities. Meanwhile, biogeographic factors and the distance among sites limited the distribution of molecular-based composition, resulting in strong differences among the floodplain lakes analyzed. This finding suggests that ecological research and biomonitoring activities should find an equilibrium between morphological and molecular approaches because each approach provides unique insights.

Microbial degradation influences the quality of oil resources. The environmental factors that shape the composition of oil microbial communities are largely unknown because most samples from oil fields are impacted by anthropogenic oil production, perturbing the native ecosystem with exogenous fluids and microorganisms. We investigated the relationship between formation water geochemistry and microbial community composition in undisturbed oil samples. We isolated 43 microliter-sized water droplets naturally enclosed in the heavy oil of the Pitch Lake, Trinidad and Tobago. The water chemistry and microbial community composition within the same water droplet were determined by ion chromatography and 16S rRNA gene amplicon sequencing, respectively. The results revealed a high variability in ion concentrations and community composition between water droplets. Microbial community composition was mostly affected by the chloride concentration, which ranged from freshwater to brackish-sea water. Remarkably, microbial communities did not respond gradually to increasing chloride concentration but showed a sudden change to less diverse and uneven communities when exceeding a chloride concentration of 57.3 mM. The results reveal a threshold-regulated response of microbial communities to salinity, offering new insights into the microbial ecology of oil reservoirs., Microbial communities from Pitch Lake oil show a sudden threshold-regulated response to salinity, leading to less diverse and uneven communities

THE International Society of Protistologists (ISOP) met online from 26-30 July, 2021. The Program and Abstract book for this is available here (File S1). An in-person international meeting was not possible due to the continued COVID-19 pandemic. The meeting therefore took place using the society's Zoom license, which is available for use by ISOP members for other online protist meetings.

The biodiversity and biogeography of protists inhabiting many ecosystems have been intensely studied using different sequencing approaches, but tropical ecosystems are relatively under-studied. Here, we sampled planktonic waters from 32 lakes associated with four different river-floodplains systems in Brazil, and sequenced the DNA using a metabarcoding approach with general eukaryotic primers. The lakes were dominated by the largely free-living Discoba (mostly the Euglenida), Ciliophora, and Ochrophyta. There was low community similarity between lakes even within the same river-floodplain. The protists inhabiting these floodplain systems comprise part of the large and relatively undiscovered diversity in the tropics.

Amazonia encompasses forests that grow in areas that are periodically inundated by overflowing rivers. The inundation depth and duration vary according to the slope of the terrain, creating a flooding gradient. This gradient directly affects the biota, but the effect on soil organisms remains elusive. Here, we use DNA metabarcoding to estimate prokaryote and eukaryote diversity from soil and litter samples in a seasonally flooded forest and its adjacent unflooded forest in central-western Amazonia using 16S and 18S gene sequences, respectively. We characterize the below-ground diversity and community composition based on Amplicon Sequence Variants (ASVs) along the flooding gradient. We test for the relationship of soil biota with the flooding gradient, soil properties and above-ground woody plant diversity. The flooding gradient did not explain below-ground biodiversity. Nor was the below-ground diversity explained by the above-ground woody plant diversity. However, we found taxonomic groups not previously reported in Amazonian seasonally flooded forests. Also, the flooding gradient and woody plant diversity did, in part, explain the community composition of soil bacteria. Although the effects of the flooding gradient, soil properties and above-ground woody plant diversity is hard to quantify, our results thus indicate that flood stress could influence below-ground bacterial community composition.

Microsporidia are obligate parasites that are closely related to Fungi. While the widely known “long-branch” Microsporidia infect mostly metazoans, the hosts of “short-branch” Microsporidia are only partially characterized or not known at all. Here, we used network analyses from Neotropical rainforest soil metabarcoding data, to infer co-occurrences between environmental lineages of short-branch microsporidians and their potential hosts. We found significant co-occurrences with several taxa, especially with Apicomplexa, Cercozoa, and Fungi, as well as some Metazoa. Our results are the first step to identify potential hosts of the environmental lineages of short-branch microsporidians, which can be targeted in future molecular and microscopic studies. Supplementary Information The online version contains supplementary material available at 10.1007/s00248-020-01657-9.

Protists dominate eukaryotic diversity and play key functional roles in all ecosystems, particularly by catalyzing carbon and nutrient cycling. To date, however, a comparative analysis of their taxonomic and functional diversity that compares the major ecosystems on Earth (soil, freshwater and marine systems) is missing. Here, we present a comparison of protist diversity based on standardized high throughput 18S rRNA gene sequencing of soil, freshwater and marine environmental DNA. Soil and freshwater protist communities were more similar to each other than to marine protist communities, with virtually no overlap of Operational Taxonomic Units (OTUs) between terrestrial and marine habitats. Soil protists showed higher γ diversity than aquatic samples. Differences in taxonomic composition of the communities led to changes in a functional diversity among ecosystems, as expressed in relative abundance of consumers, phototrophs and parasites. Phototrophs (eukaryotic algae) dominated freshwater systems (49% of the sequences) and consumers soil and marine ecosystems (59% and 48%, respectively). The individual functional groups were composed of ecosystem- specific taxonomic groups. Parasites were equally common in all ecosystems, yet, terrestrial systems hosted more OTUs assigned to parasites of macro-organisms while aquatic systems contained mostly microbial parasitoids. Together, we show biogeographic patterns of protist diversity across major ecosystems on Earth, preparing the way for more focused studies that will help understanding the multiple roles of protists in the biosphere.

Operational taxonomic units (OTUs) are usually treated as if they are internally uniform in environmental metabarcoding studies of microbial and macrobial eukaryotes, even when the OTUs are being used to infer biogeographic patterns. The OTUs constructed by the program Swarm have underlying network topologies in which nodes represent amplicons and edges represent 1 nucleotide differences between nodes. Such networks can be exploited to search for biogeographic patterns within each OTU. To do this, here we used an available protistan metabarcoding dataset consisting of the hypervariable V4 region of the 18S rRNA locus amplified from 77 lakes collected across Norway and Sweden. The 82 most abundant and wide-spread OTUs constructed by Swarm were evaluated using shortest path, assortativity, and geographical analyses. We found that while pairs of amplicons from the same lake were usually connected directly to each other within the OTUs, these pairs of amplicons from the same lake did not form assortative clusters within the OTUs, and amplicons were not more connected with other amplicons occurring in neighboring lakes than expected by chance. This new approach to looking at within-OTU is applicable to other metabarcoding datasets and we provide code to perform these analyses.

The Neotropics is one of the most diverse regions of the globe in terms of plants and animal species. Regarding the microbial world, however, little is known about the diversity and biogeography patterns of microorganisms in the Neotropics. The biogeography of several microbial taxonomic groups is still missing and/or incomplete, such as the protists. Despite the hard taxonomic identification of protists, the advance of molecular techniques (e.g., metabarcoding) have allowed to better explore the distribution of several protistan groups. Our goal here was to summarize the available information of Neotropical protists, focusing on metabarcoding studies, to explore what these data evidence on their ecology and biogeography. For this, we reviewed the findings from all articles that focused on or included the terrestrial protists using a metabarcoding approach and identified the gaps and future perspectives in this research field. We found that Neotropical protistan diversity patterns seem to be, at least in part, congruent with that of macro-organisms and, different than plants and bacteria, just weakly explained by environmental variables. We argue that studies with standardized protocols including different ecoregions are necessary, such as temperate forests, grasslands, and savannas from Southern of South America and Northern Atlantic Forest, to fully characterize the ecology and biogeography on Neotropical protists. Furthermore, dismembering evolutionary lineages and functional guilds of protists are important to better understand the relationship between diversity, dispersal abilities, and functionality of particular taxa of protists in their habitats. Resumo: A região Neotropical é uma das mais diversas regiões do globo em termos de espécies vegetais e animais. Em relação ao mundo microbiano, entretanto, pouco se sabe sobre a diversidade e os padrões biogeográficos dos microrganismos no Neotrópico. Nesse contexto, a biogeografia de diversos grupos taxonômicos microbianos ainda é escasso e/ou incompleto como os protistas, devido à difícil identificação taxonômica de tais microscópicos organismos. Neste contexto, o avanço dos dados moleculares de amostras ambientais (por exemplo, metabarcoding) permitiu explorar a distribuição de vários grupos de protistas. Nosso objetivo aqui foi resumir as informações disponíveis dos protistas neotropicais, com foco em metabarcoding, para explorar o que esses dados evidenciam sobre sua ecologia e biogeografia. Para isso, revisamos os resultados de todos os artigos que enfocavam ou incluíam os protistas terrestres usando uma abordagem de metabarcoding e identificamos as lacunas e as perspectivas futuras neste campo de pesquisa. Os padrões de diversidade dos protistas Neotropicais parecem ser, pelo menos em parte, congruentes com os de macroorganismos e, diferentes das plantas e bactérias, sendo pouco explicados por variáveis ambientais. Estudos com protocolos padronizados incluindo diferentes Ecorregiões são necessários, como em florestas temperadas, campos nativos e savanas no sul da América do Sul e no norte da Mata Atlântica, para melhor caracterizar a ecologia e biogeografia de protistas Neotropicais. Além disso, é importante diferenciar linhagens evolutivas e guildas funcionais de protistas para entender melhor a relação entre diversidade, capacidade de dispersão e funcionalidade de determinados táxons de protistas em seus habitats.

The Colpodea form a major clade of ciliates that are often found in environmental DNA sequencing studies. They are united by similar somatic ciliature, but differentiated by complex oral structures. Although there are four well supported colpodean subclades, there is disagreement in molecular phylogenetic inferences about their branching order. Using available nuclear SSU-rRNA sequences, we evaluated if the bursariomorphids or the platyophryids are sister to the remaining colpodeans. We inferred the "platyophryids-early" topologies using different alignment and masking methods, but constrained analyses could not reject the "bursariomorphids-early" topology. Both bursariomorphids and platyophryids clades have a similar number of nucleotide positions shared with the outgroup, and both are interconnected with the outgroup in phylogenetic networks. Based on these discordant results, it is hard to determine which clade branched off first, although the "platyophryids-early topology" is also supported by mitochondrial SSU-rRNA data. We also offer different reference alignments that can be used to phylogenetically place short- and long-read data from environmental DNA sequencing studies, and we propose some tentative evolutionary and ecological interpretations of those placements.

The predominance of sex in eukaryotes is still enigmatic. Sex, a composed process of meiosis and mixis cycles, confers high costs but the selective advantages remain unclear. In this review, we focus on potentially detrimental effects of asexuality on genome evolution. Theory predicts that asexual lineages should suffer from lack of meiotic DNA repair, accumulation of deleterious mutations, proliferation of transposable elements, among others. Here, we compare the different genomic features, life cycles, developmental pathways, and cytological mechanisms in the major eukaryotic groups, i.e., in protists, animals, fungi, and plants. In general, it is difficult to disentangle lineage-specific features from general features of asexuality. In all groups, forms of asexuality are predominantly facultative or cyclical. A variety of mixed or partial sexual developmental pathways exists, maintaining some components of sexuality, while obligate asexuality appears to be rare in eukaryotes. The strongest theoretical prediction for negative consequences of asexuality is decreased effectiveness of selection compared to sexuality. While some studies have shown increased rates of mutation accumulation in asexuals, others using whole-genome comparisons did not find this pattern. Various mechanisms exist that can alleviate the negative consequences of accumulation of negative mutations. More empirical data are needed to understand comprehensively the role of genome evolution for the maintenance of sex.

Forest soils typically harbour a vast diversity of fungi, but are usually dominated by filamentous (hyphae‐forming) taxa. Compared to temperate and boreal forests, though, we have limited knowledge about the fungal diversity in tropical rainforest soils. Here we show, by environmental metabarcoding of soil samples collected in three Neotropical rainforests, that Yeasts dominate the fungal communities in terms of the number of sequencing reads and OTUs. These unicellular forms are commonly found in aquatic environments, and their hyperdiversity may be the result of frequent inundation combined with numerous aquatic microenvironments in these rainforests. Other fungi that are frequent in aquatic environments, such as the abundant Chytridiomycotina, were also detected. While there was low similarity in OTU composition within and between the three rainforests, the fungal communities in Central America were more similar to each other than the communities in South America, reflecting a general biogeographic pattern also seen in animals, plants and protists.

116 pages, 1 figure, 4 tables, 3 appendices, 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, Research support was provided as follows: SMA by NSERC 249889‐2007; DB by NERC NE/H009426/1 and NE/H000887/1; MWB by NSF 1456054; FB by a Fellowship from Science for Life Laboratory and VR/2017‐04563; PC by EU‐Horizon 2020 research and innovation program through the SponGES project 679849 [...]; IC by CSF 18‐18699S; BE by RCN TaxMArc 268286/GMR; LG by ANR HAPAR (ANR‐14‐CE02‐0007); VH MK JL by ERDF; MEYS with ERC 771592 CZ 1.05/1.1.00/02.0109 BIOCEV; SK by RSF 16‐14‐10302; MK by CSF GA18‐28103S; CEL by NSF 1541510 and NIH‐AI124092; EL by CAM: 2017‐T1/AMB‐5210; and by grant 2017‐T1/AMB‐5210 from the program >Atracción de talentos> from the Consejería de Educación, Juventud y Deporte, Comunidad de Madrid; JL by ERC CZ LL1601 and OPVVV 16_019/0000759; MP by NSF DEB‐1455611; DJR by the Beatriu de Pinós postdoctoral programme of the Government of Catalonia's Secretariat for Universities and Research of the Ministry of Economy and Knowledge; CLS by the intramural research program of the National Library of Medicine, National Institutes of Health; AS by RSF 17‐14‐01391 and RFBR 16‐04‐01454 NY by NSF DEB 1557102; VZ by RFBR 16‐34‐60102 mol‐a‐dk; UniEuk and EukRef by the Gordon and Betty Moore Foundation

Piredda, Roberta ... et al.-- 12 pages, 5 figures, 1 table, supporting information https://doi.org/10.1038/s41598-018-36345-9, Diatoms constitute a diverse lineage of unicellular organisms abundant and ecologically important in aquatic ecosystems. Compared to other protists, their biology and taxonomy are well-studied, offering the opportunity to combine traditional approaches and new technologies. We examined a dataset of diatom 18S rRNA- and rDNA- (V4 region) reads from different plankton size-fractions and sediments from six European coastal marine sites, with the aim of identifying peculiarities and commonalities with respect to the whole protistan community. Almost all metabarcodes (99.6%) were assigned to known genera (121) and species (236), the most abundant of which were those already known from classic studies and coincided with those seen in light microscopy. rDNA and rRNA showed comparable patterns for the dominant taxa, but rRNA revealed a much higher diversity particularly in the sediment communities. Peculiar to diatoms is a tight bentho-pelagic coupling, with many benthic or planktonic species colonizing both water column and sediments and the dominance of planktonic species in both habitats. Overall metabarcoding results reflected the marked specificity of diatoms compared to other protistan groups in terms of morphological and ecological characteristics, at the same time confirming their great potential in the description of protist communities, Financial support to the project BioMarKs was provided by the EU (2008-6530, ERA-net BIODIVERSA, EU) and, for the Italian participation, by Stazione Zoologica Anton Dohrn and the projects FIRB Biodiversitalia-Approccio multitaxa allo studio delle risposte della biodiversità italiana al cambiamento climatico (RBAP10A2T4), funded by the Italian Ministry of Education, University and Research (MIUR), which also supported Roberta Piredda’s fellowship

Some protists with microsporidian‐like cell biological characters, including Mitosporidium, Paramicrosporidium, and Nucleophaga, have SSU rRNA gene sequences that are much less divergent than canonical Microsporidia. We analysed the phylogenetic placement and environmental diversity of microsporidian‐like lineages that group near the base of the fungal radiation and showed that they group in a clade with metchnikovellids and canonical microsporidians to the exclusion of the clade including Rozella, in line with what is currently known of their morphology and cell biology. These results show that the phylogenetic scope of Microsporidia has been greatly underestimated. We propose that much of the lineage diversity previously thought to be cryptomycotan/rozellid is actually microsporidian, offering new insights into the evolution of the highly specialized parasitism of canonical Microsporidia. This insight has important implications for our understanding of opisthokont evolution and ecology, and is important for accurate interpretation of environmental diversity. Our analyses also demonstrate that many opisthosporidian (aphelid+rozellid+microsporidian) SSU V4 OTUs from tropical forest soils group with the short‐branching Microsporidia, consistent with the abundance of their protist and arthropod hosts in soils. This novel diversity of environmental Microsporidia provides a unique opportunity to investigate the evolutionary origins of a highly specialised clade of major animal parasites.

Species in the genera Hexasterias and Halodinium have been recorded over the last decades as acritarchs in palynological and/or plankton studies. In paleoenvironmental studies, these resting stages are often interpreted as indicators of freshwater input. The biological affinity of these genera has never been definitely established. Here, a new species, Halodinium verrucatum sp. nov., is described and molecular evidence (single specimen SSU and LSU rDNA sequencing) reveals that both this new species and Hexasterias problematica, collected from sediment samples in the Skagerrak and Baltic Sea, are resting stages of prorodontid ciliates. Additionally, infrared spectroscopic analysis (micro-FTIR) of Hexasterias problematica and Halodinium spp. specimens indicates a carbohydrate-based composition of the cyst wall with evidence for nitrogen-containing functional groups. A similar composition is recorded for tintinnid loricae, which further supports the placement of Hexasterias and Halodinium as ciliate cysts, and the composition is consistent with the heterotrophic nature of ciliates. The morphologically similar species Radiosperma corbiferum has a comparable composition, suggesting a similar ciliate affinity and indicating the utility of micro-FTIR in understanding acritarch affinity. Hexasterias problematica typically occurs in coastal waters from temperate to arctic regions. Halodinium verrucatum sp. nov. is observed in temperate estuarine sediments in the northern hemisphere.

Tropical animals and plants are known to have high alpha diversity within forests, but low beta diversity between forests. By contrast, it is unknown whether microbes inhabiting the same ecosystems exhibit similar biogeographic patterns. To evaluate the biogeographies of tropical protists, we used metabarcoding data of species sampled in the soils of three lowland Neotropical rainforests. Taxa–area and distance–decay relationships for three of the dominant protist taxa and their subtaxa were estimated at both the OTU and phylogenetic levels, with presence–absence and abundance‐based measures. These estimates were compared to null models. High local alpha and low regional beta diversity patterns were consistently found for both the parasitic Apicomplexa and the largely free‐living Cercozoa and Ciliophora. Similar to animals and plants, the protists showed spatial structures between forests at the OTU and phylogenetic levels, and only at the phylogenetic level within forests. These results suggest that the biogeographies of macro‐ and micro‐organismal eukaryotes in lowland Neotropical rainforests are partially structured by the same general processes. However, and unlike the animals and plants, the protist OTUs did not exhibit spatial structures within forests, which hinders our ability to estimate the local and regional diversity of protists in tropical forests.

While the biodiversity and biogeography of protists inhabiting many ecosystems have been intensely studied using different sequencing approaches, tropical ecosystems are relatively under-studied. Here we sampled planktonic waters from 32 lakes associated with four different river-floodplains systems in Brazil, and sequenced the DNA using a metabarcoding approach with general eukaryotic primers. The lakes were dominated by the largely free-living Discoba (mostly the Euglenida) and Ciliophora unlike previously sampled Neotropical environments, bu the community similarities between samples were likewise low. These protists inhabiting these floodplains potentially form part of the large diversity of unknown diversity in the tropics.

Protists include all eukaryotes except plants, fungi and animals. They are an essential, yet often forgotten, component of the soil microbiome. Method developments have now furthered our understanding of the real taxonomic and functional diversity of soil protists. They occupy key roles in microbial foodwebs as consumers of bacteria, fungi and other small eukaryotes. As parasites of plants, animals and even of larger protists, they regulate populations and shape communities. Pathogenic forms play a major role in public health issues as human parasites, or act as agricultural pests. Predatory soil protists release nutrients enhancing plant growth. Soil protists are of key importance for our understanding of eukaryotic evolution and microbial biogeography. Soil protists are also useful in applied research as bioindicators of soil quality, as models in ecotoxicology and as potential biofertilizers and biocontrol agents. In this review, we provide an overview of the enormous morphological, taxonomical and functional diversity of soil protists, and discuss current challenges and opportunities in soil protistology. Research in soil biology would clearly benefit from incorporating more protistology alongside the study of bacteria, fungi and animals.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Animal and plant communities in tropical rainforests are known to have high alpha diversity within forests, but low beta diversity between forests. By contrast, it is unknown if the microbial protists inhabiting the same ecosystems exhibit similar biogeographic patterns. To evaluate the biogeographies of soil protists in three lowland Neotropical rainforests using metabarcoding data, we estimated taxa-area and distance-decay relationships for three large protist taxa and their subtaxa, at both the OTU and phylogenetic levels, with presence-absence and abundance based measures, and compared the estimates to null models. High local alpha and low regional beta diversity patterns were persistently found for both the parasitic Apicomplexa and the free-living Cercozoa and Ciliophora, even though they have different ecological functions and different dispersal modes. In comparison to the null models, both OTU and phylogenetic diversities showed spatial structures between forests, but only phylogenetic diversity showed spatial patterns within forests. These results suggest that the biogeographies of macro-and micro-organismal eukaryotes in lowland Neotropical rainforests are partially structured by the same general processes. As with arthropods, the protists’ high alpha diversity within forests presents problems for estimating their local diversity, and shows that regional diversity cannot be easily estimated because of low turnover between forests.

Forest soils typically harbour a vast diversity of fungi, but are usually dominated by filamentous (hyphae-forming) taxa. Compared to temperate and boreal forests, though, we have limited knowledge about the fungal diversity in tropical rainforest soils. Here we show, by environmental metabarcoding of soil samples collected in three Neotropical rainforests, that Yeasts dominate the fungal communities in terms of the number of sequencing reads and OTUs. These unicellular forms are commonly found in aquatic environments, and their hyperdiversity may be the result of frequent inundation combined with numerous aquatic microenvironments in these rainforests. Other fungi that are frequent in aquatic environments, such as the abundant Chytridiomycotina, were also detected. While there was low similarity in OTU composition within and between the three rainforests, the fungal communities in Central America were more similar to each other than the communities in South America, reflecting a general biogeographic pattern also seen in animals, plants, and protists.Contactdunthorn@rhrk.uni-kl.de

Colpodean ciliates potentially pose a problem to macro-organismic theories of evolution: they are putatively asexual and extremely ancient, and yet there is one apparently derived sexual species. If macro-organismic theories of evolution also broadly apply to microbial eukaryotes, though, then most or all of the colpodean ciliates should merely be secretively sexual. Here we show using de novo genome sequencing, that colpodean ciliates have the meiotic genes required for sex and these genes are under functional constraint. Along with these genomic data, we argue that these ciliates are sexual given the cytological observations of both micronuclei and macronuclei within their cells, and the behavioral observations of brief fusions as if the cells were mating. The challenge that colpodean ciliates pose is therefore not to evolutionary theory, but to our ability to induce microbial eukaryotic sex in the laboratory.Contact:dunthorn@rhrk.uni-kl.de

Symbiodinium encompasses a diverse clade of dinoflagellates that are ecologically important as symbionts of corals and other marine organisms. Despite decades of study, cytological evidence of sex (karyogamy and meiosis) has not been demonstrated in Symbiodinium, although molecular population genetic patterns support the occurrence of sexual recombination. Here, we provide additional support for sex in Symbiodinium by uncovering six meiosis-specific and 25 meiosis-related genes in three published genomes. Cryptic sex may be occurring in Symbiodinium's seldom-seen free-living state while being inactive in the symbiotic state.

Using 11 new SSU-rDNA sequences, we analyze relationships within the class Colpodea, especially of some uncommon taxa, such as Kalometopia duplicata, Cyrtolophosis minor, and Jaroschia sumptuosa. The sequences do not change the basic structure of the molecular Colpodea tree, i.e., all belong to one of the four molecular clades recognized by Foissner et al. (2011): Colpodida, Cyrtolophosidida, Bursariomorphida, and Platyophryida. The addition of three Colpoda sequences strengthens the observation that species of this genus are distributed over the whole molecular Colpodea tree. Very likely, this is caused by a fast radiation of Colpoda, several species of which then evolved independently, forming new genera and families. Cyrtolophosis minor, which belongs to the molecular Pseudocyrtolophosis clade, is referred to a new genus, Apocyrtolophosis nov. gen., characterized by a comparably large, deltoid oral opening, an unciliated posterior region, and the absence of an oblique kinety in the left oral polykinetid. Bryometopus triquetrus does not erase the paraphyly of its genus. Platyophrya vorax, P. spumacola, and P. bromelicola form a highly supported clade in the order Platyophryida. Platyophryides and Ottowphrya are close genetically but differ in the silverline pattern (colpodid vs. platyophryid).

The Haptoria are free-living predatory ciliates living in terrestrial and aquatic habitats all around the world. They belong to a highly diverse class, Litostomatea, whose morphological and molecular classifications harmonize poorly since both approaches produce rather different frameworks. In the present study, we analyzed the genealogy of the litostomateans, including eight new haptorian 18S rRNA gene sequences. Apart from traditional tree-building methods, we also applied phylogenetic networks, split spectrum analysis and quartet likelihood mapping to assess the information content of alignments. These analyses show that: (1) there are several strongly supported monophyletic litostomatean lineages — Rhynchostomatia, Trichostomatia, Haptorida, Lacrymariida, Pleurostomatida, and Didiniida; (2) the Rhynchostomatia are the best candidates for a basal litostomatean group; (3) sister relationship of the Trichostomatia and Haptoria is very likely, which well corroborates the traditional morphology-based classifications; (4) molecular phylogeny of the order Spathidiida is only poorly resolved very likely due to one or several rapid radiation events or due to the incomplete lineage sorting at the rRNA locus; and (5) the basal position of the genera Chaenea and Trachelotractus in molecular trees and phylogenetic networks is very likely a result of class III long-branch effects.

International audience; The dinoflagellate microalga Symbiodinium sustains coral reefs, one of the most diverse ecosystems of the biosphere, through mutualistic endosymbioses with a wide diversity of benthic hosts [1]. Despite its ecological and economic importance, the presence of Symbiodinium in open oceanic waters remains unknown, which represents a significant knowledge gap to fully understand the eco-evolutionary trajectory and resilience of endangered Symbiodinium-based symbioses. Here, we document the existence of Symbiodinium (i.e., now the family Symbiodiniaceae [2]) in tropical- and temperate-surface oceans using DNA and RNA metabarcoding of size-fractionated plankton samples collected at 109 stations across the globe. Symbiodinium from clades A and C were, by far, the most prevalent and widely distributed lineages (representing 0.1% of phytoplankton reads), while other lineages (clades B, D, E, F, and G) were present but rare. Concurrent metatranscriptomics analyses using the Tara Oceans gene catalog [3] revealed that Symbiodinium clades A and C were transcriptionally active in the open ocean and expressed core metabolic pathways (e.g., photosynthesis, carbon fixation, glycolysis, and ammonium uptake). Metabarcodes and expressed genes of clades A and C were detected in small and large plankton size fractions, suggesting the existence of a free-living population and a symbiotic lifestyle within planktonic hosts, respectively. However, high-resolution genetic markers and microscopy are required to confirm the life history of oceanic Symbiodinium. Overall, the previously unknown, metabolically active presence of Symbiodinium in oceanic waters opens up new avenues for investigating the potential of this oceanic reservoir to repopulate coral reefs following stress-induced bleaching.

High animal and plant richness in tropical rainforest communities has long intrigued naturalists. It is unknown if similar hyperdiversity patterns are reflected at the microbial scale with unicellular eukaryotes (protists). Here we show, using environmental metabarcoding of soil samples and a phylogeny-aware cleaning step, that protist communities in Neotropical rainforests are hyperdiverse and dominated by the parasitic Apicomplexa, which infect arthropods and other animals. These host-specific parasites potentially contribute to the high animal diversity in the forests by reducing population growth in a density-dependent manner. By contrast, too few operational taxonomic units (OTUs) of Oomycota were found to broadly drive high tropical tree diversity in a host-specific manner under the Janzen-Connell model. Extremely high OTU diversity and high heterogeneity between samples within the same forests suggest that protists, not arthropods, are the most diverse eukaryotes in tropical rainforests. Our data show that protists play a large role in tropical terrestrial ecosystems long viewed as being dominated by macroorganisms.