O short-branch Microsporidia, where art thou? Identifying diversity hotspots for future sampling.

Short-branch Microsporidia were previously shown to form a basal grade within the expanded Microsporidia clade and to branch near the classical, long-branch Microsporidia. Although they share simpler versions of some morphological characteristics, they do not show accelerated evolutionary rates, making them ideal candidates to study the evolutionary trajectories that have led to long-branch microsporidian unique characteristics. However, most sequences assigned to the short-branch Microsporidia are undescribed, novel environmental lineages for which the identification requires knowledge of where they can be found. To direct future isolation, we used the EukBank database of the global UniEuk initiative that contains the majority of the publicly available environmental V4 SSU rRNA gene sequences of protists. The curated OTU table and corresponding metadata were used to evaluate the occurrence of short-branch Microsporidia across freshwater, hypersaline, marine benthic, marine pelagic, and terrestrial environments. Presence-absence analyses infer that short-branch Microsporidia are most abundant in freshwater and terrestrial environments, and alpha- and beta-diversity measures indicate that focusing our sampling effort on these two environments would cover a large part of their overall diversity. These results can be used to coordinate future isolation and sampling campaigns to better understand the enigmatic evolution of microsporidians' unique characteristics.

How marine are Marine Stramenopiles (MAST)? A cross-system evaluation.

Marine Stramenopiles (MAST) were first described two decades ago through ribosomal RNA gene (rRNA gene) sequences from marine surveys of microbial eukaryotes. MAST comprise several independent lineages at the base of the Stramenopiles. Despite their prevalence in the ocean, the majority of MAST diversity remains uncultured. Previous studies, mainly in marine environments, have explored MAST's cell morphology, distribution, trophic strategies, and genomics using culturing-independent methods. In comparison, less is known about their presence outside marine habitats. Here, we analyse the extensive EukBank dataset to assess the extent to which MAST can be considered marine protists. Additionally, by incorporating newly available rRNA gene sequences, we update Stramenopiles phylogeny, identifying three novel MAST lineages. Our results indicate that MAST are primarily marine with notable exceptions within MAST-2 and MAST-12, where certain subclades are prevalent in freshwater and soil habitats. In the marine water column, only a few MAST species, particularly within clades -1, -3, -4, and -7, dominate and exhibit clear latitudinal distribution patterns. Overall, the massive sequencing dataset analysed in our study confirms and partially expands the previously described diversity of MASTs groups and underscores the predominantly marine nature of most of these uncultured lineages.

Predicting global distributions of eukaryotic plankton communities from satellite data.

Satellite remote sensing is a powerful tool to monitor the global dynamics of marine plankton. Previous research has focused on developing models to predict the size or taxonomic groups of phytoplankton. Here, we present an approach to identify community types from a global plankton network that includes phytoplankton and heterotrophic protists and to predict their biogeography using global satellite observations. Six plankton community types were identified from a co-occurrence network inferred using a novel rDNA 18 S V4 planetary-scale eukaryotic metabarcoding dataset. Machine learning techniques were then applied to construct a model that predicted these community types from satellite data. The model showed an overall 67% accuracy in the prediction of the community types. The prediction using 17 satellite-derived parameters showed better performance than that using only temperature and/or the concentration of chlorophyll a. The constructed model predicted the global spatiotemporal distribution of community types over 19 years. The predicted distributions exhibited strong seasonal changes in community types in the subarctic-subtropical boundary regions, which were consistent with previous field observations. The model also identified the long-term trends in the distribution of community types, which suggested responses to ocean warming.

Interpreting phylogenetic placements for taxonomic assignment of environmental DNA.

Taxonomic assignment of operational taxonomic units (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 OTU placements within a subtaxon, and the branch distances between equally likely placement locations. We also inferred the evolutionary and ecological characteristics of the colpodean OTUs using their placements within subtaxa. This study demonstrates how to fully analyze the output of EPA-ng, by using GAPPA in conjunction with knowledge of the taxonomic diversity of the clade of interest.

Fruit fly phylogeny imprints bacterial gut microbiota.

One promising avenue for reconciling the goals of crop production and ecosystem preservation consists in the manipulation of beneficial biotic interactions, such as between insects and microbes. Insect gut microbiota can affect host fitness by contributing to development, host immunity, nutrition, or behavior. However, the determinants of gut microbiota composition and structure, including host phylogeny and host ecology, remain poorly known. Here, we used a well-studied community of eight sympatric fruit fly species to test the contributions of fly phylogeny, fly specialization, and fly sampling environment on the composition and structure of bacterial gut microbiota. Comprising both specialists and generalists, these species belong to five genera from to two tribes of the Tephritidae family. For each fly species, one field and one laboratory samples were studied. Bacterial inventories to the genus level were produced using 16S metabarcoding with the Oxford Nanopore Technology. Sample bacterial compositions were analyzed with recent network-based clustering techniques. Whereas gut microbiota were dominated by the Enterobacteriaceae family in all samples, microbial profiles varied across samples, mainly in relation to fly identity and sampling environment. Alpha diversity varied across samples and was higher in the Dacinae tribe than in the Ceratitinae tribe. Network analyses allowed grouping samples according to their microbial profiles. The resulting groups were very congruent with fly phylogeny, with a significant modulation of sampling environment, and with a very low impact of fly specialization. Such a strong imprint of host phylogeny in sympatric fly species, some of which share much of their host plants, suggests important control of fruit flies on their gut microbiota through vertical transmission and/or intense filtering of environmental bacteria.

Seasonal dynamics of marine protist communities in tidally mixed coastal waters.

Major seasonal community reorganizations and associated biomass variations are landmarks of plankton ecology. However, the processes of plankton community turnover rates have not been fully elucidated so far. Here, we analyse patterns of planktonic protist community succession in temperate latitudes, based on quantitative taxonomic data from both microscopy counts (cells >10 µm) and ribosomal DNA metabarcoding (size fraction >3 µm, 18S rRNA gene) from plankton samples collected bimonthly over 8 years (2009-2016) at the SOMLIT-Astan station (Roscoff, Western English Channel). Based on morphology, diatoms were clearly the dominating group all year round and over the study period. Metabarcoding uncovered a wider diversity spectrum and revealed the prevalence of Dinophyceae and diatoms but also of Cryptophyta, Chlorophyta, Cercozoa, Syndiniales and Ciliophora in terms of read counts and or richness. The use of morphological and molecular analyses in combination allowed improving the taxonomic resolution and to identify the sequence of the dominant species and OTUs (18S V4 rDNA-derived taxa) that drive annual plankton successions. We detected that some of these dominant OTUs were benthic as a result of the intense tidal mixing typical of the French coasts in the English Channel. Our analysis of the temporal structure of community changes point to a strong seasonality and resilience. The temporal structure of environmental variables (especially Photosynthetic Active Radiation, temperature and macronutrients) and temporal structures generated by species life cycles and or species interactions, are key drivers of the observed cyclic annual plankton turnover.

pr2-primers: An 18S rRNA primer database for protists.

Metabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost solely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, an informed primer choice is necessary, as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few online resources available to list existing primers. We built a database listing 285 primers and 83 unique primer pairs that have been used for eukaryotic 18S rRNA gene metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR2 version 4.12.0 for eukaryotes and a subset of silva version 132 for bacteria and archaea. We developed an R-based web application enabling browsing of the database, visualization of the taxonomic distribution of the amplified sequences with the number of mismatches, and testing any user-defined primer or primer set (https://app.pr2-primers.org). Taxonomic specificity of primer pairs, amplicon size and location of mismatches can also be determined. We identified universal primer sets that matched the largest number of sequences and analysed the specificity of some primer sets designed to target certain groups. This tool enables guided primer choices that will help a wide range of researchers to include protists as part of their investigations.

Genetic Variation in Host-Specific Competitiveness of the Symbiont Rhizobium leguminosarum Symbiovar viciae.

Legumes of the Fabeae tribe form nitrogen-fixing root nodules resulting from symbiotic interaction with the soil bacteria Rhizobium leguminosarum symbiovar viciae (Rlv). These bacteria are all potential symbionts of the Fabeae hosts but display variable partner choice when co-inoculated in mixture. Because partner choice and symbiotic nitrogen fixation mostly behave as genetically independent traits, the efficiency of symbiosis is often suboptimal when Fabeae legumes are exposed to natural Rlv populations present in soil. A core collection of 32 Rlv bacteria was constituted based on the genomic comparison of a collection of 121 genome sequences, representative of known worldwide diversity of Rlv. A variable part of the nodD gene sequence was used as a DNA barcode to discriminate and quantify each of the 32 bacteria in mixture. This core collection was co-inoculated on a panel of nine genetically diverse Pisum sativum, Vicia faba, and Lens culinaris genotypes. We estimated the relative Early Partner Choice (EPC) of the bacteria with the Fabeae hosts by DNA metabarcoding on the nodulated root systems. Comparative genomic analyses within the bacterial core collection identified molecular markers associated with host-dependent symbiotic partner choice. The results revealed emergent properties of rhizobial populations. They pave the way to identify genes related to important symbiotic traits operating at this level.

Periodontitis associates with species-specific gene expression of the oral microbiota.

The purpose of the present investigation was to characterize species-specific bacterial activity of the oral microbiota in periodontitis. We tested the hypotheses that chronic inflammation, i.e., periodontitis, associates with bacterial gene expression of the oral microbiota. Oral microbial samples were collected from three oral sites-subgingival plaque, tongue, and saliva from patients with periodontitis and healthy controls. Paired metagenomics and metatranscriptomics were used to perform concomitant characterization of taxonomic composition and to determine species-specific bacterial activity as expressed by the ratio of specific messenger RNA reads to their corresponding genomic DNA reads. Here, we show the association of periodontitis with bacterial gene expression of the oral microbiota. While oral site was the main determinant of taxonomic composition as well as bacterial gene expression, periodontitis was significantly associated with a reduction of carbohydrate metabolism of the oral microbiota at three oral sites (subgingival plaque, tongue, and saliva). Data from the present study revealed the association of periodontitis with bacterial gene expression of the oral microbiota. Conditions of periodontitis was associated with bacterial activity of local subgingival plaque, but also on tongue and the salivary microbiota. Collectively, data suggest that periodontitis associates with impaired carbohydrate metabolism of the oral microbiota. Future longitudinal and interventional studies are warranted to evaluate the potential pathogenic role of impaired bacterial carbohydrate metabolism not only in periodontitis but also in other diseases with low-grade inflammation, such as type 2 diabetes mellitus.

Swarm v3: towards tera-scale amplicon clustering.

MOTIVATION: Previously we presented swarm, an open-source amplicon clustering programme that produces fine-scale molecular operational taxonomic units (OTUs) that are free of arbitrary global clustering thresholds. Here, we present swarm v3 to address issues of contemporary datasets that are growing towards tera-byte sizes. RESULTS: When compared with previous swarm versions, swarm v3 has modernized C++ source code, reduced memory footprint by up to 50%, optimized CPU-usage and multithreading (more than 7 times faster with default parameters), and it has been extensively tested for its robustness and logic. AVAILABILITY AND IMPLEMENTATION: Source code and binaries are available at https://github.com/torognes/swarm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High and specific diversity of protists in the deep-sea basins dominated by diplonemids, kinetoplastids, ciliates and foraminiferans.

Heterotrophic protists (unicellular eukaryotes) form a major link from bacteria and algae to higher trophic levels in the sunlit ocean. Their role on the deep seafloor, however, is only fragmentarily understood, despite their potential key function for global carbon cycling. Using the approach of combined DNA metabarcoding and cultivation-based surveys of 11 deep-sea regions, we show that protist communities, mostly overlooked in current deep-sea foodweb models, are highly specific, locally diverse and have little overlap to pelagic communities. Besides traditionally considered foraminiferans, tiny protists including diplonemids, kinetoplastids and ciliates were genetically highly diverse considerably exceeding the diversity of metazoans. Deep-sea protists, including many parasitic species, represent thus one of the most diverse biodiversity compartments of the Earth system, forming an essential link to metazoans.

metaXplor: an interactive viral and microbial metagenomic data manager.

BACKGROUND: Efficiently managing large, heterogeneous data in a structured yet flexible way is a challenge to research laboratories working with genomic data. Specifically regarding both shotgun- and metabarcoding-based metagenomics, while online reference databases and user-friendly tools exist for running various types of analyses (e.g., Qiime, Mothur, Megan, IMG/VR, Anvi'o, Qiita, MetaVir), scientists lack comprehensive software for easily building scalable, searchable, online data repositories on which they can rely during their ongoing research. RESULTS: metaXplor is a scalable, distributable, fully web-interfaced application for managing, sharing, and exploring metagenomic data. Being based on a flexible NoSQL data model, it has few constraints regarding dataset contents and thus proves useful for handling outputs from both shotgun and metabarcoding techniques. By supporting incremental data feeding and providing means to combine filters on all imported fields, it allows for exhaustive content browsing, as well as rapid narrowing to find specific records. The application also features various interactive data visualization tools, ways to query contents by BLASTing external sequences, and an integrated pipeline to enrich assignments with phylogenetic placements. The project home page provides the URL of a live instance allowing users to test the system on public data. CONCLUSION: metaXplor allows efficient management and exploration of metagenomic data. Its availability as a set of Docker containers, making it easy to deploy on academic servers, on the cloud, or even on personal computers, will facilitate its adoption.

Identifying Potential Hosts of Short-Branch Microsporidia.

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.

The Windblown: Possible Explanations for Dinophyte DNA in Forest Soils.

Dinophytes are widely distributed in marine- and fresh-waters, but have yet to be conclusively documented in terrestrial environments. Here, we evaluated the presence of these protists from an environmental DNA metabarcoding dataset of Neotropical rainforest soils. Using a phylogenetic placement approach with a reference alignment and tree, we showed that the numerous sequencing reads that were phylogenetically placed as dinophytes did not correlate with taxonomic assignment, environmental preference, nutritional mode, or dormancy. All the dinophytes in the soils are rather windblown dispersal units of aquatic species and are not biologically active residents of terrestrial environments.

Heterogeneity of the rice microbial community of the Chinese centuries-old Honghe Hani rice terraces system.

The Honghe Hani rice terraces system (HHRTS) is a traditional rice cultivation system where Hani people cultivate remarkably diverse rice varieties. Recent introductions of modern rice varieties to the HHRTS have significantly increased the severity of rice diseases within the terraces. Here, we determine the impacts of these recent introductions on the composition of the rice-associated microbial communities. We confirm that the HHRTS contains a range of both traditional HHRTS landraces and introduced modern rice varieties and find differences between the microbial communities of these two groups. However, this introduction of modern rice varieties has not strongly impacted the overall diversity of the HHRTS rice microbial community. Furthermore, we find that the rice varieties (i.e. groups of closely related genotypes) have significantly structured the rice microbial community composition (accounting for 15%-22% of the variance) and that the core microbial community of HHRTS rice plants represents less than 3.3% of all the microbial taxa identified. Collectively, our study suggests a highly diverse HHRTS rice holobiont (host with its associated microbes) where the diversity of rice hosts mirrors the diversity of their microbial communities. Further studies will be needed to better determine how such changes might impact the sustainability of the HHRTS.

Diversity and dynamics of relevant nanoplanktonic diatoms in the Western English Channel.

In the ocean, Bacillariophyta are one of the most successful protistan groups. Due to their considerable biogeochemical implications, diatom diversity, development, and seasonality have been at the center of research, specifically large-sized species. In comparison, nanoplanktonic diatoms are mostly disregarded from routine monitoring and are often underrepresented in genetic reference databases. Here, we identified and investigated the temporal dynamics of relevant nanodiatoms occurring in the Western English Channel (SOMLIT-Astan station). Coupling in situ and laboratory approaches, we revealed that nano-species from the genera Minidiscus and Thalassiosira are key components of the phytoplankton community that thrive in these coastal waters, but they display different seasonal patterns. Some species formed recurrent blooms whilst others were persistent year round. These results raise questions about their regulation in the natural environment. Over a full seasonal cycle at the monitoring station, we succeeded in isolating viruses which infect these minute diatoms, suggesting that these mortality agents may contribute to their control. Overall, our study points out the importance of considering nanodiatom communities within time-series surveys to further understand their role and fate in marine systems.

Controlled sampling of ribosomally active protistan diversity in sediment-surface layers identifies putative players in the marine carbon sink.

Marine sediments are one of the largest carbon reservoir on Earth, yet the microbial communities, especially the eukaryotes, that drive these ecosystems are poorly characterised. Here, we report implementation of a sampling system that enables injection of reagents into sediments at depth, allowing for preservation of RNA in situ. Using the RNA templates recovered, we investigate the 'ribosomally active' eukaryotic diversity present in sediments close to the water/sediment interface. We demonstrate that in situ preservation leads to recovery of a significantly altered community profile. Using SSU rRNA amplicon sequencing, we investigated the community structure in these environments, demonstrating a wide diversity and high relative abundance of stramenopiles and alveolates, specifically: Bacillariophyta (diatoms), labyrinthulomycetes and ciliates. The identification of abundant diatom rRNA molecules is consistent with microscopy-based studies, but demonstrates that these algae can also be exported to the sediment as active cells as opposed to dead forms. We also observe many groups that include, or branch close to, osmotrophic-saprotrophic protists (e.g. labyrinthulomycetes and Pseudofungi), microbes likely to be important for detrital decomposition. The sequence data also included a diversity of abundant amplicon-types that branch close to the Fonticula slime moulds. Taken together, our data identifies additional roles for eukaryotic microbes in the marine carbon cycle; where putative osmotrophic-saprotrophic protists represent a significant active microbial-constituent of the upper sediment layer.

Global Trends in Marine Plankton Diversity across Kingdoms of Life.

The ocean is home to myriad small planktonic organisms that underpin the functioning of marine ecosystems. However, their spatial patterns of diversity and the underlying drivers remain poorly known, precluding projections of their responses to global changes. Here we investigate the latitudinal gradients and global predictors of plankton diversity across archaea, bacteria, eukaryotes, and major virus clades using both molecular and imaging data from Tara Oceans. We show a decline of diversity for most planktonic groups toward the poles, mainly driven by decreasing ocean temperatures. Projections into the future suggest that severe warming of the surface ocean by the end of the 21st century could lead to tropicalization of the diversity of most planktonic groups in temperate and polar regions. These changes may have multiple consequences for marine ecosystem functioning and services and are expected to be particularly significant in key areas for carbon sequestration, fisheries, and marine conservation. VIDEO ABSTRACT.

How deep can ectomycorrhizas go? A case study on Pisolithus down to 4 meters in a Brazilian eucalypt plantation.

Despite the strong ecological importance of ectomycorrhizal (ECM) fungi, their vertical distribution remains poorly understood. To our knowledge, ECM structures associated with trees have never been reported in depths below 2 meters. In this study, fine roots and ECM root tips were sampled down to 4-m depth during the digging of two independent pits differing by their water availability. A meta-barcoding approach based on Illumina sequencing of internal transcribed spacers (ITS1 and ITS2) was carried out on DNA extracted from root samples (fine roots and ECM root tips separately). ECM fungi dominated the root-associated fungal community, with more than 90% of sequences assigned to the genus Pisolithus. The morphological and barcoding results demonstrated, for the first time, the presence of ECM symbiosis down to 4-m. The molecular diversity of Pisolithus spp. was strongly dependent on depth, with soil pH and soil water content as primary drivers of the Pisolithus spp. structure. Altogether, our results highlight the importance to consider the ECM symbiosis in deep soil layers to improve our understanding of fine roots functioning in tropical soils.

Worldwide Occurrence and Activity of the Reef-Building Coral Symbiont Symbiodinium in the Open Ocean.

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.