Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

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.

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.S ince the works of early naturalists such as von Humboldt and Bonpland 1 , we have known that animal and plant communities in tropical rainforests are exceedingly species rich. For example, one hectare can contain more than 400 tree species 2 and one tree can harbour more than 40 ant species 3 . This hyperdiversity of trees has been partially explained by the Janzen-Connell model 4,5 , which hypothesizes that host-specific predators and parasites reduce plant population growth in a density-dependent manner 6,7 . Sampling up in the tree canopies and below on the ground has further led to the view that arthropods are the most diverse eukaryotes in tropical rainforests 8,9 .The focus on eukaryotic macroorganisms in these studies is primarily because they are familiar and readily observable to us. We do not know whether the less familiar and less readily observable protists-microbial eukaryotes that are not animals, plants or fungi 10 -inhabiting these same ecosystems exhibit similar diversity patterns. To evaluate if macroorganismic diversity patterns are reflected at the microbial scale with protists, we conducted an environmental DNA metabarcoding study by sampling soils in 279 locations in a variety of lowland Neotropical forest types in La Selva Biological Station, Costa Rica, Barro Colorado Island, Panama and Tiputini Biodiversity Station, Ecuador. This metabarcoding approach has the power to uncover known and new taxa on a massive scale 11 . By amplifying DNA extracted from the soils with broadly targeted primers for the V4 region of 18S rRNA and sequencing it using the Illumina MiSeq platform, we were able to detect most eukaryotic lineages, and assess the diversity and relative dominance of free-living and parasitic lineages.

Summary 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.

Fungi appear to be rare in marine environments. There are relatively few marine isolates in culture, and fungal small subunit ribosomal DNA (SSU rDNA) sequences are rarely recovered in marine clone library experiments (i.e., culture-independent sequence surveys of eukaryotic microbial diversity from environmental DNA samples). To explore the diversity of marine fungi, we took a broad selection of SSU rDNA data sets and calculated a summary phylogeny. Bringing these data together identified a diverse collection of marine fungi, including sequences branching close to chytrids (flagellated fungi), filamentous hypha-forming fungi, and multicellular fungi. However, the majority of the sequences branched with ascomycete and basidiomycete yeasts. We discuss evidence for 36 novel marine lineages, the majority and most divergent of which branch with the chytrids. We then investigate what these data mean for the evolutionary history of the Fungi and specifically marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine ecosystems.

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Fungi are the principal degraders of biomass in most terrestrial ecosystems. In contrast to surface environments, deep-sea environmental gene libraries have suggested that fungi are rare and non-diverse in high-pressure marine environments. Here, we report the diversity of fungi from 11 deep-sea samples from around the world representing depths from 1500 to 4000 m (146-388 atm) and two shallower water column samples (250 and 500 m). We sequenced 239 clones from 10 fungal-specific 18S rRNA gene libraries constructed from these samples, from which we detected only 18 fungal 18S-types in deep-sea samples. Our phylogenetic analyses show that a total of only 32 fungal 18S-types have so far been recovered from deep-sea habitats, and our results suggest that fungi, in general, are relatively rare in the deep-sea habitats we sampled. The fungal diversity detected suggests that deep-sea environments host an evolutionarily diverse array of fungi dominated by groups of distantly related yeasts, although four putative filamentous fungal 18S-types were detected. The majority of our new sequences branch close to known fungi found in surface environments. This pattern contradicts the proposal that deep-sea and hydrothermal vent habitats represent ancient ecosystems, and demonstrates a history of frequent dispersal between terrestrial and deep-sea habitats.

This study presents the first 18S rRNA multi-library environmental PCR survey of a single protozoan phylum, Cercozoa Cavalier-Smith 1998, from a range of different habitats. Phylogenetic analysis reveals at least nine novel clades within the phylum, several possibly at the level of order or above. Further experiments are described to ascertain the true ecological and geographical distributions of some clades that might be inferred from the tree to be restricted in either or both ways. These results suggest that the diversity of cercozoan taxa may run into thousands of lineages, making it comparable in diversity to the largest better-characterized protozoan phyla, e.g. Ciliophora (ciliates and suctorians) and Foraminifera. New sequences of cultured Spongomonas, Metromonas and Metopion are also presented. In the light of these additions, and the increased taxon sampling from the environmental libraries, some revisions of cercozoan classification are made: the transfer of Spongomonadea from Reticulofilosa to Monadofilosa; the removal of Metopiida from Sarcomonadea; and the creation of the new order Metromonadida, currently containing the single genus Metromonas. Although Metromonas groups with weak to moderate support with Chlorarachnea, it is here placed in superclass Monadofilosa, to which it is morphologically more similar.