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.

A decade after environmental scientists integrated high‐throughput sequencing technologies in their toolbox, the genomics‐based monitoring of anthropogenic impacts on the biodiversity and functioning of ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental genomics to this end, technical limitations and conceptual issues still stand in the way of its broad application by end‐users. In addition, the multiplicity of potential implementation strategies may contribute to a perception that the routine application of this methodology is premature or “in development”, hence restraining regulators from binding these tools into legal frameworks. Here, we review recent implementations of environmental genomics‐based methods, applied to the biomonitoring of ecosystems. By taking a general overview, without narrowing our perspective to particular habitats or groups of organisms, this paper aims to compare, review and discuss the strengths and limitations of four general implementation strategies of environmental genomics for monitoring: (A) Taxonomy‐based analyses focused on identification of known bioindicators or described taxa; (B) De novo bioindicator analyses; (C) Structural community metrics including inferred ecological networks; and (D) Functional community metrics (metagenomics or metatranscriptomics). We emphasise the utility of the three latter strategies to integrate meiofauna and microorganisms that are not traditionally utilised in biomonitoring because of difficult taxonomic identification. Finally, we propose a roadmap for the implementation of environmental genomics into routine monitoring programs that leverage recent analytical advancements, while pointing out current limitations and future research needs.

Background: Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.Results: The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs) far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i) BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA) gene sequences for taxonomic assignments of tags; (ii) a clustering of tags at k differences (Levenshtein distance) with a newly developed algorithm enabling very fast OTU clustering for large tag sequence data sets; and (iii) a novel parsing procedure to combine the data from individual analyses.Conclusion: Our data highlight the magnitude of the under-sampled 'protistan gap' in the eukaryotic tree of life. This study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.

Summary: Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small‐subunit rDNA) of an intertidal protistan model community, using the Roche GS‐FLX and the most recent GS‐FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2039 unique tags (from 3879 V4 GS‐FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS‐FLX). Pyrosequencing error rates were higher for V4 fragments compared with the V9 domain and for the GS‐FLX Titanium compared with the GS‐FLX system. Furthermore, this experiment revealed that error rates are taxon‐specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data‐processing pipelines.

Summary: The frontiers of eukaryote life in nature are still unidentified. In this study, we analysed protistan communities in the hypersaline (up to 365 g l ⁻¹ NaCl) anoxic L'Atalante deep‐sea basin located in the eastern Mediterranean Sea. Targeting 18S ribosomal RNA retrieved from the basin's lower halocline (3501 m depth) we detected 279 protistan sequences that grouped into 42 unique phylotypes (99% sequence similarity). Statistical analyses revealed that these phylotypes account only for a proportion of the protists inhabiting this harsh environment with as much as 50% missed by this survey. Most phylotypes were affiliated with ciliates (45%), dinoflagellates (21%), choanoflagelates (10%) and uncultured marine alveolates (6%). Sequences from other taxonomic groups like stramenopiles, Polycystinea , Acantharea and Euglenozoa , all of which are typically found in non‐hypersaline deep‐sea systems, are either missing or very rare in our cDNA clone library. Although many DHAB sequences fell within previously identified environmental clades, a large number branched relatively deeply. Phylotype richness, community membership and community structure differ significantly from a deep seawater reference community (3499 m depth). Also, the protistan community in the L'Atalante basin is distinctively different from any previously described hypersaline community. In conclusion, we hypothesize that extreme environments may exert a high selection pressure possibly resulting in the evolution of an exceptional and distinctive assemblage of protists. The deep hypersaline anoxic basins in the Mediterranean Sea provide an ideal platform to test for this hypothesis and are promising targets for the discovery of undescribed protists with unknown physiological capabilities.