Thorsten Stoeck

Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water

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.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms

A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.

Novel Eukaryotic Lineages Inferred from Small-Subunit rRNA Analyses of Oxygen-Depleted Marine Environments

ABSTRACT Microeukaryotes in oxygen-depleted environments are among the most diverse, as well as the least studied, organisms. We conducted a cultivation-independent, small-subunit (SSU) rRNA-based survey of microeukaryotes in suboxic waters and anoxic sediments in the great Sippewisset salt marsh, Cape Cod, Mass. We generated two clone libraries and analyzed approximately 300 clones, which contained a large diversity of microeukaryotic SSU rRNA signatures. Only a few of these signatures were closely related (sequence similarity of >97%) to the sequences reported earlier. The bulk of our sequences represented deep novel branches within green algae, fungi, cercozoa, stramenopiles, alveolates, euglenozoa and unclassified flagellates. In addition, a significant number of detected rRNA sequences exhibited no affiliation to known organisms and sequences and thus represent novel lineages of the highest taxonomical order, most of them branching off the base of the global phylogenetic tree. This suggests that oxygen-depleted environments harbor diverse communities of novel organisms, which may provide an interesting window into the early evolution of eukaryotes.

Patterns of Rare and Abundant Marine Microbial Eukaryotes

BACKGROUND Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.

Novel Eukaryotes from the Permanently Anoxic Cariaco Basin (Caribbean Sea)

ABSTRACT Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000). Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S. L. Baldauf, Science 300:1703-1706, 2003). The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery. Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast. On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages. These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups. Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches. These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s).

Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions

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.

Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic Fjord (Framvaren, Norway)

ABSTRACT To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O2/H2S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O2/H2S gradient.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

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.

A Molecular Approach to Identify Active Microbes in Environmental Eukaryote Clone Libraries

A rapid method for the simultaneous extraction of RNA and DNA from eukaryote plankton samples was developed in order to discriminate between indigenous active cells and signals from inactive or even dead organisms. The method was tested using samples from below the chemocline of an anoxic Danish fjord. The simple protocol yielded RNA and DNA of a purity suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR) and PCR, respectively. We constructed an rRNA-derived and an rDNA-derived clone library to assess the composition of the microeukaryote assemblage under study and to identify physiologically active constituents of the community. We retrieved nearly 600 protistan target clones, which grouped into 84 different phylotypes (98% sequence similarity). Of these phylotypes, 27% occurred in both libraries, 25% exclusively in the rRNA library, and 48% exclusively in the rDNA library. Both libraries revealed good correspondence of the general community composition in terms of higher taxonomic ranks. They were dominated by anaerobic ciliates and heterotrophic stramenopile flagellates thriving below the fjord’s chemocline. The high abundance of these bacterivore organisms points out their role as a major trophic link in anoxic marine systems. A comparison of the two libraries identified phototrophic dinoflagellates, “uncultured marine alveolates group I,” and different parasites, which were exclusively detected with the rDNA-derived library, as nonindigenous members of the anoxic microeukaryote community under study.