Dominik Forster

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.

Testing ecological theories with sequence similarity networks: marine ciliates exhibit similar geographic dispersal patterns as multicellular organisms

BackgroundHigh-throughput sequencing technologies are lifting major limitations to molecular-based ecological studies of eukaryotic microbial diversity, but analyses of the resulting millions of short sequences remain a major bottleneck for these approaches. Here, we introduce the analytical and statistical framework of sequence similarity networks, increasingly used in evolutionary studies and graph theory, into the field of ecology to analyze novel pyrosequenced V4 small subunit rDNA (SSU-rDNA) sequence data sets in the context of previous studies, including SSU-rDNA Sanger sequence data from cultured ciliates and from previous environmental diversity inventories.ResultsOur broadly applicable protocol quantified the progress in the description of genetic diversity of ciliates by environmental SSU-rDNA surveys, detected a fundamental historical bias in the tendency to recover already known groups in these surveys, and revealed substantial amounts of hidden microbial diversity. Moreover, network measures demonstrated that ciliates are not globally dispersed, but are structured by habitat and geographical location at intermediate geographical scale, as observed for bacteria, plants, and animals.ConclusionsCurrently available ‘universal’ primers used for local in-depth sequencing surveys provide little hope to exhaust the significantly higher ciliate (and most likely microbial) diversity than previously thought. Network analyses such as presented in this study offer a promising way to guide the design of novel primers and to further explore this vast and structured microbial diversity.

Benthic protists: the under-charted majority

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBIs Short Read Archive. We suggest that the benthic realm may therefore be the worlds largest reservoir of marine protist diversity, with most taxa at present undescribed.

Meta-analyses of environmental sequence data identify anoxia and salinity as parameters shaping ciliate communities

Nearly 6000 SSU rRNA sequences of ciliated protists, compiled from 50 marine and terrestrial sampling sites worldwide were analysed to identify environmental barriers spatially relating ciliate communities. The hypotheses were: (i) oxygen is a dispersal barrier for strict anaerobes, allowing allopatric speciation in isolated habitats and resulting in high diversity; (ii) as salinity affects metabolism it may lead to distinct clusters with evolutionarily separated lineages; (iii) different oceanic realms support significantly different ciliate communities. Based on the α-diversity of the ciliate sequences in each habitat and the shared diversity among different habitats, ecological and geographic boundaries were revealed. Community similarities between pairs of habitats and all habitats were depicted with a metric distance matrix. Anoxia and salinity emerged as decisive determinants structuring the communities, with anoxia largely overlaying the effect of other environmental parameters. Measurements of α-diversity suggest a tremendous diversity of ciliates in anoxic environments, exceeding the one in normoxic environments. Salinity also exerts a high selection pressure on ciliates, contributing to community structure, composition and distribution patterns. However, marine–freshwater transitions seem to be more recent in ciliates compared with most other protistan taxa. The theory of island biogeography seems to apply to anoxic habitats, making these sites promising targets for the discovery of novel diversity.

Comparison of three clustering approaches for detecting novel environmental microbial diversity

Discovery of novel diversity in high-throughput sequencing studies is an important aspect in environmental microbial ecology. To evaluate the effects that amplicon clustering methods have on the discovery of novel diversity, we clustered an environmental marine high-throughput sequencing dataset of protist amplicons together with reference sequences from the taxonomically curated Protist Ribosomal Reference (PR2) database using three de novo approaches: sequence similarity networks, USEARCH, and Swarm. The potentially novel diversity uncovered by each clustering approach differed drastically in the number of operational taxonomic units (OTUs) and in the number of environmental amplicons in these novel diversity OTUs. Global pairwise alignment comparisons revealed that numerous amplicons classified as potentially novel by USEARCH and Swarm were more than 97% similar to references of PR2. Using shortest path analyses on sequence similarity network OTUs and Swarm OTUs we found additional novel diversity within OTUs that would have gone unnoticed without further exploiting their underlying network topologies. These results demonstrate that graph theory provides powerful tools for microbial ecology and the analysis of environmental high-throughput sequencing datasets. Furthermore, sequence similarity networks were most accurate in delineating novel diversity from previously discovered diversity.

Molecular Diversity of Fungi from Marine Oxygen-Deficient Environments (ODEs)

Molecular diversity surveys of marine fungi have demonstrated that the species richness known to date is just the tip of the iceberg and that there is a large extent of unknown fungal diversity in marine habitats. Reports of novel fungal lineages at higher taxonomic levels are documented from a large number of marine habitats, including the various marine oxygen-deficient environments (ODEs). In the past few years, a strong focus of eukaryote diversity research has been on a variety of ODEs, as these environments are considered to harbor a large number of organisms, which are highly divergent to known diversity and could provide insights into the early eukaryotic evolution. ODEs that have been targeted so far include shallow water sediments, hydrothermal vent systems, deep-sea basins, intertidal habitats, and fjords. Most, if not all, molecular diversity studies in marine ODEs have shown, that contrary to previous assumptions, fungi contribute significantly to the micro-eukaryotic community in such habitats. In this chapter, we have reanalyzed the environmental fungal sequences obtained from the molecular diversity survey in 14 different sites to obtain a comprehensive picture of fungal diversity in these marine habitats. The phylogenetic analysis of the fungal environmental sequences from various ODEs have grouped these sequences into seven distinct clades (Clade 1-7) clustering with well-known fungal taxa. Apart from this, four environmental clades (EnvClade A, B, C, and D) with exclusive environmental sequences were also identified. This has provided information on the positioning of the environmental sequences at different taxonomic levels within the major fungal phylums. The taxonomic distribution of these environmental fungal sequences into clusters and clades has also shown that they are not restricted by geographical boundaries. The distribution pattern together with the reports on the respiratory abilities of fungi under reduced oxygen conditions shows that they are highly adaptive and may have a huge ecological role in these oxygen deficient habitats.

Transition boundaries for protistan species turnover in hypersaline waters of different biogeographic regions

The identification of environmental barriers which govern species distribution is a fundamental concern in ecology. Even though salt was previously identified as a major transition boundary for micro- and macroorganisms alike, the salinities causing species turnover in protistan communities are unknown. We investigated 4.5 million high-quality protistan metabarcodes (V4 region of the SSU rDNA) obtained from 24 shallow salt ponds (salinities 4%-44%) from South America and Europe. Statistical analyses of protistan community profiles identified four salinity classes, which strongly selected for different protistan communities: 4-9%, 14-24%, 27-36% and 38-44%. The proportion of organisms unknown to science is highest in the 14-24% salinity class, showing that environments within this salinity range are an unappreciated reservoir of as yet undiscovered organisms. Distinct higher-rank taxon groups dominated in the four salinity classes in terms of diversity. As increasing salinities require different cellular responses to cope with salt, our results suggest that different evolutionary lineages of protists have evolved distinct haloadaptation strategies. Salinity appears to be a stronger selection factor for the structuring of protistan communities than geography. Yet, we find a higher degree of endemism in shallow salt ponds compared with less isolated ecosystems such as the open ocean. Thus, rules for biogeographic structuring of protistan communities are not universal, but depend on the ecosystem under consideration.

Environmental DNA metabarcoding of benthic bacterial communities indicates the benthic footprint of salmon aquaculture

We evaluated benthic bacterial communities as bioindicators in environmental impact assessments of salmon aquaculture, a rapidly growing sector of seafood industry. Sediment samples (n=72) were collected from below salmon cages towards distant reference sites. Bacterial community profiles inferred from DNA metabarcodes were compared to reference data from standard macrofauna biomonitoring surveys of the same samples. Deltaproteobacteria were predominant in immediate vicinity of the salmon cages. Along the transect, significant shifts in bacterial community structures were observed with Gammaproteobacteria dominating the less-impacted sites. Alpha- and beta-diversity measures of bacterial communities correlated significantly with macrofauna diversity metrics and with five ecological status indices. Benthic bacterial communities mirror the reaction of macrofauna bioindicators to environmental disturbances caused by salmon farming. The implementation of bacterial eDNA metabarcoding in future Strategic Framework Directives is an alternative cost-effective high-throughput biomonitoring solution, providing a basis for management strategies in a matter of days rather than months.

Towards an eDNA metabarcode-based performance indicator for full-scale municipal wastewater treatment plants

Knowledge-driven management for wastewater treatment plant (WWTP) performance becomes increasingly important considering the globally growing production of wastewater and the rising demand of clean water supply. Even though the potential of microbial organisms (bacteria and protists) as bioindicators for WWTP performance is well known, it is far from being fully exploited for routine monitoring programs. Therefore, we here used massive sequencing of environmental (e)DNA metabarcodes from bacterial (V3-V4 region of the SSU rRNA gene) and eukaryote (V9 region of the SSU rRNA gene) communities in 21 activated sludge samples obtained from full-scale municipal WWTPs in Germany. Microbial community patterns were compared to standard WWTP operating parameters and two traditionally used WWTP performance indicators (Sludge Biotic Index and Sludge Index). Both indices showed low concordance and hardly correlated with chemical WWTP performance parameters nor did they correlate with microbial community structures. In contrast, microbial community profiles significantly correlated with WWTP performance parameters and operating conditions of the plants under study. Therefore, eDNA metabarcode profiles of whole microbial communities indicate the performance of WWTP and can provide useful information for management strategies. We here suggest a strategy for the development of an eDNA metabarcode based bioindicator system, which can be implemented in future standard monitoring programs for WWTP performance and effluent quality.

Supervised machine learning outperforms taxonomy-based environmental DNA metabarcoding applied to biomonitoring

Biodiversity monitoring is the standard for environmental impact assessment of anthropogenic activities. Several recent studies showed that high‐throughput amplicon sequencing of environmental DNA (eDNA metabarcoding) could overcome many limitations of the traditional morphotaxonomy‐based bioassessment. Recently, we demonstrated that supervised machine learning (SML) can be used to predict accurate biotic indices values from eDNA metabarcoding data, regardless of the taxonomic affiliation of the sequences. However, it is unknown to which extent the accuracy of such models depends on taxonomic resolution of molecular markers or how SML compares with metabarcoding approaches targeting well‐established bioindicator species. In this study, we address these issues by training predictive models upon five different ribosomal bacterial and eukaryotic markers and measuring their performance to assess the environmental impact of marine aquaculture on independent data sets. Our results show that all tested markers are yielding accurate predictive models and that they all outperform the assessment relying solely on taxonomically assigned sequences. Remarkably, we did not find any significant difference in the performance of the models built using universal eukaryotic or prokaryotic markers. Using any molecular marker with a taxonomic range broad enough to comprise different potential bioindicator taxa, SML approach can overcome the limits of taxonomy‐based eDNA bioassessment.