Alexandra Stock

Delimiting operational taxonomic units for assessing ciliate environmental diversity using small-subunit rRNA gene sequences

Delineating operational taxonomic units (OTUs) is a central element in any culture-independent analysis of environmental microbial eukaryotic diversity. Previous studies either have not justified their choice in sequence distance used to bin small-subunit ribosomal RNA (SSU rRNA) gene sequences amplified from environmental samples into OTUs, or have used a value based on the average across a broad sampling of microbial eukaryotes. Here, we analyse distances (320 922 pairwise comparisons) among sequences just from identified ciliates, and compare these with their taxonomic hierarchy. Our results show that no single sequence similarity value can always and unambiguously delineate species boundaries and higher taxa. Nevertheless, we suggest the use of 98% similarity to delineate ciliate OTUs because this threshold at least accounts for intra-specific polymorphism among multiple rRNA cistron copies. However, we suggest refraining from reconciling SSU rRNA gene-based OTUs and ciliate morphotypes; these OTUs should be used to analyse ciliate phylotype diversity, not ciliate species diversity.

Placing environmental next generation sequencing amplicons from microbial eukaryotes into a phylogenetic context

Nucleotide positions in the hypervariable V4 and V9 regions of the small subunit (SSU)-rDNA locus are normally difficult to align and are usually removed before standard phylogenetic analyses. Yet, with next-generation sequencing data, amplicons of these regions are all that are available to answer ecological and evolutionary questions that rely on phylogenetic inferences. With ciliates, we asked how inclusion of the V4 or V9 regions, regardless of alignment quality, affects tree topologies using distinct phylogenetic methods (including PairDist that is introduced here). Results show that the best approach is to place V4 amplicons into an alignment of full-length Sanger SSU-rDNA sequences and to infer the phylogenetic tree with RAxML. A sliding window algorithm as implemented in RAxML shows, though, that not all nucleotide positions in the V4 region are better than V9 at inferring the ciliate tree. With this approach and an ancestral-state reconstruction, we use V4 amplicons from European nearshore sampling sites to infer that rather than being primarily terrestrial and freshwater, colpodean ciliates may have repeatedly transitioned from terrestrial/freshwater to marine environments.

Evidence for isolated evolution of deep-sea ciliate communities through geological separation and environmental selection

BackgroundDeep hypersaline anoxic basins (DHABs) are isolated habitats at the bottom of the eastern Mediterranean Sea, which originate from the ancient dissolution of Messinian evaporites. The different basins have recruited their original biota from the same source, but their geological evolution eventually constituted sharp environmental barriers, restricting genetic exchange between the individual basins. Therefore, DHABs are unique model systems to assess the effect of geological events and environmental conditions on the evolution and diversification of protistan plankton. Here, we examine evidence for isolated evolution of unicellular eukaryote protistan plankton communities driven by geological separation and environmental selection. We specifically focused on ciliated protists as a major component of protistan DHAB plankton by pyrosequencing the hypervariable V4 fragment of the small subunit ribosomal RNA. Geospatial distributions and responses of marine ciliates to differential hydrochemistries suggest strong physical and chemical barriers to dispersal that influence the evolution of this plankton group.ResultsCiliate communities in the brines of four investigated DHABs are distinctively different from ciliate communities in the interfaces (haloclines) immediately above the brines. While the interface ciliate communities from different sites are relatively similar to each other, the brine ciliate communities are significantly different between sites. We found no distance-decay relationship, and canonical correspondence analyses identified oxygen and sodium as most important hydrochemical parameters explaining the partitioning of diversity between interface and brine ciliate communities. However, none of the analyzed hydrochemical parameters explained the significant differences between brine ciliate communities in different basins.ConclusionsOur data indicate a frequent genetic exchange in the deep-sea water above the brines. The “isolated island character” of the different brines, that resulted from geological events and contemporary environmental conditions, create selective pressures driving evolutionary processes, and with time, lead to speciation and shape protistan community composition. We conclude that community assembly in DHABs is a mixture of isolated evolution (as evidenced by small changes in V4 primary structure in some taxa) and species sorting (as indicated by the regional absence/presence of individual taxon groups on high levels in taxonomic hierarchy).

Living at the Limits: Evidence for Microbial Eukaryotes Thriving under Pressure in Deep Anoxic, Hypersaline Habitats

The advent of molecular tools in microbial ecology paved the way to exploit the diversity of microbes in extreme environments. Here, we review these tools as applied in one of the most polyextreme habitats known on our planet, namely, deep hypersaline anoxic basins (DHABs), located at ca. 3000–3500 m depth in the Eastern Mediterranean Sea. Molecular gene signatures amplified from environmental DHAB samples identified a high degree of genetic novelty, as well as distinct communities in the DHABs. Canonical correspondence analyses provided strong evidence that salinity, ion composition, and anoxia were the strongest selection factors shaping protistan community structures, largely preventing cross-colonization among the individual basins. Thus, each investigated basin represents a unique habitat (“isolated islands of evolution”), making DHABs ideal model sites to test evolutionary hypotheses. Fluorescence in situ hybridization assays using specifically designed probes revealed that the obtained genetic signatures indeed originated from indigenous polyextremophiles. Electron microscopy imaging revealed unknown ciliates densely covered with prokaryote ectosymbionts, which may enable adaptations of eukaryotes to DHAB conditions. The research reviewed here significantly advanced our knowledge on polyextremophile eukaryotes, which are excellent models for a number of biological research areas, including ecology, diversity, biotechnology, evolutionary research, physiology, and astrobiology.

Environmental selection of protistan plankton communities in hypersaline anoxic deep‐sea basins, Eastern Mediterranean Sea

High salt concentrations, absence of light, anoxia, and high hydrostatic pressure make deep hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea one of the most polyextreme habitats on Earth. Taking advantage of the unique chemical characteristics of these basins, we tested the effect of environmental selection and geographic distance on the structure of protistan communities. Terminal restriction fragment length polymorphism (T‐RFLP) analyses were performed on water samples from the brines and seawater/brine interfaces of five basins: Discovery, Urania, Thetis, Tyro, and Medee. Using statistical analyses, we calculated the partitioning of diversity among the ten individual terminal restriction fragment (T‐RF) profiles, based on peak abundance and peak incidence. While a significant distance effect on spatial protistan patterns was not detected, hydrochemical gradients emerged as strong dispersal barriers that likely lead to environmental selection in the DHAB protistan plankton communities. We identified sodium, magnesium, sulfate, and oxygen playing in concerto as dominant environmental drivers for the structuring of protistan plankton communities in the Eastern Mediterranean DHABs.

Deep Hypersaline Anoxic Basins as Model Systems for Environmental Selection of Microbial Plankton

Biodiversity and biogeographic patterns of microbial organisms in general and microbial plankton specifically fuel a multitude of scientific surveys and discussions. In this context, a central question addresses the influence of environmental variables versus historical contingencies on the evolution and composition of microbial plankton communities. Such and similar fundamental questions in microbial ecology can be addressed with the benefits of model systems, such as deep hypersaline anoxic basins (DHABs). DHABs occur in different oceanic regions, for example, in the Gulf of Mexico, the Red Sea, and the Mediterranean Sea. They are characterized by a salt-induced stratification of the water column with a stable, polyextreme brine lake with highest salt concentrations, anoxia, and high pressure in complete darkness. Their unique hydrochemistries and physical separation for thousands of years give these brine lakes an “island character” with little or no genetic exchange with surrounding habitats. This makes DHABs ideal model systems in biogeography and evolutionary research. After the discovery of diverse planktonic life in the DHAB brines (see the chapter “ Microbial Eukaryotes in Marine Oxygen Minimum Zones” by Orsi and Edgcomb in this volume), studies have been conducted on the distribution of prokaryotes and eukaryotes in these environments. We here review our current knowledge on this subject and emphasize the model character of DHABs for future research.