Steffen Jost

Diversity in a hidden world: potential and limitation of next-generation sequencing for surveys of molecular diversity of eukaryotic microorganisms.

With the delivery of millions of sequence reads in a single experiment, next‐generation sequencing (NGS) is currently revolutionizing surveys of microorganism diversity. In particular, when applied to Eukaryotes, we are still lacking a rigorous comparison of morphological and NGS‐based diversity estimates. In this report, we studied the diversity and the seasonal community turnover of alveolates (Ciliophora and Dinophyceae) in an oligotrophic freshwater lake by SSU amplicon sequencing with NGS as well as by classical morphological analysis. We complemented the morphological analysis by single‐cell PCR followed by Sanger sequencing to provide an unambiguous link to the NGS data. We show that NGS and morphological analyses generally capture frequency shifts of abundant taxa over our seasonal samples. The observed incongruencies are probably largely due to rDNA copy number variation among taxa and heterogeneity in the efficiency of cell lysis. Overall, NGS‐based amplicon sequencing was superior in detecting rare species. We propose that in the absence of other nuclear markers less susceptible to copy number variation, rDNA‐based diversity studies need to be adjusted for confounding effects of copy number variation.

Contrasting seasonal niche separation between rare and abundant taxa conceals the extent of protist diversity

With the advent of molecular methods, it became clear that microbial biodiversity had been vastly underestimated. Since then, species abundance patterns were determined for several environments, but temporal changes in species composition were not studied to the same level of resolution. Using massively parallel sequencing on the 454 GS FLX platform we identified a highly dynamic turnover of the seasonal abundance of protists in the Austrian lake Fuschlsee. We show that seasonal abundance patterns of protists closely match their biogeographic distribution. The stable predominance of few highly abundant taxa, which previously led to the suggestion of a low global protist species richness, is contrasted by a highly dynamic turnover of rare species. We suggest that differential seasonality of rare and abundant protist taxa explains the—so far—conflicting evidence in the ‘everything is everywhere’ dispute. Consequently temporal sampling is basic for adequate diversity and species richness estimates.

Protistan community analysis: key findings of a large-scale molecular sampling

Protists are perhaps the most lineage-rich of microbial lifeforms, but remain largely unknown. High-throughput sequencing technologies provide opportunities to screen whole habitats in depth and enable detailed comparisons of different habitats to measure, compare and map protistan diversity. Such comparisons are often limited by low sample numbers within single studies and a lack of standardisation between studies. Here, we analysed 232 samples from 10 sampling campaigns using a standardised PCR protocol and bioinformatics pipeline. We show that protistan community patterns are highly consistent within habitat types and geographic regions, provided that sample processing is standardised. Community profiles are only weakly affected by fluctuations of the abundances of the most abundant taxa and, therefore, provide a sound basis for habitat comparison beyond random short-term fluctuations in the community composition. Further, we provide evidence that distribution patterns are not solely resulting from random processes. Distinct habitat types and distinct taxonomic groups are dominated by taxa with distinct distribution patterns that reflect their ecology with respect to dispersal and habitat colonisation. However, there is no systematic shift of the distribution pattern with taxon abundance.

Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria.

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.

AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

CULTIVATION-INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE-CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.

Hydrurus-related golden algae (Chrysophyceae) cause yellow snow in polar summer snowfields

In polar regions, melting snow fields can be occupied by striking blooms of chrysophycean algae, which cause yellowish slush during summer. Samples were harvested at King George Island (South Shetland Islands, Maritime Antarctica) and at Spitsbergen (Svalbard archipelago, High Arctic). The populations live in an ecological niche, where water‐logged snow provides a cold and ephemeral ecosystem, possibly securing the survival of psychrophilic populations through the summer. A physiological adaptation to low temperatures was shown by photosynthesis measurements. The analysis of soluble carbohydrates showed the occurrence of glycerol and sugars, which may play a role in protection against intracellular freezing. Although both populations were made of unicells with Ochromonas‐alike morphology, investigation by molecular methods (18S rDNA sequencing) revealed unexpectedly a very close relationship to the mountain‐river dwelling Hydrurus foetidus (Villars) Trevisan. However, macroscopic thalli typical for the latter species were never found in snow, but are known from nearby localities, and harvested samples of snow algae exposed to dryness evolved a similar pervading, ‘fishy’ smell. Moreover, in both habitats tetrahedal zoospores with four elongate spikes were found, similar to what is known from Hydrurus. Our molecular results go along with earlier reports, where chrysophycean sequences of the same taxonomic affiliation were isolated from snow. This points to a distinct group of photoautotrophic, Hydrurus‐related chrysophytes, which are characteristic for long‐lasting, slowly melting snow packs in certain cold regions of the world.

Magnesiothermic conversion of the silica-mineralizing golden algae Mallomonas caudata and Synura petersenii to elemental silicon with high geometric precision

Summary Chrysophyceae, also known as golden algae, contain characteristic, three-dimensional biomineralized silica structures. Their chemical composition and microscopic structure was studied. By high-temperature conversion of the skeleton of Mallomonas caudata and Synura petersenii into elementary silicon by magnesium vapour, nanostructured defined replicates were produced which were clearly seen after removal of the formed magnesium oxide with acid.