Summary: Analyses of high‐throughput environmental sequencing data have become the ‘gold‐standard’ to address fundamental questions of microbial diversity, ecology and biogeography. Findings that emerged from sequencing are, e.g. the discovery of the extensive ‘rare microbial biosphere’ and its potential function as a seed‐bank. Even though applied since several years, results from high‐throughput environmental sequencing have hardly been validated. We assessed how well pyrosequenced amplicons [the hypervariable eukaryotic V 4 region of the small subunit ribosomal RNA ( SSU rRNA ) gene] reflected morphotype ciliate plankton. Moreover, we assessed if amplicon sequencing had the potential to detect the annual ciliate plankton stock. In both cases, we identified significant quantitative and qualitative differences. Our study makes evident that taxon abundance distributions inferred from amplicon data are highly biased and do not mirror actual morphotype abundances at all. Potential reasons included cell losses after fixation, cryptic morphotypes, resting stages, insufficient sequence data availability of morphologically described species and the unsatisfying resolution of the V 4 SSU rRNA fragment for accurate taxonomic assignments. The latter two underline the necessity of barcoding initiatives for eukaryotic microbes to better and fully exploit environmental amplicon data sets, which then will also allow studying the potential of seed‐bank taxa as a buffer for environmental changes.

Ciliates represent central nodes in freshwater planktonic food webs, and many species show pronounced seasonality, with short-lived maxima of a few dominant taxa while many being rare or ephemeral. These observations are primarily based on morphospecies counting methods, which, however, have limitations concerning the amount and volume of samples that can be processed. For high sampling frequencies at large scales, high throughput sequencing (HTS) of freshwater ciliates seems to be a promising tool. However, several studies reported large discrepancy between species abundance determinations by molecular compared to morphological means. Therefore, we compared ciliate DNA metabarcodes (V9 regions of the 18S rRNA gene) with morphospecies counts for a 3-year study (Lake Zurich, Switzerland; biweekly sampling, n = 74). In addition, we isolated, cultivated and sequenced the 18S rRNA gene of twelve selected ciliate species that served as seeds for HTS analyses. This workflow allowed for a detailed comparison of V9 data with microscopic analyses by quantitative protargol staining (QPS). The dynamics of V9 read abundances over the seasonal cycle corroborated well with morphospecies population patterns. Annual successions of rare and ephemeral species were more adequately characterized by V9 reads than by QPS. However, numbers of species specific sequence reads only partly reflected rank orders seen by counts. In contrast, biomass-based assemblage compositions showed higher similarity to V9 read numbers, probably indicating a relation between cell sizes and numbers / sizes of macronuclei (or 18S rRNA operons). Full-length 18S rRNA sequences of ciliates assigned to certain morphospecies are urgently needed for barcoding approaches as planktonic taxa are still poorly represented in public databases and the interpretation of HTS data depends on profound reference sequences. Through linking operational taxonomic units (OTUs) with known morphospecies, we can use the deep knowledge about the autecology of these species.

We followed the changes in the protist assemblage over an annual cycle at 3 sites and different depths of Traunsee in the Austrian Alps and quantified the variability of the ciliate assemblage along successive depth and time intervals, respectively. More than 60 ciliate species were identified alive and after quantitative protargol staining (QPS). The ciliate diversity was high, and is described in detail for 50 taxa in time¿depth intervals. Rimostrombidium brachykinetum/Rimostrombidium hyalinum and Balanion planctonicum were the most frequent species, accounting for 44% of the annual mean abundance and 15% of the annual mean biovolume, respectively. Our results suggested a stronger variability in the ciliate assemblage structure within seasons than along the depth gradient. Gradual changes in the assemblage structure with depths: (1) were accompanied by a decrease of algivorous and mixotrophic and an increase of bacterivorous ciliates from surface to deeper layers; (2) were highly significantly altered with steep depth gradients of their potential food resources, i.e. the biomass of heterotrophic flagellates, bacteria and algae; and (3) were related with lower significance to environmental parameters. High similarity (>80%) between successive months was reached only when net changes in the total ciliate abundance were negligible, while a strong increase or decrease in the ciliate abundance was associated with pronounced changes in the species composition. These seasonal changes in the ciliate assemblage structure (4) were linked to shifts of algivorous and mixotrophic, but not of bacterivorous ciliates and (5) were less predictable with food resources compared to the depth gradient. The phagotrophic flagellates generally followed the seasonal and vertical patterns described for ciliates, and were shown to be important members of the planktonic food web in a cold, deep, oligotrophic lake

We analyzed the genetic diversity (V4 region of the 18S rRNA) of planktonic microbial eukaryotes in four high mountain lakes including two remote biogeographic regions (the Himalayan mountains and the European Alps) and distinct habitat types (clear and glacier-fed turbid lakes). The recorded high genetic diversity in these lakes was far beyond of what is described from high mountain lake plankton. In total, we detected representatives from 66 families with the main taxon groups being Alveolata (55.0% OTUs_97%, operational taxonomic units), Stramenopiles (34.0% OTUs_97%), Cryptophyta (4.0% OTUs_97%), Chloroplastida (3.6% OTUs_97%) and Fungi (1.7% OTUs_97%). Centrohelida, Choanomonada, Rhizaria, Katablepharidae and Telonema were represented by <1% OTUs_97%. Himalayan lakes harbored a higher plankton diversity compared to the Alpine lakes (Shannon index). Community structures were significantly different between lake types and biogeographic regions (Fisher exact test, P < 0.01). Network analysis revealed that more families of the Chloroplastida (10 vs 5) and the Stramenopiles (14 vs 8) were found in the Himalayan lakes than in the Alpine lakes and none of the fungal families was shared between them. Biogeographic aspects as well as ecological factors such as water turbidity may structure the microbial eukaryote plankton communities in such remote lakes.