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 V4 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 V4 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.

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% OTUs97%, operational taxonomic units), Stramenopiles (34.0% OTUs97%), Cryptophyta (4.0% OTUs97%), Chloroplastida (3.6% OTUs97%) and Fungi (1.7% OTUs97%). Centrohelida, Choanomonada, Rhizaria, Katablepharidae and Telonema were represented by <1% OTUs97%. 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.

Summary Climate warming is accelerating the retreat of glaciers and recently, many ‘new’ glacial turbid lakes have been created. In the course of time, the loss of the hydrological connectivity to a glacier causes, however, changes in their water turbidity and turns these ecosystems into clear ones.To understand potential differences in the food‐web structure between glacier‐fed turbid and clear alpine lakes, we sampled ciliates, phyto‐, bacterio‐ and zooplankton in one clear and one glacial turbid alpine lake, and measured key physicochemical parameters. In particular, we focused on the ciliate community and the potential drivers for their abundance distribution.In both lakes, the zooplankton community was similar and dominated by the copepod Cyclops abyssorum tatricus and rotifers including Polyarthra dolichoptera, Keratella hiemalis, Keratella cochlearis and Notholca squamula. The phytoplankton community structure differed and it was dominated by the planktonic diatom Fragilaria tenera and the cryptophyte alga Plagioselmis nannoplanctica in the glacial turbid lake, while chrysophytes and dinoflagellates were predominant in the clear one.Ciliate abundance and richness were higher in the glacial turbid lake (∼4000–27 800 Ind L−1, up to 29 species) than in the clear lake (∼570–7150 Ind L−1, up to eight species). The dominant species were Balanion planctonicum, Askenasia cf. chlorelligera, Urotricha cf. furcata and Mesodinium cf. acarus. The same species dominated in both lakes, except for Mesodinium cf. acarus and some particle‐associated ciliates, which occurred exclusively in the glacial turbid lake. The relative underwater solar irradiance (i.e. percentage of PAR and UVR at depth) significantly explained their abundance distribution pattern, especially in the clear water lake. In the glacial turbid lake, the abundance of the dominating ciliate taxa was mainly explained by the presence of predatory zooplankton.Our results revealed an unexpected high abundance and richness of protists (algae, ciliates) in the glacial turbid lake. This type of lake likely offers more suitable environmental conditions and resource niches for protists than the clear and highly UV transparent lake.

Species of the genus Coleps are one of the most common planktonic ciliates in lake ecosystems. The study aimed to identify the phenotypic plasticity and genetic variability of different Coleps isolates from various water bodies and from culture collections. We used an integrative approach to study the strains by (i) cultivation in a suitable culture medium, (ii) screening of the morphological variability including the presence/absence of algal endosymbionts of living cells by light microscopy, (iii) sequencing of the SSU and ITS rDNA including secondary structures, (iv) assessment of their seasonal and spatial occurrence in two lakes over a one-year cycle both from morphospecies counts and high-throughput sequencing (HTS), and, (v) proof of the co-occurrence of Coleps and their endosymbiotic algae from HTS-based network analyses in the two lakes. The Coleps strains showed a high phenotypic plasticity and low genetic variability. The algal endosymbiont in all studied strains was Micractinium conductrix and the mutualistic relationship turned out as facultative. Coleps is common in both lakes over the whole year in different depths and HTS has revealed that only one genotype respectively one species, C. viridis, was present in both lakes despite the different lifestyles (mixotrophic with green algal endosymbionts or heterotrophic without algae). Our results suggest a future revision of the species concept of the genus Coleps.

Microbial planktonic communities are the basis of food webs in aquatic ecosystems since they contribute substantially to primary production and nutrient recycling. Network analyses of DNA metabarcoding data sets emerged as a powerful tool to untangle the complex ecological relationships among the key players in food webs. In this study, we evaluated co‐occurrence networks constructed from time‐series metabarcoding data sets (12 months, biweekly sampling) of protistan plankton communities in surface layers (epilimnion) and bottom waters (hypolimnion) of two temperate deep lakes, Lake Mondsee (Austria) and Lake Zurich (Switzerland). Lake Zurich plankton communities were less tightly connected, more fragmented and had a higher susceptibility to a species extinction scenario compared to Lake Mondsee communities. We interpret these results as a lower robustness of Lake Zurich protistan plankton to environmental stressors, especially stressors resulting from climate change. In all networks, the phylum Ciliophora contributed the highest number of nodes, among them several in key positions of the networks. Associations in ciliate‐specific subnetworks resembled autecological species‐specific traits that indicate adaptions to specific environmental conditions. We demonstrate the strength of co‐occurrence network analyses to deepen our understanding of plankton community dynamics in lakes and indicate biotic relationships, which resulted in new hypotheses that may guide future research in climate‐stressed ecosystems.

Ciliates in shallow alpine lakes are exposed to high levels of incident solar ultraviolet radiation (UVR). We observed the presence of specific sunscreen compounds, the mycosporine-like amino acids (MAAs), in several populations of Bursaridium, a relatively large ciliate species found in such lakes. The populations from 3 highly UV transparent lakes revealed the presence of 7 MAAs (MG, SH, PR, PI, AS, US, and PE) in total concentrations of 3.6–52.4 10−5 μg μg−1 dry weight (DW) per individual, whereas in one glacially turbid and less UV transparent lake, no MAAs were detected in the Bursaridium population. The MAAs in the ciliates generally reflected the composition and relative amounts of the lakes’ seston MAAs, assuming that the ciliates fed on MAA-rich plankton. We experimentally found that naturally acquired MAAs prevented ciliate mortality under simulated UVR and photosynthetically active radiation (PAR) conditions. We further tested the dietary regulation of the MAAs-content in the ciliates under artificial UVR and PAR exposure and found an increase in MAAs concentrations in all treatments. Our assumption was that several stress factors other than irradiation were involved in the synthesis or up-regulation of MAAs.

Since ciliates rarely possess structures that easily fossilize, we are limited in our ability to use paleontological studies to reconstruct the early evolution of this large and ecologically important clade of protists. Tintinnids, a group of loricate (house-forming) planktonic ciliates, are the only group that has a significant fossil record. Putative tintinnid fossils from rocks older than Jurassic, however, possess few to no characters that can be found in extant ciliates; these fossils are best described as 'incertae sedis eukaryotes'. Here, we review the Devonian fossil Nassacysta reticulata and propose that it is likewise another incertae sedis eukaryote due to the lack of any unambiguous ciliate characters. Future tintinnid fossil descriptions would be most helpful if: (i) neutral terminology is used in the descriptions but ciliate-specific terminology in the interpretations; (ii) the current ciliate classification is used, although fossil data may expand or modify classifications based on modern forms; (iii) close collaboration with specialists studying extant ciliates is done; and (iv) editors include an expert of extant ciliates in the review process.

Network analyses of biological communities allow for identifying potential consequences of climate change on the resilience of ecosystems and their robustness to resist stressors. Using DNA metabarcoding datasets from a three-year-sampling (73 samples), we constructed the protistan plankton co-occurrence network of Lake Zurich, a model lake ecosystem subjected to climate change. Despite several documentations of dramatic lake warming in Lake Zurich, our study provides an unprecedented perspective by linking changes in biotic association patterns to climate stress. Water temperature belonged to the strongest environmental parameters splitting the data into two distinct seasonal networks (October–April; May–September). The expected ecological niche of phytoplankton, weakened through nutrient depletion because of permanent thermal stratification and through parasitic fungi, was occupied by the cyanobacterium Planktothrix rubescens and mixotrophic nanoflagellates. Instead of phytoplankton, bacteria and nanoflagellates were the main prey organisms associated with key predators (ciliates), which contrasts traditional views of biological associations in lake plankton. In a species extinction scenario, the warm season network emerged as more vulnerable than the cold season network, indicating a time-lagged effect of warmer winter temperatures on the communities. We conclude that climate stressors compromise lake ecosystem robustness and resilience through species replacement, richness differences, and succession as indicated by key network properties.