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.

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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.

Ciliated protists (ciliates) are widely used for investigating evolution, mostly due to their successful radiation after their early evolutionary branching. In this study, we employed high-throughput sequencing technology to reveal the phylogenetic position of Synhymenia, as well as two classes Nassophorea and Phyllopharyngea, which have been a long-standing puzzle in the field of ciliate systematics and evolution. We obtained genomic and transcriptomic data from single cells of one synhymenian (Chilodontopsis depressa) and six other species of phyllopharyngeans (Chilodochona sp., Dysteria derouxi, Hartmannula sinica, Trithigmostoma cucullulus, Trochilia petrani, and Trochilia sp.). Phylogenomic analysis based on 157 orthologous genes comprising 173,835 amino acid residues revealed the affiliation of C. depressa within the class Phyllopharyngea, and the monophyly of Nassophorea, which strongly support the assignment of Synhymenia as a subclass within the class Phyllopharyngea. Comparative genomic analyses further revealed that C. depressa shares more orthologous genes with the class Nassophorea than with Phyllopharyngea, and the stop codon usage in C. depressa resembles that of Phyllopharyngea. Functional enrichment analysis demonstrated that biological pathways in C. depressa are more similar to Phyllopharyngea than Nassophorea. These results suggest that genomic and transcriptomic data can be used to provide insights into the evolutionary relationships within the "Nassophorea-Synhymenia-Phyllopharyngea" assemblage.