Seasonality of Planktonic Freshwater Ciliates: Are Analyses Based on V9 Regions of the 18S rRNA Gene Correlated With Morphospecies Counts?

Abstract

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.

Integrated Space-Time Dataset Reveals High Diversity and Distinct Community Structure of Ciliates in Mesopelagic Waters of the Northern South China Sea

“…Comparison of composition at lower taxonomic levels as revealed by the two approaches therefore becomes inappropriate and inaccurate under these circumstances. This also corresponds with the findings of previous studies where both methods have been applied, i.e., on tintinnid ciliates from coastal waters (Bachy et al, 2013; Santoferrara et al, 2014) or planktonic ciliates from mountain lakes (Stoeck et al, 2014; Pitsch et al, 2019), which have also reported a gap between molecular and morphological methods for revealing community composition at lower taxonomic ranks.…”

Metabarcoding Insights Into the Trophic Behavior and Identity of Intertidal Benthic Foraminifera

“…The V9 region of the 18S rRNA gene was targeted with the 1389F/1510R primers described by Amaral-Zettler et al (2009), and widely used in ecological studies for the investigation of eukaryotic diversity (e.g., de Vargas et al, 2015; Sawaya et al, 2019; Pitsch et al, 2019). Primers were modified at the 5′ end to include overhang sequences (Illumina adapters) for the downstream sequencing (forward overhang (37 bp): 5′-ATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; reverse overhang (34 bp): 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′).…”

Perspectives from Ten Years of Protist Studies by High‐Throughput Metabarcoding

“…) or for tracking the distribution of particular lineages in space and time (Pitsch et al. ; Santoferrara et al. ).…”