From Source to Mouth: Unveiling the Diversity and Distribution of the Smallest Eukaryotes in the Longest European River Using High-Throughput rRNA Gene Sequencing

1. Protist communities are key components of riverine ecosystems, making critical contributions to their structure and functioning. However, their distribution in riverine systems remains poorly studied.
2. The Volga River, the longest river in Europe, has previously been investigated mainly using microscopy, which limits the resolution of protist community assessments. Here, we report the first description of the longitudinal distribution of planktonic protists in the Volga River using V4 18S rRNA gene metabarcoding, with amplicon sequence variants (ASVs) inferred via the DADA2 pipeline and taxonomic assignment against the PR2 database. Diversity patterns and their environmental drivers were assessed by modelling the Shannon diversity index using generalised least squares (GLS) modelling with stepwise variable selection. Beta-diversity analysis was performed based on Bray–Curtis dissimilarity and visualised using non-metric multidimensional scaling (NMDS) and tested for significance with permutational multivariate analysis of variance (PERMANOVA).
3. We detected high eukaryotic diversity, spanning representatives of seven supergroups comprising 25 phyla. Alpha diversity was primarily associated with water transparency, which decreased downstream, and distance from the river source, while community composition was influenced by multiple environmental factors, although a substantial fraction of variation remained unexplained. Contrary to expectations, alpha diversity declined downstream, and the impact of the hydropower plant cascade on protist communities was negligible. The large number of unclassified ASVs highlights the considerable unknown phylogenetic diversity of riverine protists.
4. These findings provide a comprehensive overview of protist diversity along the Volga River and highlight the importance of molecular approaches in uncovering cryptic biodiversity in large river systems. They also reveal general environmental patterns shaping protist communities along river gradients in highly regulated river systems, with implications for biodiversity studies in other major rivers.