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Abstract Background Anthropogenic impacts on freshwater habitats are causing a recent biodiversity decline far greater than that documented for most terrestrial ecosystems. However, knowledge and description of freshwater biodiversity is still limited, especially targeting all size classes to uncover the distribution of biodiversity between different trophic levels. We assessed the biodiversity of the Lower Rhine and associated water bodies in the river’s flood plain including the river’s main channel, oxbows and gravel-pit lakes, spanning from the level of protists up to the level of larger invertebrate predators and herbivores organized in size classes (nano-, micro, meio- and macrofauna). Morphological diversity was determined by morphotypes, while the molecular diversity (amplicon sequencing variants, ASVs) was assessed through eDNA samples with metabarcoding targeting the V9 region of the 18S rDNA. Results Considering all four investigated size classes, the percentage of shared taxa between both approaches eDNA (ASVs with 80–100% sequence similarity to reference sequences) and morphology (morphotypes), was always below 15% (5.4 ± 3.9%). Even with a more stringent filtering of ASVs (98–100% similarity), the overlap of taxa could only reach up to 43% (18.3 ± 12%). We observed low taxonomic resolution of reference sequences from freshwater organisms in public databases for all size classes, especially for nano-, micro-, and meiofauna, furthermore lacking metainformation if species occur in freshwater, marine or terrestrial ecosystems. Conclusions In our study, we provide a combination of morphotype detection and metabarcoding that particularly reveals the diversity in the smaller size classes and furthermore highlights the lack of genetic resources in reference databases for this diversity. Especially for protists (nano- and microfauna), a combination of molecular and morphological approaches is needed to gain the highest possible community resolution. The assessment of freshwater biodiversity needs to account for its sub-structuring in different ecological size classes and across compartments in order to reveal the ecological dimension of diversity and its distribution.

IntroductionHeterotrophic protists colonizing microbial mats have received little attention over the last few years, despite their importance in microbial food webs. A significant challenge originates from the fact that many protists remain uncultivable and their functions remain poorly understood.MethodsMetabarcoding studies of protists in microbial mats across high-altitude lagoons of different salinities (4.3–34 practical salinity units) were carried out to provide insights into their vertical stratification at the millimeter scale. DNA and cDNA were analyzed for selected stations.ResultsSequence variants classified as the amoeboid rhizarian Rhogostoma and the ciliate Euplotes were found to be common members of the heterotrophic protist communities. They were accompanied by diatoms and kinetoplastids. Correlation analyses point to the salinity of the water column as a main driver influencing the structure of the protist communities at the five studied microbial mats. The active part of the protist communities was detected to be higher at lower salinities (

Heterotrophic protists are key components of marine ecosystems. They act as controllers of bacterial and microphytobenthos production and contribute significantly to the carbon flux to higher trophic levels. Still, metabarcoding studies on benthic protist communities are much less frequent than for planktonic organisms. Especially in the Baltic Sea, representing the largest brackish water environment on earth, so far, no extensive metabarcoding studies have been conducted to assess the diversity of benthic protists in this unique and diverse habitat. This study aims to give first insights into the diversity of benthic protist communities in two different regions of the Baltic Sea, Fehmarnbelt, and Oderbank. Using amplicon sequencing of the 18S rDNA V9 region of over 100 individual sediment samples, we were able to show significant differences in the community composition between the two regions and to give insights into the vertical distribution of protists within the sediment (0–20 cm). The results indicate that the differences in community composition in the different regions might be explained by several abiotic factors such as salinity and water depth, but are also influenced by methodological aspects such as differences between DNA and RNA results.

Protists are key components of the microbial food web in marine pelagic systems because they link algal and bacterial production to higher trophic levels. However, their functioning and bathymetric distribution in benthic deep-sea ecosystems are still only poorly understood. However, biogeographical patterns of communities can be coupled to the functioning of ecosystems and are therefore important to understand ecological and evolutionary processes. In this study, we investigated the diversity and distribution of benthic protist communities from the sublittoral down to the deep seafloor (50–2000 m) around three islands of the Azores in the North Atlantic Ocean. Using amplicon sequencing of the V9 region (18S rDNA) of 21 samples, we found that protist community compositions from different depths were significantly different. Three assemblages were separated along the following depths: 50 m, 150–500 m and 1000–2000 m, which indicate that deep-sea areas surrounding islands might act as isolating barriers for benthic protist species. A limited gene flow between the communities could favor speciation processes, leading to the unique protist communities found at the different investigated islands.

Land use intensification is a major driver of biodiversity change. Here the authors measure diversity across multiple trophic levels in agricultural grassland landscapes of varying management, finding decoupled responses of above- and belowground taxa to local factors and a strong impact of landscape-level land use.

Alexandra Schoenle et al. use DNA metabarcoding and cultivation-based surveys of 11 regions in the Pacific and Atlantic Oceans encompassing the bathyal, abyssal, and hadal zones to identify the role of deep-sea heterotrophic protists relative to other trophic levels. Their results demonstrate that protistan species richness was locally specific and greater than that of metazoans and that measured in pelagic samples, and that deep-sea protist communities are relatively distinct from their pelagic counterparts.

Protists are the most diverse eukaryotes on our planet and metabarcoding has revealed an enormous diversity even from deep-sea environments. A range of different species has also been isolated from the deep sea and some have proven able to survive and even grow under deep-sea conditions. However, little is known about how the community structure of benthic protists changes from sublittoral down to abyssal depths. This is especially important regarding island and seamount communities which are surrounded by deep-sea areas potentially isolating them. Using a combination of live-counting and cultivation techniques, we investigated the abundance and taxonomic composition of benthic protist communities in sediments from sublittoral to abyssal depths around three islands and two seamounts of the Azores’ archipelago in the North Atlantic. Protist abundance decreased significantly and community composition changed with increasing depth. While some species were found at all depths, others were only detected in sublittoral or lower bathyal depths, indicating that some benthic taxa are limited in their distribution to a certain depth, whereas others are also present at the deep-sea floor. The proportion of unidentified specimens increased with depths pointing towards a high number of so far undetected species in the deep-sea realm.

Extreme environmental conditions in the deep sea hamper access to protist communities. In combination with the potentially highly diverse species composition, it demands a wide range of methods to be applied at the same time to guarantee a high resolution of quantitative and qualitative studies of deep-sea heterotrophic flagellates (HF). Within this study, we present a possible combination of several culture-independent and culture-dependent methods available for investigating benthic deep-sea HF communities. Besides live-counting and fixation of HF, we refer to cultivation methods and molecular surveys using next generation sequencing. Laboratory ecological experiments under deep-sea conditions (high pressure, low temperature) could allow the approval of the potential deep-sea origin of sampled HF. The combination of different methods offers a unique possibility to receive detailed information on nanofaunal life in the deep sea. Specific fixation techniques to preserve samples directly at the sampling depth must be applied in further studies to reflect the real biodiversity of the largest habitat on earth.