Protists influence ecosystems by modulating microbial population size, diversity, metabolic outputs and gene flow. In this study we used eukaryotic ribosomal amplicon diversity from 218 European freshwater lakes sampled in August 2012 to assess the effect of mountain ranges as biogeographic barriers on spatial patterns and microbial community structure in European freshwaters. The diversity of microbial communities as reflected by amplicon clusters suggested that the eukaryotic microbial inventory of lakes was well-sampled at the European and at the local scale. Our pan-European diversity analysis indicated that biodiversity and richness of high mountain lakes differed from that of lowland lakes. Further, the taxon inventory of high-mountain lakes strongly contributed to beta-diversity despite a low taxon inventory. Even though ecological factors, in general, strongly affect protist community pattern, we show that geographic distance and geographic barriers significantly contribute to community composition particularly for high mountain regions which presumably act as biogeographic islands. However, community composition in lowland lakes was also affected by geographic distance but less pronounced as in high mountain regions. In consequence protist populations are locally structured into distinct biogeographic provinces and community analyses revealed biogeographic patterns also for lowland lakes whereby European mountain ranges act as dispersal barriers in particular for short to intermediate distances whereas the effect of mountain ranges levels off on larger scale.protists (algae) are the dominant primary producers in aquatic ecosystems and contribute roughly 50 % to global primary production, i.e. they are of similar importance as land plants. Heterotrophic protists, on the other hand, are the primary agents of the top-down control of bacteria and primary producers and are thus key organisms in determining the fate and transport of organic matter in nature (Boenigk and Arndt 2002, Caron 2001, Foissner 1987, Sherr et al. 2007) .Increasing our knowledge of biodiversity distribution patterns at any level of the tree of life is of paramount ecological and evolutionary significance (Campo et al.
Metagenomics and total RNA sequencing (total RNA-Seq) have the potential to improve the taxonomic identification of diverse microbial communities, which could allow for the incorporation of microbes into routine ecological assessments. However, these target-PCR-free techniques require more testing and optimization. In this study, we processed metagenomics and total RNA-Seq data from a commercially available microbial mock community using 672 data-processing workflows, identified the most accurate data-processing tools, and compared their microbial identification accuracy at equal and increasing sequencing depths. The accuracy of data-processing tools substantially varied among replicates. Total RNA-Seq was more accurate than metagenomics at equal sequencing depths and even at sequencing depths almost one order of magnitude lower than those of metagenomics. We show that while data-processing tools require further exploration, total RNA-Seq might be a favorable alternative to metagenomics for target-PCR-free taxonomic identifications of microbial communities and might enable a substantial reduction in sequencing costs while maintaining accuracy. This could be particularly an advantage for routine ecological assessments, which require cost-effective yet accurate methods, and might allow for the incorporation of microbes into ecological assessments.
Freshwaters face some of the highest rates of species loss, caused by strong human impact. To decrease or even revert this strong impact, ecological restorations are increasingly applied to restore and maintain the natural ecological status of freshwaters. Their ecological status can be determined by assessing the presence of indicator species (e.g., certain fish species), which is called biomonitoring. However, traditional biomonitoring of fish, such as electrofishing, is often challenging and invasive. To augment traditional biomonitoring of fish, the analysis of environmental DNA (eDNA) has recently been proposed as an alternative, sensitive approach. The present study employed this modern approach to monitor the Rhine sculpin (Cottus rhenanus), a fish species that has been reintroduced into a recently restored stream within the Emscher catchment in Germany, in order to validate the success of the applied restorations and to monitor the species' dispersal. We monitored the dispersal of the Rhine sculpin using replicated 12S end-point nested PCR eDNA surveillance at a fine spatial and temporal scale. In that way, we investigated if eDNA analysis can be applied for freshwater assessments. We also performed traditional electrofishing in one instance to validate our eDNA-based approach. We could track the dispersal of the Rhine sculpin and showed a higher dispersal potential of the species than we assumed. eDNA detection indicated the species' dispersal across a potential dispersal barrier and showed a steep increase of positive detections once the reintroduced population had established. In contrast to that, false negative eDNA results occurred at early reintroduction stages. Our results show that eDNA detection can be used to confirm and monitor reintroductions and to contribute to the assessment and modeling of the ecological status of streams.
Metagenomics and total RNA sequencing (total RNA-Seq) have the potential to improve the taxonomic identification of diverse microbial communities, which could allow for the incorporation of microbes into routine freshwater assessments. However, these targeted-PCR-free techniques require more testing and optimization. In this study, we processed metagenomics and total RNA-Seq data from a commercially available microbial mock community using 768 data-processing workflows, identified the most accurate data-processing tools, and compared their microbial identification accuracy at equal and increasing sequencing depths. The accuracy of data-processing tools substantially varied among replicates. Total RNA-Seq was more accurate than metagenomics at equal sequencing depths and even at sequencing depths almost one order of magnitude lower than those of metagenomics. We show that while data-processing tools require further exploration, total RNA-Seq might be a favorable alternative to metagenomics for targeted-PCR-free taxonomic identifications of microbial communities and might enable a substantial reduction in sequencing costs while maintaining accuracy. This could be particularly an advantage for routine freshwater assessments, which require cost-effective yet accurate methods, and might allow for the incorporation of microbes into freshwater assessments.
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