Metagenomic barcode approach of the ExStream Project 2014 for eukaryotes

Sampling

Freshwater fungi have a crucial role in decomposition of organic material in aquatic ecosystems. Nevertheless they remain poorly investigated compared to fungi in other habitats like soil. Especially the community response to anthropogenic stressors is addressed in a very limited number of publications only.Therefore, we created a mesocosm experiment along an artificial sidearm of a freshwater stream in Germany, to test the effects of three stressors on the community composition. 64 mesocosms were equipped with litterbags (filled with leaf litter from riparian vegetation) and former sterilized ceramic tiles, which serve as a standardized sampling area for biofilm communities. An initial period of two weeks for colonization of respective compartments was followed by two weeks with stressor exposure. As stressors we chose fine sediment, salt (NaCl) and a reduction of flow velocity and all possible combinations. Each stressor and combination was applied in eight replicates. In contrast to the majority of hitherto published fungal freshwater studies we did not use morphological criteria to assess fungal communities but used a metagenomic barcode approach of the ITS nrDNA marker.Clustering of raw reads into Operational Taxonomic Units (OTUs) with CD-HIT-OTU and usearch revealed highly identical results between the two different algorithms. However, the detected 570 OTUs exceed the number of species detected with bait- and cultivation-based methods of former studies by factor three to ten. The unique study design with two different compartments enabled the identification of three different ecological gilds according to their compartment preferences. The first gild represents aquatic fungi, the second comprises amphibious fungi, while the third contains terrestrial fungi. Each gild is characterized by a unique stressor response supporting the idea to divide the total community of freshwater fungi into specific sub-communities. While aquatic and terrestrial fungi show no significant effect caused by the applied stressors, the amphibious fungi respond significantly towards the enriched salt concentrations. However we assume this signal to be a secondary effect since the amphibious fungi are highly correlated to algal communities, which are more prone to applied stressors than the fungal communities.

Method steps

1. Pipeline used: https://www.ebi.ac.uk/metagenomics/pipelines/5.0