Metagenomic barcode approach of the ExStream Project 2014 for eukaryotes

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.

淡水真菌在水生生态系统中有机物质分解过程中扮演着至关重要的角色。然而，相较于土壤等其他生境中的真菌，它们的研究仍然相对匮乏。尤其是针对人类活动带来的压力因子对真菌群落反应的研究，仅在有限的几篇出版物中有所涉及。因此，我们于德国一条淡水溪流的人工支流中建立了一个中宇宙实验，以检验三种压力因子对群落组成的影响。64个中宇宙装置配备了装有河岸植被落叶的腐殖质袋以及经过灭菌的陶瓷瓷砖，这些瓷砖作为标准化采样区域用于生物膜群落。实验分为两个阶段，首先为期两周的殖民化阶段，随后为两周的压力因子暴露阶段。我们选取了细颗粒沉积物、盐（NaCl）以及流速降低作为压力因子，并测试了所有可能的组合。每个压力因子及其组合均以八个重复进行应用。与以往多数已发表的淡水真菌研究不同，我们未采用形态学标准来评估真菌群落，而是采用了ITS nrDNA标记的宏基因组条形码方法。使用CD-HIT-OTU和usearch算法将原始读数聚类为操作分类单元（OTUs），结果显示两种算法得到了高度一致的结果。然而，检测到的570个OTUs的数量是之前研究中基于诱饵和培养方法检测到的物种数量的三到十倍。本研究的独特设计，即采用两个不同的区域，使得根据区域偏好识别出三种不同的生态阶层成为可能。第一种阶层代表水生真菌，第二种包括两栖真菌，而第三种则包含陆生真菌。每个阶层均以其独特的压力反应为特征，支持将淡水真菌总体群落划分为特定的亚群落。尽管水生和陆生真菌对施加的压力因子没有显示出显著影响，但两栖真菌对盐浓度增高的反应则显著。然而，我们推测这种信号是一种次级效应，因为两栖真菌与藻类群落高度相关，而藻类群落相较于真菌群落更易受到施加的压力因子的影响。