Phillip Pham ^1,2 Yichao Shi ² Izhar Khan ² Mark Sumarah ² Justin Renaud ² Mark Sunohara ² Emilia Craiovan ² David Lapen ² Stéphane Aris-Brosou ¹ Wen Chen ^1,2*

• ¹ University of Ottawa, Ottawa, Ontario, Canada
• ² Agriculture and Agri-Food Canada (AAFC), Ottawa, Ontario, Canada

Fungi are essential to the aquatic food web, nutrient cycling, energy flow, and ecosystem regulation. Fungal community structures in water can be influenced by adjacent terrestrial environments, which drive and control some ecosystem services they provide. However, the roles of freshwater fungal communities remain underexplored compared to bacterial communities in this context. Here, we assessed the impact of anthropological and environmental factors on freshwater mycobiota in an agriculturally dominated water basin in eastern Ontario, Canada. We undertook bi-weekly surface water sampling from 2016-2021 and conducted fungal internal transcribed spacer 2 (ITS2) metabarcoding on the samples, complemented by ancillary data, including water physicochemical properties, upstream land use, hydrology, and weather conditions. Our study yielded 6,571 OTUs from 503 water samples, spanning 15 fungal phyla, dominated by Ascomycota, Basidiomycota, and Chytridiomycota. Agricultural land use was associated with decreased mycobiota alpha diversity and distinct fungal communities were observed at agricultural drainage ditch and mixed-land use sites compared to a forested site that had minimal anthropogenic activities in catchment. Notably, river discharge emerged as a predominant influencer of both community diversity and composition, likely amplified by precipitation-induced erosion and drainage from adjacent terrestrial environments. Water physicochemical properties, including stream fungicide levels, explained a small proportion of the variation in mycobiota communities, underscoring the significance of unmeasured factors, alongside stochastic community assembly processes. Nevertheless, stream mycobiota demonstrated functional resilience for critical ecological processes under different environmental conditions. Altogether, these results highlight the complex interplay of factors influencing the freshwater mycobiota, which is essential for elevated understanding of the ecosystem services these fungi provide.