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Urban-rural gradients are important ecological settings to investigate how human land use impacts habitats and, indirectly, inherent organisms. Seasonally flooded vernal pools are com- mon throughout temperate forest landscapes and may represent significant sites of forest nutrient cycling. The effects of urbanization on vernal pool microbial communities, important drivers of nutri- ent cycling, are largely unknown; thus studies to improve our understanding of microbes and their functional roles in these habitats are needed. Eukaryotic microbial communities sampled from 30 vernal pools of the Cuyahoga River watershed (USA), located along a gradient of urban land use, were profiled with denaturing gradient gel electrophoresis, and were compared between pools using nonmetric multidimensional scaling (NMS). Microbial diversity, and, specifically, the richness and diversity of the fungal operational taxonomic units (OTUs), increased with urbanization. Vernal pool eukaryotic microorganisms formed 2 NMS clusters that differed significantly in sub-watershed urban area. However, the significance of urbanization disappeared when fungal and algal communities were analyzed separately. Water conductivity was consistently correlated with different microbial communities (e.g. eukaryotic, fungal, and algal). Fungal communities also appeared related to the carbon content of the substrate, indicating that vegetation at a local scale may be important for com- munity structure. Almost half of the OTUs matched fungal species, which provides taxonomic evi- dence that the eukaryotic microbial communities of vernal pools are dominated by fungal species. Overall, our data suggest that the eukaryotic microbes of vernal pools are influenced by a variety of factors of the surrounding landscape, including urbanization, water chemistry, and vegetation type.

Changes in eukaryotic microbial communities of vernal pools along an urban–rural land use gradient