Insect gut-dwelling fungi, also known as trichomycete fungi, are obligate symbionts that reside in the digestive tracts of freshwater insect larvae or nymphs. Trichomycete fungi have been extensively documented worldwide and hold significant ecological and evolutionary importance. However, their distribution in Rouge National Urban Park (RNUP) and Greater Toronto Area (GTA), Ontario, Canada, remains underexplored. To enhance our understanding of microbial fungal resources in Canada and to examine trichomycetes diversity in urban environments, a comprehensive study was conducted in RNUP and associated watersheds in GTA, including Highland Creek, Little Rouge Creek, and West Duffins Creek. A total of 28 collection sites were sampled, leading to the identification of various species new to the area, including <i>Caudomyces</i> sp., <i>Ejectosporus trisporus</i>, <i>Genistelloides</i> sp., <i>Stachylina paucispora</i>, <i>Stachylina penetralis</i>, <i>Trichozygospora chironomidarum</i>, <i>Smittium caudatum</i>, <i>Smittium culicis</i>, and <i>Smittium simulii</i>. Our findings significantly expand the known distribution of these symbiotic fungi in urban environments and underscore the importance of these habitats as reservoirs of microbial fungal diversity.
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