Abstract
Non-Dikarya fungi remain poorly characterized due to their cryptic morphology, cultivation difficulties, and limited representation in reference databases. To investigate their diversity and environmental distribution at a global scale, we reanalyzed over 6000 environmental samples using metabarcoding targeting the V4 region of the 18S rRNA gene, encompassing marine, freshwater, soil, hypersaline, polar, and other habitats. We constructed reference phylogenetic trees based on near full-length 18S rRNA gene sequences to enable accurate placement of short-read amplicon sequence variants (ASVs). This approach yielded robust classification at the phylum level and provided finer-scale clade resolution within major non-Dikarya groups. We delineated precise clades within Chytridiomycota, Microsporidia, Rozellida, and Aphelidea, and unveiled several novel ones. Our results show strong ecological structuring of fungal communities across habitats, with inland systems harboring greater fungal abundance and broader phylogenetic diversity than marine systems. Non-Dikarya fungi were consistently detected across diverse environments, including extreme habitats such as hypersaline lakes, deep sediments, and polar regions, where they were often the dominant fungal taxa. Although most ASVs tended to occur in a limited number of ecologically related habitats, phylogenetically related ASVs within the same clade were often adapted to different environments, indicating ecological diversity within clades. Our findings underscore both the ecological relevance and the cryptic diversity of non-Dikarya fungi in globally distributed environments, including extreme ones. Improved taxonomic resolution and broader reference dataset coverage are required to fully integrate these newly characterized lineages into fungal systematics and environmental surveys.