Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation

In order to study fungal diversity in oxygen minimum zones of the Arabian Sea, we analyzed 1440 cloned small subunit rRNA gene (18S rRNA gene) sequences obtained from environmental samples using three different PCR primer sets. Restriction fragment length polymorphism (RFLP) analyses yielded 549 distinct RFLP patterns, 268 of which could be assigned to fungi (Dikarya and zygomycetes) after sequence analyses. The remaining 281 RFLP patterns represented a variety of nonfungal taxa, even when using putatively fungal-specific primers. A substantial number of fungal sequences were closely related to environmental sequences from a range of other anoxic marine habitats, but distantly related to known sequences of described fungi. Community similarity analyses suggested distinctively different structures of fungal communities from normoxic sites, seasonally anoxic sites and permanently anoxic sites, suggesting different adaptation strategies of fungal communities to prevailing oxygen conditions. Additionally, we obtained 26 fungal cultures from the study sites, most of which were closely related (> 97% sequence similarity) to well-described Dikarya. This indicates that standard cultivation mainly produces more of what is already known. However, two of these cultures were highly divergent to known sequences and seem to represent novel fungal groups on high taxonomic levels. Interestingly, none of the cultured isolates is identical to any of the environmental sequences obtained. Our study demonstrates the importance of a multiple-primer approach combined with cultivation to obtain deeper insights into the true fungal diversity in environmental samples and to enable adequate intersample comparisons of fungal communities.