Beyond dikarya: 28S metabarcoding uncovers cryptic fungal lineages across a tidal estuary.

Fungi are key drivers of biogeochemical processes, yet marine fungi remain understudied and under-characterized due to primer biases and database gaps. In this study, we conducted a metabarcoding survey targeting the small and large subunit rRNA genes and the internal transcribed spacer region of fungi (18S, 28S, and ITS2) in the sediment and surface water of salt and brackish marshes in the North Inlet-Winyah Bay estuarine system (Georgetown, South Carolina, USA). The universal 18S/16S primer set (515F-Y and 926R) identified few fungal taxa. The ITS2 primer set (ITS3mix and ITS4) revealed high diversity among Dikarya but failed to capture the full extent of early diverging fungi (EDF). In contrast, the 28S primer set (LR0R and LF402) excelled at identifying EDF lineages, including Chytridiomycota, Mucoromycota, Zoopagomycota, and Blastocladiomycota, many of which dominated the brackish marsh sampling site but were less prevalent in the salt marsh sampling sites. Over half of the fungal OTUs identified by the 28S primer set were from EDF lineages. Copy-normalized 28S qPCR showed that EDF were more abundant in brackish sediments than in the salt marsh. Several putative denitrifying fungi, primarily species from Trichoderma and Purpureocillium, were also detected, suggesting overlooked functional guilds that may contribute to estuarine nitrogen cycling. A FUNGuild analysis found that most lineages were saprotrophic. Overall, our findings show that EDF are key contributors to community differences across salinity gradients and may play more important functional roles in coastal biogeochemistry than is currently understood. The 28S primer set is ideal for marine fungal metabarcoding because it provides comprehensive taxonomic coverage and enables phylogenetic analysis.