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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. 

Microbial communities play a fundamental role in biogeochemical cycling within salt and brackish marsh ecosystems, yet fungal‐prokaryotic interactions in these environments remain poorly understood. This study employed metabarcoding of the 16S and 28S rRNA genes to investigate prokaryotic and fungal communities across four locations in sediments and surface waters of the North Inlet salt marsh and Winyah Bay brackish marsh (South Carolina, USA) over four time points from 2020 to 2021. Co‐occurrence network analyses were used to identify potential microbial interactions and their ecological implications. Distinct fungal and prokaryotic communities were observed between the two marsh types. From the 16S prokaryotic primer set, Proteobacteria, Bacteroidota, and Cyanobacteriota dominated both marshes. Early diverging fungi and Actinomycetota (bacteria) were prevalent in the brackish marsh, whereas salt marsh communities were primarily composed of Dikarya fungi (Ascomycota and Basidiomycota) and Desulfobacteria. Network analyses revealed contrasting interactions between surface water and sediment. In brackish marsh sediments, fungi and prokaryotes exhibited nearly exclusively negative connections, suggesting strong resource competition. In contrast, Dikarya fungi in brackish marsh surface water displayed numerous positive connections with bacteria, suggesting potential cross‐feeding interactions. In the salt marsh, fungi and prokaryotes exhibited potential cooperative and competitive/antagonistic interactions. Ascomycota were positively connected with Desulfobacteria, suggesting a role in complex organic matter degradation. Conversely, negative connections between Chytridiomycota (early diverging fungi) and Cyanobacteriota (bacteria) implied parasitic interactions. These findings highlight the dynamic nature of fungal‐prokaryotic interactions in coastal ecosystems. By analyzing potential microbial relationships in salt and brackish marshes, this study provides new insights into the ecological roles of fungi in estuarine environments, particularly their contributions to nutrient cycling and organic matter decomposition. Understanding these interactions is crucial for generating hypotheses and predicting microbial responses to environmental changes—such as shifts in salinity and nutrient availability—and their potential impacts on marsh ecosystem functioning. 

Abstract Marine microorganisms are drivers of biogeochemical cycles in the world’s oceans, including oxygen minimum zones (OMZs). Using a metabarcoding survey of the 16S rRNA gene, we investigated prokaryotic communities, as well as their potential interactions with fungi, at the coastal, offshore, and peripheral OMZ of the eastern tropical North Pacific. Water samples were collected along a vertical oxygen gradient, and large volumes were filtered through three size fractions, 0.22, 2, and 22 µm. The changes in community composition along the oxygen gradient were driven by Planctomycetota, Bacteroidota, Verrucomicrobiota, and Gammaproteobacteria; most are known degraders of marine polysaccharides and usually associated with the large particle-associated (LPA) community. The relative abundance of Nitrososphaerota, Alphaproteobacteria, Actinomycetota, and Nitrospinota was high in free-living and small particle-associated (SPA) communities. Network analyses identified putative interactions between fungi and prokaryotes in the particle-associated fractions, which have been largely overlooked in the ocean. In the SPAnetwork analysis, fungal amplicon sequence variants (ASVs) had exclusively negative connections with SAR11 nodes. In the LPA network analysis, fungal ASVs displayed both negative and positive connections with Pseudomonadota, SAR324, and Thermoplasmatota. Our findings demonstrate the utility of three-stage size-fractioned filtration in providing novel insights into marine microbial ecology.