Microbial diversity of Atlantic Rainforest ponds assessed by nanopore sequencing

Despite their limited size, ponds are ecologically important habitats that harbor rich microbial communities and play key roles in global biodiversity and biogeochemical cycling. In this study, we conducted a metagenomic survey of microbial diversity in three ponds - Vermelha, Grande, and Furnas - situated within the Atlantic Rainforest biome in Brazil. Vermelha and Grande are natural, remote, and minimally impacted by human activity, whereas Furnas is an artificial pond with greater human accessibility and long-term anthropogenic influence. Using a long-read nanopore shotgun metagenomics approach, we sequenced DNA extracted from pond water, assembled metagenomes, and performed taxonomic classification and functional annotation. From these datasets, 21 metagenome-assembled genomes (MAGs) were recovered. Our results reveal pond-specific microbial community compositions and functional profiles. Although a direct temporal comparison would require synchronized sampling, the current data offer compelling snapshots of microbial diversity. Vermelha was characterized by high cyanobacterial diversity and exclusive Planctomycetes, while Furnas displayed broader taxonomic diversity, and more notable Actinobacteria and Patescibacteria. Viral communities, including dsDNA bacteriophages and eukaryotic viruses, also varied across sites, with Prasinovirus consistently detected in all ponds. Functional annotation identified putative antibiotic resistance genes (ARGs), most of them in Furnas, potentially reflecting human impact. Besides ARGs, cyanotoxin biosynthesis genes— especially those linked to microcystin production— were predominantly found in Vermelha. Our findings underscore the ecological distinctiveness of each pond and demonstrate the utility of nanopore-based metagenomics for investigating microbial biodiversity in understudied freshwater ecosystems. This work provides knowledge on microbial community structure and anthropogenic influence in tropical pond environments, with implications for conservation and biotechnological exploration.