Illuminating the Dark: Using eDNA Metabarcoding to Study the Unique Ichthyofauna of the Furnas Formation Sinkholes

Abstract

Environmental DNA (eDNA) metabarcoding is a powerful tool for uncovering hidden biodiversity and identifying underestimated taxa in aquatic ecosystems. In this study, we applied eDNA metabarcoding to assess fish species in isolated environments of the Tibagi River basin, specifically sinkholes. Water samples were collected from three sinkholes (Sites 1, 2, and 4), one site at Dourada Lake within the Vila Velha State Park (VVSP), and two sites along the main channel of the Tibagi River. A fragment of the mitochondrial 12S rDNA gene was amplified and sequenced, yielding a final dataset of 975,197 reads after filtering. We identified 31 Molecular Operational Taxonomic Units (MOTUs) belonging to Actinopterygii, distributed across four taxonomic orders: Characiformes, Cichliformes, Gymnotiformes, and Siluriformes. No MOTUs were detected in sinkhole 1, indicating local extinction. A single MOTU (Psalidodon fasciatus) was identified in sinkhole 2. In sinkhole 4, two distinct MOTUs were found, corresponding to the family Heptapteridae and the genus Cambeva, but the potential presence of P. fasciatus was not confirmed. As expected, the highest species richness was observed in the Tibagi River. Despite the lack of a local reference database, our findings offer valuable insights into the detection of diverse taxa in isolated aquatic environments using eDNA metabarcoding. This study underscores the potential of eDNA to inform conservation strategies in ecologically unique and underexplored habitats and highlights the need for developing region-specific genetic reference databases to enhance taxonomic resolution and monitoring efforts in the future.