Environmental DNA/RNA metabarcoding in estuaries of São Paulo, Brazil, reveals fish diversity and the presence of invasive species

ABSTRACT Tropical estuaries within the Brazilian Atlantic Forest are biodiversity hotspots facing escalating anthropogenic pressures, yet their ichthyofaunal assemblages remain incompletely documented. We evaluated the combined use of environmental DNA (eDNA) and environmental RNA (eRNA) metabarcoding to characterize fish communities in two estuaries with contrasting levels of urbanization (the Juqueriquerê and Escuro rivers) on the northern coast of São Paulo, Brazil. Targeting the mitochondrial 12S rRNA (MiFish) fragment, we detected a diverse vertebrate assemblage totaling 93 species. eDNA identified 32 fish species across both systems, while eRNA detected 22 species in the preserved estuary, providing robust signals of metabolically active assemblages. The less impacted estuary exhibited significantly higher diversity indices and a more heterogeneous taxonomic composition. In contrast, the urbanized system displayed clear molecular signatures of anthropogenic influence, including the presence of invasive species ( Oreochromis niloticus, O. aureus , and Clarias gariepinus ) and domestic animals. This study constitutes the first application of fish eRNA metabarcoding in Brazil and demonstrates that integrating eDNA and eRNA refines ecological interpretation by coupling biodiversity detection with improved inference about contemporary community composition. Our findings highlight the potential of multi-molecule metabarcoding for routine, non-invasive biodiversity assessment in megadiverse and conservation-priority coastal ecosystems.