From fish to invertebrates: multi-marker eDNA metabarcoding for monitoring wetland biodiversity and non-indigenous species in Macao SAR China

Urban wetlands provide crucial ecosystem services but are increasingly threatened by urbanization. In Macao, a densely populated city on the Pearl River Estuary, roughly half of the historical wetland habitats have been lost, yet they remain vital for the East Asian-Australasian Flyway. To assess biodiversity in the remaining wetlands, this study applied environmental DNA (eDNA) metabarcoding targeting 12S rRNA, 18S rRNA, and COI genes with a primary focus on fish and invertebrates. The results revealed 85 fish, 298 invertebrate and 9 non-fish chordate species, including 18 non-indigenous fish and several invertebrates. The communities were highly site-specific, showing clear distinctions between inland and coastal wetlands, but non-indigenous fish were widespread, reflecting strong anthropogenic pressure. Moreover, while not observed in fish, coastal invertebrate communities showed strong seasonal turnover. Nevertheless, 56% of COI-derived ESVs could only be assigned to higher taxonomic levels, suggesting substantial diversity remains uncharacterized due to incomplete reference databases. Collectively, these findings demonstrate how fragmentation and seasonal dynamics shape biodiversity differently across taxonomic groups. This study establishes the first comprehensive eDNA baseline for Macao's wetlands, highlighting the need to expand local reference databases and integrate molecular techniques with traditional surveys to improve monitoring and conservation of urban ecosystems.