Abstract
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.
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Data availability
Raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject **PRJNA1344804 (** https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1344804). Supplementary Information provides full lists of detected taxa, including taxonomic identification, primers, and the sites where each taxon was detected, as well as tables summarizing raw reads, merged reads, and reads retained after quality filtering for each library.
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Acknowledgements
We acknowledge members of the ME-BARCODE team from the University of Minho, particularly Tadeu Fontes, Jorge Moutinho, and André Ferreira, for their valuable ideas and support during the data analysis process as well as Darwi Htoo Saw and Steve Si Tou for the assistance during the field sampling.
Funding
This research was funded by the Drop by Drop Project through the WASH Foundation and Las Vegas Sands, grant number WASH/2023.
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M.K. Leong¹ prepared the Introduction and Methods sections, formatted the manuscript, conducted sampling and experiments, and assisted in figure production. I.H. Lau¹ was responsible for data collection and analysis, prepared the Results and Discussion sections, performed sampling and experiments, and produced figures. F.O. Costa² provided advice on the metabarcoding approach and data analysis techniques and contributed to reviewing the manuscript. M.P. Cabezas² offered guidance on metabarcoding data analysis methodologies and participated in reviewing and refining the manuscript. K. Tagulao¹ conceptualized the project, designed the sampling/experiments, secured funding, coordinated research activities, and contributed to reviewing and finalizing the manuscript.
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Leong, M.K., Lau, I.H., Costa, F.O. et al. From fish to invertebrates: multi-marker eDNA metabarcoding for monitoring wetland biodiversity and non-indigenous species in Macao SAR China. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39652-8
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DOI: https://doi.org/10.1038/s41598-026-39652-8
