The Strait of Malacca supports diverse marine fish communities and provides important habitat for small cetaceans, yet information on fish diversity and cetacean occurrence outside Matang, Perak remains limited. This study used environmental DNA (eDNA) metabarcoding to characterise marine fish communities and examine their potential associations with water conditions and small cetacean occurrence. A total of 122 seawater samples were collected from Klang (Selangor), Penang, Tanjung Piai (Johor), and Yan (Kedah) in 2025 and 2026. Depth, pH, dissolved oxygen, salinity, turbidity, and temperature were measured at each sampling station, while small cetacean occurrence and behaviour were recorded during visual surveys. A partial mitochondrial 12S rRNA region was amplified using MiFish-U primers and sequenced on the Illumina MiSeq i100 platform. Fish diversity and community composition were assessed using alpha- and beta-diversity analyses. A total of 65 unique fish families were detected across all sampling locations and both years. Based on pooled read composition, the leading species were Deveximentum hanedai (13.2%), Johnius belangerii (10.8%), and Coilia dussumieri (10.2%) in Klang; Leiognathus brevirostris (13.5%), Encrasicholina devisi (8.7%), and Hilsa kelee (8.4%) in Penang; Encrasicholina devisi (10.2%), Lutjanus malabaricus (5.9%), and Leiognathus ruconius (4.6%) in Tanjung Piai; and Stolephorus insularis (12.9%), Sardinella brachysoma (11.9%), and Rastrelliger brachysoma (10.5%) in Yan. Effective Shannon diversity was significantly higher in 2026 than in 2025 (Wilcoxon rank-sum test, q = 0.0006), with Tanjung Piai consistently recording the highest mean diversity in both years. Penang was the only location with a significant within-location increase (p = 0.017). Fish-community composition differed significantly among locations and between years based on Bray-Curtis and Jaccard dissimilarities (permutation p = 0.0001 for all comparisons). These patterns indicate strong spatial structuring of coastal fish assemblages and suggest that interannual environmental variability may influence local diversity. A total of 18 small-cetacean sighting records were documented, comprising Irrawaddy dolphins, Indo-Pacific humpback dolphins, and Indo-Pacific finless porpoises. At the species level, Neenchelys buitendijki and Arius maculatus showed positive associations with cetacean occurrence, but neither remained significant after multiple-testing correction. Temperature showed the strongest positive association with cetacean occurrence (OR = 2.07 per standard deviation, q = 0.118), while lower turbidity during cetacean-observed events in Klang represented a site-specific pattern (q = 0.0096). Overall, 12S eDNA metabarcoding effectively revealed location-based and interannual variation in coastal fish communities and provides a promising non-invasive approach for long-term marine ecosystem monitoring.
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