Indonesia is the heart of the Coral Triangle, the worlds most diverse marine ecosystem. Preserving the biological and economic value of this marine biodiversity requires efficient and economical ecosystem monitoring, yet our understanding of marine biodiversity in this region remains limited. This study uses environmental DNA (eDNA) to survey fish communities across a pronounced biodiversity gradient in Indonesia. A total of 12,939,690 sequence reads of MiFish 12S rRNA from 39 sites spanning 7 regions of Indonesia revealed 4,146 Amplified Sequence Variants (ASVs). Regional patterns of fish diversity based on eDNA broadly conformed to expectations based on traditional biodiversity survey methods, with the highest fish biodiversity in Raja Ampat and generally lower diversity in Western Indonesia. However, eDNA performed relatively poorly compared to visual survey methods in site-by-site comparisons, both in terms of total number of taxa recovered and ability to assign species names to ASVs. This result stands in a stark contrast to eDNA studies of temperate and tropical ecosystems with lower diversity. Analyses show that while sequencing depth was sufficient to capture all fish diversity within individual seawater samples, variation among samples from individual localities was high, and sampling effort was insufficient to capture all fish diversity at a given sampling site. Interestingly, mean ASVs recovered per one-liter seawater was surprisingly similar across sites, despite substantial differences in total diversity, suggesting a limit to total ASVs (~200) per one-liter eDNA sample. Combined, results highlight two major challenges of eDNA in highly diverse ecosystems such as the Coral Triangle. First, reference databases are incomplete and insufficient for effective ASV taxonomic assignment. Second, eDNA sampling design developed from lower diversity temperate marine ecosystems are inadequate to fully capture diversity of biodiversity hotspots like the Coral Triangle.

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