Environmental <scp>DNA</scp> Reveals Diverse and Depth‐Stratified Biodiversity in East Indian Ocean Submarine Canyons

ABSTRACT Submarine canyons are globally recognized as biodiversity hotspots, yet logistical challenges in accessing deep‐sea environments hinder comprehensive surveys of their biota. This study presents the first extensive environmental DNA (eDNA) survey of the Cape Range and Cloates submarine canyons in the East Indian Ocean, integrating eDNA metabarcoding, remotely operated vehicle (ROV) imagery, and specimen collection to develop curated DNA reference sequences. Two metabarcoding assays (COI Leray and 16S Fish) were applied to 178 ten‐liter water samples collected across 5 depths: surface, 200, 500, 1000 m, and bottom (1750–4540 m). These assays detected 226 species spanning 126 families, with each canyon revealing unique species. Notably, our study identified 83 putative new records or range extensions, including a range extension for the sleeper shark ( Somniosus ) and the elusive giant squid ( Architeuthis dux ), and the first Western Australian record of the faceless cusk eel ( Typhlonus nasus ). We also detected deep‐diving mammals, including the pygmy sperm whale ( Kogia breviceps ) and Cuvier's beaked whale ( Ziphius cavirostris ). Our results revealed strong assay‐specific differences in vertical stratification patterns, with clear depth‐related signals in the COI Leray dataset and weaker, broad‐scale separation in the 16S Fish dataset. These disparities likely reflect the biological characteristics of the target taxa for each assay, as well as the dynamic nature of submarine canyons influencing vertical mixing. Together, these findings highlight the complexity of interpreting spatial biodiversity patterns using eDNA and the necessity of considering biological, ecological, and physical context of both target taxa and environment in future studies. Our work demonstrates the efficacy of eDNA metabarcoding in exploring submarine canyon biodiversity and its potential for characterizing biodiversity in unexplored deep‐sea habitats. The implementation of eDNA metabarcoding in deep‐sea research shows promise as a tool for establishing ecological baselines and informing conservation practices as growing anthropogenic pressures threaten these unique ecosystems.