The deep sea, the largest biome on Earth, is the least explored and understood. This lack of knowledge hampers our ability to understand and protect this important environment. In this study, water and sediment samples were collected at different depths in the central Mediterranean (224–780 m), specifically, within the Dohrn Canyon and the Palinuro Seamount, to investigate the diversity of cephalopods and establish a baseline knowledge of their distribution in these sites to preserve their habitats and estimate the impacts of human-driven environmental changes. Key taxa identified included Heteroteuthis sp., Loligo sp., and Histioteuthis sp., which were the most abundant across all sampling stations. A low overlap in species detection was observed between water and sediment samples, confirming previous findings that the typology of environmental matrices used in eDNA metabarcoding has a significant impact on the organisms detected and, therefore, the integrated use of different matrices to better represent local biodiversity is recommended. Furthermore, this study highlights the limitations posed by gaps in reference databases, particularly for deep-sea organisms, and addresses these by emphasising the need for improved multi-marker approaches and expanded reference databases to enhance the accuracy of eDNA-based biodiversity assessment.
