Seeing Through the “Clouds” With Molecular “Eyes”. First eDNA-Based Detections of Pygmy Sperm Whale (Kogia breviceps) in the Mediterranean Sea

Introduction: The pygmy sperm whale (Kogia breviceps) is globally distributed but considered absent in the Mediterranean Sea, with no confirmed sightings to date. Environmental DNA (eDNA) analysis offers a non-­ invasive, cost-­ effective, and highly sensitive tool for detecting marine species where direct observation is challenging. Methods: An extensive eDNA metabarcoding analysis was conducted on 393 samples collected during the LIFE-­ CONCEPTU MARIS multidisciplinary monitoring programme (October 2022–October 2024). Mitochondrial 12S-­ rDNA and 16S-­ rDNA amplicons were compared with reference databases and with sequences from stranded Kogia individuals from Atlantic regions adjacent to the Mediterranean and from the Mediterranean itself to ensure robust species identification. Results: At least five independent detections of K. breviceps were recorded from Gibraltar to the Tyrrhenian Sea. Repeated signals and high read abundance support detection reliability. Spatio-­ temporal patterns and haplotype diversity suggest the presence of multiple individuals, possible seasonal occurrence, with detections significantly associated with nocturnal sampling. Discussion: The study provides the first molecular detection of K. breviceps in the Mediterranean and shows that eDNA is effective in detecting the species, possibly facilitated by the release of DNA-­ rich defensive fluids characteristic of the genus. The lack of visual records likely reflects limited surface activity and historical exclusion from Mediterranean cetacean check-­ lists, suggesting that some unidentified small-­ cetacean sightings may correspond to this species. Synthesis and Recommendations: This study highlights the value of integrated molecular, acoustic and visual monitoring and recommends enhancing multidisciplinary monitoring frameworks to better understand the distribution and ecology of this poorly known, rare and elusive species.