INTRODUCTION
The Mediterranean monk seal (Monachus monachus ) is the only pinniped historically and permanently present in the Mediterranean basin. Originally Mediterranean monk seals were found regularly throughout the Mediterranean, Marmara and Black Seas, along the West African coast to as far south as Cap Blanc, and in Cape Verde, Canary, Madeira and Azores Islands. In the 20th century, the species was drastically reduced, primarily by fishermen, over most of its range, leading to severe bottleneck and inbreeding depression signatures in the refugial populations in eastern Mediterranean (Stoffelet al. , 2018; Karamanlidis et al. , 2021). However, in the last decade, occasional sightings on Italian coastal waters have been recorded (Figure S1 in Supporting Information).
The Mediterranean monk seal current distribution is only partially known with most available data retrieved from sites where adult females are known to return every year to reproduce. Thanks to the predictability of these events, some of the delivery caves are monitored year-round using camera-trap (e.g. Martinez-Jauregui et al ., 2012), allowing us to learn much about mother/pup relation. Yet, the home ranges of these marine mammals are wide, as adult individuals are known to be able to cover several tens of kilometers per day (Adamantopoulou et al ., 2011), and little is known about milling and foraging areas and which is the actual distributional range during the non-breeding season. This kind of information is difficult to retrieve as monk seals are often elusive. The recently become available possibility to molecularly monitor the presence of any target species through the analysis of traces of its DNA released in the environment (eDNA) provides a precious and absolutely non-invasive means by which unveiling and oversee the Mediterranean monk seal population.
In this letter we desire to make available a triple set of species-specific molecular assays able to detect the presence of residual Mediterranean monk seal DNA both from soil and water environmental matrices. The developed assays were validated both in silico and in the wet lab on a wide range of DNA templates, including tissue (n=3) and residual (n=12) extracts and marine eDNA samples (n=58). The overall sample (n=73) was split into 7 categories: 3 positive and 2 negative controls and 2 sets (offshore/coastal) of marine eDNA samples. Our results show that 1) the assays are successful in identifying the smallest amount of monk seal eDNA (up to 5.7*10-8 mg/L); 2) the screening on both Mediterranean marine eDNA sample sets (from previous and ongoing studies) detected the presence of monk seal in about 50% of the samples of either set, and showed that eDNA analysis can anticipate the detection of the monk seal presence in the study areas before human eye; 3) finally, the proposed approach can potentially unveil still unknown aspects of the biology of this charismatic marine mammal species, by allowing its identification in inaccessible contexts (i.e. offshore waters and during night-time).