Relevance of nocturnal samples
This study provides the first example not only of molecular detection of the monk seal presence based on marine water analysis, but it also represents the first case where monk seals are looked for in open waters and, moreover, at any time of day. Despite diurnal (n=17, 47.2%) and nocturnal (n=19, 52.8%) ferry samples were roughly equally represented, positive monk seal DNA detections were more commonly found at night (64.7%, n=11) than in samples collected during light hours (35.3%, n=6). Although we have denominated the ferry samples as “offshore eDNA” (Category 5), the surveyed Livorno-Golfo Aranci route crosses inevitably twice the continental shelf. Thus, 22 (61.1%) of our 36 ferry-based samples were collected off the continental shelf (depth>200mt). These included all the 7 samples collected in fix stations 3 and 4 (n=14), plus all the sighting samples (n=8), all occurring along the study route between fix stations 3 and 4 (Figure 3). About half (n=12, 54.5%) of these 22 deep water samples were collected at night. Interestingly, despite the sample size is not appropriate to establish statistical significance, the instances (n=9) in which monk seal traces were found in offshore waters originated prevalently from nocturnal samples (n=6). These DNA recoveries were however all weak (DBNQ), denoting either diluted (relatively far from the point of shedding) or faded (not freshly released in the environment) signals. Interestingly however, in most of the 22 truly pelagic samples (off the continental shelf) positivity was given by locus MarVer3 (the largest in amplicon size), thus suggesting the presence of not too degraded (recently shed?) eDNA. If confirmed by future offshore screening, the finding would indicate that monk seals are likely to frequent deep waters -probably foraging- at night, while they have more coastal habits during day time. Interestingly, the metabarcoding analysis on the 2018 ferry-sample subset showed significantly higher vertebrate read counts (>95% attributable to bony fish) in nocturnal-vs-diurnal samples (Valsecchi et al ., in prep.). The sample recording the highest monk seal signal (MmoMo17) was diurnal, but collected on the continental shelf, at about ten kilometers from two islands. This might explain why this species is more commonly seen by the human eye (thus during day-light hours) in coastal water rather than in offshore waters, which are probably more frequently attended by monk seals at night.