Conclusions
We present three molecular assays tailored for the detection of monk seal DNA, validated on three sets of positive control samples, including eDNA samples collected during the 2020 monk seal breeding season off Madeira (Desertas Islands). The eDNA-based assays have proven to be able to successfully detect the presence of this threatened species in both tested sets of coastal and offshore Mediterranean eDNA, witnessing its presence long before being recorded by human eyes. The outcomes also suggest that this emblematic species is progressively re-appropriating its original range. Not only could this approach represent an innovative and absolutely non-invasive way to monitor the status of this unique pinniped, but it also has the potential to highlight still uninvestigated aspects of its behavior, such as its nocturnal hunting nature. Coupled with traditional ecological surveys, the proposed approach appears an ideal molecular surveillance tool valuable in many applications relevant to the monk seal conservation/study (reviewed in Appendix S1).
Figure legends
Figure 1 Log2 DNA-copies distribution across positive quantifiable and detectable (PQD) samples. Values (expressed per liter of marine water in the case of eDNA samples) for all three markers across the entire set of positive control samples are shown in comparison with the same values recorded in the 7 Mediterranean eDNA samples (last ones on the right) reporting unambiguous signals of the monk seal presence (i.e. above the detectable threshold).
Figure 2 Madeira eDNA positive-control (sample category 3) sampling sites and results. In the map the eight sampled sites are indicated with color-coded dots according to which marker detected monk seal DNA in that specific site. The result table reports on the left-hand side the field notes of each sampling site as reported from the rangers at the moment of sample collection and release to the molecular team after disclosure of results. On the right-hand side are shown the molecular results for each of the three assays expressed in ranges of logarithmic scale of number of DNA copies per liter of sampled marine water.
Figure 3 Calendar of sample collection and sightings along with molecular results in the two tested Mediterranean areas for which eDNA samples were available from on-going projects: offshore Tyrrhenian (above) and coastal Strait of Sicily, Pelagie archipelago (bottom section). In the tables black squares indicate positive and quantifiable (PQD) monk seal DNA detection by one or more of the three loci (red symbols in the map). The grey squares show samples reporting weak -below the detection threshold- monk-seal signals (yellow symbols in the map), defined in the text as DBNQ (detectable but not quantifiable). The number of squares reflects the number of the sample replicas with positive detection. Coloured cells indicate sampling events, with darker shading showing nocturnal samples. Positions and dates of recent monk seal sightings in the two study areas are reported (in red in the calendar tables).
Box 1 Details of the seven sample categories. ā€œ+Cā€ and ā€œ-C ā€œ denote positive and negative control sample sets respectively.