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+Cā and ā-C
ā denote positive and negative control sample sets respectively.