Traditional PCR and assays’ multiplexing 
The possibility to visualize the assays’ outcomes using the traditional PCR, rather than qPCR, is a desirable feature, as it allows the test to be carried out in any structure equipped with essential molecular biology laboratory apparatus, besides being cheaper. Although the diagnostic sensitivity decreases in the case of traditional PCR, our study indicates that when the signal is not too weak (see below), even basic PCR allows the identification of traces of monk seal DNA. The possibility of using the traditional PCR, in turn, entails further advantages, such as the possibility of multiplex run, particularly advantageous when multiple assays are available. This procedure reserves more plusses. Firstly, it allows saving on the amount of eDNA template (precious as typically limited) to be used. Another advantage is the possibility of amplifying the template molecule with different probes at once, a strategy that may reveal useful when dealing with highly-diluted eDNA of the desirable target species and especially for large body size species which are likely to shed “clustered” eDNA (e.g. only one of the three replica-filters, may contain the target species DNA, although they all come from the same water sample). In these instances, multiplexing could minimize the possibility of false negatives while possibly gathering clues about the signal age (see above), although the latter aspect needs further deepening. A preliminary multiplex test indicates that all combinations of the three proposed assays are compatible for multiplexing and that the approach is efficient also with eDNA samples, providing a relatively high (say >10 log2DNA copies/liter) monk seal DNA content such as in Madeira control sample MmoM+01 (Figure S5).