Species specific primer sets design
Candidate regions for designing monk seal specific primers were identified within the mtDNA regions targeted by MarVer primers (Valsecchi et al ., 2020), as the amplicons produced by the three primer sets -MarVer1, MarVer2 and MarVer3 - were all highly polymorphic also between the two sister species of the Mediterranean (Monachus monachus ) and the Hawaiian (Neomonachus schauinslandi ) monk seals: 12 (out of 199bp), 14 (out of 87bp) and 24 (out of 237bp) variable sites were found in the fragments amplified by MarVer1, MarVer2 and MarVer3 respectively. Internally to each amplicon, one monk-seal specific primer was designed from the stretch of sequence showing the largest number of diagnostic sites. Each monk-seal specific primer was paired for amplification to the corresponding MarVer universal primer (Valsecchi et al ., 2020) on the opposite strand. In those instances where the universal primer presented one degenerate base, the mammalian variant was used (Valsecchi et al ., 2020). For the reasons exposed in the Discussion, another prerequisite we included in the search for adequate priming sites was that of aiming at producing amplicons sufficiently different in size between the three loci. Hereafter we use the terms “MarVer1”, “MarVer2” and “MarVer3” to designate the three loci, rather than the primer sets.