PCR-RFLP: A practical method for the identification of specimens of Patella ulyssiponensis s.l. (Gastropoda: Patellidae)

"Patella ulyssiponensis s.l. is widely distributed in the Mediterranean as well as in the north-eastern Atlantic, from southern Norway to north-western Africa, including Macaronesia. Throughout its range, P. ulyssiponensis s.l. shows a high degree of variability in the shape and colour pattern of the shell and in the colour pattern of the foot. In addition, the species demonstrates widely overlapping morphological parameters with the coexisting P. caerulea (in the Mediterranean), P. vulgata (in north-eastern Atlantic) and P. candei (in the Macaronesian islands). Thus P. ulyssiponensis s.l. identification proves to be difficult. So far, method to unequivocally identify uncertain specimens of P. ulyssiponensis s.l. from the coexisting species was using a molecular taxonomy approach based on the Cytochrome c Oxidase subunit I (COI) sequences analysis. The aim of the present study was to identify specific PCR-RFLP (Restriction Fragment Length Polymorphism PCR-based) markers on the COI sequences which could be used as an alternative and fast diagnostic tool for the discrimination of P. ulyssiponensis s.l. from P. caerulea, P. vulgata and P. candei, when the morphology of the coexisting individuals provided equivocal information. First, we sampled 65 Patella spp. and classified these by means of the COI sequencing. Then, we selected the TaqI endonuclease for the specific RFLP reaction, which produced a combination of RFLP electrophoretic bands, allowing an unequivocal identification of the individuals of P. ulyssiponensis s.l. "