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distinguish P. ulyssiponensis s. l. not only from P. caerulea, P. vulgata and P. candei, but
also from an enlarged number of Patella species. We expected optimal results by the use of
the TCGA restriction sites, cut by the endonuclease Taq l (Figures l and 2).

2.2 Sampling
A total of 65 specimens of Patella spp. were collected from the lower intertidal zone of
different localities in the Mediterranean (Sardinia, Sicily, Tuscany, northern Adriatic Sea,
32 specimens), north-eastern Atlantic (Brittany, 18 specimens), and Canaries (Tenerife, 15
specirnens) (see Table II for details). Sampling efforts mainly focused on the collection of
individuals whose morphology was not conclusive for the taxonomic attribution (Table II).
The shells of the specimens analysed were deposited in the Zoologica] Museurn of the
Dipartimento di Zoologia e Genetica Evoluzionistica (University of Sassari, Italy).

2.3. COI analysis
Ali 65 Patella specimens collected were identified by a molecular taxonomy approach,
using the COI Folmer region. DNA was extracted using the DNeasy®Tissue Kit (QIAGEN
Inc.®). The PCR, carried out in a total volume of25 !J.l, contained about lO ng/!J.l oftotal
genomic DNA on average, 2.5 U ofTaq DNA polymerase (Eurotaq® by Euroclone®), l x
reaction buffer, 1.25 mM of MgClz, 0.4 11M of both primers, and 200 11M of dNTPs mix.
PCR amplification was performed in a MJ PTC-1 00®Thermal Cycler (MJ research®)
programmed as follows: a preheating of2 min at 94° C, 35 cycles of 40 s at 94° C, 45 s at
55° C and l min and 40 s at 72° C. At the end, a post-treatment of 5 min at 72° Canda
final cooling at 4° C were performed. Both positive and negative controls were used to test
the effectiveness of the PCR reagents. Electrophoresis was carried out on a 2% agarose

104

Gìan Luca Dedola: ANALISI DELLA VARIABILITÀ GENETICA DI PATELLA FéRRUGINEA, PATEL-LA UL l'SSIPONENSIS (MOLLUSCA: GASTROPODA) E PINNA
NOBILIS (MOLLUSCA: BIVALVIA): lLCONTRJBUTO DEl DATI MOLECOLARI ALLA CONSERVAZIONE DI SPECIE MINACCIATE - Tesi di douorato in Scienze
della natura e delle sue risorse • indiri7.zo BK>logi.a Ambientale, Università degli Studi di Sassarl