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Materials and methods

Sampling and DNA extraction

A total of 213 specimens of Patella f erruginea were collected from the intertidal zones
of33 localities ofthe Western Mediterranean (see Table l , Fig. l for details). For some
sampling sites, the number of individuals analysed is very small due to the extremely
low density of P. f erruginea at those localities (e.g., Nido d'Aquila, Tizzano, Bonifacio,
Pantelleria, Marettimo, Favignana) (Table 1). Whenever possible, at least ten
individuals were collected from each sampling site using the following nonlethal
protocol: the individuai was gently removed from the substrate by means of a wood
chisel, and a 30-60 mg sample of foot muscle was excised using a sterilised surgical
forceps. The individuai was then repositioned in its so-called ' home scar', a depression

in the rock formed by abrasion by the shell, resulting in a tighter fitto the rock and

reduced risk of desiccation. Genomic DNA was extracted from the tissue using the
QIAGEN DNeasy Tissue kit.

ISSR analysis

Seven primers tested in a previous study (Casu et al. 2006) were used for genotyping
the 213 specimens (Table 2). The PCR mixture, amplification and electrophoretic
techniques were performed as described in Casu et al. (2006).

To overcome potential problems due to small sampling size at a number of
localities, the underlying genetic population structure was inferred using an individuai-
based approach (Luikart et al. 2003) using the Bayesian model-based clustering

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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 MOLECOLARl 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