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1294 Genetica (2011) 139:1293–1308

The occurrence of shells in Palaeolithic and Neolithic To perform a comprehensive assessment of the genetic
deposits suggests that the species was commonly distrib- population structure of P. ferruginea, we sampled indi-
uted in the Western Mediterranean throughout the Pleis- viduals across the entire extant range. In doing so, we
tocene to modern times (Espinosa and Ozawa 2006). Since investigated the presence of genetic discontinuities across
prehistoric times, intense human exploitation of P. fer- the sampled area and explored the potential causes of any
ruginea has led to the fragmentation of the species’ range genetic structuring. To achieve these goals, we used both
(Paracuellos et al. 2003). It is currently restricted to the nuclear ISSR markers and partial sequences of the fol-
following areas: Sardinia, Corsica, Tuscan Archipelago, lowing three mtDNA genes: COI, 12S (small-subunit
few scattered sites in Sicily, Pantelleria, and the North ribosomal RNA gene) and 16S (large-subunit ribosomal
African shoreline from Tunisia to the Strait of Gibraltar RNA gene).
(Guerra-Garcı ´a et al. 2004).
The extent to which this fragmentation has affected the
genetic population structure of the species is not clear Materials and methods
(Espinosa and Ozawa 2006; Casu et al. 2006). An analysis
of the mitochondrial DNA (mtDNA) region coding for Sampling and DNA extraction
Cytochrome c Oxidase subunit I (COI) has shown a lack of
genetic population structure in the populations located in A total of 213 specimens of P. ferruginea were collected
Southern Spain and North Africa (Espinosa and Ozawa from the intertidal zones of 33 localities of the Western
2006). In contrast, Inter-simple sequence repeat (ISSR) Mediterranean (see Table 1 and Fig. 1 for sampling details;
markers have shown significant genetic structuring Fig. 2 for currents patterns). For some sampling sites, the
between populations in two Sardinian marine protected number of individuals analysed is very small due to the
areas located only 150 km apart (Casu et al. 2006). extremely low density of P. ferruginea at those localities
The lack of genetic data from across the entire extant (e.g., Nido d’Aquila, Tizzano, Bonifacio, Pantelleria,
range of P. ferruginea represents an issue in managing its Marettimo, Favignana) (Table 1). Whenever possible, at
conservation because the knowledge of the amount of least ten individuals were collected from each sampling site
genetic variability and distribution in space and time is using the following nonlethal protocol: the individual was
critical for a correct diagnosis of the status, threats and gently removed from the substrate by means of a wood
viability of populations (Frankham 1995; Escudero et al. chisel, and a 30–60 mg sample of foot muscle was excised
2003). Indeed, genetic approaches are particularly useful using a sterilised surgical forceps. The individual was then
in addressing issues such as the determination of the repositioned in its so-called ‘‘home scar’’, a depression in
potential of dispersal of marine species. In fact, a direct the rock formed by abrasion by the shell, resulting in a
determination of dispersal ability in marine species is tighter fit to the rock and reduced risk of desiccation.
difficult to quantify (Kinland and Gaines 2003) and Genomic DNA was extracted from the tissue using the
therefore estimates of dispersal are often indirectly QIAGEN DNeasy Tissue kit.
inferred by assessing the genetic differentiation among
populations (Chambers et al. 2006; Pannacciulli et al. ISSR analysis
2009).
A low potential for dispersal may enhance the percep- As molecular markers of choice we used ISSRs because
tion to geographic barriers (Baus et al. 2005). Different this technique is affordable and less laborious compared
studies (see Mejri et al. 2009 and references therein) have with other fingerprinting methods. Simulations indicate
found evidences of population genetic structuring in rela- that dominant markers, such as ISSRs, may be as efficient
tion to the presence of straits and passages in the Medi- as the co-dominant ones in estimating genetic differentia-
terranean. In the study area, a well known genetic break is tion (Mariette et al. 2002) and may give reliable results as
located at the Almeria-Oran oceanographic front (AOF), microsatellites (Evanno et al. 2005). Furthermore, ISSRs
which is a major dispersal barrier for many marine detected high levels of genetic variability in populations of
organisms (Patarnello et al. 2007, Galarza et al. 2009). marine organisms, even at a small spatial scale (e.g., Casu
Additionally, the Algerian Current which flows eastward et al. 2005; De Aranzamendi et al. 2008; Lai et al. 2008;
near the north-African coast most of the year (Rio et al. Machkour-M’Rabet et al. 2009), and were successfully
2007), may act as a north–south dispersal barrier across the used in P. ferruginea (Casu et al. 2006). Although the
Sardinian Channel. For instance, distinct genetic lineages presence of homoplasious bands (not identical by descent)
between adjacent localities of Tunisia and Sicily were may be an issue, numerous studies have shown that
found in the vermetid gastropod Dendropoma petreum homoplasy is unlikely to distort analysis restricted to
(Calvo et al. 2009). intraspecific level or to groups of closely related species

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