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gels in a 0.5 x TBE buffer ethidium bromide stained (IO mg/ml), at 4 V/cm for 20 min.
Purification and sequencing were performed as reported in Casu et al. (2010).
The 65 new COI sequences (GenBank Accession Nos. are in Table I) were aligned
using Clustal W with the 131 sequences of P. ulyssiponensis s.l. , P. caerulea, P. vulgata
and P. candei as in Section 2.1 (Table I). Cy mbula nigra (da Costa, 1771) (GenBank
Accession No.: AB445098) was used as confamilial outgroup.
Molecular taxonomic attribution was done by cluster analysis applying the Maximum
Likelihood (ML) and the Bayesian Inference (BI) methods. For ML, the best of SPR
and NNI branch rearrangements were applied on a starting tree obtained using the
BioNJ. Following Sa-Pinto et al. (2005), the generai time reversible (GTR) model was
used, allowing the estimation of proportion of invariable sites and the gamma
distribution. Branch support for the nodes with the highest likelihood score was
assessed by a non-parametric bootstrap (1 ,000 replicates). For BI, two independent runs
each consisting of seven Metropolis-coupled MCMC chains were run simultaneously.
Parameters were specified according to GTR model by setting NsT= 6, rates =
invgamma, assuming a Dirichlet distribution. Each analysis was run for 5,000,000
generations sampling trees every 500 generations in each of the two runs. The first 25%
ofsampled trees were discarded. Nodes ofthe ML and BI trees with bootstrap higher
than 70% and posterior probability higher than 90% respectively are considered highly
supported. All the above-mentioned bioinformatic analyses were carried out on the
freely available website Bioportal (www.bioportal.uio.no). Intraspecific and
interspecific average number of pairwise differences were calculated by means of
Arlequin 3.0 (Excoffier et al. 2005) using the Kimura 2-parameter (K2P) model
(Kimura 1980).

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Gìan Luca Dedola: ANALISI DELLA VARIABILITÀ GENETICA DI PATELLA FéRRUGINEA, PATEL.LA ULl'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