For molecular phylogeny reconstructions, tissue samples from the foot of the snails were used to extract DNA with the E.Z.N.A. Mollusc DNA Kit (Omega Biotek). The primer sets published by Meyer (2003) and Schiaparelli et al. (2005) were used to amplify the mtDNA markers CO-I and 16S marker, respectively. The PCR reaction mixtures were composed after Gittenberger et al. (2006). For 16S, 0,005 ml MilliQ was replaced by an equal volume of Qsolution (Qiagen). The annealing temperature used for 16S was set at 52°, whereas for CO-I a ramp was used, starting at 40° and ending at 44°, increasing with 0.1° s-1.
Sequencing was performed on a MegaBace 1000, 96 capillary sequencer at Leiden University, and on an Automatic Sequencer 3730xl by Macrogen, Korea. The raw sequence data were assembled and edited using Sequencher 4.2 (Gene Codes Corporation®) and aligned with ClustalX. The sequences in the CO-I dataset were all checked for stop codons. All sequences were referenced against GenBank (National Center for Biotechnology Information, NCBI) to ensure that non-targeted DNA had not been sequenced. For the phylogeny reconstruction, 17 GenBank sequences for both 16S and CO-I (Table 1), representing the same species, were combined with sequences of the Atlantic species. For the single individual of Cyphoma signatum, CO-I could not be amplified. Therefore, this species was excluded from the dataset. The final alignment consisted of 28 sequences containing 961 base pairs. Newly sequenced species are deposited in GenBank under accession numbers GU363427 – GU363450.
Within 16S highly variable regions exist, formed by either insertions or deletions. Aligning this region proved to be very difficult and the unalignable region, consisting of 75 base pairs in length (position 166 till 241), was deleted. To check whether both datasets could be combined, an incongruence length difference test (ILD-test) was performed. This test resulted in a P-value (P = 1.00) allowing the data combination. To determine the optimal evolutionary models the combined molecular dataset was subjected to Modeltest (Posada and Crandall, 1998). This resulted in the Generalised Time Reversible evolutionary model + invariable sites + gamma (GTR I+G). The obtained parameters were used to run a maximum likelihood search in PAUP* 4.0b (Swofford, 2003). No fewer than a 100 bootstrap replicates were used to evaluate the robustness of the nodes. The search was carried out with tree bisection-reconnection (TBR). Gaps were in all cases treated as missing character and not as a fifth character state. The selected outgroup sequence was that of the cypraeid Cypraea tigris (Linnaeus, 1758).