**2. Methods and tools**

#### **2.1 Sampling**

Specimens were collected from three different regions of the Gulf of Thessaloniki, northern Aegean Sea (Fig. 3). In total, 78 adult individuals of *F. glaber* and 57 adult individuals of *F. proteus* from the three different locations, were collected (Table 1). Specimens were classified according to the number of dominant ribs.

Fig. 3. Sampling sites of the analyzed population for both *F. glaber* and *F. proteus*. A: Airport; P: Paliomana; N: Naziki.

#### **2.2 DNA extraction and PCR amplification**

Total DNA was extracted from the anterior adductor muscle according to Hillis et al. (1996). A universal primer set (Palumbi, 1996) was used for the amplification of the 16S rDNA gene in both *F. glaber* and *F. proteus*. The reaction mixture contained template DNA (approximately 100 ng), 1X PCR buffer, 2.2 mM MgCl2, 20 pmol of each primer, 0.25 mM of each dNTP and 0.5 U of Promega polymerase. Amplification was started at 94°C for 3 min, followed by 31 cycles at 94°C for 50 s, 50°C for 50 s, 72°C for 50 s and a final extension at 72oC for 5 min.

Electrophoresis of 3 μl of the PCR product was performed in 1XTBE buffer for 1 h at 150 V, in 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. The size of the PCR products was checked against a 100 bp DNA ladder and was approximately 500 bp for both taxa. The resulting DNA fragments were visualized by UV transilumination and photographed.

### **2.3 DNA sequencing**

112 Aquaculture

16S rDNA gene. A parallel and similar study (Pujolar et al., 2010), was also made for the

Specimens were collected from three different regions of the Gulf of Thessaloniki, northern Aegean Sea (Fig. 3). In total, 78 adult individuals of *F. glaber* and 57 adult individuals of *F. proteus* from the three different locations, were collected (Table 1). Specimens were classified

Fig. 3. Sampling sites of the analyzed population for both *F. glaber* and *F. proteus*. A: Airport;

Total DNA was extracted from the anterior adductor muscle according to Hillis et al. (1996). A universal primer set (Palumbi, 1996) was used for the amplification of the 16S rDNA gene in both *F. glaber* and *F. proteus*. The reaction mixture contained template DNA (approximately 100 ng), 1X PCR buffer, 2.2 mM MgCl2, 20 pmol of each primer, 0.25 mM of each dNTP and 0.5 U of Promega polymerase. Amplification was started at 94°C for 3 min, followed by 31 cycles at 94°C for 50 s, 50°C for 50 s, 72°C for 50 s and a final extension at

Electrophoresis of 3 μl of the PCR product was performed in 1XTBE buffer for 1 h at 150 V, in 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. The size of the PCR products

Adriatic populations of the *Flexopecten* complex.

according to the number of dominant ribs.

**2. Methods and tools** 

P: Paliomana; N: Naziki.

72oC for 5 min.

**2.2 DNA extraction and PCR amplification** 

**2.1 Sampling** 

A sequencing analysis on a 3730Xl DNA Analyzer (Applied Biosystems) was followed using both forward and reverse primers for crosschecking. DNA sequences were deposited to GenBank (accession numbers GU320272 – GU320288; HM 627014 – HM627051).
