2.2 DNA extraction, PCR and sequencing
Total DNA was extracted from up to 25 mg mantle muscle tissue using the DNeasy Mini Kit (Qiagen, Germany) according to the standard tissue protocol but reducing the final elution volume to 100μl. In order to obtain mantle tissue, dissection was carried out using sterilized forceps and scalpels, isolating the mantle from the rest of the tissues such as intestine and tunic.
Cytochrome c oxidase subunit I (COI) PCR. The tunicate primers pair Tun_reverse2 (Rev) (Stefaniak et al., 2009) and Cve-CO1-F54 (Fwd) 5´ AGTGTTTTAATTCGAACAGA 3´, and the primers pair Deg COI F2 (Fwd) and Deg COI R2 (Rev) (Reem, Douek, Paz, Katzir, & Rinkevich, 2017) were used for amplification. The primer Cve-CO1-F54 (Fwd) was designed within this study as consequence of bad quality (double peaks, ill-defined or garbled peaks in the chromatograms) forward sequences obtained with the Stefaniak-primer, our primer was designed using the software Geneious version R8 (Kearse et al., 2012), and based on good quality forward sequences from this work. Reactions were carried out in 25 μl volumes, using 0.025 U/µl of Promega GoTaq G2 Flexi DNA Polymerase, 30 ng of DNA, 0.5 µM of each primer and 2 mM of MgCl2. The amplification protocol was 2 min at 94°C for initial denaturation followed by 36 cycles of 60 s at 94°C, 50 s at 46°C, 50 s at 72°C, and a final elongation step of 8 min at 72°C.
Nuclear Ribosomal RNA Gene (18S rDNA) PCR. Primers 18S1 (Fwd) and 18S4 (Rev) (Tsagkogeorga et al., 2009) were used for amplification. Reactions were carried out in 25 μl volumes, using 0.03 U/µl of TaKaRa LA Taq HS, 30 ng of DNA, 0.5 µM of each primer and 0.05 mM of Betaine. The amplification protocol was 1 min at 94°C for initial denaturation followed by 30 cycles of 10 s at 98°C, 50 s at 50°C, 2 min at 72°C, and a final elongation step of 10 min at 72°C.
PCR products were visualized on a 1 % TAE agarose gel stained with GelRed (Nucleic Acid Gel Stain) under UV illumination. PCR products were outsourced for sequencing to Eurofins MWG Operon (Germany) on an ABI3730XL automatic DNA sequencer, using either of the two primers used for amplification.