DNA extraction, COI and rDNA amplification and sequencing
Analyses of sequences from the mitochondrial cytochrome c oxidase subunit I gene (COI, partially) and a selection of nuclear rDNA (the entire ITS1, 5.8S, ITS2 and a fragment of 18S and 28S) were used to infer phylogenetic relationships between the examined taxa. COI and rDNA were both amplified from most, but not for all, specimens of Psammocora explanulata and Coscinaraea wellsi analyzed in this study. The list of examined samples and successful amplifications is reported in Table 1. Both markers have been previously used to assess evolutionary relationships among the Anthozoa (Benzoni et al., 2007, 2010; Stefani et al., 2008; Forsman et al., 2009; Gittenberger et al., 2011). The DNA was extracted from ethanol-preserved tissues using a DNeasy® Tissue Kit (QIAGEN, Qiagen Inc., Valencia, CA, USA). Each extract was quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific).
A COI fragment of ca. 500 bp was amplified using fungiid-specific COI primers fungCOIfor1 (5’- CTG CTC TTA GTA TGC TTG TA -3’) and fungCOIrev2 (5’- TTG CAC CCG CTA ATA CAG -3’) by Gittenberger et al. (2011). A PCR mix (50 µl) consisted of 1X Buffer, 2 mM MgCl2 , 0.2 µM of forward and reverse primer, 0.1 mM dNTPs, 2 units of Taq DNA polymerase and ~30 ng DNA. The protocol was 94°C (4 min), followed by 30 cycles of 94°C (1 min), 53°C (30 sec) and 72°C (1 min), followed by 72°C (5 min). An approximately 800 bp long region of rDNA was amplified and sequenced with the universal primer ITS4 (5’- TCC TCC GCT TAT TGA TAT GC -3’) (White et al., 1990) and coral-specific primer A18S (5’- GAT CGA ACG GTT TAG TGA GG -3’) (Takabayashi et al., 1998). Amplifications were performed in a 50 µl volume, using 1X Buffer, 2 mM MgCl2 , 0.4 µM of forward and reverse primer, 0.1 mM dNTPs, 2 units of Taq DNA polymerase and ~30 ng DNA. PCR cycling was as follows: 96°C (2 min), followed by 30 cycles of 96°C (10 sec), 50°C (30 sec) and 72°C (4 min), followed by 72°C (5 min). PCR products were purified and directly sequenced using an automated 3730xl DNA Analyzer (Applied Biosystem, Foster City, CA, USA). Sequences obtained in this study have been deposited in GenBank, and accession numbers are listed in Table 1.