Material and methods
Sampling took place at various localities in the Indo-Pacific (Fig. 2). Specimens were collected for molecular analyses in Egypt (Gulf of Aqaba, Red Sea), Yemen (Gulf of Aden), Socotra Island, Kenya, Mayotte Island, La Réunion, Chagos (Indian Ocean), Indonesia, and New Caledonia (Pacific Ocean). Digital images of living corals in the field were taken with a Canon G9 in an Ikelite underwater housing. Coral specimens were collected, labelled, and fragments of ca 1 cm2 were subsampled and preserved in absolute ethanol for molecular analysis. The remaining corallum was placed in sodium hypochlorite for 48 hours to remove all soft tissue, rinsed in freshwater and dried for microscopic examination. Images of cleaned skeletons were taken with a Canon G9 digital camera.
Museum collections and other examined specimens
Type material and specimens examined for this study are deposited in the following institutes.
Type specimens of Psammocora explanulata were examined in the BMNH and ZMA collections. Three of them (BMNH 19188.8.131.52, ZMA Coel. 1072, and ZMA Coel. 1071) are indicated as ‘Syntype’ on their label, while for specimen BMNH 19184.108.40.206 the indication ‘Type’ is given. The holotype of Coscinaraea wellsi USNM 44818 was examined, as well as the specimens of C. wellsi at MTQ that are depicted with the original species description (Veron and Pichon, 1980).
The specimens were identified by use of the original descriptions and illustrations of Psammocora explanulata and Coscinaraea wellsi by Van der Horst (1922) and Veron and Pichon (1980), respectively. Species descriptions and illustrations in other widely used references were also examined (Wells, 1954; Veron and Pichon, 1976; Scheer and Pillai, 1983; Veron, 1986, 2000; Sheppard and Sheppard, 1991; Hoeksema and van Ofwegen, 2004; Fenner, 2005; Dai and Horng, 2009; Pichon et al., 2010). The morphological terms used follow the terminology explained and illustrated by Hoeksema (1989).
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).
Table 1. List of specimens from which sequences and morphological data were obtained. For each specimen the code, identification, sampling locality, and Genbank accession code for sequences generated in this study are provided. * For sampling details, see lists of examined material.
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.
The obtained COI and rDNA sequences were aligned with other available homologues from the families Siderastreidae and Fungiidae (Benzoni et al., 2007; Gittenberger et al., 2011). In particular all the genera of the former family, and 14 out of 15 of the latter were included in the analyses, thus excluding only the genus Cantharellus Hoeksema and Best, 1984. Among the 11 species currently recognized in the genus Cycloseris, only the six analyzed by Gittenberger et al. (2011) were included in the present analyses. Sequences were viewed, edited and assembled using CodonCode Aligner 2.0.6 (CodonCode Corporation, Dedham, MA, USA). Multiple alignments were finally adjusted using BioEdit 220.127.116.11 (Hall, 1999). Identification of invariable, polymorphic and parsimony informative sites was conducted with DnaSP 5.10.01 (Librado and Rozas, 2009). Intra and interspecific pairwise distances (uncorrected p-distances) were calculated in MEGA 4.0.2 (Tamura et al., 2007).
Phylogenetic relationships were reconstructed using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). For both markers MP analyses were performed with PAUP* 4.0b10 (Swofford, 2003) using a heuristic search with starting trees obtained by random stepwise addition, with 10 replicates, and the tree bisection-reconnection (TBR) branch swapping algorithm. Bootstrap replicates (n=500) were used to assess the robustness of the internal nodes of the trees. For the ML and BI analyses, nucleotide substitution model parameters were determined by using MrModeltest2.3 (Nylander, 2004). Based on arguments presented by Posada and Buckley (2004), we used the Akaike Information Criterion (AIC) to select best-fit models. The model GTR+I+G (gamma=0.4791 and p-invar=0.4595) was suggested as best fit for COI, and for the rDNA, the model SYM+I+G (gamma=0.6387 and p-invar=0.5340) was selected instead. ML reconstructions were performed with PhyML 3.0 (Guindon and Gascuel, 2003) using the default parameters. The reliability of the ML tree was assessed by bootstrap analyses, with 500 replications. BI analyses were conducted with MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003), using the previously determined models of nucleotide evolution. In the case of COI, a Markov Chain Monte Carlo analysis was applied with four chains running for 1,500,000 generations, saving the current tree every 10 generations. Subsequently, a consensus tree was produced (with a burnin of 37500 trees) indicating the Bayesian posterior probabilities of each node. The rDNA phylogeny was obtained on the basis of 6,000,000 generations, sampling trees every 100 generations. The first 30,000 trees were discarded as burnin. Convergence of parameters estimates were monitored using Tracer 1.5 (Drummond and Rambaut, 2007) and by using the statistics provided by MrBayes.