*2.5. DNA Extraction, Amplification, Sequencing, and Barcoding of Coral Species*

A commercially available spin-column-based DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany) was used for DNA extraction from coral tissues, according to the manufacturer's instructions. Small pieces of the coral samples were excised with sterile forceps, and 20–25 mg samples were placed in a sterile 1.5 mL microcentrifuge tube and subjected to extended lysis for 24 h or longer. The extracted DNA was stored at −20 ◦C. A specific region of the 658 bp fragment of the cytochrome oxidase gene (subunit I) (COI) was amplified by polymerase chain reaction (PCR) using the universal primers LCO 1490 and HCO 2198 [45]. The PCR reaction mix consisted of 1 × PCR colourless buffer (pH 8.3) (GoTaq, Promega), 4 mM MgCl2, 0.2 mM dNTPs, 0.25 μM of each primer, 0.01 mg mL−<sup>1</sup> bovine serum albumin, 0.625 U Taq polymerase (GoTaq, Promega), and 50 ng μL−<sup>1</sup> genomic DNA in a 25 μL reaction volume. The PCR thermal regime consisted of one initial denaturation cycle for 2 min at 95 ◦C; five 1-minute denaturation cycles at 95 ◦C, annealing for 1.5 min at 45 ◦C and an extension for 1.5 min at 72 ◦C; 35 1-minute denaturation cycles at 95 ◦C, annealing for 1.5 min at 50 ◦C and an extension for 1 min at 72 ◦C, followed by a final extension for 5 min at 72 ◦C.

The PCR products were sequenced in both the forward and reverse directions by Macrogen Inc. (Seoul, Korea). All DNA sequence chromatograms were assembled and edited using Unipro (Novosibirsk, Russia), UGENE 1.29.0. The sequences were then aligned using ClustalW [46], and the aligned outputs queried to identify the species, along with the reference sequences of various coral species that were retrieved from the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov, accessed on 25 July 2021) [47], GenBank and Barcode of Life Data System (BOLD) databases, using the Basic Local Alignment Search Tool (BLAST).
