*2.3. DNA Extraction, PCR and ITS 2*

A total of 21 zoantharian polyps were analyzed. Deoxyribonucleic acid (DNA) was extracted from specimens (30–50 mg) of zoantharian tissue following the manufacturer's protocol of an E.Z.N.A. Tissue DNA Kit (Omega BIO-TEK. Model no. D3396-02 Norcross, GA, USA). Mitochondrial cytochrome oxidase subunit 1 (COI) was amplified using the following zoantharian-specific primers (LCOant 5 -TTTTCYACTAATCATAAAGATAT 3 , COIantr 5 -GCCCACACAATAAAGCCCAATAYYCCAAT 3 ) (see [21]). Polymerase chain reaction (PCR) amplifications from template DNA were carried out in a BIORAD 96-well thermocycler (Model No. MyCycler Thermal Cycler; Series No. 580BR 7657, Hercules, CA, USA) performed under the following conditions: initial set up at 95 ◦C for 3 min, followed by 40 cycles of denaturation at 94 ◦C for 30 s, annealing at 52 ◦C at 1 min, extension at 72 ◦C at 2 min, and final extension at 72 ◦C for 5 min (see [22]). Aliquots from PCR amplification were checked by 1.7% agarose gel electrophoresis. Each PCR product was enzymatically purified with 1.8 μL Exonuclease I, and 3.6 μL Shrimp Alkaline Phosphatase (ExoSAP, ThermoFisher Scientific, Santa Clara, CA, USA), and incubated in the PCR thermocycler at 37 ◦C for 30 min, followed by 95 ◦C for 5 min (see [20]).

The internal transcribed spacer (ITS) 2 region of Symbiodiniaceae samples were amplified using the following primers: ITSintfor2, 5 -GAATTGCAGAACTCCGTG-3 , ITS2 clamp, 5 -CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCGGGATCCATATGCTTA AGTTCAGCGGGT-3 , with a "Touchdown" protocol [22–25]. Products from the PCR were electrophoresed overnight in gradient gels between 45–80% and stained with sybergreen. PCR-denaturation gradient gel electrophoresis (DGGE) gels were photographed, and distinct gel bands were excised using a scapula, then transferred to 1.5 mL Eppendorf tubes. Gel bands were excised to determine Symbiodiniaceae genera identities.
