Computational protocol: Local adaptation constrains the distribution potential of heat-tolerant Symbiodinium from the Persian/Arabian Gulf

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Protocol publication

[…] The non-coding region of the chloroplast psbA gene (psbAncr) was amplified from zooxanthellae of 109 P. lobata, P. lutea and P. harrisoni coral colonies collected at sites along ~1000 km of UAE and Oman coastline from September 2012 to March 2013. Out of 109 coral colonies analysed for this study, 22 were previously phylotyped () and included for comparison.The psbAncr was amplified from frozen DNA using the primers psbAFor_1 and psbARev_1 with thermal cycling as described in . PCR reaction conditions were as described for the SYM_VAR_FWD and SYM_VAR_REV primer pair in .Amplicons were directly sequenced using the internal primer psbA_int_Fwd, 5′-CTAGGTATGGAAGTGATGCATG-3' sequencing services were provided by Eurofins Genomics. Sequence chromatograms were checked by eye for potential sequencing errors. Any sequence with a chromatogram characteristic of multiple PCR products (for example, where several peaks were registered for a single nucleotide position or where reading frame shifts were apparent) was discarded from further analysis (~7.5% of all directly sequenced PCR amplicons).The phylogenetic resolution of psbAncr sequences of ITS2-type C3 zooxanthellae associated with Porites spp. from the PAG and the Gulf of Oman (127 sequences) was assessed in relation to psbAncr sequences of ITS2-type C3 zooxanthellae and closely related variants from corals elsewhere retrieved from databases (266 sequences). Sequences were first aligned using ClustalW in MEGA6 (Molecular Evolutionary Genetics Analysis software, http://www.megasoftware.net/mega.php), checked by eye and edited manually. Phylogenies were estimated through Bayesian inference using MrBayes 3.2.24 (MrBayes:Bayesian Inference of Phylogeny software, http://mrbayes.sourceforge.net/index.php) applying the Jukes-Cantor model with a gamma-shaped distribution with invariable sites. The MCMC (Markov Chain Monte Carlo) analyses were run for 7.0 × 106 generations, sampling every 500 generations. A relative burn-in of 0.25 was used in calculating a 50% majority rule consensus tree. Nodal support was estimated using posterior probabilities (PPs). A non-Gulf C3-type psbAncr sequence collected in the Great Barrier Reef was used as an outgroup (accession number JQ043643). Pairwise genetic distances between psbAncr haplotypes were calculated with MEGA6 using all substitutions and with gaps considered via pairwise deletion. […]

Pipeline specifications

Software tools Clustal W, MEGA, MrBayes
Application Phylogenetics
Organisms Thermothelomyces thermophila