Computational protocol: Multi gene analysis of Symbiodinium dinoflagellates: a perspective on rarity, symbiosis, and evolution

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[…] DNA sequences were inspected and assembled using Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI, USA) and manually aligned with BioEdit v5.0.9 sequence alignment software (). Thirteen distinct DNA alignments were generated: six alignments corresponding to individual gene alignments, one fully concatenated alignment of all six genes (ALL Concat), and six partially concatenated alignments including all possibilities of five genes each (i.e., each alignment excluded one of the six gene candidates). Concatenated alignments were created using the ‘join sequence files’ option in TREEFINDER v12.2.0 (). elf2 was included in these analyses despite two missing samples (see samples #27 and #30; ), which were coded as missing data in all concatenated alignments. GenBank accession numbers for all investigated sequences are shown in .Each DNA alignment was analyzed independently under both Maximum-likelihood (ML) and Bayesian environments. Best-fit models of evolution were estimated for each alignment (see ) using Modeltest v3.7 (). ML analyses were carried out using PhyML v3.0 (), and the reliability of internal branches was assessed using 100 bootstraps with subtree pruning-regrafting branch swapping. Bayesian tree reconstructions with posterior probabilities were inferred using MrBayes v3.2 (), using the same model of DNA evolution as for the ML analyses. Four simultaneous Markov chains were run for 1,000,000 generations with trees sampled every 10 generations, with 50,000 initial trees discarded as “burn-in”, based on visual inspections. Concatenated alignments were run under ML and Bayesian environments as described above, with the alignments partitioned so that the specific model of evolution corresponded to each gene fragment. [...] To compare the topology of the various trees, approximately unbiased (AU) topological congruency tests () were performed using site likelihood calculation in RaxML v7.2.5 (), followed by AU tests using CONSEL () with default scaling and replicate values. elf2 was excluded from the single gene analyses due to missing data (samples #27 and #30; ), but was included in the concatenated analyses (see above).In order to determine evolutionary rates among Symbiodinium lineages for each of the six investigated genes, relative-rate tests (RRT) were performed using the program RRTREE v1.1 (). Clades and sub-clades were compared in a pair-wise fashion with G. simplex as the outgroup. Relative rates of evolution (K-scores from RRTREE analysis above) were compared among clades and among cellular organelles using a two way ANOVA, followed by post hoc analysis with Tukey’s Honestly Significant Difference (THSD) test. […]

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