Symbiodinium Taxonomic Diversity Assessment

To assess which of the three primer pairs performed the best, each primer pair was assessed for specificity (i.e. preferential amplification of Symbiodinium DNA) and sensitivity (i.e. ability to amplify Symbiodinium from Symbiodinium-rare environments). Additionally, potential within-Symbiodinium taxonomic biases were assessed by comparing the proportions of sequences for each primer pair that belonged to each of the nine major lineages of Symbiodinium, clades A–I (Pochon & Gates, 2010 (link)). Our assumption was that if no or only small clade biases exist, the proportion of sequences from each clade should be similar between primers. Consequently, a large deviation in clade proportions by any one of the primer pairs was regarded as indicative of taxonomic bias.
The specific PCR conditions used for each primer pair are given in Table 1 with PCR reagents as detailed above. The sequences returned from each PCR were annotated using blastn and the NCBI ‘nt’ database according to their closest match to one of the following categories: Symbiodinium, dinoflagellate, stony coral, Hydrozoan, uncultured eukaryote, other, or ‘no match’. To do this, the .fasta file produced from the initial QC from each sample was run against NCBI’s ‘nt’ database with the max_target_seqs argument set to 1 and an output format string of ‘6 qseqid sseqid staxids sscinames sblastnames evalue’. For sequences to be categorised as Symbiodinium, an e-value >50 was required (representing approximately a 100% coverage match at 80–85% nt identity) in addition to the closest match being of Symbiodinium origin. Additionally, closest match subject sequences were screened for two sequences in particular (JN406302 and JN406301), which are mis-annotated as Symbiodinium (highly divergent from any other Symbiodinium sequences) in the ‘nt’ database. Thus, query sequences matching these sequences were annotated as ‘Unclutured eukaryote.’ Notably, before controlling for this, 58% of all ‘Symbiodinium’ sequences amplified by the ITSintfor2 and ITS-DINO primer pairs (0% for the SymVar primer pair) in the SRF-OO samples matched these sequences as their closest match (Fig. S2).
blastn was also used to associate Symbiodinium sequences to one of the nine clades. To do this, a .fasta file was created for each sample that contained all sequences that had previously been categorised as Symbiodinium. This file was used as an input for blastn with the max_target_seqs argument set to 1 and an output format string of ‘6 qseqid sseqid evalue pident gcovs’. A custom BLAST database was used that contained a single representative sequence for each of the nine clades (Data S1).