Efficacities of specific-clade primer sets
Standard curves for different primers, corresponding to Ct values versus logarithmic 10 fold dilution of purified 28S PCR products. Each plot corresponds to individual Ct values obtained from three technical replicates. Primer efficacities were deduced by linear regression: a) mix of purified 28S PCR products from Symbiodinium A-F each concentrated at equal concentration, 1•1011 x e(-0.681x) (clade A, R2= 0.999), 1•1011 x e(-0.676x) (clade B, R2= 0.999), 1•1012 x e(-0.687x) (clade C, R2= 0.983), 1•1012 x e(-0.694x) (clade D, R2= 0.998), 6•1010 x e(-0.663x) (clade E, R2= 0.998) and 8•1010 x e(-0.694x) (clade F, R2= 0.997); and b) mix of coral DNA each concentrated at equal concentration, universal coral primer set, 1.25•104 x e(-0.694x) (mix of 10 coral species: R2=0.999), 9.02•105 x e(-0.705x) (mix of A.cytherea: R2=1.000), 6.58•105 x e(-0.692x) (mix of P.rus: R2=0.999) and 1.0•106 x e(-0.629x) (mix of P.damicronis: R2=0.996).
Efficacities of specific-clade primer sets on Symbiodinium cells isolated from corals
Standard curves for clade-specific primer sets for clades A, C and D corresponding to cell densities versus logarithmic 10 fold-dilution of isolated coral-symbiotic Symbiodinium. Each plot corresponds to individual Ct values obtained from three technical replicates. Primer efficacities were deduced by linear regression: 1•1010 x e(-0.628x) (clade A, R2=0.85), 1•108 x e(-0.45x) (clade C, R2=0.91) and 4•106 x e(-0.363x) (clade D, R2=0.92).
Phylogenetic tree of Symbiodinium using sequences of 28S rDNA
Phylogenetic tree of Symbiodinium clades A-F derived from bayesian analyses using sequences of 28S rDNA from Moorea (in bold black), BURR collection strains described in Table S2 (in bold grey), and published genetic sequence from GenBank (regular font). Bayesian posterior probabilities (first values) and Maximum Likelihood bootstrap support values (second value) are presented following the substitution model of Kimura 2-parameter with a proportion of invariable sites.
Symbiodinium clades C and F in seawater samples of Moorea
Presence of Symbiodinium clade C, E and F in seawater samples of Moorea. PCR amplification (primer sets clade-specific of Yamashita et al. 2011) on DNA extracts from saturated 0.2μm filters (filtration volume: 6-9 L) of 4 seawater samples (M1, M2, M3 and M4)., T-: negative control (no DNA); TD: positive control (clade D DNA). A 100bp amplicon characterizes a positive amplification. Uppercase letters indicate the corresponding clade.
Characteristics of primer sets used for identification of Symbiodinium clades associated to corals with qPCR assay on 28S amplicons (a) or coral DNA extract (b).
qPCR assays on cultures Symbiodinium strains
Values of Ct obtained by qPCR assays of the corresponding clade-specific primer sets on different DNAs issued from cultured Symbiodinium strains (clades A to F; BURR collection). Each DNA was tested at an equal amount of 10ng per reaction. (-) means no amplification or unspecific amplifications based on dissociation curves analysis, and (*) symbiotic strains used in mix at same concentration and tested with each primer sets.