ESM Figure 1: Phylogenetic Methods and Results
Taxon sampling and tree building To further demonstrate that amplicons generated using the primers 18N-F2 and 18N-R1 (Toller et al. 2002) were apicomplexan in origin, the 19 haplotypes recovered in this study were included in a phylogenetic analysis with other alveolate sequences. These included sequences of apicomplexans previously isolated from corals (“genotype N”) and 21 additional known apicomplexans, 3 colpodellids, and 8 dinoflagellates (including representatives of Symbiodinium clades A-D) acquired from GenBank. Two additional alveolates, Chromera velia and Perkinsus marinus, were also included in the phylogenetic analysis due to their close relationships to apicomplexans and/or dinoflagellates. The ciliate Oxytricha nova was the chosen outgroup taxa.
The partial 18S rDNA sequence dataset was aligned in Mafft v6.861b using the L-INS-in algorithm with 1000 iterative refinements (Katoh and Toh 2008). The alignment was then examined by eye and manually adjusted (to fix errors introduced during automated alignment) with Mesquite v2.75 (Maddison and Maddison 2011). The (GTR+!+I) model of evolution was selected for the alignment using hierarchical likelihood ratio tests (hLRT) in jModelTest v0.1.1 (Posada 2008). This model was employed to infer a maximum likelihood (ML) phylogenetic tree, with 1000 bootstrap replicates, in RAxML v7.0.4 (Stamatakis 2006).
ESM Fig 1. Proposed phylogeny of the 19 unique haplotypes generated from the 100 randomly sequenced amplicons from this study, and supplemented with other known alveolates, inferred from a maximum likelihood analysis (-ln L= -19290.44, shape parameter (") = 0.44, and proportion of invariable sites = 0.23). Nodes with bootstrap support <50% were collapsed. Two specific nodes are denoted by dots: red indicates the 19 haplotypes fall within Apicomplexa with 100% bootstrap support and yellow indicates all apicomplexans amplified from coral hosts with the 18N-F2 and 18N-R1 primer set form a monophyletic clade with 100% bootstrap support. Haplotypes are numbered and correspond to samples detailed in ESM Table 3. Numbers in parentheses following the names refer to the number of times that haplotype was recovered out of 100. Parallel lines indicate a break in the branch length (i.e., the mean number of substitutions per site) to aid in visualization.
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References for ESM Fig. 1
Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286-298
Maddison WP, Maddison DR (2011) Mesquite: a modular system for evolutionary analysis. Version 2.75. http://mesquiteproject.org
Posada D (2008) JModeltest: phylogenetic model averaging. Mol Biol Evol 25:12531256
Toller WW, Rowan R, Knowlton N (2002) Genetic evidence for a protozoan (phylum Apicomplexa) associated with corals of the Montastraea annularis species complex. Coral Reefs 21:143-146
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688-2690