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Thus, a Perl script from the Pigeon package (hfs-finder.pl2) was used to generate a list of high-frequency sequences by counting the number of occurrences of each unique sequence. Unless otherwise specified, high-frequency sequences were defined as unique sequence types with >50 identical representatives across all samples (96 DNA and 17 cDNA), which included the dominant variants of each PE identified in Becraft et al. (2011). High-frequency sequences showed no evidence of recombination in the region studied using the RDP3 RRAD software package (Martin et al., 2010). Each high-frequency sequence was assigned as either Synechococcus A-like or B��-like if it was ��95% identical to the respective psaA genomic homologs in Synechococcus strain A (JA-3-3Ab) or B�� (JA-2-3B��a (2-13)) (Bhaya et al., 2007). Because no genome sequence was available for the A�� lineage [as defined by 16S rRNA sequence variation (Ferris and Ward, 1997)], we classified psaA sequences to the A�� lineage if they showed the highest similarity to 68��C metagenomic library sequences, which contain predominantly A��-like variants (see Supplementary Information in Becraft et al., 2011; Klatt et al., 2011), and these were included in the A-like phylogeny. Separate analyses were performed on the pool of A��-like plus A-like high-frequency sequences and for B��-like high-frequency sequences using Ecotype Simulation (with 1.5x sorting) to predict the number of PEs (Cohan and Perry, 2007; Koeppel et al., 2008). Neighbor-joining trees were constructed and uploaded into Ecotype Simulation as Newick files for ecotype demarcation. Two methods for Ecotype Simulation ecotype demarcation have been developed (Becraft et al., 2011; Melendrez et al., 2011; Francisco et al., 2012; Kashtan et al., 2014), both of which were used. The more conservative approach tends to yield PEs that are more inclusive clades; here a PE is demarcated as the most inclusive phylogenetic group for which the confidence interval for the number of predicted ecotypes includes the value 1. In our alternative fine-scale demarcation, PEs are demarcated as the largest groups for which the maximum likelihood solution for the number of predicted PEs equals the value 1. The Ecotype Simulation software and instructions for its use are freely available online3 Determination of All Variants within Putative Ecotype Populations The set of high-frequency sequences was de-replicated (i.e., to include only one example of each sequence). Two neighbor-joining trees were created, one for all unique A-like plus A��-like sequences, and the other for B��-like sequences, using Phylip��s dnadist and neighbor-joining programs (Felsenstein, 1989). When a low-frequency sequence fell within a clade of high-frequency sequences that were demarcated to a single PE, it was classified to that PE.