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5 (Origin Lab Corp., Northampton, MA, USA). Results Ecotype Simulation  Analysis and Sequence Variation within and Among Putative Ecotypes A total of 119 unique high-frequency sequences were found in the variation detected in 113 environmental samples (96 DNA; 17 cDNA), and these sequences were analyzed using Ecotype Simulation to predict PEs (Figure ?Figure11). Ecotype Simulation analysis of high-frequency sequences from the clade of A-like and A��-like lineages predicted 22 PEs using fine-scale demarcation (15 and 7 PEs, respectively) and analysis of the B��-like lineage predicted 24 PEs in conservative demarcation analyses (Tables ?Tables11 and ?22, and Figure ?Figure11). Results from the alternative demarcation approaches are also shown in Figure ?Figure11. The choice of demarcation approach was initially guided by PE [http://www.selleckchem.com/products/ABT-888.html http://www.selleckchem.com/products/ABT-888.html] distributions identified in Becraft et al. (2011) and high-frequency sequence distributions in the current study. Table 2 Summary of non-singleton conservatively demarcated B��-like PE average population percentages for individual habitat samples corresponding to distributions, expression timing and responses to environmental perturbations. FIGURE 1 Comparison of putative ecotypes (PEs) demarcated from high-frequency psaA sequences by Ecotype Simulation using the fine-scale and conservative demarcation approaches in the (A) A/A�� lineage and the (B) B�� lineage. Black vertical bars ... Many PEs predicted from sequences obtained by pyrosequencing (2008 collections) corresponded to PEs previously  demarcated in cloning and sequencing  studies (2006 collections; Becraft et al., 2011) and this correspondence is shown in Tables ?Tables11 and ?22. All 22 of the newly discovered PEs were either based on a single sequence or were in low frequency (ranging from 0.05 to 3.8% of total sequences; average 0.8% �� 0.14 SE). Among these only PE B��15 had a frequency greater than 1% of the total sequences (Supplementary Tables S4�CS13). Newly discovered PEs in pyrosequencing analyses were labeled sequentially following the PEs previously identified and numbered in Becraft et al. (2011). Ecotype Simulation calculated the following rates of periodic selection (sigma) and ecotype formation (omega): 2.71 (sigma) and 0.94 (omega) for the A-like plus A��-like sequences, and 1.86 and 0.94 for B��-like sequences, parameterized as the number of events (periodic selection or ecotype formation) per nucleotide substitution in the 324 bp of the psaA gene. These results indicated that the rate of periodic selection was higher than the ecotype formation rate. Each high-frequency sequence within a PE was associated with a set of low-frequency sequences (