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T. Madigan, personal communication). [Note: We will use the term acclimation to mean the physiological response of an organism to an environmental change; we will use adaptation to mean an alteration in the structure or function of an organism or any of its parts that results from natural selection and by which the organism becomes better fitted to survive and multiply in its environment.] If evolutionarily adapted Synechococcus ecotypes exist, changes in the relative abundances of differently adapted Synechococcus ecotypes, such as those observed in light alteration experiments by Becraft et al. (2015), would provide an alternate explanation of the responses observed in earlier studies. Previous studies of evolutionary adaptation to light were performed on Synechococcus strains obtained from low-dilution enrichments with 16S rRNA sequences representative of predominant natural populations (Allewalt et al., 2006) or substrains therefrom (Kilian et al., 2007). As such, these strains might contain multiple ecotypes with the same 16S rRNA sequence, which were shown to exist by higher-molecular resolution, theory-based analyses (Becraft et al., 2011, 2015). Here we report on Synechococcus strains obtained from more highly diluted mat samples, which are representative of PEs designated from psaA sequence variation. These Synechococcus cultures are not axenic because they presently contain heterotrophic contaminants, but we refer to them as strains since we will show that they represent individual genotypes of distinct Synechococcus PEs (i.e., with respect to the cultivated Synechococcus, they are strains of a known species). We will demonstrate that strains representative of PEs that are known to predominate at different positions along the vertical light Ixazomib purchase gradient in the mat have different evolutionary adaptations and acclimative responses to light, even though they have identical or nearly identical 16S rRNA sequences. The genomic sequences of these strains are described in the third paper of this series (Olsen et al., 2015). Materials and Methods Sample Collection Samples for cultivation were collected from Mushroom Spring, YNP, at sites with temperatures of 60, 63, and 65��C on September 7, 2010 using a #4 cork borer (8 mm diameter). The top green layer, ?1 mm in thickness, of each mat sample was removed with a razor blade, placed in a 1.5-mL microcentrifuge tube, and then returned to the lab in a thermos containing Mushroom Spring source water (cooled to the temperature at which the sample was collected). The time from sampling to the lab was ?2 h, and the temperature in the thermos was ?7 to 10��C degrees cooler upon arrival at the lab than that measured at the field site.