A Couple Of Methods To Play With RRAD And In Fact Profit From It!

, 2000). The latter cores were subsequently dissected at ~80 ��m intervals using a cryotome, as RRAD described in Becraft et al. (2011). Samples for transcript analysis over a diel cycle were taken at hourly intervals starting at 1700 h on 11 September 2009, and continuing until 1600 h on 12 September 2009 using a #4 cork borer (38.5 mm2). Duplicate samples were collected from within a ~1 m2 area at a 60��C site and pooled. Perturbation Experiments Temperature Shift Samples for temperature-shift experiments were collected in duplicate using a #2 cork borer, placed in their original vertical orientation into 3-ml glass vials that were filled with spring water, capped, and suspended in the effluent channel at a higher temperature site by aluminum wires attached to wooden stakes on either side of the channel (see Supplementary Figure S1A). Duplicate samples were retrieved 2 and 4 days after the disturbance was initiated on 27 October 2008. Previous studies showed that confining samples in this way did not alter the initial population structure of samples when incubated at the collection site (i.e., not shifted in temperature; Ruff-Roberts et al., 1994). Some bleaching was noticed in later stages of the incubation period, likely due to the lack of flow in the closed vials, which severely impacts diffusion, or possibly to other inhibiting factors, such as the accumulation of toxic metabolites. Light Alteration On 27 October 2008, a 254 cm2 rectangular wooden frame covered with four layers of stretched muslin (Supplementary Figure S1B) was placed ~2.5 cm above the mat surface at a ~63��C site to reduce ambient solar irradiance by ~92%. Duplicate samples were retrieved 2 and 4 days after the disturbance was initiated on 27 October 2008. All samples were immediately frozen on dry ice (-78.5��C) in the field and kept frozen at -80��C until analysis. Molecular Methods DNA was extracted and purified as described in Becraft et al. (2011), and RNA was extracted and purified as described in Liu et al. (2011). Primers for the amplification of Synechococcus A/B-lineage psaA genes (psaAcenterforward: 5��-TTCCACTACCACAAGCGGGCTCC-3��, psaAreverse: 5��-CAGGCCACCCTTGAAGGTG-3��) were designed to yield a 324 bp segment to maximize the number of single-nucleotide polymorphisms (SNPs) that could be used to differentiate PEs, and were tested for specificity to A/B��-like Synechococcus sequences as described in Becraft et al. (2011). The SNPs in this region of the psaA gene are unlikely to be under positive selection, as described in Supplementary Information Section I. The reduced sequence length in this study eliminated nucleotide diversity in some cases. This caused sequences representative of subclades PE A1-3 and A1-4, identified in Becraft et al.