Pooled DNA from Hypolith Sample Type III
To assign phylogenetic affiliation, 454 pyrosequencing was conducted. In order to reduce the number of samples, equal amounts of DNA from each of the 9 samples were pooled according to type. Multiplex identifiers were added to the primers with a 4 base tag for each sample. The V3 16S rRNA genes were amplified using the primers 341F (5’-CCTACGGGAGGCAGCAG-3’) and 805R (5’- CCGTCAATTCCTTTRAGTTT-3’), containing the 454 FLX adaptors, with the sample-specific multiple identifier.    A PCR mixture contained each primer at 0.5 umol l-1, each deoxynucleoside triphosphate at 0.25 mmol-1, 1.25 U of Phusion High-Fidelity DNA Polymerase DNA Polymerase and HF buffer (Finzymes). PCRs were conducted in triplicate and pooled, purified using the NucleoSpin Kit™ (Clonetech, Japan)and quantified using the Picogreen assay kit(Invitrogen)Samples were then submitted to the GATC (Germany) for pyrosequencing on a 454 GS FLX. Hypolith Type I: Barcode CATA TitA_8-28fB 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGCATAAGAGTTTGATCCTGGCTCAG-3’  Hypolith Type II: ACAG TitA_8-28fC 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGACAGAGAGTTTGATCCTGGCTCAG-3’   Hypolith Type III: TTCA TitA_8-28fE 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCAAGAGTTTGATCCTGGCTCAG-3’   Open Soil Type IV AGCG TitA_8-28fF 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCGAGAGTTTGATCCTGGCTCAG-3’   