TOPO vectors and cloned by using a TOPO-TA cloning kit (Invitrogen). Ninety-six clones from each soil sample were selected for sequencing. Plasmid purification and sequencing of cloned PCR products was done at the Qiagen Genomic Services/Sequencing facility with an ABI 377 or 377xl sequencer (Applied Biosystems).
A total of 2,239 cloned 16s sequences were aligned with the NAST alignment tool (DeSantis et al., 2006b), and the alignments were manually edited based on conserved primary sequence and secondary structure information in the ARB software package (Ludwig et al., 2004). The phylum affiliation of each sequence was checked by using the BLAST tool within the National Center for Biotechology Information (Altschul et al., 1990). Potentially chimeric sequences were identified by using the Bellerophon server (Huber et al., 2004). Putative chimeric sequences were manually assessed by building trees in ARB that contained a set of reference sequences obtained from the Greengenes database (DeSantis et al., 2006a) and the 5′ and 3′ sides of the putative chimeras. Sequences were removed from the analysis that had 5′ and 3′ ends affiliating with different groups of reference sequences in the tree (Horner-Devine et al., 2004a).
There is no standard definition of microbial species. Therefore we grouped our 2,196 nonchimeric Acidobacteria sequences into phylotypes with a <99% sequence similarity cutoff by using the programs PHYLIP (Felsenstein, 1989) and DOTUR (Schloss and Handelsman, 2005). This is a commonly used phylotype designation (Kroes et al., 1999), which provides high phylogenetic resolution. One sequence was randomly chosen to represent each phylotype. The representative sequences were used to build a phylogenetic tree by maximum-likelihood methods using the program phyML (Guindon and Gascuel, 2003).We used Jukes–Cantor and gamma substitution models where the gamma distribution parameter was estimated from the data. Only informative base positions were used to bin sequences into phylotypes and build the microbial phylogenetic tree. All diversity analyses were later repeated by using 97%, 94%, and 90% sequence similarity cutoffs (Figs. 7.4–7.6).
Angiosperms within each quadrat were identified to species level and checked against Rocky Mountain Biological Laboratory (RMBL) Herbarium specimens jointly by B.J.E., A.J.K., and C.L. Vouchers are being prepared for deposition in the RMBL Herbarium and the University of Arizona Herbarium. All plants were identified in 2005, except for the plants at the lowest site, which were sampled in 2006. Plots were sampled near the peak of the growing season, and thus some individuals with later phenologies could not be identified to species. We staggered the plant