Pyrosequencing Data Analysis

Pyrosequencing data were processed with the aid of the mothur software package, version 1.31.1 (Schloss et al., 2009) as described previously (Wald et al., 2015). Operational taxonomic units (OTUs) were defined at 3% dissimilarity. Validation of OTUs detected in ¹³C-DNA was performed as follows: if the mean number plus standard deviation of OTU sequences in triplicate T0 samples was higher than the mean number of OTU sequences in triplicate ¹³C-DNA samples, the OTU was considered as non-labeled background contamination of ¹³C-DNA and was excluded from further analyses. Non-metric multidimensional scaling ordination analysis (NMDS) with vector fitting was performed in the vegan package (Oksanen et al., 2013) in R (R Development Core Team, 2009). Major OTUs responsible for the differences in the communities were identified with mothur-implemented version of metastats (White et al., 2009) and depicted in a heatmap, which was constructed in R using heatmap2 function with a dendrogram created based on Bray–Curtis dissimilarity matrix and average linkage hierarchical clustering. PERMANOVA was performed using the adonis function in R to statistically evaluate the significance of treatment on the microbial communities (Anderson, 2001).