Ashley Campbell added supporting references for bulk community succession  almost 10 years ago

Commit id: 1225d66724682890729031fc2cf1bc999b0a6322

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\section{Results and Discussion}  \textbf{Temporal microbial succession during C degradation.} With the rapid advancement and declining costs of high throughput sequencing, it has become increasingly easy to investigate microbial communities. In this study, we couple stable-isotope probing with 454 pyrosequencing in order to better understand organic matter decomposition dynamics as a function of soil microbial community C utilization. We ran a temporal series of parallel microcosms and measured the changes in the microbial community as result of the addition of a complex carbon mixture using 454 pyrosequencing of the bulk microbial community and fractions from CsCl gradient fractionation (Fig. S1). Overall, temporal changes in microbial community composition are consistent with C decomposition being accompanied by a microbial community succession. Analysis of the sequenced bulk community DNA demonstrates Proteobacteria (26-35\%), Actinobacteria (19-26\%), and Acidobacteria (12-21\%) as the most dominant phyla throughout the duration of the experiment. This is consistent with previous observations\cite{Goldfarb_2011}\cite{Fierer_2007}. We found trends of Proteobacteria and Actinobacteria decreasing and Acidobacteria increasing as C availability declines (TableS1). This is congruent with findings in soils sampled from a wide range of ecosystems in the US\cite{Fierer_2007}.  At days 1, 3, and 7 the bulk community was composed of ~12-18\% Bacteriodetes and Firmicutes combined. At days 14 and 30, these phyla declined to a combined 7-9\% of the whole community accompanied with an increase in Plancktomycetes, Verrucomicrobia, and Chloroflexi (2-3\% at day 1 to 5-7\% at day 30). Abundance of Bacteriodetes have been shown to be positively correlated with C availability\cite{Fierer_2007}.  Despite these changes, the rank abundance of the community fluctuates minimally over time (Fig S2A). accompanied with time associated community shifts (FigS2B). Twenty fractions from a CsCl gradient fractionation for each treatment at each time point were sequenced (Fig. S1). Using NMDS analysis from weighted unifrac distances, the relationship between microbial communities at each buoyant density from all treatments and time points are plotted (Fig 1). Each point on the NMDSrepresents the microbial community based on 16S sequencing from a single fraction where the size of the point is representative of the denisty of that fraction and the colors represent the treatments (Fig1A) or days (Fig1B). The high-density fractions that are differentiating from the control along NMDS2 correspond to fractions that control \textsuperscript{13}C-labeled OTUs (herein called 'responders'). The differential separation of high density fractions in the \textsuperscript{13}C-xylose treatment compared to the \textsuperscript{13}C-cellulose treatment is indicative of a difference in the responders for each of the substrates (Fig 1A). There is an observable time signature of responders at days 1,3, and 7 for the xylose treatment and days 14 and 30 for the cellulose treatment (Fig1B). This demonstrates that different microbial community members are responsible for the consumption of these two substrates; xylose is consumed quickly, whereas, cellulose decomposition takes longer. This supports the hypothesis of a microbial community succession during the decomposition process. Furthermore, this demonstrates the sensitivity of this technique by being able to detect \textsuperscript{13}C-label incorporation in samples with low C additions (blah mg g\textsuperscript{-1} soil).   "Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin–protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6percent of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates" \cite{Goldfarb_2011}  \textbf{Differential taxa C utilization.} Using fractions from within a denisty range of 1.7125-1.755 g/ml, relative abundances of phyla in the experimental treatments were compared to the respective relative abundances in the control treatment to calculate the log\textsubscript{2} fold change (Fig2). The log\textsubscript{2} fold change demonstrates the boom and bust of phyla with time. More notably, it shows that phylum level inferences are misrepresentative, as only a few OTUs within a phylum are responders. To portray the response of a few OTUs or clades as a phylum level response would be overreaching. For OTUs passing a conservative threshold of \textit{p}-value = <0.10 for log\textsubscript{2} fold change (FigSx), we measured the density shift in the experimental treatment compared to the control (Fig Sx). Those OTUs with a center of mass shift greater than zero were considered 'responders'.