After adding the organic matter amendment to soil, we tracked the flow of \(^{13}\)C from \(^{13}\)C-xylose or \(^{13}\)C-cellulose into microbial DNA over time using DNA-SIP (Figure \ref{fig:setup}). The amendment consisted of compounds found in of plant biomass including cellulose, lignin, sugars found in hemicellulose, amino acids, and inorganic nutrients (see Supplemental Information (SI)). The amendment was added at 2.9 mg C g\(^{-1}\) soil dry weight (d.w.), and this comprised 19% of the total C in the soil. The cellulose-C (0.88 mg C g\(^{-1}\) soil d.w.) and xylose-C (0.42 mg C g\(^{-1}\) soil d.w.) in the amendment comprised 6% and 3% of the total C in the soil, respectively. The soil microbial community respired 65% of the xylose within one day and 29% of the added xylose remained in the soil at day 30 (Figure \ref{fig:13C}). In contrast, cellulose-C declined at a rate of approximately 18 \(\mu\)g C d \(^{-1}\) g \(^{-1}\) soil d.w. and 40% of added cellulose-C remained in the soil at day 30 (Figure \ref{fig:13C}).
We assessed assimilation of \(^{13}\)C into microbial DNA by comparing the SSU rRNA gene sequence composition of SIP density gradient fractions between \(^{13}\)C treatments and the unlabeled control (see Methods and SI). Our main focus is to identify evidence of isotope incorporation into the DNA of specific OTUs (as described below), but it is instructive to begin by observing overall patterns of variance in the SSU rRNA gene sequence composition of gradient fractions. In the unlabeled control treatment, fraction density represented the majority of the variance in SSU rRNA gene composition (Figure \ref{fig:ord}). This result is expected because Genome G\(+\)C content correlates positively with DNA buoyant density and influences SSU rRNA gene composition in gradient fractions \citep{Buckley_2007}. In contrast, isotope assimilation into DNA will cause variation in gene sequence composition between corresponding density fractions from controls and labeled treatments. For example, the SSU rRNA gene composition in gradient fractions from the \(^{13}\)C-cellulose treatment deviated from corresponding control fractions on days 14 and 30 and this difference was observed only in the high density fractions (\(>\)1.7125 g mL\(^{-1}\), Figure \ref{fig:ord}). Likewise, SSU rRNA gene composition in gradient fractions from the \(^{13}\)C-xylose treatment also deviated from corresponding control fractions but on days 1, 3, and 7 as opposed to 14 and 30 (Figure \ref{fig:ord}). The \(^{13}\)C-cellulose and \(^{13}\)C-xylose treatments also differed from each other in corresponding high density gradient fractions indicating that different microorganisms were labeled across time these treatments (Figure \ref{fig:ord}). These results are generally consistent with predictions of the degradative succession hypothesis.
We can observe further differences in the pattern of isotope incorporation over time for each treatment. For example the SSU rRNA gene sequence composition in the \(^{13}\)C-cellulose treatment was similar on days 14 and 30 in corresponding high density fractions indicating similar patterns of isotope incorporation into DNA on the days. In contrast, in the \(^{13}\)C-xylose treatment, the SSU rRNA gene composition varied between days 1, 3, and 7 in corresponding high density fractions indicating different patterns of isotope incorporation into DNA on these days. In the \(^{13}\)C-xylose treatment on days 14 and 40 the SSU gene composition was similar to control on days 14 and 30 for corresponding high density fractions (Figure \ref{fig:ord}) indicating that \(^{13}\)C was no longer detectable in bacterial DNA on these days for this treatment. These results show that the dynamics of isotope incorporation into DNA varied considerably for organisms that assimilated C from either xylose or cellulose.
We monitored the soil microbial community over the course of the experiment by surveying SSU rRNA genes in non-fractionated DNA from the soil. The SSU rRNA gene composition of the non-fractionated DNA changed with time (Figure \ref{fig:bulk_ord}, P-value \(=\) 0.023, R\(^{2}\) \(=\) 0.63, Adonis test \citep{Anderson2001a}). In contrast, the microbial community could not be shown to change with treatment (P-value 0.23, Adonis test) (Figure \ref{fig:bulk_ord}). The latter result demonstrates the substitution of \(^{13}\)C-labeled substrates for unlabeled equivalents could not be shown to alter the soil microbial community composition. Twenty-nine OTUs exhibited sufficient statistical evidence (adjusted P-value \(<\) 0.10, Wald test) to conclude they changed in relative abundance in the non-fractionated DNA over the course of the experiment (Figure \ref{fig:time}). When SSU rRNA gene abundances were combined at the taxonomic rank of “class”, the classes that changed in abundance (adjusted P-value \(<\) 0.10, Wald test) were the Bacilli (decreased), Flavobacteria (decreased), Gammaproteobacteria (decreased), and Herpetosiphonales (increased) (Figure \ref{fig:time_class}). Of the 29 OTUs that changed in relative abundance over time, 14 putatively incorporated \(^{13}\)C into DNA (see below and Figure \ref{fig:time}). OTUs that likely assimilated \(^{13}\)C from \(^{13}\)C-cellulose tended to increase in relative abundance with time whereas OTUs that assimilated \(^{13}\)C from \(^{13}\)C-xylose tended to decrease (Figure \ref{fig:babund}). OTUs that responded to both substrates did not exhibit a consistent relative abundance response over time as a group (Figure \ref{fig:time} and \ref{fig:babund}).
If an OTU exhibited strong evidence for assimilating \(^{13}\)C into DNA, we refer to that OTU as a “responder” (see Methods and SI for our operational definition of “responder”). The SSU rRNA gene sequences produced in this study were binned into 5,940 OTUs and we assessed evidence of \(^{13}\)C-labeling from both \(^{13}\)C-cellulose and \(^{13}\)C-xylose for each OTU. Forty-one OTUs responded to \(^{13}\)C-xylose, 55 OTUs responded to \(^{13}\)C-cellulose, and 8 OTUs responded to both xylose and cellulose (Figure \ref{fig:l2fc}, Figure \ref{fig:tiledtree}, Figure \ref{fig:genspec}, Table \ref{tab:xyl}, and Table \ref{tab:cell}). The number of xylose responders peaked at days 1 and 3 and declined with time. In contrast, the number of cellulose responders increased with time peaking at days 14 and 30 (Figure \ref{fig:rspndr_count}).
The phylogenetic composition of xylose responders changed with time (Figure \ref{fig:l2fc} and Figure \ref{fig:xyl_count}) and 86% of xylose responders shared \(>\) 97% SSU rRNA gene sequence identity with bacteria cultured in isolation (Table \ref{tab:xyl}). On day 1, Bacilli OTUs represented 84% of xylose responders (Figure \ref{fig:xyl_count}) and the majority of these OTUs were closely related to cultured representatives of the genus Paenibacillus (Table \ref{tab:xyl}, Figure \ref{fig:tiledtree}). For example, “OTU.57” (Table \ref{tab:xyl}), annotated as Paenibacillus, had a strong signal of \(^{13}\)C-labeling at day 1 coinciding with its maximum relative abundance in non-fractionated DNA. The relative abundance of “OTU.57” declined until day 14 and “OTU.57” did not appear to be \(^{13}\)C-labeled after day 1 (Figure \ref{fig:example}). On day 3, Bacteroidetes OTUs comprised 63% of xylose responders (Figure \ref{fig:xyl_count}) and these OTUs were closely related to cultured representatives of the Flavobacteriales and Sphingobacteriales (Table \ref{tab:xyl}, Figure \ref{fig:tiledtree}). For example, “OTU.14”, annotated as a flavobacterium, had a strong signal for \(^{13}\)C-labeling in the \(^{13}\)C-xylose treatment at days 1 and 3 coinciding with its maximum relative abundance in non-fractionated DNA. The relative abundance of “OTU.14” then declined until day 14 and did not show evidence of \(^{13}\)C-labeling beyond day 3 (Figure \ref{fig:example}). Finally, on day 7, Actinobacteria OTUs represented 53% of the xylose responders (Figure \ref{fig:xyl_count}) and these OTUs were closely related to cultured representatives of Micrococcales (Table \ref{tab:xyl}, Figure \ref{fig:tiledtree}). For example, “OTU.4”, annotated as Agromyces, had signal for \(^{13}\)C-labeling in the \(^{13}\)C-xylose treatment on days 1, 3 and 7 with the strongest evidence of \(^{13}\)C-labeling at day 7 and did not appear \(^{13}\)C-labeled at days 14 and 30. The relative abundance of “OTU.4” in non-fractionated DNA increased until day 3 and then declined until day 30 (Figure \ref{fig:example}). Proteobacteria were also common among xylose responders at day 7 where they comprised 40% of xylose responder OTUs. Notably, Proteobacteria represented the majority (6 of 8) of OTUs that responded to both cellulose and xylose (Figure \ref{fig:genspec}).
The phylogenetic composition of cellulose responders did not change with time to the same extent as the xylose responders. Also, in contrast to xylose responders, cellulose responders often were not closely related (\(<\) 97% SSU rRNA gene sequence identity) to cultured isolates. Both the relative abundance and the number of cellulose responders increased over time peaking at days 14 and 30 (Figure \ref{fig:l2fc}, Figure \ref{fig:rspndr_count}, and Figure \ref{fig:babund}). Cellulose responders belonged to the Proteobacteria (46%), Verrucomicrobia (16%), Planctomycetes (16%), Chloroflexi (8%), Bacteroidetes (8%), Actinobacteria (3%), and Melainabacteria (1 OTU) (Table \ref{tab:cell}).
The majority (85%) of cellulose responders outside of the Proteobacteria shared \(<\) 97% SSU rRNA gene sequence identity to bacteria cultured in isolation. For example, 70% of the Verrucomicrobia cellulose responders fell within unidentified Spartobacteria clades (Figure \ref{fig:tiledtree}), and these shared \(<\) 85% SSU rRNA gene sequence identity to any characterized isolate. The Spartobacteria OTU “OTU.2192” exemplified many cellulose responders (Table \ref{tab:cell}, Figure \ref{fig:example}). “OTU.2192” increased in non-fractionated DNA relative abundance with time and evidence for \(^{13}\)C-labeling of “OTU.2192” in the \(^{13}\)C-cellulose treatment increased over time with the strongest evidence at days 14 and 30 (Figure \ref{fig:example}). Most Chloroflexi cellulose responders belonged to an unidentified clade within the Herpetosiphonales (Figure \ref{fig:tiledtree}) and they shared \(<\) 89% SSU rRNA gene sequence identity to any characterized isolate. Characteristic of Chloroflexi cellulose responders, “OTU.64” increased in relative abundance over 30 days and evidence for \(^{13}\)C-labeling of “OTU.64” in the \(^{13}\)C-cellulose treatment peaked days 14 and 30 (Figure \ref{fig:example}). Bacteroidetes cellulose responders fell within the Cytophagales in contrast with Bacteroidetes xylose responders that belonged instead to the Flavobacteriales or Sphingobacteriales (Figure \ref{fig:tiledtree}). Bacteroidetes cellulose responders included one OTU that shared 100% SSU rRNA gene sequence identity to a Sporocytophaga species, a genus known to include cellulose degraders. The majority (86%) of cellulose responders in the Proteobacteria were closely related (\(>\) 97% identity) to bacteria cultured in isolation, including representatives of the genera: Cellvibrio, Devosia, Rhizobium, and Sorangium, which are all known for their ability to degrade cellulose (Table \ref{tab:cell}). Proteobacterial cellulose responders belonged to Alpha (13 OTUs), Beta (4 OTUs), Gamma (5 OTUs), and Delta-proteobacteria (6 OTUs).
Cellulose responders, relative to xylose responders, tended to have lower relative abundance in non-fractionated DNA, demonstrated signal consistent with higher atom % \(^{13}\)C in labeled DNA, and had lower estimated rrn copy number (Figure \ref{fig:shift}). OTUs that assimilated C from either cellulose or xylose were also clustered phylogenetically (see below) indicating that these traits were not dispersed randomly across bacterial species.
In the non-fractionated DNA, cellulose responders had lower relative abundance (1.2 x 10\(^{-3}\) (s.d. 3.8 x 10\(^{-3}\))) than xylose responders (3.5 x 10\(^{-3}\) (s.d. 5.2 x 10\(^{-3}\))) (Figure \ref{fig:xyl_count}, P-value \(=\) 1.12 x 10\(^{-5}\), Wilcoxon Rank Sum test). Six of the ten most common OTUs observed in the non-fractionated DNA responded to xylose, and, seven of the ten most abundant responders to xylose or cellulose in the non-fractionated DNA were xylose responders.
DNA buoyant density (BD) increases in proportion to atom % \(^{13}\)C. Hence, the extent of \(^{13}\)C incorporation into DNA can be evaluated by the difference in BD between \(^{13}\)C-labeled and unlabeled DNA. We calculated for each OTU its mean BD weighted by relative abundance to determine its “center of mass” within a given density gradient. We then quantified for each OTU the difference in center of mass between control gradients and gradients from \(^{13}\)C-xylose or \(^{13}\)C-cellulose treatments (see SI for the detailed calculation, Figure \ref{fig:c1}). We refer to the change in center of mass position for an OTU in response to \(^{13}\)C-labeling as \(\Delta\hat{BD}\). This value can be used to compare relative differences in \(^{13}\)C-labeling between OTUs. \(\Delta\hat{BD}\) values, however, are not comparable to the BD changes observed for DNA from pure cultures both because they are based on relative abundance in density gradient fractions (and not DNA concentration) and because isolated strains grown in uniform conditions generate uniformly labeled molecules while OTUs composed of heterogeneous strains in complex environmental samples do not. Cellulose responder \(\Delta\hat{BD}\) (0.0163 g mL\(^{-1}\) (s.d. 0.0094)) was greater than that of xylose responders (0.0097 g mL\(^{-1}\) (s.d. 0.0094)) (Figure \ref{fig:shift}, P-value \(=\) 1.8610 x 10\(^{-6}\), Wilcoxon Rank Sum test).
We predicted the rrn gene copy number for responders as described \citep{Kembel_2012}. The ability to proliferate after rapid nutrient influx correlates positively to a microorganism’s rrn copy number \citep{Klappenbach_2000}. Cellulose responders possessed fewer estimated rrn copy numbers (2.7 (1.2 s.d.)) than xylose responders (6.2 (3.4 s.d.)) ( P = 1.878 x 10\(^{-9}\), Wilcoxon Rank Sum test, Figure \ref{fig:shift} and Figure \ref{fig:copy}). Furthermore, the estimated rrn gene copy number for xylose responders was inversely related to the day of first response (P = 2.02 x 10\(^{-15}\), Wilcoxon Rank Sum test, Figure \ref{fig:copy},Figure \ref{fig:shift}).
We assessed phylogenetic clustering of \(^{13}\)C-responsive OTUs with the Nearest Taxon Index (NTI) and the Net Relatedness Index (NRI) \citep{Webb2000}. We also quantified the average clade depth of cellulose and xylose responders with the consenTRAIT metric \citep{Martiny2013}. Briefly, the NRI and NTI evaluate phylogenetic clustering against a null model for the distribution of a trait in a phylogeny. The NRI and NTI values are z-scores or standard deviations from the mean and thus the greater the magnitude of the NRI/NTI, the stronger the evidence for clustering (positive values) or overdispersion (negative values). NRI assesses overall clustering whereas the NTI assesses terminal clustering \citep{Evans2014a}. The consenTRAIT metric is a measure of the average clade depth for a trait in a phylogenetic tree. NRI values indicate that cellulose responders clustered overall and at the tips of the phylogeny (NRI: 4.49, NTI: 1.43) while xylose responders clustered terminally (NRI: -1.33, NTI: 2.69). The consenTRAIT clade depth for xylose and cellulose responders was 0.012 and 0.028 SSU rRNA gene sequence dissimilarity, respectively. As reference, the average clade depth as inferred from genomic analyses or growth in culture is approximately 0.017, 0.013 and 0.034 SSU rRNA gene sequence dissimilarity for arabinase (arabinose like xylose is a five C sugar found in hemicellulose), glucosidase and cellulase, respectively \citep{Martiny2013,Berlemont2013}. These results indicate xylose responders form terminal clusters dispersed throughout the phylogeny while cellulose responders form deep clades of terminally clustered OTUs.