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emi12209-sup-0002-si.ppt692K

Fig. S1. Relative abundances of major family level 16S rRNA derived taxa (relative abundance ≥3%) in amplicon libraries of density fractionated RNA, ‘heavy’ RNA of [13C]- and [12C]-2,4-DCP treatments (13 and 12°C, respectively) from soil columns (A) and soil slurries (B) was analysed. 0–5 and 5–10 cm indicate soil depth.

Fig. S2. Dominant 2,4-DCP-[13C] ‘labelled’ genus-level taxa derived from 16S rRNA amplicon libraries as indicated by CAP-SIP. Only sequences affiliating with labelled families (see Fig. 4) were considered and genera with labelling intensities greater than 5% are shown to emphasize highly labelled genera. Bars represent ‘labelling intensities’ indicative of the relative importance of a certain taxon in assimilating 2,4-DCP-derived carbon (see Experimental procedures) for details on CAP-SIP approach and calculation of ‘labelling intensities’, as well as Fig. S1 and Tables S3–S6). (A) Soil column incubations; (B) soil slurries. 0–5 and 5–10 cm indicate soil depth.

Fig. S3. Relative abundances of representative 16S rRNA derived taxa in amplicon libraries along the whole gradient of density resolved RNA. RNA was extraced from the upper 0–5 cm soil of soil columns treated with [13C]- and [12C]-2,4-DCP (13 and 12°C, respectively) in the absence of earthworms. Numbers indicate RNA fractions with a buoyant density ranging from 1.8297 to 1.7606 g ml−1.

Fig. S4. Relative abundances of representative 16S rRNA derived taxa affiliating with Comamonadaceae (A) and Sphingomonadaceae (B) in amplicon libraries from density resolved RNA. RNA was extraced from the upper 0–5 cm soil of soil columns treated with [13C]- and [12C]-2,4-DCP (closed and open symbols, respectively) in the absence of earthworms. Solid and dashed lines represent regressions of data from [13C]- and [12C]-2,4-DCP treatments, respectively.

Fig. S5. Relative abundance of family level OTUs in pooled heavy fractions (P, buoyant density of 1.82–1.84 g ml−1) and in heaviest fraction only (H, buoyant density of 1.84 g ml−1) of field fresh soil prior to soil column incubation at time point t0. 12 and 13°C indicate treatments with [12C]- and [13C]2,4-DCP, respectively.

emi12209-sup-0001-si.doc752K

Table S1. Samples and barcodes that were fused to 16S rRNA specific PCR primers used for generation of amplicons. PCR conditions: 100 μl PCR reactions consisted of 40 μl 2.5 × 5 Prime Master Mix solution (5 Prime GmbH, Hamburg, Germany), 4 μl MgCl2 solution (25 mM), 6 μM of each primers (Microsynth AG, Balgach, Switzerland), 7 μl of cDNA template and 37 μl of deionized water. PCR was performed in a SensoQuest Thermo Cycler (SensoQuest GmbH, Göttingen, Germany) using the following programme: initial denaturation at 94°C for 3 min, then 19 cycles of 94°C for 30 s, 65°C for 30 s (touch down PCR, 0.5°C per cycle), 72°C for 90 s, followed by 14 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 90 s. Final extension was at 72°C for 3 min. 12C, treatment with [12C]2,4-DCP; 13C, treatment with [13C]2,4-DCP; Bw, burrow wall; Bu, upper layer of bulk soil (0–5 cm); Bl, lower layer of bulk soil (5–10 cm); Sc, soil column; Mc, microcosm; t0 soil prior to incubation; +/+, material with earthworms and 2,4-DCP; −/+, material with 2,4-DCP; −/−, unsupplemented control.

Table S2. Diversity and richness estimators of 16S rRNA sequences obtained from amplicon 454 pyrosequencing. Values in brackets indicate lower and upper limits of 95% confidence intervals. Bu, upper layer of bulk soil (0–5 cm); Bl, lower layer of bulk soil (5–10 cm); +/+, material pretreated with earthworms and 2,4-DCP; −/+, material pretreated with 2,4-DCP; −/− untreated control; 12C, treatment with [12C]2,4-DCP; 13C, treatment with [13C]2,4-DCP. A and B indicate data derived from soil columns and soil slurry incubations, respectively.

Table S3. Relative abundance of 16S rRNA defined taxa with a minimal relative abundance greater than 1% in one of the amplicon libraries, and total number of sequences retrieved from ‘heavy’ fractions of soil column experiments during CAP-SIP. Only taxa with a relative abundance of greater than 1% in one of the amplicon libraries are reported.

Table S4. Relative abundance of 16S rRNA defined taxa with a minimal relative abundance greater than 1% in one of the amplicon libraries and total number of sequences retrieved from ‘heavy’ fractions of soil slurry experiments during CAP-SIP. Only taxa with a relative abundance of greater than 1% in one of the amplicon libraries are reported.

Table S5. Taxa that were enriched in ‘heavy’ fractions of [13C]- relative to [12C]2,4-DCP control treatments during soil column incubations derived from comparative analysis of tend versus t0. Classified taxa with at least one positive value greater than 1.18 in one of the treatments (see Experimental Procedures) are shown.

Table S6. Taxa that were enriched in ‘heavy’ fractions of [13C]- relative to [12C]2,4-DCP control treatments during soil slurry incubations derived from comparative analysis of tend versus t0. Classified taxa with at least one positive value greater than 1.18 in one of the treatments (see Experimental Procedures) are shown.

Table S7. Phylogenetic affiliation and sequence length of 16S rRNA fragments representing major family-level taxa identified during CAP-SIP with [U-13C]2,4-DCP.

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