Transcript Supplemental Figures (ppt 4523K)

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narG
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Number of OTUs
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nirK
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nirS
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nosZ
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Threshold distance (%)
Figure S1. Effect of threshold distance on the number of OTUs. The grey box indicates
maximal pyrosequencing and PCR-noise (Behnke et al., 2010; Quince et al. 2011), and
arrows indicate species-level threshold distances used for the assignment of sequences
to OTUs in further analyses.
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N2O (N2+N2O) (%)
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Unturbated + Nitrate
Cryoturbated + Nitrate
Unturbated + Nitrite
Cryoturbated + Nitrite
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Nitrate/Nitrite (µM)
Figure S2. Effect of supplemental nitrate or nitrite on the ratio of N2O to total N gases.
Mean values and standard errors of three replicates are shown. The Spearman
correlation coefficient r for nitrate and nitrite to the ratio of N2O to total N gases was 1.0
and 0.9, respectively.
Figure S3. Concentrations of organic acids and ethanol during anoxic microcosm
incubations of peat soil after anoxic pre-incubation for 9 days to deplete nitrate and
nitrite. Electron donors and nitrite were supplied at 0 and 1080 h as indicated by arrows.
(1) unturbated and (2) cryoturbated peat soil; (A) Unsupplemented, (B) nitrite only, (C)
nitrite and acetate, (D) nitrite and ethanol, (E) nitrite and formate, (F) nitrite and
propionate, (G) nitrite and butyrate, and (H) nitrite and lactate. Samples of duplicate
incubations were pooled per timepoint prior to analysis. Concentrations of acetate
(closed squares), ethanol (open squares), formate (closed circles), propionate (open
circles), butyrate (closed triangles), and lactate (open triangles) were measured.
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Number of Sequences
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B
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Sequence Length (bp)
Figure S4. Distribution of sequence read lenghts obtained by pyrosequencing of
structural genes. A: narG, B: nirK, C: nirS, D: nosZ. Only sequences longer than 300 bp
(nirK, nirS) and 350 bp (narG, nosZ) are shown..
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Number of Sequences
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D
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Sequence Length (bp)
Figure
S4 (continued). Distribution of sequence read lenghts obtained by
pyrosequencing of structural genes. A: narG, B: nirK, C: nirS, D: nosZ. Only sequences
longer than 300 bp (nirK, nirS) and 350 bp (narG, nosZ) are shown..
Figure S5. Rarefaction analysis of narG forward (A) and reverse (B), nirK (C), nirS (D), nosZ
foward (E) and reverse (F) sequences retrieved from unturbated (dashed lines) and cryoturbated
(solid lines). Rarefaction curves and 95% confidence intervals are shown. Vertical lines indicate
250 sequence-level for comparisons of rarified number of OTUs [n(OTUs)]. Such comparisons of
the rarified number of OTUs are less sensitive to accumulating PCR- and Pyrosequencing noise
than utilizing all sequences obtained (Dickie, 2010).
Figure S6. Phylogenetic tree of narG reverse reads retrieved from unturbated and
cryoturbated peat soil. The tree is based on translated amino acid sequences.
Representatives for each OTU are shown. Relative abundances of sequences from
unturbated and cryoturbated peat soil are shown in parentheses per OTU. Codes
preceeding sequence names represent sequence accession numbers in public
databases. In total, 2 044 and 3 906 sequences from reverse reads were obtained from
unturbated and cryoturbated peat soil, respectively. Grey boxes indicate branches where
the majority of sequences are derived from reference strains of a certain phylogenetic
class. The percentage of replicate trees in which the associated taxa clustered together
in the bootstrap test (10 000 replicates) are shown next to the branches. Values below
50% have been omitted. The outgroup was narG of Haloarcula marismortui ATCC 43049
(NC 006396).
Figure S7. Phylogenetic tree of nosZ reverse reads retrieved from unturbated and cryoturbated
peat soil. The tree is based on translated amino acid sequences. Representatives for each OTU
are shown. Relative abundances of sequences from unturbated and cryoturbated peat soil are
shown in parentheses per OTU. Codes preceeding sequence names represent sequence
accession numbers in public databases. In total, 1 919 and 3 664 sequences from reverse reads
were obtained from unturbated and cryoturbated peat soil, respectively. Grey boxes indicate
branches where the majority of sequences are derived from reference strains of a certain
phylogenetic class; white boxes indicate minority sequences from genera not affiliated with the
indicated class. The percentage of replicate trees in which the associated taxa clustered together
in the bootstrap test (10 000 replicates) are shown next to the branches. Values below 50% have
been omitted. The outgroup was nosZ of Haloarcula marismortui ATCC 43049 (NC 006396).