1. Article title: Archaea and bacteria respectively dominate nitrification in lightly and heavily grazed soil in a grassland system
Author names: Hong Pan1, Kexin Xie1, Qichun Zhang1, Zhongjun Jia2, Jianming Xu1, Hongjie Di1, Yong Li1*
1Institute of Soil and Water Resources and Environmental Science, College of Environmental and Resource Sciences, Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, Zhejiang University, Hangzhou, , China
2State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, , China
*Corresponding author:
Yong Li
Tel ;
Fax: or

2. Fig. S1 The 13C in soils immediately after labeling (Day 0) and at the end of incubation (Day 56). The black squares represent total 13C derived from 13C-CO2 and 13C-Urea at day 56, the white ones represent 13C derived from 13C-Urea at day 56, the black triangles represent 13C amount at day 0. The error bars represent the standard errors (P<0.05)

3. Fig. S2 Changes in the soil NO3--N (a-c) and exchangeable NH4+-N (d-f) concentrations over a 56-day incubation period in the SIP microcosms of four soils incubated with 12CO2,13CO2 or 13CO2+C2H2. The error bars represent the standard errors of the mean of triplicate microcosms

4. Fig. S3 Relationships between nitrification and stocking rates in the soils. The trend line indicates the fitted relationship.

5. Fig. S4 Phylogenetic analysis of archaeal 16S rRNA genes in the SIP microcosms. The designation ‘Total DNA-AOA-16SrRNA- OTU ’ indicates that OTU-1 contains S rRNA archaeal amoA reads with a 97% sequence similarity. All the sequences were pooled from the soil microcosms at day-0 and day-56. Bootstrap values higher than 50% are indicated at the branch nodes. The scale bars represent 0.5% nucleic acid sequence divergence for the 16S rRNA genes

6. Fig. S5 Phylogenetic analysis of bacterial 16S rRNA genes in the SIP microcosms. The designation ‘Total DNA-AOB-16SrRNA-OTU-1- 25’ indicates that OTU-1 contains 25 amoA reads with a 97% sequence similarity. All the sequences were pooled from the soil microcosms at day-0 and day-56. Bootstrap values higher than 50% are indicated at the branch nodes. The scale bars represent 2% nucleic acid sequence divergence for the 16S rRNA genes

7. Fig. S6 Phylogenetic analysis of the 16S rRNA genes from soil nitrite-oxidizing bacteria (NOB) in SIP microcosms. The designation ‘Total DNA-NOB-16SrRNA-OTU-1-81’ indicates that OTU-1 contains 81 NOB 16S rRNA reads with a 97% sequence similarity. All the sequences were pooled from the soil microcosms at day-0 and day-56. The scale bars represent a 5% nucleic acid sequence divergence

8. Fig. S7 Rarefaction curves of 16S rRNA genes for the total DNA extracted from soil microcosms in the presence of 13CO2 at day-0 and day-56 (a). Rarefaction curves of 16S rRNA genes for the heavy fractions (fractions 3-7) of the labeled (day-56-13CO2) and control (day 56-12CO2 and day-56-13CO2+C2H2) microcosms (b).

9. Fig. S8 Proportional changes of active nitrifying phylotypes in SIP microcosms after an incubation period of 56 days. The changes in AOA (a), AOB (b) and NOB (c) composition were analyzed by target 16S rRNA genes in the active DNA from labeled microcosms at day 56

10. Table S1 Primers and conditions used in this study
Primer Name
Primer sequence (5′-3′)
Target gene
Thermal Profile
Molecular analysis
Reference
Arch-amoAF
STA ATG GTC TGG CTT AGA CG
archaeal amoA gene
95℃/2m;40cycles of 95℃/20s,55 ℃/20s,72℃/30s
Real-Time PCR in Fig. 1-2
(Francis et al 2005)
Arch-amoAR
GCG GCC ATC CAT CTG TAT GT
amoA-1F
GGG GTT TCT ACT GGT GGT
bacterial amoA gene
95℃/2m;40cycles of 95℃/20s,57℃/30s,72℃/30s
(Rotthauwe et al 1997)
amoA-2R
CCC CTC KGS AAA GCC TTC TTC
515F
GTG CCA GCM GCC GCG G
universal 16S rRNA genes
95°C, 3min; 32×(95°C, 30s; 55°C, 30s; 72°C, 30s); 72°C, 10min
The high-throughput sequencing in Fig. 1, 3, 4, 5, 6; Fig. S4-S8; Table S2-3
(Stubner 2002)
907R
CCG TCA ATT CMT TTR AGT TT
CrenamoA 23f
ATG GTC TGG CTW AGA CG
95°C/90s;25 cycles of 95°C/30s,55°C/30s,72°C/90s;72°C/6m
Clone library in
Fig. 3-5
(Tourna et al 2008)
CrenamoA 616f
GCC ATA CAB CKR TAN GTC CA
95°C/5m;38cycles of 95°C/45s,55°C/30s,72°C/1m;72°C/10m

11. Table S2 Sequencing summary of the total microbial communities in SIP microcosms using the universal primers 515F-907R of the total 16S rRNA genes
Soil
Treatment
16S rRNA genes
Bacteria
Archaea R1 R2 R3
SR0
Day-0
30263
41203
33121
16143
(97.7%)
16205
(98.0%)
16284
(98.5%)
388
(2.3%)
326
(2.0%)
247
(1.5%)
Day-56-13CO2
43831
42958
33073
14652
(88.6%)
15693
(94.9%)
16039
(97.0%)
1879
(11.4%)
838
(5.1%)
492
(3.0%)
SR3
44462
31836
38065
16138
(97.6%)
16244
(98.3%)
16167
(97.8%)
393
(2.4%)
287
(1.7%)
364
(2.2%)
39798
43368
37336
16001
(96.8%)
16011
(96.9%)
16027
530
(3.2%)
520
(3.1%)
504
SR6
30302
39230
40042
16169
16055
(97.1%)
15807
(95.6%)
362
476
(2.9%)
724
(4.4%)
39323
38823
42472
15850
(95.9%)
16060
(97.2%)
15749
(95.3%)
681
(4.1%)
471
(2.8%)
782
(4.7%)
SR9
32204
34987
36048
15967
(96.6%)
16440
(99.4%)
16415
(99.3%)
564
(3.4%) 91
(0.6%)
116
(0.7%)
38737
30410
34970
15821
(95.7%)
15170
(91.8%)
15984
(96.7%)
710
(4.3%)
1361
(8.2%)
547
(3.3%)
Total
896862
383091
13653

12. Table S3 The putative contribution of AOA and AOB to nitrification activity in the soils tested
Microbes
Treatment
Copy numbers of genes
(g-1 d.w.s)
Ratios of gene copy numbers in heavy DNA to all DNA fractions
copy number of genes in heavy DNA
Number of labeled cells a
Ratio of AOA:AOB in heavy DNA
Nitrification rate (μg NO3- -N g-1 d.w.s day-1)
femto mol NH3 oxidized cell-1 hour-1 b
SR0
AOA
(amoA gene)
13CO2
1.53×108
3.6%
5.51×106
0.12
12.23
6.61
AOB
1.68×108
67%
1.13×108
4.52×107
0.81
SR3
1.64×108
20%
3.28×107
1.15
13.45
1.22
1.03×108
69%
7.11×107
2.84×107
1.41
SR6
1.67×108
3.3%
0.21
12.53
6.77
8.68×107
75%
6.51×107
2.60×107
1.43
SR9
1.26×108
3.7%
4.66×106
0.24
10.62
6.78
6.35×107
80%
4.76×107
1.91×107
1.65
a :represents the labeled cells of AOA and AOB assuming that each AOA and AOB cell contains 1.0 and 2.5 of amoA gene, respectively
b :represents cell-specific rate of ammonia oxidation by AOA or AOB, assuming that all the nitrate is solely transferred from either AOA or AOB alone and each cell has equal activity

13. References
Francis CA, Roberts KJ, Beman JM, Santoro AE, Oakley BB (2005) Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. P Natl Acad Sci USA 102:14683–14688
Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microb 63:4704–4712
Stubner S (2002) Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen™ detection. J Microbiol Meth 50:155–164
Tourna M, Freitag TE, Nicol GW, Prosser JI (2008) Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms. Environ Microbiol 10:1357–1364