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, 310058, China 2State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, 210008, China   *Corresponding author: Yong Li Tel. +86-571-88982410; Fax: +86-571-88982410 e-mail: yongl@zju.edu.cn or liyongcn@zju.edu.cn

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)

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

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

Fig. S4 Phylogenetic analysis of archaeal 16S rRNA genes in the SIP microcosms. The designation ‘Total DNA-AOA-16SrRNA- OTU-1-3201’ indicates that OTU-1 contains 3201 16S 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

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

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

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).

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

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

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

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

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