Response of Microbial Diversity to C: N: P Stoichiometry in Fine Root and Microbial Biomass

Supplementary Information

Response of microbial diversity to C: N: P stoichiometry in fine root and microbial biomass following afforestation

1. ChengjieRen

College of Agronomy, Northwest A&F University, Yangling, 712100 Shaanxi, China

The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100 Shaanxi, China

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2. Ji Chen

Center for Ecological and Environmental Sciences, Key Laboratory for Space Bioscience & Biotechnology, Northwestern Polytechnical University, Xi’an, 710072, China

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3. Jian Deng

College of Agronomy, Northwest A&F University, Yangling, 712100 Shaanxi, China

The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100 Shaanxi, China

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4. Fazhu Zhao

College of Urban and Environmental Science, Northwest University, Xi'an, Shaanxi, China

712100 Shaanxi, China

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5. Xinhui Han (Corresponding author)

College of Agronomy, Northwest A&F University, Yangling, 712100 Shaanxi, China

The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100 Shaanxi, China

Tel:13892872667

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6.Gaihe Yang (Corresponding author)

College of Agronomy, Northwest A&F University, Yangling, 712100 Shaanxi, China

The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100 Shaanxi, China

Tel: 13709129773

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7. Xiaogang Tong

College of Natural Resources and Environment, Northwest A&F University, Yangling, 712100 Shaanxi, China.

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8. YongzhongFeng

College of Agronomy, Northwest A&F University, Yangling, 712100 Shaanxi, China

The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100 Shaanxi, China

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9. Shelby Shelton

Milken Institute of Public Health, The George Washington University, Washington, 20052, USA

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10. GuangxinRen

College of Agronomy, Northwest A&F University, Yangling, 712100 Shaanxi, China

The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100 Shaanxi, China

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For field sampling, environmental parameter measurements and MiSeqsequencing, please contact:

Fig. S1Rarefaction curves for Shannon index were calculated using Mothur (v1.27.0) with reads normalized to 10,000 for bacteria (Fig. S1a) and to 19,830 for fungi (Fig.S1b) in each sample considering a 97% sequence identity level.

Fig. S2 Distribution of 16S rRNA sequences across bacterial taxa (class and order level)in thethreeland use typesand at twosampling periods.Differentletters indicate significant differences (ANOVA, P < 0.05, Tukey’s HSDpost-hoc analysis) amongthree land use in June and October. The abbreviations are for the class and order level.

The class level (Fig. S2a): Alphaproteobacteria (Alpha), Betaproteobacteria (Betap), Gammaproteobacteria (Gamma), Deltaproteobacteria (Delta), Sphingobacteriia (Sphin), Acidimicrobiia (Acidi), Cytophagia (Cytop), Anaerolineae (Anaer), Bacilli (Bacil), Thermomicrobia (Therm), Flavobacteriia (Flavo), and Nitrospira (Nitro).

The order Level (Fig. S2b): Rhizobiales (Rhizo), Solirubrobacterales (Solir),Xanthomonadales(Xanth), Burkholderiales (Burkh), Nitrosomonadales (Nitro), Gaiellales (Gaiel) Sphingobacteriales(Sphin),Rhodospirillales (Rhodo),Myxococcales (Myxoc),Propionibacteriales (Propio),Acidimicrobiales (Acidi)Cytophagales(Cytop),Micrococcal(Micro),Pseudonocardiales(Pseud),Gemmatimonadales(Gemma),Rhodobacterales (Rhodo), Frankiales (Frank), Micromonosporales (Micro),Desulfobacterales (Desul), Flavobacteriales (Flavo), Chloroflexales (Chlor), Chromatiales (Chrom), Streptomycetales (Strep),Sphingomonadales (Sphin), Corynebacteriales (Coryn), Chlorobiales(Chlor), Desulfurellales (Desul),Legionellales (Legio),Lactobacillales (Lacto), Pseudomonadales (Pseud),Opitutales(Opitu), and Streptosporangiales (Strep)

Fig. S3 Distribution of ITS sequences across fungaltaxa (class and order level)in the threeland use types.Differentletters indicate significant differences (ANOVA, P < 0.05, Tukey’s HSDpost-hoc analysis) amongthree land use in June and October. The abbreviations are for the class and order level.

The class Level: Dothideomycetes(Dothi), Sordariomycetes (Sorda), Eurotiomycetes (Eurot),Leotiomycetes (Leoti),Tremellomycetes (Treme), Agaricomycetes (Agari), Orbiliomycetes (Orbil), and Pezizomycetes (Peziz)

The order Level: Agaricales (Agari), Xylariales (Xylar),Pezizales (Peziz), Cantharellales (Canth), Sebacinales (Sebac), Microascales (Micro), Cystofilobasidiales (Cysto), Onygenales (Onyge), Verrucariales (Verru),Botryosphaeriales (Botry), Trichosphaeriales (Trich), Orbiliales (Orbil), Agaricostilbales (Agari), and Glomerellales (Glome).

Fig. S4Ordination plots of the redundancy analysis (RDA) to identify the relationships between the abundance of bacterialtaxa (Black arrows) and soil properties (Red arrows) at the class level (Fig. S4a) andthe order level (Fig. S4b),respectively.The abbreviations are for the class and the order level.

The class level (Fig. S4a): Alphaproteobacteria (Alpha), Betaproteobacteria (Betap), Gammaproteobacteria (Gamma), Deltaproteobacteria (Delta), Sphingobacteriia (Sphin), Acidimicrobiia (Acidi), Cytophagia (Cytop), Anaerolineae (Anaer), Bacilli (Bacil), Thermomicrobia (Therm), Flavobacteriia (Flavo), and Nitrospira (Nitro).

The order Level (Fig. S4b): Rhizobiales (Rhizo), Solirubrobacterales (Solir),Xanthomonadales(Xanth), Burkholderiales (Burkh), Nitrosomonadales (Nitro), Gaiellales (Gaiel) Sphingobacteriales(Sphin),Rhodospirillales (Rhodo),Myxococcales (Myxoc),Propionibacteriales (Propio),Acidimicrobiales (Acidi)Cytophagales(Cytop),Micrococcal(Micro),Pseudonocardiales(Pseud),Gemmatimonadales(Gemma),Rhodobacterales (Rhodo), Frankiales (Frank), Micromonosporales (Micro), Desulfobacterales (Desul), Flavobacteriales (Flavo), and Chloroflexales (Chlor), Chromatiales (Chrom), Streptomycetales (Strep),Sphingomonadales (Sphin), Corynebacteriales (Coryn), Chlorobiales(Chlor), Desulfurellales (Desul),Legionellales (Legio),Lactobacillales (Lacto), Pseudomonadales (Pseud),Opitutales(Opitu), and Streptosporangiales (Strep)

Fig. S5Ordination plots of the redundancy analysis (RDA) to identify the relationships between the abundance of fungal taxa (Black arrows) and soil properties (Red arrows)at theclass level (Fig. S5a) and the order level (Fig. S5b),respectively.The abbreviations are for the class and the order level.

Table S1Changes in C, N, P contents in soil at 0-20cm afterafforestation.

Factors / June in 2009a / June in 2011a / June in 2013a
Landuseb / AL / CK / RP / AL / CK / RP / AL / CK / RP
C (g/kg)c / 2.347 / 5.823 / 7.971 / 2.461 / 6.176 / 8.386 / 2.909 / 6.506 / 9.006
N (g/kg)d / 0.430 / 0.547 / 0.678 / 0.434 / 0.580 / 0.742 / 0.471 / 0.612 / 0.819
P (g/kg)e / 0.462 / 0.558 / 0.644 / 0.515 / 0.583 / 0.695 / 0.523 / 0.622 / 0.738

a means that specific sampling time in three land use; Landuseb=three land use types, including Robiniapseudoacacia L., CaraganaKorshinskiiKom and Abandoned land; C (g/kg)c= soil organic C; N (g/kg)d = soil total N; P (g/kg)e = soil total P.

Table S2 Characteristics of thethree landuse

Land use types / Location / elevation/m / Slope aspect/° / Mainly Vegetation
Types of Herb / Important valuea
Robiniapseudoacacia L.
(RP40a) / 36.87N, 109.34E / 1320 / NbyE45 / Ulmuspumila L
Pennisetumcentrasiaticum
Poasphondylodes
Drabanemorosa
Tripoliumvulgare
Melicascabrosa / 10.09
8.66
8.22
6.73
6.47
5.87
5.81
CaraganaKorshinskiiKom
(CK40a) / 36.87 N, 109.35 E / 1318 / NbyE10 / Tripoliumvulgare
Heteropappusaltaicus
Roegneriakamoji
Dendranthemaindicum
Rubiacordifolia
Melicascabrosa
Viola dissecta / 14.91
12.33
9.57
8.90
7.28
5.17
5.04
Abandoned land
(AL40a) / 36.87 N, 109.35 E / 1308 / NbyE30 / Bidenspilosa
Roegneriakamoji
Lespedeza davurica
Tripoliumvulgare
Thermopsislupinoides
Artemisia scoparia
Bothriochloaischaemum
Phragmitesaustralis / 8.22
7.64
7.47
7.26
6.93
6.46
6.40
6.20

aHerb species with important value5%, Important value is the average of relative coverage, relative frequency, and relative density.

Table S3Phylum-level composition of bacteria communities underthreeland use types in June and October. The community composition is presented as relative abundance (%).

Variable / June / October
RP40a / CK40a / AL40a / RP40a / CK40a / AL40a
Proteobacteria / 38.22±0.84 A / 38.24±0.92 A / 29.83±0.48 B / 40.07±1.53 A / 38.47±1.71 A / 29.31±0.99 B
Actinobacteria / 21.65±0.89 B / 20.83±1.07 B / 26.38±0.30 A / 18.43±0.84 B / 20.30±1.61 B / 27.27±0.91 A
Acidobacteria / 19.25±0.75 A / 18.58±0.76 A / 19.63±0.30 A / 17.26±2.02 A / 17.50±0.67 A / 17.18±0.75 A
Chloroflexi / 4.36±0.18 B / 4.20±0.13 B / 6.13±0.21 A / 4.29±0.65 B / 5.21±0.51 B / 7.01±0.58 A
Planctomycetes / 5.74±0.37 A / 5.66±0.20 A / 6.32±0.18 A / 6.58±0.41 A / 6.06±0.43 A / 6.27±0.32 A
Gemmatimonadetes / 1.24±0.07 B / 1.21±0.11 B / 1.80±0.18 A / 1.62±0.29 A / 1.74±0.27 A / 1.88±0.31 A
Bacteroidetes / 5.77±0.33 A / 6.32±0.68 A / 2.42±0.34 B / 7.40±1.59 A / 6.55±1.24 A / 2.27±0.32 B
Cyanobacteria / 0.35±0.04 B / 0.48±0.09 B / 2.22±0.54 A / 1.08±0.71 B / 0.29±0.04 B / 4.65±0.86 A
Nitrospirae / 0.93±0.14 B / 0.74±0.10 B / 1.69±0.24 A / 0.84±0.16 A / 1.00±0.17 A / 1.26±0.10 A
Firmicutes / 0.74±0.08 C / 1.70±0.17 A / 1.16±0.11 B / 0.36±0.04 C / 0.89±0.09 A / 0.60±0.06 B
Armatimonadetes / 0.31±0.04 B / 0.36±0.03 B / 0.57±0.06 A / 0.37±0.05 B / 0.44±0.02 B / 0.80±0.06 A
Verrucomicrobia / 0.09±0.02 A / 0.17±0.04 A / 0.10±0.03 A / 0.13±0.04 B / 0.21±0.03 A / 0.10±0.02 AB

All values are presented as Means ± Standard error (means ± SE) (n=6). Capital letter next to the Means ±SE indicates significant differences among three land use in each period (June and October) at P< 0.05 using Duncan's Multiple Range Test (DMRT) following ANOVA.; Three land use types: RobiniapseudoacaciaL. (RP); CaraganakorshinskiiKom (CK);abandoned land (AL)

Table S4 Phylum-level composition of fungal communities underthreeland use types in June and October. The community composition is presented as relative abundance (%).

Variable / June / October
RP40a / CK40a / AL40a / RP40a / CK40a / AL40a
Ascomycota / 78.53±0.861A / 77.29±0.951 A / 71.52±0.717B / 76.34±1.061A / 74.04±1.385A / 168.87±1.557B
Basidiomycota / 6.82±0.674B / 7.42±0.562B / 12.57±0.977A / 6.78±0.888C / 11.47±0.813B / 15.45±0.554A
Zygomycota / 11.61±0.388A / 11.84±0.442A / 12.57±1.215A / 14.19±0.871A / 12.10±1.211A / 12.48±1.652A
Chytridiomycota / 0.01±0.007A / 0.03±0.020A / 0.08±0.037A / 0.02±0.003B / 0.03±0.005B / 0.10±0.029A
Glomeromycota / 0.01±0.002A / 0.01±0.002A / 0.04±0.040A / 0.02±0.006A / 0.05±0.004A / 0.14±0.106A

All values are presented as Means ± Standard error (means ± SE) (n=6). Capital letter next to the Means ±SE indicates significant differences among three land use in each period (June and October) at P< 0.05 using Duncan's Multiple Range Test (DMRT) following ANOVA.Three land use types: RobiniapseudoacaciaL. (RP); CaraganakorshinskiiKom (CK);abandoned land (AL).
Table S5The C, N, P of soil microbial biomass and fine root biomassas affected by afforestationin June and October.

Variable / June / October
RP40a / CK40a / AL40a / RP40a / CK40a / AL40a
Fine root C (g/kg) ** / 361.36±5.887 A / 352.10±0.918 A / 242.41±7.990 B / 310.09±1.14 A / 302.91±3.54 A / 145.39±5.41 B
Fine root N (g/kg)** / 18.54±0.434 A / 17.56±0.030 B / 8.79±0.073 C / 13.80±0.19 A / 13.43±0.05 B / 6.94±0.03 C
Fine root P (g/kg)** / 1.90±0.040 A / 1.97±0.030 A / 1.42±0.017 B / 1.56±0.03 A / 1.61±0.02 A / 1.17±0.04 B
MBC (mg/kg)** / 444.77±3.365 A / 427.29±8.79 A / 191.45±7.762 B / 398.70±5.34 A / 319.72±9.54 B / 167.56±2.71 C
MBN (mg/kg)** / 57.10±1.301 A / 52.66±1.541 B / 23.90±0.347 C / 71.45±1.07 A / 55.05±1.36 B / 21.93±0.14 C
MBP (mg/kg)** / 13.09±0.278 A / 10.65±0.222 B / 9.54±0.461 C / 13.48±0.32 B / 17.74±0.70 A / 8.65±0.49 C

All values are presented as Means ± Standard error (means ± SE) (n=6). Capital letter next to the Means ±SE indicates significant differences among three land use in each period (June and October) at P< 0.05 using Duncan's Multiple Range Test (DMRT) following ANOVA; ** P <0.01, * P <0.05; Three land use types: RobiniapseudoacaciaL. (RP); CaraganakorshinskiiKom (CK);abandoned land (AL). MBC, MBN, MBPrepresents the abbreviations ofmicrobial biomass carbon, microbial nitrogen, microbial phosphorus, respectively.