The vegetation characteristics and soil properties across N addition treatments.
The N addition had a positive effect on LNCWM, and foliar N/P (Table 1). For instance, LNCWM and foliar N/P increased by 54% and 93% at N5 plots, respectively. The increase in foliar N/P of dominant species in grassland communities indicates that dominant plants gradually shifted from N-limited to P-limited. The N addition had a significantly negative influence on diversity (Splant and Hplant). In addition, N addition had a positive effect on biomass, and but negatively affected LPCWM and SLACWM, though the effect was not significant. Increasing the level of N resulted in an inconsistent response in LCCWM and FDis; however, no significant effects were recorded for both parameters across all N addition treatments. The results of PCoA and PERMANOVA showed that N addition did not cause significant changes in plant community compositions (Fig. S2a, Table S1). An evaluation of soil properties showed that N addition had a small effect on soil properties (Table 2). The results showed that increasing the level of N resulted in no significant changes in OC, TN, AN, TP, AP, pH, SMC, and these soil properties almost remained unaffected with N addition.
Table 1
The vegetation characteristics across N addition treatments. Values are means ± standard error (n=6). Different letters within each row indicate significant differences (P < 0.05). Splant represents the species richness of plants, Hplant represents Shannon index of plants.
Parameters
|
N0
|
N1
|
N2
|
N3
|
N4
|
N5
|
F
|
P
|
LCCWM(g kg−1)
|
490.3±3.3
|
480.3±5.1
|
474.5±3.8
|
470.0±3.9
|
486.7±5.5
|
483.0±7.2
|
2.318
|
0.068
|
LNCWM (g kg−1)
|
15.0±0.3e
|
17.9±0.8cd
|
18.3±0.5c
|
20.3±0.4bc
|
21.8±0.6ab
|
23.1±1.1a
|
19.240
|
0.000
|
LPCWM (g kg−1)
|
1.32±0.04
|
1.22±0.11
|
1.16±0.03
|
1.09±0.05
|
1.08±0.06
|
1.08±0.10
|
2.013
|
0.105
|
SLACWM (cm−2 g−1)
|
143.2±11.0
|
126.8±8.5
|
131.2±5.2
|
116.2±6.0
|
109.6±2.8
|
127.0±10.0
|
2.263
|
0.074
|
Foliar N/P ratio
|
11.3±0.5d
|
15.0±1.8c
|
15.8±1.7c
|
18.8±2.2b
|
20.3±1.6ab
|
21.8±2.6a
|
26.845
|
0.000
|
FDis
|
1.4±0.1
|
1.3±0.1
|
1.3±0.1
|
1.6±0.1
|
1.3±0.2
|
1.2±0.2
|
0.869
|
0.513
|
Splant
|
13.3±1.0a
|
11.5±1.1ab
|
13.3±1.0a
|
9.5±1.2b
|
9.7±0.9b
|
8.7±1.2b
|
3.704
|
0.010
|
Hplant
|
2.3±0.1a
|
2.0±0.1ab
|
2.2±0.1a
|
1.9±0.1bc
|
1.8±0.1bc
|
1.7±0.1c
|
4.023
|
0.007
|
Biomass (g m−2)
|
202.0±17.7
|
280.0±37.0
|
304.6±41.8
|
296.1±71.1
|
484.5±108.6
|
330.2±25.4
|
2.492
|
0.053
|
Table 2
Soil properties across N application treatments. Values are means ± standard error (n=3). Different letters within each row indicate significant differences (P < 0.05).
Parameters
|
N0
|
N1
|
N2
|
N3
|
N4
|
N5
|
F
|
P
|
OC(g kg−1)
|
24.90±1.55
|
24.88±1.91
|
27.80±1.43
|
26.37±2.58
|
27.13±1.14
|
26.38±0.49
|
0.508
|
0.765
|
TN(g kg−1)
|
2.63±0.32
|
2.60±0.33
|
2.93±0.30
|
2.77±0.36
|
2.61±0.58
|
2.71±0.15
|
0.367
|
0.862
|
AN(mg kg−1)
|
67.90±4.28
|
70.00±4.50
|
78.40±5.30
|
82.37±5.75
|
77.70±2.52
|
72.37±6.96
|
1.177
|
0.376
|
TP(g kg−1)
|
0.62±0.04
|
0.62±0.02
|
0.62±0.01
|
0.60±0.04
|
0.62±0.01
|
0.61±0.02
|
0.260
|
0.926
|
AP(mg kg−1)
|
3.10±0.07
|
2.90±0.40
|
3.17±0.20
|
2.01±0.09
|
3.16±0.35
|
2.82±0.32
|
2.673
|
0.076
|
pH
|
8.25±0.05
|
8.26±0.04
|
8.22±0.04
|
8.30±0.03
|
8.27±0.04
|
8.26±0.05
|
0.393
|
0.845
|
SWC(%)
|
21.55±0.90
|
21.53±0.75
|
23.95±0.51
|
23.34±1.43
|
23.41±1.21
|
22.52±1.88
|
0.677
|
0.649
|
The microbial communities across N addition treatments.
Diversity of microbial communities.
A total of 1,064,911 bacterial sequences were obtained from all soil samples, with an average of 59,162 sequences per sample, and the sequencing results yielded a total of 10,650 bacterial OTUs. There was also a total of 1,079,258 fungal sequences (59,959 on average) from all samples, which were clustered into 6900 OTUs. According to the results, there were no significant changes in bacterial α-diversity (ACE, Chao 1 and Shannon) across all N addition treatments (Table 3). Also, there was no significant change in fungal α-diversity across all N addition treatments. Spearman’s correlation coefficients showed that soil microbial α-diversity was weakly correlated with soil properties and vegetation characteristics (Fig. S1). Changes in soil bacterial richness (ACE, and Chao1 index) exhibited a negative correlation with SLACWM (R2=-0.58, P=0.014; R2=-0.60, P=0.011) only (Fig. S1a). However, soil fungal diversity (ACE, Chao1 and Shannon indices) was weakly correlated with all factors (Fig. S1b).
Table 3
Soil bacterial (B) and fungal (F) α-diversity of different N application gradients. Values are means ± standard error (n=3). Different letters indicate significant differences (P < 0.05).
Parameters
|
N0
|
N1
|
N2
|
N3
|
N4
|
N5
|
F
|
P
|
B_ACE
|
4140.0±61.1
|
4115.7±166.2
|
4270.7±54.5
|
4312.7±26.4
|
4266.3±83.5
|
4125.1±162.7
|
0.670
|
0.654
|
B_Chao1
|
4034.7±59.3
|
4000.8±160.2
|
4158.5±46.6
|
4193.2±42.9
|
4184.4±71.9
|
4041.1±159.5
|
0.692
|
0.639
|
B-Shannon
|
9.70±0.02
|
9.53±0.16
|
9.71±0.05
|
9.72±0.06
|
9.69±0.07
|
9.66±0.10
|
0.650
|
0.667
|
F_ACE
|
2460.9±207.2
|
2051.2±272.7
|
2109.3±158.8
|
2738.5±445.2
|
2508.7±253.7
|
2263.5±298.8
|
0.833
|
0.551
|
F_Chao1
|
2382.1±195.0
|
2005.3±307.0
|
2024.5±142.5
|
2699.5±469.5
|
2453.7±264.5
|
2207.0±267.1
|
0.843
|
0.545
|
F-Shannon
|
7.30±0.43
|
6.74±0.30
|
6.52±0.13
|
7.81±0.30
|
6.93±0.55
|
6.84±0.55
|
1.304
|
0.325
|
Composition of microbial communities.
The dominant phyla of the bacterial community were Proteobacteria (34.3-40.5%), Acidobacteria (22.5-25.2%), Actinobacteria (9.6-15.5%) and Gemmatimonadetes (6.3-7.9%) (Fig. 1a). There were no significant changes in relative abundance of the bacterial community across all N addition treatments. The dominant phyla of the fungal community were Ascomycetes (21.7-42.8%), Mortierellomycota (9.6-22.0%) and Basidiomycetes (5.9-24.2%) (Fig. 1b). The results showed that N addition increased the relative abundance of Ascomycetes while it decreased the relative abundance of Basidiomycetes compared with the control.
LEfSe was used to determine the taxa that significantly differed in abundance under varied levels of N addition. For fungal communities, the significantly abundant taxa were Periconiaceae (at the family level) and Periconia (at the genus level) at N4 plots (Fig. S2b). However, Helotiales (at the order level) and Roesleria (at the genus level) were significantly abundant at N5 plots. The biomarkers of fungal community were associated with the Phylum Ascomycetes. However, no biomarkers were observed in the bacterial community across all N addition treatments (Fig. S2a).
PCoA was used to analyze changes in microbial community compositions across all N addition treatments, and PERMANOVA analysis was used to determine significant differences in microbial community compositions. PCoA showed that all plots were clustered together without significant separations (Fig. S3). PERMANOVA analysis showed that there was no significant change in bacterial (P=0.879) and fungal (P=0.060) community compositions across all N addition treatments (Table S1). Therefore, N addition did not change microbial community compositions.
Assembly and species turnover of microbial communities.
The niche width of microbial communities almost remained unchanged under different levels of N addition (Fig. S4). However, N altered the balance between random and deterministic processes in the microbial community (Fig. 2). C-score results showed that the value of standardized effect size (SES) changed significantly with increasing the level of N. Bacterial (N5, P<0.001) (Fig. 2a, Table S2) and fungal (N3, P<0.005; N4, P<0.001; N5, P<0.001) (Fig. 2b, Table S2) communities were transformed from random to deterministic processes in high levels of N.
Richness-based species exchange ratio (SERr) was used to quantify species turnover in microbial communities (Fig. 3a). The SERrs of bacterial and fungal communities ranged from 0.40-0.41 and 0.56-0.63, respectively. The SERrs of fungal communities were greater than those of bacterial communities, suggesting that the fungal communities were more susceptible to N addition. To explore the effects of species turnover on microbial community formation, the contribution of extinct and immigrated OTUs to the changes in microbial richness and community structure was assessed. In the bacterial communities, the immigrated OTUs accounted for more than 23% of the OTU richness under N addition, with the lowest (23.91%) and highest (29.59%) proportions recorded at N1 and N3 plots, respectively (Fig. 3b). In addition, 22.18 to 25.48% of the native OTUs were categorized as extinct OTUs, with the lowest and highest proportions recorded at N4 and N1 plots, respectively. In the fungal communities, the proportion of immigrated OTUs in the OTU richness under N addition varied from 28.04 to 52.00%, with the lowest and highest proportions recorded at N2 and N3 plots, respectively. The rate of OTUs extinction ranged from 30.17 to 52.93%, with the lowest and highest proportions recorded at N3 and N2 plots, respectively. These results indicated that N addition altered the OTU richness of microbial community. Also, it was noted that N addition led to higher proportions of immigrated and extinct OTUs in the fungal communities than the bacterial communities, indicating that fungal community compositions were more sensitive to N addition. However, the immigrated and extinct OTUs accounted for a small proportion of the relative abundance in microbial communities, and the proportions in bacterial and fungal communities varied from 1.25 to 1.85% and 2.13 to 5.56%, respectively. A high proportion of microbial richness along with a low proportion of community compositions indicate that species turnover had a greater contribution to the microbial richness than the microbial community structure. Similar results were also demonstrated by the SIMPER analysis, which showed that the immigrated and extinct OTUs contributed no more than 8% to the variation in microbial communities between the N treatments and control (Fig. 3c).
Drivers of microbial communities.
The structural equation model (SEM) illustrating the effects of soil properties and vegetation characteristics on soil microbial compositions and diversity is illustrated in Fig. 4. According to the results, 85 and 44% of the variance in bacterial and fungal community compositions, respectively, was explained by the fitted model (Fig. 4a). The results showed that TN and plant community, respectively, had significantly positive and negative effects on the bacterial community, whereas pH and SLACWM had significantly negative influences on the fungal community. Overall, the results showed that plant community compositions (P < 0.05) and TN (P < 0.001) were important driving factors for bacterial community compositions, whereas fungal community compositions were mainly regulated by pH (P < 0.05) and SLACWM (P < 0.05). A significant negative effect of SLACWM on bacterial diversity was revealed by the model (Fig. 4b). However, none of the factors associated with soil properties and vegetation characteristics had a significant effect on fungal diversity, which was consistent with the results illustrated in Figure S1. Nitrogen addition had no significant effects on microbial community diversity and compositions. Nitrogen addition significantly affected LNCWM and Hplant, though they had no significant impacts on microbial community diversity and compositions. However, the factors (i.e. plant community compositions, TN and SLACWM) that significantly affected the diversity and compositions of microbial community were not significantly influences by N addition.