Effect of vegetation loss on abundant and rare microbial phylotypes across N-enrichment levels
We found that vegetation loss increased the total relative abundance of abundant bacterial phylotypes, while a corresponding decrease in the total relative abundance of rare bacterial phylotypes at the medium and high N-enrichment levels. An increase in the total relative abundance of abundant bacterial phylotypes was due to a greater percentage of the abundant phylotypes with positive than negative responses to the vegetation loss (22-31 vs. 4-5%). Our results support the idea that vegetation loss-induced increase in the soil pH at medium or high N-enrichment levels may favor soil bacteria. Substantial data indicate that relatively high soil pH favors the growth and activity of bacteria in various soil ecosystems [9, 31, 32]. However, the vegetation loss did not alter the total relative abundance of abundant or rare fungal phylotypes at any N-enrichment level. This was true because there was no difference in the percentages of abundant fungal phylotypes with positive than negative responses to the vegetation loss, thus implying that most of the abundant fungal phylotypes (77-84%) did not respond to vegetation loss at any N-enrichment levels. The different responses of abundant soil bacterial and fungal phylotypes to vegetation loss were consistent with a previous study of the Mongolian grasslands that also showed positive or no effect of vegetation loss on abundant bacteria and fungi, respectively . While, a higher ratio of the abundant bacteria to abundant fungi caused by vegetation loss in current study was also consistent with other plant diversity experiments [23, 33], which reported that biomass-based ratio of bacteria to fungi increased with a decrease in the input of plant material into soil. The difference in soil bacterial and fungal responses to vegetation loss may be due to their differential sensitivity to the environmental disturbance in the form of vegetation loss, and due to the high requirements of nutrients per unit C for bacteria than fungi .
Interestingly, we found that N-enrichment substantially reduced the relative abundance of abundant bacterial and fungal phylotypes but it increased the relative abundance of rare bacterial and fungal phylotypes. However, these trends differ from those exhibited by the plant communities, i.e., the N-enrichment generally increased the biomass of dominant plant species but it reduced the biomass of rare species [9, 31]. The responses of abundant vs. rare microbial phylotypes to the N-enrichment differ probably because the former has evolved strategies to reduce the resource use under water- and nutrient-limited conditions, while these strategies may not be sufficient to deal with the strong environmental disturbances (e.g. low soil pH) at medium or high N-enrichment levels [9, 14]. Our findings are in line with the recent theoretical and empirical predictions [18, 34] that the abundance of rare bacterial phylotypes is usually low but it can increase under suitable conditions. Overall, the vegetation loss had a much stronger effect on the relative abundance of soil bacterial than fungal phylotypes.
Effects of vegetation loss on microbial alpha diversity across N-enrichment levels
Unlike the relative abundance, the alpha-diversity of soil bacterial or fungal phylotypes did not respond to the vegetation loss, which is contrasted with recent reports that bacterial and fungal alpha-diversity increased with plant species richness in the grassland ecosystems at local  and regional [36, 37] scales. The lack of response of the alpha-diversity of all phylotypes to the vegetation loss strongly supports the view that plant and soil organisms are largely uncoupled [38-41]. However, a recent research have suggested that soil bacterial than fungal community properties are more closely associated with the plant biomass , in addition, we also found that the vegetation loss reduced the alpha-diversity of rare fungal phylotypes at low and medium N-enrichment levels. The stronger responses of alpha-diversity of fungal than bacterial phylotypes to the vegetation loss are partly in line with the recent empirical evidences predicting variable responses of soil microbial groups to the vegetation loss [13, 22, 23]. In the current study, the decline in alpha-diversity of rare fungal phylotypes with vegetation loss were mainly explained by the variables related to soil nutrients and ANPP, which is reasonable, because fungi are the first consumers of belowground plant-derived inputs to the soil [12, 43]. Overall, our results indicate that the alpha-diversity of soil fungal than bacterial phylotypes is more susceptible to the vegetation loss; in terms of relative abundance, however, the fungal than bacterial phylotypes are less sensitive to the vegetation loss.
We also found that the alpha-diversity of all bacterial phylotypes was determined by both abundant and rare bacterial phylotypes, while the abundant fungal phylotypes explained the alpha-diversity of all fungal phylotypes. These results indicate that, for bacteria, the rare phylotypes serve as a reservoir of species and can maintain the alpha-diversity of the bacterial community [16, 17]. We propose that the conflicting results, particularly those, describing the relationships between plant and soil microbial alpha-diversity within single sites [41, 44] can be explained by the differences between the abundant and rare microbial phylotypes, while these differences mainly result from differences in the environmental contexts under investigation. Furthermore, our results revealed that N-enrichment strongly reduced the diversity of both abundant and rare bacterial phylotypes but it decreased the diversity of rare fungal phylotypes, which nevertheless suggests that fungi than bacteria have higher environmental tolerance [9, 45, 46]. These findings differ from results reported for the plant communities, which showed that species richness was more sensitive to the chronic N-enrichment for rare than common species in the grasslands of Asia, America and Europe under arid and non-arid conditions [26, 47]. Overall, our findings provide direct evidence that soil microbial groups differ in their responses to a reduction in plant-derived inputs to soil and that the responses of alpha-diversity depend on the microbial phylotype abundance (rare vs. abundant) and soil nutrient status.
Effects of vegetation loss on microbial beta diversity across N-enrichment levels
The vegetation loss significantly altered the beta diversity of abundant and rare phylotypes of soil bacteria and soil fungi, while these effects were relatively consistent across all N-enrichment levels. However, previous research has reported weaker responses of the bacterial alpha- than beta-diversity to the vegetation loss [5, 48]. These substantial changes in the bacterial beta-diversity could be due to the substantial percentage of the abundant (19-35%) and rare (21-30%) phylotypes that responded to the vegetation loss across the N-enrichment levels, although previous studies have reported such changes at the phylum level . The large changes in the fungal beta-diversity could be due to the substantial percentage of rare (35-48%) than abundant fungal phylotypes that responded to vegetation loss across N-enrichment levels. While, the changes in the fungal beta-diversity could also be ascribed to the declines in plant-associated classes such as Dothideomycetes and Sordariomycetes . However, the lack of response of the beta-diversity of abundant fungal phylotypes to the vegetation loss might be due to their non-responsiveness to the vegetation loss across the N-enrichment levels. The SEM analysis further showed that the beta-diversity of all bacterial and fungal phylotypes was mainly explained by the plant-induced changes in the SOC at the low and medium N-enrichment levels , and by the plant-related variables  and soil pH  at the high N-enrichment level. We also found large differences in the determinants of the beta-diversity of abundant vs. rare phylotypes at each N-enrichment level. Previous studies on macro-organisms showed that the occurrence of abundant and rare species is often regulated by different ecological processes . In the case of microbial communities, relatively little is known about how ecological processes regulate the occurrence of dominant and rare phylotypes [18, 34, 52]. Our results, therefore, suggest that vegetation loss-induced changes in the beta-diversity of soil bacteria and fungi at different N levels arises from niche and stochastic processes; however, further research is needed to partition the relative influence of either ecological process.
The Mantel test showed that the beta-diversity of all bacterial phylotypes was explained by both abundant and rare phylotypes but that of all fungal phylotypes was determined by only abundant phylotypes. Our results evidenced that rare phylotypes make substantial contributions to beta-diversity [16, 17]. For instance, Gobet et al.  showed that, after removing 50% of the rare phylotypes, a significant change in the beta-diversity of soil bacterial community cross coastal sands was disappeared. However, our results indicate that a substantial contribution of rare phylotypes to the community structure was not evident in either bacteria or fungi. Furthermore, our results indicate that the effects of N-enrichment on bacterial and fungal beta-diversity could be explained by the fact that N-enrichment altered 82-94% of bacterial and 29-43% of fungal phylotypes. However, previous studies have mostly described such changes at the phylum or class level [14, 15, 45]. Although strong effects of N-enrichment on the beta-diversity of microbial communities are previously studied, our findings, for the first time, show phylotype level responses of soil bacterial and fungal phylotypes and their beta diversity to the vegetation loss and N enrichment.
Consistent effects of vegetation loss on microbial properties across N-enrichment levels
We did not find a statistically significant interaction between the effects of vegetation loss and N-enrichment levels on microbial community properties at the phylotype level, thus suggesting that the vegetation loss-induced changes in the structure of microbial communities is relatively not obvious across N-enrichment levels, though changes in the N-enrichment levels substantially affected soil fertility and plant productivity (Fig. 7). Our finding is supported by a recent study, which reported that the effects of vegetation loss on soil microbial functional groups did not differ among 30 island ecosystems differing in the soil fertility and plant productivity . Our results differed, however, from those of several previous studies, which indicated that the effects of vegetation loss on microbial ecosystems can be strongly influenced by environmental contexts [53, 54]. The interaction between vegetation loss and N-enrichment level in the current study was not statistically significant probably because the interaction altered only 13-15% of bacterial and 9-10% of fungal phylotypes. Another possible explanation for the lack of this interaction might be that the soil microbial properties are regulated more by the soil N availability and pH than by vegetation loss under various levels of N-enrichment [9, 30]. There is often some decoupling between the above- and below-ground biota because the soil biota can be driven by abiotic factors independent of vegetation [12, 15]. The current results revealed that the effects of vegetation loss were relatively consistent across N-enrichment levels, i.e., the effects were largely independent of the environmental contexts (Fig. 7). Our findings also suggest that the belowground functions of different ecosystems may have similar negative responses to the vegetation loss irrespective of the large differences in their productivity or fertility.