**4. Discussion**

The composition and diversity of soil bacterial and fungal communities varied significantly with forest succession. The alpha diversity of soil bacteria and fungi decreased from initial to terminal succession stages except for Shannon-Wiener and Simpson indices of soil fungal community, which was in contrast to our first hypothesis. *Proteobacteria* and *Acidobacteria* dominated soil bacterial communities, and *Ascomycota* and *Basidiomycota* dominated soil fungal communities in all six successional stages. However, a significant difference was observed in the relative abundance of dominant soil microbial phyla among the six successional stages. Meanwhile, the ANOSIM results demonstrated that soil microbial community structure was substantially different among the six successional stages. These results were consistent with our second hypothesis. RDA showed that soil pH had the greater effect on the differences in the composition of soil communities across the forest successional series, while non-woody debris stock was correlated with only the composition of soil bacterial community, partly supporting our third hypothesis. Generally, these results confirm that forests in different successional stages not only nursed the same dominant microbial communities but also support unique biomarkers and that the biodiversity in soil bacterial and fungal communities differed greatly in response to forest vegetation succession.

#### *4.1. Changes in the Diversity of Soil Microbial Community with Forest Succession*

Soil microbial diversity demonstrated a global tendency to decrease from initial to terminal succession stages; an exception to this finding was demonstrated by the Shannon-Wiener and Simpson indices for the fungal communities, which was supported by the findings of several studies [58,59]. However, the changes in soil bacterial and fungal alpha diversity varied greatly with forest succession from those reported previously (Table 6). A considerable increase in bacterial alpha diversity and an increase followed by a decrease in fungal diversity were found with forest succession in different regions [25,30,32]. Meanwhile, Zhang et al. [31] and Liu et al. [25] observed a trend of decreasing first and then increasing. In addition, the alpha diversity of soil bacterial and fungal communities

did not show significant changes with succession [30,42,59]. As succession progressed, the aboveground plant community changed significantly. However, at middle and late successional stages, the changes in plant species and the intensification of competition led to a loss of soil nutrients and a decrease in the diversity of soil microbial communities [25]. Moreover, the decreased alpha diversity index may be due to the different growth strategies of microbes; in particular, at initial succession stages, the soil microbial population lives in harsh and unpredictable conditions and has a high reproductive rate but a low survival rate, resulting in high diversity but low abundance. At terminal succession stages, the microbial population lives in a favorable and predictable environment, with a low reproductive rate but a high survival rate, resulting in increased competitiveness and a stable population number [31]. In addition, the differences in soil bacterial and fungal alpha diversity may have occurred since soil microorganisms have differing nutrient preferences [60].

**Table 6.** Comparisons of forest soil microbial community alpha diversity index across forest succession series in different regions.


ns: no significance; –: no data.

#### *4.2. Changes in Soil Microbial Community Composition with Forest Succession*

*Proteobacteria*, *Acidobacteria* and *Actinobacteria* were the dominant bacterial phyla in the six forest successional stages, but their relative abundance differed significantly, which was consistent with the findings of a growing number of studies in different ecosystems [62–64]. Fierer et al. [65] analyzed the extent to which these phyla were dominant in many soil bacterial communities based on phylogenetic analysis. The abundance of *Proteobacteria* increased with increasing vegetation successional stage, and this result was generally consistent with findings in New Zealand [66]. These findings suggested that *Proteobacteria* likely play a functional role in the restoration of soil [67]. Many soil *Proteobacteria* are copiotrophic and become abundant when labile substrates are available during secondary forest succession [65]. However, a higher abundance of *Actinobacteria* was observed at initial successional stages in our study since *Acidobacteria* are oligotrophic and prefer nutrient-poor environments. Notably, *Planctomycetes*, the dominant phylum in other forest succession ecosystems [31,68,69], was not dominant in these subalpine successional forests. This phenomenon suggested that serious water and soil erosion during subalpine forest succession might limit habitat availability for *Planctomycetes*, which is mainly present in oceans, marine sediments, freshwater lakes and wastewater [70].

*Ascomycota* and *Basidiomycota* were two dominant phyla in soil fungal communities among the six successional stages, but slight changes were observed in their abundance

among the stages. This result was consistent with the findings of previous studies [25,71] but was contradictory to the observation in a *Pinus yunnanensis* forest [42]. Meanwhile, the abundance of *Mortierellomycota*, *Rozellomycota* and clade GS01 at terminal succession stages (S5 and S6) were higher than those in the other stages. Generally, the members of *Ascomycota* are common in extreme environments, while *Mortierellomycota*, *Rozellomycota* or clade GS01 members may have a strong preference for host plants in infertile soil, as these fungi exhibited environmental selection pressure and dispersal limitation with forest succession. Therefore, the abundance of these taxa in soil fungal communities increased with forest succession, indicating the increased accumulation of soil nutrients and maturation of the ecosystem.

The structure of soil bacterial and fungal communities differed substantially among the six successional stages based on ANOSIM analyses. This result was also observed in previous research [32]. However, other observations showed a lack of significant difference of community structure among successional stages [72]. These results suggested that the development pattern is consistent between plant and microbial communities and showed that above- and underground communities follow a synchronous succession process [73]. This further verified the importance of plant succession in microbial community structure.

#### *4.3. The Roles of Forest Variables in Microbial Community Composition and Forest Succession*

Our observations showed that the composition of soil microbial communities across the successional series was significantly affected by forest variables (e.g., soil pH, soil organic carbon, non-woody debris stock, altitude and soil temperature), which was consistent with the findings of previous studies [61,74,75]. Soil pH is a critical factor that affects the structure of soil bacterial and fungal communities [76–78]. Landesman et al. [27] have confirmed that the changes in tree species caused by secondary succession can lead to the acidification of forest soil, which was in line with our first result. Meanwhile, the members of *Acidobacteria*, *Mortierellomycota* and *Rozellomycota* [79,80], can physiologically tolerate acidic pH levels [81,82]. Therefore, the increase in the number of these microbial communities at terminal succession stages could be explained by the acidification of soil pH in this period. In addition to soil pH, soil temperature was also strongly correlated with the characteristics of soil microbial communities [83,84]. Soil temperature can not only directly accelerate microbial metabolic rates and biochemical processes [85–87] but also indirectly affect activity levels by changing the temperature dependency of the community [88]. In addition, plant successional processes, including nutrient and water uptake, as well as the initiation, branching, and orientation of root growth, also result in changes in soil temperature [89]. These factors could result in microbial communities performing variably over the succession period in response to soil temperatures.

Our results also found that the composition of soil bacterial communities, but not the composition of soil fungal communities, was significantly correlated with SOC and non-woody debris stock across the forest successional series, which was inconsistent with the findings of some previous studies [90,91]. Soil bacterial and fungal communities utilize soil organic carbon at different rates [92,93]. Variations in NWD among successional stage and vegetation type evoke different microbial responses to the quantitative and qualitative differences in plant compounds [94]. Meanwhile, our results showed that the soil concentration of SOC increased with plant community succession, which may be due to the decomposition of plant NWD. The concentration of soil organic matter strongly affects the structure and function of soil microbial communities [95,96]. Our results further demonstrated that distinct soil and vegetation properties could induce different soil microbial composition across forest succession series.
