**3. Results**

#### *3.1. Changes in Soil Properties and Stocks of Woody Debris and Non-Woody Debris with Succession*

The stock of woody debris (WD), soil concentrations of SOC, TN and C:N showed a significant tendency of increase from the S1 to S6 succession stages (all *p* < 0.05). On the contrary, the stock of non-woody debris (NWD), soil temperature (T) and pH were observed significantly higher in initial than terminal stages of succession (all *p* < 0.05; Table 1). Especially, the S4 succession stage had the lowest soil concentrations of SOC and TN and the highest soil C:N and soil pH (Table 1).

**Table 1.** Soil physicochemical properties and the stocks of woody debris and non-woody debris among different succession stages.


SOC: the concentrations of soil organic carbon; TN: the concentrations of total nitrogen; C:N: the mass ratio of SOC and TN; T: soil temperature; pH: soil pH; WD: the stock of woody debris; NWD: the stock of non-woody debris; Values followed by different lowercase letters mean significant difference *(p* < 0.05) among six successional series based on Wilcoxon rank-sum test. S1 to S6 represent successional stages from 1 to 6, respectively.

#### *3.2. Changes in the Sequence Data and Soil Microbial Alpha Diversity*

A total of 3,004,539 and 2,845,500 high-quality effective sequences of bacteria and fungi were acquired across the subalpine forest successional series; the sequences were clustered with 97% identity into 13,684 and 12,233 OTUs, respectively. Among these OTUs, 3490 bacterial and 761 fungal OTUs were common in soil microbial communities at six successional stages (Figure 1). Soil bacterial and fungal communities at the initial succession stage (S1) had the most independent OTUs (1225 and 1572, respectively) (Figure 1). The alpha diversity of soil bacterial community at the initial successional stage (S1) was significantly higher than that at the terminal successional stages (S5 and S6) (*p* < 0.05; Figure 3.3), although there was not a remarkable difference in fungal alpha diversity among the six forest successional stages according to these two indices (*p* > 0.05; Figure 3). However, ACE and Species richness indices in fungal communities were significantly higher at the S1 succession stage than at the S6 succession stage (*p* < 0.05; Section 3.3 and Figure 3).

**Figure 1.** Flower diagram of the common and unique bacterial (**a**) and fungal (**b**) OTUs across a subalpine forest succession series. S1 to S6 represent successional stages from 1 to 6, respectively.

**Figure 2.** Differences in bacterial alpha diversity across a subalpine forest succession series. Calculations based on the OTUs at 97% sequence identity. Shannon-Wiener index (**a**), Simpson index (**b**), ACE index (**c**) and Species richness (**d**) represent the indices of alpha diversity. Values followed by different lowercase letters mean significant difference (*p* < 0.05) among six successional series based on Wilcoxon rank-sum test. S1 to S6 represent successional stages from 1 to 6, respectively.

#### *3.3. Composition and Structure of Soil Microbial Community*

At the phylum level, soil bacterial communities at the six successional stages were dominated by *Proteobacteria* (37.7–45.2%), *Acidobacteria* (15.3–27.5%) and *Actinobacteria* (5.0–10.4%) (Table 2). The relative abundance of eight of the 10 most abundant phyla varied markedly (*p* < 0.05) with forest succession, except for that of *Proteobacteria* and *Verrucomicrobia* (Figure 4a). In particular, higher relative abundance of *Firmicutes* (*p* < 0.001), Tenericutes (*p* < 0.01) and *Acidobacteria* (*p* < 0.05) were observed at terminal succession stages (S5 or S6) (Figure 4b; Table S2). In contrast, the relative abundance of *Chloroflexi* (*p* < 0.01), *Actinobacteria* (*p* < 0.01) and *Rokubacteria* (*p* < 0.01) were markedly higher at the initial successional stages (S1, S2 and S3) (Figure 4a; Table S2). Soil fungal communities across the subalpine forest successional series were dominated by *Ascomycota* (27.7–46.4%), *Basidiomycota* (13.5–31.8%) and *Mortierellomycota* (2.0–12.5%) (Table 3). The relative abundance of the other phyla totaled less than 2% (Table 3). Among the fungal phyla, clade GS01 was not enriched in the S4 and S6 successional stages, and *Blastocladiom* was not enriched in the S4 successional stage (Table 3). Furthermore, the significantly higher relative abundance of *Mortierellomycota* (*p* < 0.05), *Rozellomycota* (*p* < 0.01) and clade GS01 (*p* < 0.05) were observed at the terminal succession stage (S5 or S6) (Figure 4b; Table S2).

**Figure 3.** Differences in fungal alpha diversity across a subalpine forest succession series. Calculations based on the OTUs at 97% sequence identity. Shannon-Wiener index (**a**), Simpson index (**b**), ACE index (**c**) and Species richness (**d**) represent the indices of alpha diversity. Values followed by different lowercase letters mean significant difference (*p* < 0.05) among six successional series based on Wilcoxon rank-sum test. S1 to S6 represent successional stages from 1 to 6, respectively.



The abundance of each taxon in relation to the abundance of all the taxa was calculated as the relative abundance (%)(basedonthedataofsequenceanalysis).S1toS6 representsuccessionalstagesfrom1to6,respectively.

**Figure 4.** Relative abundance and significance analyses of the dominant soil bacterial (**a**) and fungal (**b**) communities at phylum level across a subalpine forest succession series. Significant effect: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. Kruskal-Wallis test. S1 to S6 represent successional stages from 1 to 6, respectively.

**Table 3.** Percentage composition of the dominant phyla of soil fungal community among different succession stages.


The abundance of each taxon in relation to the abundance of all the taxa was calculated as the relative abundance (%) (based on the data of sequence analysis). S1 to S6 represent successional stages from 1 to 6, respectively.

Furthermore, based on an LDA score of 4, linear discriminant analysis effect size (LEfSe) indicated that there were a large number of significantly different taxa (biomarkers) among different succession stages (Figure 5). In total, 38 and 31 taxa (including the phylum, class, order, family, genus and species levels) were selected as biomarkers for soil bacterial and fungal communities, respectively, along the succession gradients (Figure 5).

**Figure 5.** Cladograms generated by linear discriminant analysis effect size (LEfSe) indicating differences in the bacterial (**a**) and fungal (**b**) taxa across a subalpine forest succession series. Different colored bars indicate taxa were enrichment in the corresponding succession stages. S1 to S6 represent successional stages from 1 to 6, respectively.

ANOSIM test verified that the composition of soil bacterial and fungal communities was substantially distinct among the six successional stages (all *p =* 0.001; Figure 6). Specifically, ANOSIM showed that there were significant differences in soil bacterial community structure among different succession stages, except between S1 and S2, between S1 and S4, and between S3 and S4. Slight differences in soil fungal community composition were observed between S1 and S2, between S3 and S5 and between S4 and S5, but significant differences were found between the remaining successional stages (Table S3).

**Figure 6.** Boxplot of analysis of similarities (ANOSIM) based on the Bray–Curtis distances of samples for the dissimilarities of bacterial and fungal communities across a subalpine forest succession series. S1 to S6 represent successional stages from 1 to 6, respectively.

#### *3.4. Relationships of Soil Microbial Diversity with Forest Variables*

According to the analysis of detrended correspondence analysis (DCA) (Table S4), RDA was selected to analyze the relationships of soil microbial diversity with second forest variables based on the analysis of variance inflation factor (VIF) (Table S5). RDA confirmed that the variations in soil bacterial and fungal communities across the successional series were associated with forest variables (Figure 7). Among the forest variables, soil pH had an extremely significant effect on soil bacterial (*p <* 0.01) and fungal community composition (*p <* 0.001; Tables 4 and 5). Meanwhile, soil bacterial diversity was also significantly affected by SOC, soil T, the stock of non-woody debris (NWD) and altitude (*p* < 0.05; Table 4), while soil fungal community composition was also significantly influenced by soil temperature (T) (*p* < 0.05; Table 5). In particular, among dominant bacterial phyla, soil pH was significantly and positively correlated with the relative abundance of *Gemmatimonadetes* and *Firmicutes*, but markedly and negatively correlated with the abundance of *Proteobacteria*, *Bacteroidetes* and *Verrucomicrobia* (Figure 7a). The stock of NWD and soil temperature (T) was prominently and positively correlated with the abundance of most of the bacterial phyla, but observably and negatively correlated with the abundance of *Gemmatimonadetes, Firmicutes* and *Tenericutes.* On the contrary, the correlation of altitude and most of soil bacterial community composition was negative (Figure 7a). In addition, the relative abundance of *Proteobacteria* and *Bacteroidetes* were also significantly and positively correlated with the concentration of SOC (Figure 7a). Among dominant fungal phyla, soil pH was notably positively correlated with the abundance of *Rozellomycota* and *Ascomycota* not the other dominant phyla (Figure 7b). In contrast, soil temperature (T) was significantly and positively correlated with the abundance of the most abundant phyla, except for *Rozellomycota* and *Basidiomycota* (Figure 7b).

**Figure 7.** Redundancy analysis (RDA) about the effect of forest variables on the differences in the compositions of soil bacterial (**a**) and fungal (**b**) communities across a subalpine forest succession series. SOC: the concentrations of soil organic carbon; C:N: the mass ratio of SOC and soil total nitrogen; T: soil temperature; pH: soil pH; WD: the stock of woody debris; NWD: the stock of non-woody debris. S1 to S6 represent successional stages from 1 to 6, respectively.


**Table 4.** Significance of the effects of forest variables on bacterial community composition among different succession stages.

SOC: the concentrations of soil organic carbon; C:N: the mass ratio of SOC and soil total Nitrogen; T: soil temperature; pH: soil pH; WD: the stock of woody debris; NWD: the stock of non-woody debris; Significant effect: \* *p* < 0.05, \*\* *p* < 0.01.

**Table 5.** Significance of the effects of forest variables on fungal community composition among different succession stages.


SOC: the concentrations of soil organic carbon; C:N: the mass ratio of SOC and soil total nitrogen; T: soil temperature; pH: soil pH; WD: the stock of woody debris; NWD: the stock of non-woody debris; Significant effect: \* *p* < 0.05, \*\*\* *p* < 0.001.
