Changes in Soil Bacterial Community Structure and Diversity of Pinus Tabuliformis Plantation after 65 Years of near-naturalization in North China

ABSTRACT Our study investigates the effect of near-naturalization of plantations on soil physicochemical and bacterial features and the difference between soil layers in Baxianshan National Nature Reserve. Four stands were involved, including two forest types: near-naturalized and natural secondary forests, with the former classified into three stages. Soil physicochemical and bacterial properties were determined and analyzed. TC, TN contents and C/N ratio of the surface soil were higher than the corresponding lower layer. TC, TN contents decreased first and then increased with near-naturalization, lower than the natural secondary forests, while the C/N ratio was the opposite; total and endemic OTUs quantity was more in the surface layer than the lower and both increased with near-naturalization; the dominant phyla were Proteobacteria, Acidobacteria, Gemmatimonadetes, and verrucomicrobia, the relative abundance of Proteobacteria increased with near-naturalization while that of other dominant phyla decreased; the α-diversity increased on the whole during near-naturalization and was lower than the natural secondary forests except for Simpson and Shannon index; environmental factors significantly explained the bacterial α-diversity and community structure of natural secondary forests but not near-naturalized forests. This study helps fully understand the change characteristics and response mechanisms of soil bacterial community structure to the restoration of the plantation.


Introduction
Soil microorganisms serve as an integral part of soil ecosystems in forests (J.X.Gao et al., 2020).They have been involved in the decomposition of organic matter, nutrient cycling, and energy conversion (Averill et al., 2016;Y. Li et al., 2019;Zhang et al., 2020), playing a vital role in maintaining the productivity, function, and stability of forest ecosystem (Das et al., 2017;L. Wang et al., 2019).They were reported as key measures of soil quality and productivity (Canals et al., 2019).Climate, soil characteristics, vegetation community structure, and diversity affect soil microbial community structure in the soil genetic processes.Compared with soil nutrients, plant species richness significantly impacted on soil bacterial communities (Hanif et al., 2019;Krishna et al., 2020).In particular, under the exact conditions of climate and soil type, vegetation type has been proved to be an essential factor affecting the function of underground ecosystems, including soil microbial communities (Q.K. Wang et al., 2013;X.P. Gao et al., 2016;Zhao et al., 2014).Different vegetation types influenced soil microbial communities through differences in litter composition, quality, and root exudates, enabling a strong correlation between species and abundance with aboveground plant species (Bainard et al., 2016;Horrocks et al., 2019;Z. Qu et al., 2020;Santonja et al., 2017).Vegetation types might also indirectly affect microbial composition by regulating the physicochemical properties of the soil (Yang & Zhu, 2015).Additionally, differences in the quality and quantity of litter caused by changes in plant diversity have changed the composition, amount, and community structure of underground microorganisms (T.B.Qu et al., 2016).Meanwhile, the soil bacterial communities responded to changes in the aboveground plant communities, regulating the soil nutrient circulation through its metabolism and impacting the aboveground vegetation communities.
Soil microbial communities differed greatly between coniferous and broad-leaved forests (Vuong et al., 2020).The single tree layer species of coniferous forests led to a relatively simple composition and structure of soil microbial communities.There was a more complex soil microbial community composition of coniferous broad-leaved mixed and broad-leaved deciduous forests.The composition and proportion of different tree species have affected soil microbial biomass, community structure, and metabolic activity (L.Chen et al., 2019).
To address the ecological degradation and continued decline of ecosystem services, the area of plantations has rapidly increased (C.L.C. Liu et al., 2018).The monoculture plantations have rapidly restored the above-ground biomass (Moghaddam, 2014) and carbon sequestration capacity of damaged ecosystems (X.J. Li et al., 2016).However, due to the low diversity, stability, and excessive density, the disadvantages of competitive mortality have been gradually coming to light (Zhou et al., 2019).Furthermore, monoculture forests may also suffer from the interruption of nutrient cycling, leading to soil nutrient deficiencies and detrimental to the healthy development of forest ecosystems (Yu et al., 2019).The ecosystem degradation caused by deforestation and monoculture plantations inescapably led to soil degradation (Ferraz, Fillo et al., 2014), including both degradation of soil nutrients and ecological service quality.The former is mainly due to soil nutrient deficiency caused by slow decomposition rates of litter and low nutrient return rates in monoculture plantations (Epron et al., 2015), while the latter is induced by structural and functional groups' variation of soil microorganisms (Mitchell et al., 2016), especially changes in plant-soil microbial coexistence relationships.(Liang & Balser, 2011;Zechmeister-Boltenstern et al., 2015).Near-naturalization refers to the plantation communities passively recovering into more diverse and zonal species-dominated vegetation communities relying on the natural regeneration of the forest.Near-naturalization recovery could improve the stability and health of forest ecosystems (Hou et al., 2021), maximizing the dynamic balance of forest biomes and diversity, and restoring natural species and ecological functions.(Sujii et al., 2017).
Baxianshan National Nature Reserve is located at the eastern foot of the Yanshan Mountains in North China.In 1954, Pinus tabuliformis Carr.plantations were extensively planted in areas with sparse natural vegetation in the Reserve and then closed the mountain for tending.During about 65 years of near-naturalized recovery, a restoration sequence of forests with different proportions of P. tabuliformis has been formed, providing a valuable natural laboratory for studying plant and soil dynamics during near-naturalization through the spatial instead of temporal method (G.P. Chen et al., 2017).Previous studies have focused on the relationship between soil microbial communities and altitude, above-ground plant community succession, and biodiversity (Canini et al., 2019;Mueller et al., 2014;Porazinska et al., 2018) with little attention to soil microbial changes caused by nearnaturalization of plantations.Studies only reported soil physicochemical properties and aggregate characteristics during near-naturalization of P. tabuliformis plantation in Baxianshan National Nature Reserve (G.P. Chen et al., 2017).However, there has been no report on change characteristics and response mechanisms of soil microbial to nearnaturalization in Baxianshan National Nature Reserve till now.
In this study, we selected three distinguished near-naturalized forest stages according to the coverage ratio of P. tabuliformis to tree layer coverage and natural secondary forests as a control.We investigate the effect of near-naturalization recovery and different soil layers on soil CN properties and bacterial community structure.It is of great significance to understand the changing characteristics and driving factors of soil microorganisms during nearnaturalization.Because soil microbial community characteristics dynamically respond to the change of vegetation community and soil nutrients.Clarifying the dynamics of soil microorganisms during the near-naturalization can explain the restoration of vegetation and soil scientifically and reasonably.It will also help to put forward and implement effective forest tending management measures and improve the stability of forest ecosystems.

Study sites
Baxianshan National Nature Reserve, located in the northeast of Jizhou District, Tianjin, belongs to the eastern foot of the Yanshan Mountains (Figure 1).The Reserve covers a total area of 1049 ha.Most mountains are 500 ~ 800 m above sea level, and the highest peak, Juxian peak, is 1052 m above sea level.The Reserve belongs to a temperate continental monsoon climate with an average annual temperature of 9 ~ 10°C, extreme minimum temperature of −21°C, an extreme maximum temperature of 34.5°C, January average temperature of −7.2°C, July average temperature of 23.4°C, the annual accumulative temperature of 3800 ~ 3900°C, and average annual precipitation of 986.5 mm.The vegetation type of the reserve was typical warm temperate deciduous broad-leaved forests, with a small range of evergreen coniferous forests and mixed warm coniferous broad-leaved forests.The dominant arbor layer species are mainly light-loving, deciduous broad-leaved tree species, such as Quercus mongolica Fisch.Ex ledeb, Q. wutaishansea Mary, Q. dentata Thunb, and Q. variabilis Blume.The shrub layer consists of winter deciduous species, mainly Lespedeza Michx.and Spiraea L., and the herb layer mainly comprises Poaceae Barnhart.and Cyperaceae.

Site classification and soil sampling method
According to the coverage ratio of P. tabuliformis to tree layer, we selected three distinguishable stages of near-naturalized stages: P. tabuliformis forests stage (P), mixed forests stage (M), and near-natural forests stage (NNF).P. tabuliformis forest stage represents the community with a coverage ratio of P. tabuliformis to the tree layer of more than 90%, mixed forest stage with 40%-60% and near-natural forest stage with less than 20%.Coverage was defined as the proportion of the vertical projection area of the canopy at the ground level to the total area of the quadrat.Deciduous broad-leaved zonal vegetation communities were selected to control near-naturalization, i.e., natural secondary forests (NF).Field community surveys and soil sampling were conducted in September 2020.The survey lasted for 1 week, during which the weather was cloudy and no precipitation.Basic geographical information (Table S1) and the names and numbers of tree species were recorded in a total of 27 20 m × 30 m quadrats.Surface soil from 0 to 5 cm and lower soil from 10 to 15 cm were collected in each quadrat according to the five-point sampling method with sampling points arranged at the center and four corners (3 m from the vertex of the quadrat; Bao, 2000).Samples at the same layer in each quadrat were thoroughly mixed and divided into two portions.Therefore, 54 soil samples of each portion were obtained.One part was stored in liquid nitrogen for molecular biology extraction assay, and the other was stored at room temperature and brought back to the laboratory for physicochemical analysis in time.

Determination of soil carbon and nitrogen
Samples stored at room temperature were air-dried to constant weight, gently crushed, and passed through a 60-mesh sieve (mesh size: 0.25 mm) for carbon and nitrogen determination.The contents of total carbon, total nitrogen, and carbon-nitrogen ratio were determined using the C and N element analyzer CN 802 (VELP Company, Italy).

Amplification, high-throughput sequencing of 16S rDNA gene, and splicing
We extracted total DNA from 0.5 g of each soil sample using a soil DNA kit.The primers 27 F and 1494 R were designed to amplify the hypervariable V3-V4 region of the bacterial 16S rRNA gene.The PCR instrument was BioRad S1000 (Bio-Rad Laboratory, CA, USA).The length of concentration of the PCR product was measured by 1% agarose gel electrophoresis.Mege Gene Technology Co., LTD commissioned the high-throughput sequencing operation.The library was sequenced on an Illumina Nova 600 platform to obtain 250 bp paired-end reads.Then, the paired-end raw reads were quality controlled by fastsp and the primers were removed to obtain the paired-end clean reads (https://github.com/marcelm/cutadapt/).Next, the paired-end clean reads were merged to obtain raw tags using searchfastq merge pairs.Further, raw data quality was controlled using fastsp to obtain paired-end clean tags.The high-quality sequence was clustered to generate operational taxonomic units (OTUs) at a 97% similarity level (Miao et al., 2019).

Species annotation and analysis of community structure and diversity
Each representative sequence was annotated to seven taxonomic levels: kingdom, phylum, class, order, family, genus, and species.The total number of OTUs sequences and OUT table were calculated.The core and endemic species and community were all obtained from the OUT table and annotation.Core OTUs referred to as OTUs co-occurred in all stands, and endemic OTU was only occurring in a particular stand.α-diversity indices, including Richness, Shannon-winner, ACE (Chao & S.-M, 1992), Chao1 (Chao, 1984), and Simpson (Simson, 1949) index, were discussed to analyze the complexity of species diversity (Sang et al., 2018).The Richness index was the number of OTU species in each forest type.The Shannon, Simpson, Chao1, and ACE indices were calculated with the following formulas: where H is the Shannon index, s is the number of OTUs and p i is the proportion of the community represented by OTU i.
where p i is the proportion of the community represented by OTU i where F1 and F2 are the count of singletons and doubletons, respectively.
where S abund is the number of abundant OTUs (with more than rare threshold individuals) when all samples are pooled, S rare is the number of rare OTUs (with less than or equal to rare threshold individuals) when all samples are pooled, C ace is the sample abundance coverage estimator, F 1 is the frequency of singletons, and γ ace 2 is the estimated coefficient of variation for rare OTUs.
where s is the number of OTUs and P i is the proportion of the community represented by OUT i

Data analyses
The Significance of difference in soil CN, OTUs abundance, and α-diversity indices among forest stands and soil layers were analyzed by one-way ANOVA test (SPSS 19.0).Before analysis, the data were transformed to fit the normal distribution.The difference in soil bacterial community structure was calculated by the permanova test method ("ade4" package of R software; Dray & Dufour, 2007).Pearson correlation coefficient was used to explain the relationship between environmental factors (including soil carbon and nitrogen features, slope, aspect, and elevation) and α-diversity and abundance of dominant phyla of bacteria.The conversion method for aspect was as follows: take due south as the 0 base, and the angle between the actual aspect and due south direction was considered as the conversion value of aspect.The specific method was: based on the recorded aspect in Table S1 (the aspect in the table was based on due north), if the value is greater than 180, the conversion value = table value −180; if the value is less than 180, the conversion value = 180-table value.RDA function ("vegan" package of R software) was used to analyze the effects of environmental factors on the bacterial community structure of near-naturalized forests and natural secondary forests, respectively (R Development Core Team, 2006).All the significance level was defined as P < .05.The figures were realized by excel and the ggplot2 package of R.

Effects of near-naturalization on soil carbon and nitrogen features
Total carbon, total nitrogen content and C to N ratio were significantly higher of the surface layer than the lower layer for the same forest stands (P < .05).Total carbon and total nitrogen content of both layers first decreased and then increased during nearnaturalization, showing significant difference in the surface layer (P < .05)but no significant difference in the lower layer.The content of total carbon and total nitrogen in the surface layer was lower in near-naturalized forests than the natural secondary forests, while the content of the lower soil in the near natural forest stage exceeded that of the natural secondary forests.Soil C/N ratio was higher of the surface soil than the lower soil and also higher in near-naturalized forest stages than in natural secondary forests.Along with near-naturalization, it showed a first upward and then downward trend of surface soil while an upward trend of the lower soil layer (Figure 2).

Effects of near-naturalization on the biomass and abundance of soil bacterial taxa
A total of 3,711,402 sequences were obtained by high-throughput sequencing, clustered into 132224 reads and 46873 OTUs taxa.Biomass was defined as the number of OTUs.For both total and endemic OTUs, the number of the surface layer was a bit higher than the corresponding lower layer except for P. tabuliformis forests.The number of total and endemic OTUs of surface and lower soil layers consistently enhanced with the near-naturalization with a slightly faster of surface soil than the lower soil.The number of OTUs was still a distinct disparity between near-naturalized and national secondary forests (Figure 3).
These dominant phyla of soil bacteria included Proteobacteria, Gemmatimonadetes, Acidobacteria, and Verrucomicrobia for both layers.Relative abundance was defined as the ratio of average abundance of a phylum in a stand to the sum of abundance of all phyla in the same layer and stand.The relative abundance of Proteobacteria in the surface soil was higher than the corresponding stand of lower soil (P < .05),while the relative abundance of Gemmatimonadetes and Verrucomicrobia was higher in the lower soil than the corresponding surface layer (P < .05).The relative abundance of Acidobacteria in nearnaturalized restoration forests was higher in surface soil than the lower (P > .05),but it was the opposite in the natural secondary forest (P < .05; Figure 4).During nearnaturalization, the relative abundance of Proteobacteria of the surface and lower soil showed an overall upward trend, and there were significant differences among different near-naturalization stages (P < .05).Its abundance of the surface soil in near-naturalized stands was lower than the natural secondary forest, but the opposite for the lower soil.The relative abundance of Gemmatimonadetes first raised and then fell during nearnaturalization of both soil layers and was consistently lower than that of the national secondary forest.The relative abundance of Acidobacteria first increased and then decreased during near-naturalization of the surface soil and was higher than that of the national secondary forest.The relative abundance of Verrucomicrobia declined with nearnaturalization, higher than the national secondary forest for the surface soil, while its abundance first raised and then fell in the lower surface (Figure 4).
The OTUs annotated to genera or families were further analyzed.We found that OTU1 (Bradyrhizobium) was the most abundant in the surface soil and occupied the highest relative abundance in all the stands.Therefore, OTU1 could be considered as the indicator of the bacteria of surface soil in Baxianshan National Nature Reserve.The relative abundance of OTU11 (hypomycobium) in the P. tabuliformis forest stage was second only to OTU1, significantly higher than that of other stands and other OTUs of P. tabuliformis forest stage (Figure 5).Hence, OTU11 could be regarded as an indicator to distinguish surface soil bacteria of P. tabuliformis forests stage from other stages.In the lower soil, OTU1 could still be regarded as an indicative OTU because it still occupies the highest abundance among stands and OTUs.Although the relative abundance of OTU11 in the lower soil was still greater in P. tabuliformis forests stage than in other forests stages, it was lower compared with the other OTUs of P. tabuliformis forests (Figure 6).Therefore, it could no longer be regarded as an indicator OTU of lower soil of the Pinus tabuliformis forests.

Effects of near-naturalization on α-diversity of soil bacterial community
The α-diversity of bacteria in the surface soil was lower than the lower soil in P. tabuliformis forests, while it was contrast in all other forest stands.However, only the indices of natural secondary forests were significantly different between layers (P < .05,Table 1).In the surface soil, Chao1, ACE and Richness indices showed an increasing trend with near-naturalization, but lower than the natural secondary forests  (P < .05Table 1).The above three indices increased fastest from the P. tabuliformis forest stage to the mixed forest stage.Simpson and Shannon indices also increased with near-naturalized restoration and exceeded that of the national secondary forest in the near-natural forest stage.These results suggested that the near-naturalized restoration improved the biomass and evenness of bacteria in the surface soil.In the lower soil, the α-diversity indices decreased first and then increased with near-naturalization.Chao1, ACE, and Richness indices of the near-naturalized forest restoration stages were lower than the natural secondary forests, while Simpson and Shannon indices were higher compared with the near-natural forests.This indicated that although nearnaturalization has increased the α-diversity indices of bacteria in the lower soil on the whole, it decreased in the recovery stage from the P. tabuliformis to the mixed forest stage, contrary to the change trend of surface soil (Figure 7).
Further analysis of the correlation between soil environmental factors and αdiversity indices have showed that the correlation coefficient and significance all stronger in natural secondary forests than in near-naturalized forests (Figures 8  and 9).As to near-naturalized forests, only Slope was proved to be significantly negatively related to Simpson index (P < .05, Figure 8b).Interestingly, we found an opposite correlation direction between α-diversity and elevation, aspect and slope for different soil layers.In natural secondary forests, the α-diversity indices were significantly negatively related to C/N ratio and elevation in the surface soil (P < .05 Figure 9a).In contrast, elevation was positively correlated to α-diversity in the lower soil.Beyond that, Slope was significantly positively correlated with α-indices.(Figure 9a,b).

The change of bacterial community structure and the effect of environmental factor on it
We analyzed the difference of bacterial community structures between soil layers and among forest stands by permanova test.We got the significant difference between layers  in the natural secondary forests but not in the near-naturalized forests (P < .05,Table 2).In the surface soil, the difference of bacterial community structure between P. tabuliformis forest stage and natural secondary forests was found significant.There was no significant different of community structure among other stands of the surface soil and all stands of the lower soil (Table 2).
To reveal the effect of environmental factors on soil bacterial community structure, we first studied the relationship between environmental features and the abundance of dominant phyla.The correlation between them were quite different between the two forest types.In the surface soil of near-naturalized forests, soil C-to-N ratio was positively correlated with the abundance of Acidobacteria (P < .01, Figure 10a) and the elevation was found positively correlated with the abundance of dominant phyla except Proteobacteria (P < .05, Figure 10b).For the natural secondary forests, elevation was positively correlated with the abundance of dominant phyla in the surface soil, and C/N ratio negatively correlated to their abundance (P < .05, Figure 11a,b) Then we analyzed the influence of environmental factors on the bacterial community structure.As to the near-naturalized forests, 44.47% of the variation of the bacterial community structure of surface soil was explained by soil physicochemical properties and topography (P > .05,Table 3).Elevation was proved to be significant influence factor on the community structure by perm ANOVA test (P < .05, Figure 12a, Table 3).46.24% was explained as to the   lower soil and C/N ratio was found to be a significant affecting factors.(P < .01, Figure 12b, Table 3).For the community structure of natural secondary forests, 42.24% of the surface soil and 43.52% of the lower soil was interpreted by environmental features (P < .05,Table 3, Figure 13a,  b).Aspect and slope were the significant factors related to community structure (P < .05,Table 3).

Life history strategies explained the change in dominant phyla during near naturalization
The biomass of soil microorganisms can be reflected by the abundance of OTUs, as the method is more accurate than traditional soil fumigation and cultivation (Beauregard et al., 2010).Our results showed the bacterial OTUs abundance increased along nearnaturalization and vegetation diversity (data see Table S1), consistent with the study of Hjalmarsson who proved the community with low plant diversity may lead to lower microbial biomass per unit of soil owing to a more dispersed matrix the community could provide (Hjalmarsson et al., 2013).The OTUs quantity of the near-naturalized forests was quite lower than the natural secondary forests, indicating that the near-natural restoration had not restored the soil features to the level of natural secondary forest in terms of microbial biomass.Proteobacteria represented the highest proportion of soil bacteria, and Acidobacteria, Verrucomicrobia, and Gemmatimonadetes also occupied a relatively high proportion, consistent with results of the composition of soil microbial communities in moist deciduous forests (Dai et al., 2018;Lin et al., 2017;G. Liu et al., 2019;J. Liu et al., 2014;S.F. Li et al., 2020).Proteobacteria is a kind of eutrophic bacteria with the R life strategy.The decomposition of soil organic matter promoted their growth.Here we found its abundance enhanced with the near-naturalization, indicating the accelerated decomposition of soil organic matter and improving soil nutrient quality.As reported, Acidobacteria is a kind of oligotrophic bacteria with the life strategy of K, which decomposed recalcitrant organic matters in a low-carbon environment (Chu et al., 2011;Goldfarb et al., 2011;X.Z. Li et al., 2014).Our research showed the abundance of Acidobacteria in the upper soil layer increased first and then decreased with nearnaturalization.The highest value occurred in the mixed forest stage, indicating worst soil organic nutrition, consistent with the change rule of Acidobacteria abundance during forest succession in Dinghu Mountain (G.Y.Liu et al., 2019) and the invasion of Rhus typhina L. into native plant soil in the Zhenshan Mountain in Shandong Province (Zhu et al., 2020).This was also consistent with the lowest soil carbon and nitrogen content and the highest C/N ratio in the mixed forest in our study.

α-diversity of soil bacterial communities increased during near-naturalization
The increasing trend of α-diversity of surface soil during near-naturalization indicating that the soil environment was suitable for the coexistence of more and more soil bacteria in the process of near-naturalization.As the proportion of broad-leaved tree species increased during near-naturalization, the number of fine roots and its metabolic activity increased, providing more kinds and quantities of decomposing substrates to bacteria (Teng et al., 2021).This was also consisted with the study of the effect of afforestation mode on bacterial Chao1 and ACE diversity in Xiong'an New Area (K.F.Wang et al., 2022).The authors also attributed it to the mixing afforestation meet the needs of more bacterial groups by diverse  litter and root exudates.However, some of the α-diversity was still lower than the natural secondary forests, meaning that there was still a gap between the suitability of soil environment for microorganisms in the near-naturalized and the natural secondary forests.This is consistent with the previous studies which show that soil microbial community diversity decreased significantly after natural forest was converted into artificial forest (Vitali et al., 2016).For the lower soil, the decrease of α-diversity indices in mixed forest stage may relate to low carbon, high nitrogen content and high C/N ratio.This was because the characteristics of soil nutrients in the mixed forest stage were conducive to the growth and reproduction of Acidobacteria.The positive correlation between diversity and soil carton and nitrogen of the surface soil in near-naturalized forests may attributes to the nutrient-rich soil conducive to the coexistence of more kinds of soil bacteria and the increase of bacterial biomass such as Proteobacteria.The negative correlation between diversity and soil C/N ratio means the nutrient-poor situation of soil, which benefits the growth of oligotrophic Acidobacteria, thus inhibiting the growth and reproduction of most other bacteria, resulting in a decrease in biomass and diversity

The interpretation of the environmental properties on the community structure of soil bacterial communities
We combine the correlation between environmental factors and dominant phylum abundance with the interpretation of bacterial community structure.The relationship between environmental factors and the abundance of dominant phyla was quite the opposite between forest types and between soil layers.Soil C/N ratio was proved to be positively related to the abundance of Acidobacteria in the surface soil of near-naturalized forest due to its K life strategy and oligotrophic.Because high C/N ratios often mean low nutrient availability.Elevation was positively correlated with the abundance of the dominant phyla, which was consistent with the study of characteristics of microbial communities in the black soil of Northeast China (J.Liu et al., 2014).The structure of the soil microbial community might be affected by plant species and diversity (Zhong et al., 2020), vegetation, forest types (Jassey et al., 2013), and soil types.Former studies showed that vegetation type was the main factor in the construction of soil microbial communities.Plant species diversity was also proved to be an important factor affecting soil microbial communities (Lange et al., 2015(Lange et al., , 2014)), while a single plant species had little effect on the soil bacterial community composition (Kielak et al., 2008).Significant difference only existed between P. tabuliformis forest stage and natural secondary forests of the surface soil and between two soil layers of natural secondary forests.No significant difference among other stands was due to their development toward natural secondary forest along near-naturalization weakened the differences between stands.The significant difference between the two soil layers of natural secondary forests may be related to the difference of soil nutrient level and root distribution.Our study manifested significant difference of soil C and N features between soil layers.Previous studies have also demonstrated significant differences in microbial community structure among different soil layers (Helgason et al., 2014;Yan et al., 2019).
The environmental factors significantly interpreted the soil microbial community structure of natural secondary forests.Aspect and slope were the significant factors affecting bacterial community structures.Because the soil nutrient content of natural secondary forests was sufficient, it is no longer the limiting factor of community composition, so the community structure is significantly correlated with topography.In the near-naturalized forests, there wasn't obvious difference of community structure among near-naturalized stands.The selected environmental factors did not explain the community structure significantly, indicating that more important environmental factors have not been found in this study, and there were significant differences in the factors affecting the community structure between the near-naturalized forests and the natural secondary forests This study revealed the influence and mechanism of near-naturalization on soil physicochemical features and bacterial community structures and the difference between surface and lower soil layers in the Reserve.At present, most of the remaining near-naturalized forests should be classified into near natural forest stage and there seldom community areas of P. tabuliformis forest stage and mixed forest stage, which limits our sampling of the two stages.This may affect the reliable conclusion from the perspective of data analysis, but it did actually reflect the situation of the P. tabuliformis forest and mixed forest stages.The conclusion will provide more substantial theoretical data for healthy development of P. tabuliformis forests and the soil quality toward natural secondary forests in Baxianshan Nature Reserve.

Conclusion
Our results showed the abundance of eutrophic bacteria increased and that of oligotrophic bacteria decreased during near-naturalized recovery, both consistent with the soil total carbon and total nitrogen content but contrary to the C/N ratio.Near-naturalization recovery promoted the increase of α-diversity, but still lower than that of natural secondary forests.The community structure of bacteria was not obviously affected by nearnaturalization, with only the structure between P. tabuliformis forest stage and natural secondary forests being significantly different.Environmental factors significantly interpreted the bacterial community structure in natural secondary forests, however, factors that driving the structure in near-naturalized forests need to be further explored.This contributed to a full understanding of the changes characteristics and driving environmental factors of soil bacterial diversity and community structure during near-naturalization.Meanwhile, it helped further understand the succession of aboveground vegetation by the change character of soil ecosystem, providing a scientific and reliable reference for better maintaining and promoting the species diversity and the stability of the forest ecosystems.

Figure 2 .
Figure 2. Comparison of soil total carbon and nitrogen and the ratio of carbon to nitrogen between the four forest stands.(In the legend, U: Surface soil; L: Lower soil; P: P. tabuliformis forest stage; M: Mixed forest stages; NNF: Near-natural forest stages; NF: Natural secondary forests.Different capital letters indicate significant difference between upper and lower soil in the same stand, and different lowercase letters indicate significant difference between different stands in the same soil layer).

Figure 3 .
Figure 3.The comparison of total and endemic OTUs among the four forest stands (In the legend, P: P. tabuliformis forest stage; M: Mixed forest stages; NNF: Near-natural forest stages; NF: Natural secondary forests).

Figure 4 .
Figure 4.The comparison of relative abundance of dominant phyla between soil layers and among 4 different forest stands (In the legend, U: Surface soil; L: Lower soil; P: P. tabuliformis forest stage; M: Mixed forest stages; NNF: Near-natural forest stages; NF: Natural secondary forests.Different capital letters indicate significant difference between upper and lower soil in the same stand, and different lowercase letters indicate significant difference between different stands in the same soil layer).

Figure 5 .
Figure 5.The comparison of relative abundance of dominant OTUs of the surface soil among four different forest stands (In the legend, U: Surface soil; P: P. tabuliformis forest stage; M: Mixed forest stages; NNF: Near-natural forest stages; NF: Natural secondary forests.Different capital letters indicate significant difference between upper and lower soil in the same stand, and different lowercase letters indicate significant difference between different stands in the same soil layer).

Figure 6 .
Figure 6.The comparison of relative abundance of dominant OTUs of the lower soil among four different forest stands (In the legend, L: Lower soil; P: P. tabuliformis forest stage; M: Mixed forest stages; NNF: Near-natural forest stages; NF: Natural secondary forests.Different capital letters indicate significant difference between upper and lower soil in the same stand, and different lowercase letters indicate significant difference between different stands in the same soil layer).

Figure 7 .
Figure 7.Comparison of α-diversity indices between soil layers and among 4 different forest stands (In the legend, U: Surface soil; L: Lower soil; P: P. tabuliformis forest stage; M: Mixed forest stages; NNF: Nearnatural forest stages; NF: Natural secondary forests).

Figure 8 .
Figure 8.The Pearson correlation coefficient between α-diversity indices and environmental factors of near-naturalized forests (Figure 8a indicates the surface soil, Figure 8b indicates the lower soil; N: Total nitrogen content; C: Total carbon content; C.N: the ratio of carbon to nitrogen; * represents a significant difference at the 0.05 level and ** represents a significant difference at the 0.01 level).

Figure 9 .
Figure 9.The Pearson correlation coefficient between α-diversity indices and environmental factors of natural secondary forests (A indicates the surface soil; B indicates the lower soil; N: Total nitrogen content; C: Total carbon content; C.N: the ratio of carbon to nitrogen; * represents a significant difference at the 0.05 level and ** represents a significant difference at the 0.01 level).

Figure 10 .
Figure 10.The Pearson correlation coefficient between the relative abundance of dominant phyla and environmental factors in near-naturalized forest stands (A indicates the surface soil; B indicates the lower soil; N: Total nitrogen content; C: Total carbon content; C.N: the ratio of carbon to nitrogen; P: Proteobacteria; G: Gemmatimonadetes; V: Verrucomicrobia; A: Acidobacteria; * represents a significant difference at the 0.05 level and ** represents a significant difference at the 0.01 level).

Figure 11 .
Figure 11.The Pearson correlation coefficient between the relative abundance of dominant phyla and environmental factors in natural secondary forests (A indicates the surface soil; B indicates the lower soil; N: Total nitrogen content; C: Total carbon content; C.N: the ratio of carbon to nitrogen; P: Proteobacteria; G: Gemmatimonadetes; V: Verrucomicrobia; A: Acidobacteria; * represents a significant difference at the 0.05 level and ** represents a significant difference at the 0.01 level).

Figure 12 .
Figure 12.RDA analysis on the effect of environmental factors on the bacterial community structures of the near-naturalized forest stands (A indicates the surface soil; B indicates the lower soil; The legend: P: P. tabliformis forest stage; M: Mixed forest stage; NNF: Near-natural forest stages; The arrows: N: Total nitrogen content; C: Total carbon content; C.N: the ratio of carbon to nitrogen; E: Elevation; A: Aspect; S: Slope).

Figure 13 .
Figure 13.RDA analysis on the effect of environmental factors on the bacterial community structures of the natural secondary forests (A indicates the surface soil; B indicates the lower soil; The legend: P: P. tabliformis forest stage; M: Mixed forest stage; NNF: Near-natural forest stages; The arrows: N: Total nitrogen content; C: Total carbon content; C.N: the ratio of carbon to nitrogen; E: Elevation; A: Aspect; S: Slope).

Table 1 .
P value of ANOVA test of α-diversity among forest stands and between soil layers (U: surface layer; L: lower layer; P: P. tabuliformis forest stage; M: Mixed forest stage; NNF: Nearnatural forest stage; NF: Natural secondary forests; Bold indicates significant difference).

Table 2 .
Perm ANOVA test of community structure between soil layers and among different forest stands.

Table 3 .
Anova and Monte Carlo substitution tests of the effect of environmental factors on soil bacterial community structures among stands (U: Surface soil; L: Lower soil; N: Near-naturalized forests; S: Natural secondary forests; Bold indicates the environmental factors significantly correlated with the corresponding community structure).