Variations of Bacterial and Archaeal Community Structure and Diversity Along Soil Pro les in a Peatland in Southwest China


 As bacteria and archaea are key components in the ecosystem, their alterations along soil profiles are important in understanding the biogeochemical cycles in peatland. However, little is known about the vertical distribution patterns of bacteria and archaea along the Bitahai peatland, as well as their relationship to soil chemical properties. Here, sequencing of 16S rRNA genes (Illumina, MiSeq) was used to analyze bacterial and archaeal abundance, diversity, and composition across 0-100 cm of the soil. Soil pH, total C, N, and P concentrations and stoichiometric ratios also were estimated. Results revealed that total C and total N contents, as well as C:P and N:P ratios, significantly increased with increasing peatland depths, while total P decreased. The top three dominant phyla were Proteobacteria (39.64%), Acidobacteria (12.93%), and Chloroflexi (12.81%) in bacterial communities, and were Crenarchaeota (58.67%), Thaumarchaeota (14.34%), and Euryarchaeota (10.82%) in archaeal communities in the Bitahai peatland, respectively. The total relative abundance of the methanogenic groups and ammonia-oxidizing microorganisms all significantly decreased with soil depths. Both bacterial and archaeal diversity were greatly affected by the soil depths. Soil C, N, and P concentrations and stoichiometric ratios markedly impacted the community structure and diversity just in archaea, not in bacteria. Therefore, these results highlighted that the microbial community structure and diversity depended on soil depths, and the affecting factors for bacteria and archaea were different in the peatlands.


Introduction
Peatlands cover only about 3% of the earth's land area while storing nearly 30% of the global soil carbon (Dise 2009). Peatlands are an important carbon sink due to their low rate of organic matter decomposition and high water table (Laiho 2006). Carbon accumulated in the northern, tropical, and southern peatlands are 547, 50, and 15 Gt C respectively (Yu et al. 2010). Carbon storage in peat soils is so high that carbon cycling in peatland ecosystems plays an important role in global carbon cycling.
Microbial communities, as important decomposers, play key role in the carbon cycle of peatlands and in the overall ecosystem functioning. They do not only directly control the turnover of organic carbon but also bene t nutrient mineralization and uptake (Andersen et (Wang et al. 2017). However, its belowground soil microbial communities, which play enormous roles in driving the soil biogeochemistry cycles, are rarely studied.
The studying the differences between bacterial and archaeal communities along the peat pro le and linking this to the changes in soil properties are important in the Bitahai peatland. Thus, our hypothesis was that: (1) bacterial and archaeal composition and diversity vary with soil depths and (2) soil properties affect the bacterial and archaeal communities and diversity along soil pro les in the Bitahai peatland.

Site description and soil sampling
The study site is located in the Bitahai Nature Reserve (27°46-27°57′N, 99°54′-100°09′E), Yunnan Province, Southwest China. The elevation in this region is 3512.9 m, and has a cold and humid plateau climate with an annual average temperature of 5.4°C. The hottest and coldest months are July and January, respectively, with average temperatures of 13.2 and − 3.8°C, respectively. The annual average precipitation is 617.6 mm and occurs from around June to September. Our sampling sites are distributed on the typical peat bog wetland of Bitahai, which has a soil type of peat bog soil. The peat is saturated all the year round and the peat thickness is about 3 m deep. Major vegetations in this site are Carex lehmanii, Sanguisorba liformis, Deschampsia cespitosa, and Sinocarum coloratum. Three sampling plots were set in this site during the 2017 growing season, and ten soil depths (0-10 cm, 10-20 cm, 20-30 cm, 30-40 cm, 40-50 cm, 50-60 cm, 60-70 cm, 70-80 cm, 80-90 cm, and 90-100 cm) were collected from each plot. Three replicate cores (ca., 100 g soil) were taken at each plot using a soil sampler device (diameter 52 mm, Eijkelkamp, Netherlands). Roots and stones were careful removal by hand and sieved through by 2 mm for each soil core. A total of 30 soil samples were obtained and then stored in sterilized and sealed polyethylene packages, transported on ice to the laboratory, and stored at -20°C until processing.

Soil properties analysis
Soil pH was determined using a pH meter (Orion 868, USA) with a soil-to-water ratio of 1:2.5; total carbon (TC) was measured using a Vario TOC instrument manufactured by Elementar Company; total nitrogen (TN) and total phosphate (TP) were determined by H 2 SO 4 digestion and was then measured using a Continuous Flow Analytic System (SEAL Analytical AA3, Germany).

Processing of sequencing data
The raw sequence data were processed and analyzed using the QIIME Pipeline (Caporaso et al. 2012). Brie y, sequencing reads with an average quality value ≤ Q20 were obtained, and ambiguous nucleotides in barcodes and homopolymer reads longer than 8 bp and shorter than 150 bp were removed to improve sequence quality. Paired ends were joined with FLASH (Magoc and Salzberg 2011), whereas the chimeric sequences were detected and eliminated using the USEARCH function in QIIME (Caporaso et al. 2010).
After quality control and chimeric sequence removal, a total of 886,894 bacterial 16S rRNA genes and 789,965 archaeal high quality gene sequences were obtained for community analyses of the 30 soil samples. The number of sequences per sample ranged from 7,597 to 34,181, with an average of 29,563 sequences for bacteria, whereas the number of sequences ranged from 14,008 to 33,467 with an average of 26,332 sequences for archaea. All sequences were clustered into operational taxonomic units (OTUs) at a 97% identity threshold. The Shannon's diversity index, Simpson's index, and the Ace and Chao1 estimators were generated for each sample based on the OTU table (Caporaso et al. 2010). The phylogenetic a liation of each sequence was analyzed using the RDP Classi er (http://rdp.cme.msu.edu/classi er/class_help.jsp#copynumber) at a con dence level of 80%.

Statistical analysis
PCoA was a method to visualize the similarity and difference of microbial community composition in different environment samples, and the rst axis of PCoA represented the principal coordinate component that can explain the microbial data changes maximally. Principal coordinates analysis (PCoA) in Fast UniFrac was used to show the general difference of archaeal community structure among samples based on the relative abundances of the entire archaeal community data. Regression analysis was used to determine variations in soil properties, bacterial and archaeal diversity, and relative abundances of the dominant bacterial and archaeal communities along the peatland vertical pro le. Pearson correlation analysis was used to analyze relationships among the soil properties, scores of the rst PCoA axis, and the diversity of bacteria and archaea. All statistical analyses were performed in IBM SPSS Statistics 20 (IBM Armonk, New York, U.S.), and the gures were made by the Origin 9.0. Values were considered signi cant when p < 0.05.

Vertical distribution of soil C, N, and P concentrations and stoichiometry
In this study, soil pH ranged from 5.47 to 5.60 and had no signi cant changes along the soil pro le in the Bitahai peatland. With the exception of C: N (Fig. 1d), soil depths signi cantly in uenced the C, N, and P concentrations as well as the C:P and N:P ratios (p < 0.05). Soil C and N concentrations and C:P and N:P ratios signi cantly increased (Fig. 1a, b, e, f), but P concentration decreased (Fig. 1c) with increasing soil depth.
The relative abundances of the dominant bacteria groups signi cantly changed with soil depth at the phylum and class levels (p < 0.05). As depth increasing, relative abundances of Proteobacteria and Acidobacteria signi cantly decreased, but Spirochaetae, Ignavibacteriae, Chloro exi, and Planctomycetes signi cantly increased ( Figure S1 a, b, c, d, e, f, g). At the class level, the relative abundances of Betaproteobacteria, Alphaproteobacteria, and Acidobacteria signi cantly decreased with the peatland depths, while that of Deltaproteobacteria increased ( Figure S2 a, b, c, d, e). The relative abundances of dominant archaea phyla also signi cantly varied with the soil depths (p < 0.01). As depth increased, the relative abundance of Crenarchaeota signi cantly increased (Figure S3 a), but those of Thaumarchaeota and Euryarchaeota signi cantly decreased (Figure S3 b, c).
Chao1, Ace, and Shannon values were all signi cantly higher in bacteria than in archaea (p < 0.001) (Fig. 3). As increasing soil depths, Chao1 and Ace values of bacteria and archaea show a signi cant increasing trend (Fig. 3a, b), Shannon values of bacteria and archaea possessed a signi cant and nonlinear variation trends (Fig. 3c). There was a signi cant increase in Simpson values of archaea along with soil depths (Fig. 3d).

Relationships between bacterial, archaeal communities and soil properties
The PCoA analysis showed that the rst principal coordinate axis (PCoA1) explained 39.61% and 65.84% of the overall bacterial and archaeal community in our study, respectively. For bacteria community, Pearson correlation analysis showed there was not signi cant relationships between soil properties and PCoA1, and only soil P correlated signi cantly and negatively with Ace index (Table 1). For archaea community, PCoA1 was correlated signi cantly and positively with soil C, N, N:P and C:P, and was signi cantly and negatively with soil P. Both Chao1 and Ace indexes of archaea were correlated signi cantly and positively with soil pH and negatively with C: N, while soil C, N and N:P were correlated signi cantly and positively with archaeal Shannon diversity and negatively with Simpson diversity (Table 2).  Usually, Proteobacteria involved in biogeochemical processes in various ecosystems, and was able to promote soil nutrient availability (Fierer et al. 2007). Speci cally, such sharp decline of Proteobacteria was re ected in the reduction of the relative abundances of Alphaproteobac-teria, Betaproteobacteria and Deltaproteobacteri at the class level. The decrease in Betaproteobacteria could affect N cycles as a result of their members displaying metabolic diversities in nitri cation (Prosser et al. 2014). Sulfate-reducing bacteria belonging to the class of Deltaproteobacteria, had been shown to mineralize organic carbon (Miyatake et al. 2009). Thus, decreasing trend of the relative abundance of Deltaproteobacteria along soil pro les may represent the decrease of organic carbon mineralization ). Moreover, upper soil layers with low organic carbon content had higher relative abundance of Acidobacteria than deeper soil layers. This was similar for some results have shown that the proportion of Acidobacteria in degraded wetland soil with low organic matter content was high (Peralta et al. 2013). In addition, there was a signi cantly increasing trend in abundance of Chloro exi along soil pro les. It has been detected in a wide range of anaerobic habitats, and plays an important role in degrading complex polymeric organic compounds into low molecular weight substrates (Speirs et al. 2019). Previous study also manifested that Chloro exi was survive in nutrient deprived environment (Wu et al. 2021), thus were more abundant under nutrient limitations (Hug et al. 2013). Therefore, the signi cant vertical change of these predominant groups in soil microorganisms meant soil depths would profoundly alter C and N and cycles in the Bitahai peatland.
Furtherly, the functional microbial groups related to pivotal process of C and N cycle obviously were impacted by soil depths (Figs. 4 and 5). Speci cally, the total relative abundance of the methanogenic groups signi cantly decreased with soil depth (Fig. 4), demonstrating would be restrain the methane production of the deeper layers. Methanotrophs play an important role in mitigating the release of the greenhouse gas methane (Liebner and Svenning 2013). Most methanotrophs belong to Proteobacteria, three families Methylococcaceae, Methylocystaceae, and Beijerinckiaceae are methanotrophs (Dworkin et al. 2006;Liebner et al. 2009). The decreasing trends of above three kinds thus may represent the reduction of aerobic methane oxidation with soil depths (Table S1). The ammonia-oxidizing microorganisms included Nitrospirales (bacteria) and Nitrososphaerales (archaea) and their mean relative abundances also signi cantly decreased with soil depth (Fig. 5), which meant a weak ammoniaoxidizing process may exist in deeper soil layers (Prosser et al. 2014). The signi cant vertical decline in relative abundance of methanogenic, methanotrophs and ammonia-oxidizing microorganisms probably suggested the deeper soil layers had a low intensity of methane production, oxidation and nitri cation processes in the Bitahai peatland.
In present study, there was a higher bacterial richness, evenness, and diversity compared with archaea. This result was consistent with studies performed on peatlands (Basiliko et al. 2013), anaerobic sediments of a soda lake (Rojas et al. 2018) and a deep-sea mud volcano (peatlands et al. 2011). Compared with some studies by report from Zoige peatlands (Zhong et al. 2017), both Chao1 and Ace values signi cantly increased with soil depths in the Bitahai peatlands. Our results further showed that Shannon in bacterial and Simpson in archaeal increased while Shannon in archaeal decreased with soil depths. The observed similar and obvious increasing vertical trends of diversity prokaryotic communities suggested prokaryotic diversity also played a pivotal role in peatland functions at deep soil layer. Based on the above results, therefore, our study indeed found soil depth signi cantly in uenced the structure and diversity of bacterial and archaeal in Bitahai peatland, supporting our rst hypothesis.

Relationship between bacterial and archaeal communities and soil properties along soil pro les
Our study clearly shown that various soil depths existed a distinct prokaryotic structure and diversity It was very interesting that bacterial diversity was not related to mostly soil properties except soil P ( had unexpectedly strong impacts on the soil bacterial community composition and variation (Zheng et al. 2020). In the present study, Bitahai peatland is saturated throughout the year, and this makes it very unlikely that soil moisture or water table could be undetected drivers of microbial diversity. In light of the above, despite lacking of more measurement, there was a reason to speculate these un-attended climatic factors perhaps regulate bacterial communities' diversity along the soil pro le in the Bitahai peatland. In order to better understand the distribution of bacterial communities along this soil pro le, it is urgently needed to further explore the drivers of bacterial communities along the soil depths in the Bitahai peatland. Furthermore, only soil P had a negative effect on the Bitahai peatland bacterial richness. This means natural and human activities that cause changes in soil phosphorus could affect bacterial richness in the future (Zheng et al. 2020).
Archaeal community structure was markedly impacted by soil C, N, and P concentrations and stoichiometric ratios along the peatland pro le ( , and thus partly supported our second hypothesis. It follows that the effects of soil C, N and P content and stoichiometry on community diversity were distinct between bacteria and archaea. Namely, soil properties associated with peatland depths have a stronger in uence on community structure and diversity in archaea, not in bacteria. These ndings suggested that above difference should be taken into consideration in the future works and model prediction of peatland. Additionally, dominant taxa abundance and diversity apparently increased in archaea while decreased in bacteria. Therefore, our study also highlighted that archaea community may play an increasingly vital role in biogeochemistry cycling for deeper peatland soil layers.

Conclusions
Soil depth can affect the relative abundance of C and N related microbes. As increasing soil depth, the relative abundance of methanotrophic bacteria, methanogenic groups and the ammonia-oxidizing microorganisms decreased. These higher levels of such functional groups indicated some key processes of soil C and N cycle may be more active in the upper layers. Bacterial richness, evenness, and diversity were higher compared with archaea, while their vertical changing trends were consistent. Across soil pro les, C, N, and P concentrations and their stoichiometric ratios did markedly impact the community structure and diversity for archaeal, but were not for bacteria in the Bitahai peatland. Our results highlighted the microbial community structure and diversity depended on soil depths, and the affecting factors for bacteria and archaea were different in Bitahai peatland.

Declarations
Ethical Approval and Consent to Participate Not applicable. Archaeal communities at the order level were analyzed, and the total relative abundance of the methanogenic groups (4.03-11.54%) signi cantly decreased with soil depth (R2=0.13, p<0.001) ( Figure  4).