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Applied Soil Ecology

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Moss-dominated biocrusts increase soil microbial abundance andcommunity diversity and improve soil fertility in semi-arid climates on theLoess Plateau of China

Bo Xiaoa,b,⁎, Maik Vestec,d

a Department of Soil and Water Sciences, China Agricultural University, Beijing, 100193, Chinab State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences, Yangling,712100, Chinac University of Hohenheim, Institute of Botany (210), Garbenstrasse 30, Stuttgart, 70599, Germanyd Brandenburg University of Technology Cottbus-Senftenberg, Soil Protection and Recultivation, Konrad-Wachsmann-Allee 6, Cottbus, 03046, Germany

A R T I C L E I N F O

Keywords:Biological soil crustMicrobiotic crustMicrobial community compositionMicrobial community diversityRelative abundance of speciesHigh-throughput sequencing

A B S T R A C T

Various ecological functions of biocrusts are mostly determined by their bacterial and fungal abundance andcommunity diversity, which has not yet been fully investigated. To provide more insights into this issue, wecollected samples of moss biocrusts, fixed sand, and mobile sand from a watershed with semi-arid climate on theLoess Plateau of China. The relative abundances and community diversities of soil bacteria and fungi of thesamples were determined using high-throughput DNA sequencing. Finally, we analyzed the characteristics ofbacterial and fungal community of the moss biocrusts and their relationships to the content of soil nutrients. Ourresults showed that the moss biocrusts had 1048 bacterial OTUs (operational taxonomic units) and 58 fungalOTUs, and their Shannon diversity indexes were 5.56 and 1.65, respectively. The bacterial community of themoss biocrusts was dominated by Acidobacteria (24.3%), Proteobacteria (23.8%), Chloroflexi (15.8%), andActinobacteria (14.5%), and their fungal community was dominated by Ascomycota (68.0%) and Basidiomycota(23.8%). The moss biocrusts had far more bacterial OTUs (≥ 56.9%) but similar number of fungal OTUs ascompared with the uncrusted soil, and their Sorenson’s similarity coefficients of bacterial and fungalcommunities were less than 0.768 and 0.596, respectively. Moreover, the contents of soil nutrients (C, N, P)were significantly correlated with the OTU numbers of bacteria and the relative abundances of bacteria andfungi. Our results indicated that moss biocrusts harbor a large number and high diversity of bacteria and fungi,and these diversified bacteria and fungi play important roles in ecosystem functioning through improving soilfertility.

1. Introduction

Biocrusts (also named biological soil crusts) of dry environments areformed by a highly specialized communities of moss and livingmicroorganisms (including soil lichens, green algae, cyanobacteria,fungi, and bacteria) as well by excretion of biopolymers (Belnap et al.,2016; Xiao et al., 2016). According to the dominating components andsuccessional development, biocrusts are usually classified as cyanobac-teria (blue-green algae)-, green algae-, soil lichen-, or moss-dominatedbiocrusts (Belnap et al., 2016). It has been reported that biocrusts arewidespread in arid and semi-arid climates throughout the world withvarying species composition and coverage (Belnap et al., 2003a;Bowker et al., 2016). Thus, they are considered as an importantcomponent of vegetation and land cover in dryland ecosystems

(Porada et al., 2014; Lenhart et al., 2015; Belnap et al., 2016).The dominant components of biocrusts and their small-scale dis-

tribution depend on topography, soil characteristics, climates, plantcommunities, microhabitats, successional stages, and disturbance re-gimes (Kidron et al., 2010; Bowker et al., 2016; Bu et al., 2016), but aremostly determined by the local water regimes which is regulated by soiltexture, microclimatic conditions, and precipitation (Bowker et al.,2016). Generally, biocrusts are dominated by cyanobacteria and soillichens in super-arid and arid climates with less than 250 mm of annualprecipitation (e.g., the Gurbantunggut Desert of China (Zhang et al.,2011)), while they are mostly dominated by mosses in semi-arid climatewith 250–500 mm of annual precipitation (e.g., the Loess Plateau ofChina (Xiao and Hu, 2017)). In the Negev Desert of Israel, cyanobacter-ia and green-algae are characteristic for biocrusts in areas with less than

http://dx.doi.org/10.1016/j.apsoil.2017.05.005Received 20 December 2016; Received in revised form 2 May 2017; Accepted 5 May 2017

⁎ Corresponding author at: Department of Soil and Water Sciences, China Agricultural University, Beijing, 100193, China.E-mail addresses: xiaobo@cau.edu.cn, xiaoboxb@gmail.com (B. Xiao).

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170 mm of annual precipitation (Kidron et al., 2010), while moss coverand thickness increase with increasing annual rainfall along theclimatic gradient (Yair et al., 2011).

Although the negative effects of biocrusts have been reportedseveral times (for example, they smooth the soil surface and preventplant seeds from penetrating the soil (Deines et al., 2007; Su et al.,2007; Langhans et al., 2009)), many studies have confirmed thatbiocrusts mostly perform positive roles in various ecological processessuch as preventing water and wind erosion (Bowker et al., 2008),enhancing soil water retention (Zhang et al., 2008), increasing soil Cand N (Green et al., 2008), facilitating vascular plant establishment andgrowth (Godínez-Alvarez et al., 2012), and promoting soil biodiversity(Castillo-Monroy et al., 2011a). On the other hand, they give stronginfluences on hydrological processes through enhancing or weakeningsoil infiltration and runoff production depending from the speciescomposition (Belnap, 2006; Yair et al., 2011). Particularly, mossbiocrusts attract more attention because they usually generate muchstronger influences on various ecological processes than cyanobacteria,green-algae or soil lichen biocrusts due to their greater biomass(> 10 mg cm−2) and larger thickness (> 15 mm vs. ∼3 mm), espe-cially in stabilizing soil surface and changing soil water regimes (Xiaoet al., 2016; Xiao and Hu, 2017). In general, it is believed that biocrustsare important communities for the soil processes and ecosystemfunctioning (Bowker et al., 2010), and their rehabilitation are impor-tant measures for combating land degradation and desertification (Xiaoet al., 2015).

Through stabilizing the soil surface (Zhang et al., 2006), conservingsoil water (Langhans et al., 2009; Xiao et al., 2016), and accumulatingnutrients (Li et al., 2008), biocrusts create a favorable microhabitat forother soil microorganisms in dry environments. Therefore, they usuallyharbor a large number and high diversity of soil microorganisms ascompared with uncrusted soil (Garcia-Pichel et al., 2003; Steven et al.,2014). These diversified soil microorganisms fundamentally determinethe various important ecological functions of biocrusts (Castillo-Monroyet al., 2011a), but till now we still have no detailed information aboutthem. According to the common theories of biodiversity and ecosystemfunctioning, species diversity gives positive short-term effects onecosystem processes (such as primary productivity and nutrient reten-tion) through functional niche complementarity (the complementarityeffect) and selection of extreme trait values (the selection effect), and itcontributes to the stability and maintenance of ecosystem processes inthe face of perturbations (long-term effects of biodiversity) (Loreau,2000; Loreau et al., 2001; Delgado-Baquerizo et al., 2016). In thiscontext, it is well believed that soil microbial community diversityprovides the cornerstone for support of soil ecosystem services by keyroles in soil organic matter turnover, C sequestration, and even watercycling (Nannipieri et al., 2003; Brussaard et al., 2007). In other words,various soil processes (especially C, N cycling) would possibly benefit alot from the abundant and diversified soil microorganisms inhabitedbiocrusts (Blay et al., 2017). Thus, their characteristics, particularlyabundance and community diversity, are of great importance for abetter understanding the functions of biocrusts in relation to soildevelopment in drylands and other ecosystems (Gundlapally andGarcia-Pichel, 2006; Castillo-Monroy et al., 2011a). However, up tonow only a few studies have been conducted to investigate themicrobial community composition of different types of biocrusts indifferent climate regions around the world (e.g., Soule et al., 2009;Zhang et al., 2011, 2012; Bates et al., 2012). It seems that the microbialcommunity of biocrusts could be affected by many environmentalfactors, but it mostly depends on the development of biocrusts andthe site-specific microclimatic conditions (Moquin et al., 2012). How-ever, most of the research has been restricted to arid climate regions,while the microbial community of biocrusts in semi-arid climate regions(e.g., the Loess Plateau of China) is less known.

Soil fertility (especially C and N) is essential for sustainingcryptogams and vascular plants in terrestrial ecosystems, and it is

particularly important in nutrient-limited dryland ecosystems becauseof its usual low level, susceptibility to depletion, and difficulties ofreplenishment (Ravi et al., 2010). Previous studies confirmed thatbiocrusts play a significant role in N cycling of dryland ecosystems, asthey contribute major N inputs via biological fixation (Zhao et al., 2010;Su et al., 2011) and capture of dust (Williams and Eldridge, 2011) anddepositional N, harbor intense internal N transformation processes (Huet al., 2015; Kidron et al., 2015b), and direct N losses via dissolved,gaseous (Lenhart et al., 2015), and erosional loss processes (Li et al.,2013; Barger et al., 2016). Similarly, soil C cycling is also significantlychanged by biocrusts through photosynthetic activity (Hui et al., 2014;Kidron et al., 2015a) and soil respiration (Castillo-Monroy et al., 2011b;Yu et al., 2014). On the other side, the soil microorganisms in biocrustsaccelerate the decomposition of organic matter, mainly due to theincreasing soil enzyme activities (e.g., urease, alkaline phosphatase,invertase, and protease) (Zhang et al., 2012; Liu et al., 2014). In otherwords, the mosses and other cryptogams (i.e., lichens and green-algae)in biocrusts are mainly responsible for the effects on soil formation andwater conservation (soil physical processes) through their functions instabilizing soil surface and holding soil water (Kidron and Tal, 2012;Xiao et al., 2016). Their roles in C and N cycling and improving soilfertility (soil chemical and biological processes) are mostly attributed tothe cyanobacteria, green-algae, bacteria, and fungi through theirphotosynthesis, nitrogen fixation, and effects on soil enzyme activities(Belnap et al., 2003b). It is well known that both soil bacteria and fungiare responsible for important processes (Paul, 2015) even in biocrusts(Maier et al., 2014; Steven et al., 2014; Mueller et al., 2015). For thesereasons, the bacterial and fungal abundance and community diversityof biocrusts are of very high concern owing to their significant roles inmaintaining and improving soil fertility, which are crucial for therestoration of degraded lands and vegetation in arid and semi-aridclimates (Abed et al., 2013; Steven et al., 2014; Zhang et al., 2014;Mueller et al., 2015). In addition, the bacterial and fungal communitydiversity of biocrusts are usually used to identify the successional stagesof biocrusts in natural development or restoration processes (Lan et al.,2013; Steven et al., 2015), and to evaluate their responses to dis-turbance or climate change (Ferrenberg et al., 2015; Mueller et al.,2015).

The Loess Plateau in China covers an area of 640,000 km2 and hasthe world's highest soil erosion rates (including water erosion insummer as well as wind erosion in winter and early spring) acrossthe world (Xin et al., 2008). This is due to the fact that vascular plantsare heavily degraded and cover less than 5% in some areas owing toclimate changes and human activities (e.g., agricultural overexploita-tion) (Wang et al., 2008; Xin et al., 2008). In order to combat landdegradation and desertification, the Grain for Green Project has beenimplemented to restore vegetation in recent decades (Cao et al., 2009).During the project, the agricultural activities have ceased over a largearea and a large number of native shrubs have been artificially plantedto conserve soil and water (Cao et al., 2009). Owing to stabilization ofland surface resulted from the decreasing disturbances of agriculturalactivities and increasing protection of the artificially planted shrubs,biocrusts spontaneously recolonized the soil surface and graduallydeveloped from cyanobacteria to mosses (or directly developed frombare land to moss biocrusts in the regions with abundant precipitation)over several years (Zhao et al., 2014a; Bu et al., 2016). Nowadays, mossbiocrusts are extensively developed and are widely distributed with acoverage reaching 70% in most areas of semi-arid climates on the LoessPlateau (Wang et al., 2016; Xiao et al., 2016). These moss biocrusts areable to store water and stabilize the soil surface and are an importantcontribution to combat desertification, which are serious problems inthis region (Xiao et al., 2014, 2015). Moss biocrusts have beenintensively investigated on the Loess Plateau of China, especially forsoil water cycling (Xiao et al., 2010, 2011b, 2016; Wei et al., 2014), soilfertility (Zhao et al., 2010, 2014b), and wind and water erosion (Wanget al., 2013; Zhao and Xu, 2013; Zhao et al., 2014a). However, their

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microbial community composition has not yet been fully investigated,which is important to understand the functions of biocrusts forecosystems functioning.

In this study, we hypothesized that the moss biocrusts on the LoessPlateau of China harbor a large number and high diversity of bacteriaand fungi as compared with uncrusted soil, and these diversifiedbacteria and fungi possibly play important roles in improving soilfertility including soil C, N, P. Based on these hypotheses, we collectedsamples of moss biocrusts, fixed sand, and mobile sand from arepresentative semi-arid climate on the Loess Plateau of China.Afterwards, the relative abundance and diversity of bacterial andfungal communities of these samples, at both phylum and genus levels,were determined using high-throughput DNA sequencing technique inlaboratory. We also measured the characteristics of biocrusts, thecontent of soil nutrients, and microbial densities of the samples byconventional methods. The analysis of soil nutrient content allowed usto explore relationships between the composition of bacterial/fungalcommunity of biocrusts and soil fertility. The objective of this study wasto determine the bacterial and fungal community diversity of mossbiocrusts and its implications for soil fertility on the Loess Plateau ofChina. The results will provide a better understanding of the microbialcomposition and corresponding ecological functions of moss biocrustsin semi-arid climates on the Loess Plateau of China, and comparableecoregions of the world.

2. Materials and methods

2.1. Study area

The study area located at the Liudaogou watershed(38°46′–38°51′ N and 110°21′–110°23′ E; an area of 6.89 km2 withelevation of 1081–1274 m a.s.l.) on the northern Loess Plateau ofChina. The mean annual precipitation is 409 mm (with ∼80% occur-ring during summer) and potential evaporation is 1337 mm, respec-tively (Xiao et al., 2011a). The mean annual temperature is 8.4 °C andthe mean monthly temperature ranges from −9.7 °C in winter (Dec.–-Feb.) to 23.7 °C in summer (Jun.–Aug.) (Cha and Tang, 2000). Due tothe serious degradation of natural vegetation as a result of inappropri-ate land use practices in combination with high rainfall intensities andcomplex landforms, the region is highly affected by severe soil loss. As aresult of water erosion in summer as well as wind erosion in winter andearly spring, the observed soil erosion rate varies between 15,000 and20,000 t km−2 a−1 (Cha and Tang, 2000).

For restoration of the landscape, native shrubs including Artemisiaordosica Krasch. (Asteraceae) and Caragana korshinskii Kom.(Leguminosae) were planted in the watershed about 30 years ago(Xiao and Hu, 2017). Currently, the planted shrubs are distributed inpatches and cover 20–30% of the watershed. Owing the restriction oflimited precipitation (409 mm per year in average) and soil moisture,the artificially planted shrubs are finally distributed in sparse patchesafter about 30 years (Xiao and Hu, 2017). In the study area, mossbiocrusts are extensively developed (they were initially recordedaround 30 years ago) on fallow lands, shrub lands, and grasslandsand now cover approximately 70–80% of the soil surface (Xiao et al.,2010).

2.2. Experimental design and sample collection

In the watershed, we selected four sampling sites (see Fig. 1a) withrepresentative moss biocrusts and very sparse shrub lands, composed ofartificially planted A. ordosica Krasch., C. korshinskii Kom., and Salixpsammophila C. Wang et Chang Y. Yang (Salicaceae). The soil on thesampling sites was an aeolian sandy soil (entisols in USDA soiltaxonomy or arenosols in FAO soil classification), and its texture wasloamy sand (USDA) with 81% sand, 14% silt, and 5% clay (Xiao et al.,2016). It’s field capacity, wilting point, and steady-state infiltration rate

were 12.6%, 0.8%, and 10.5 cm h−1, respectively (Xiao et al., 2016).Although the soil on the Loess Plateau is dominated by loess soil(ustochnept in USDA soil taxonomy or cambisols in FAO soil classifica-tion) with clayey or loamy texture, the aeolian sandy soil covers an areaof 79,200 km2 and accounts for 12.2% of the total area (Yamamoto andEndo, 2014). Especially in the northern Loess Plateau along the GreatWall, the aeolian sandy soil covers up to 64.6% of the area and the loesssoil covers 15.2% only (Yamamoto and Endo, 2014).

In each sampling site, the top 20 mm of the fixed sand with mossbiocrusts (moss biocrusts which was ∼30 years old with> 95% mosscoverage and naturally developed on fixed sand after the plantation ofartificially planted shrubs; see Fig. 1b), fixed sand without biocrusts(hereafter fixed sand which was constituted of abundant stabilized sandparticles, small pieces of organic materials (possibly the litters ofsurrounding vascular plants), and few moss stems (< 5%); seeFig. 1c), and mobile sand (aeolian sand with frequent disturbances ofthe surface; see Fig. 1d) were randomly sampled from 12 samplingpoints by petri dishes (90 mm diameter × 20 mm height) on Sep. 23,2014. The sub-samples collected from the 12 sampling points at eachsampling site were mixed together for each treatment, and finally therewere 12 samples in total (3 treatments × 4 sampling sites). The threetreatments were sampled in nearby locations with similar soil texture ineach sampling site, and they were mostly resulted by the differentdegree of protections from shrubs rather than their locations. In otherwords, we set three treatments and each treatment had four replicatesin this study. The samples were homogenized and sieved (< 2 mm) toremove any root material or pebbles. They were packed on dry ice andstored at −20 °C until processing.

2.3. Characterization of moss biocrusts and soil nutrients

The biocrust thickness was measured through a digital caliper (CD-6′’-ASX, Mitutoyo, Japan). Moss species in the biocrusts were visuallyidentified (e.g., shape of stems and leaves) and analyzed for size, color,and habitats; moss density was calculated from the total moss game-tophytes in a 20-mm square sample; and moss plants were washed outwith a 2-mm screen and dried at 65 °C for 24 h before measuring theirbiomass. The total chlorophyll content was also recorded throughlaboratory analysis with a UV–vis spectrophotometer (DR 5000,Hach, USA) to indicate the cryptogam biomass of biocrusts.Moreover, the bacterial, fungal, and actinobacterial densities weremeasured by the plate counts as described by Schinner et al. (1995).Additionally, the content of soil nutrients, including organic matter,total N and P (that reflect the biocrust establishment as well as biocrustbiomass), available N and P (that can explain the biocrust establish-ment), and microbial C and N, were measured according to Carter andGregorich (2006). All above measurements were conducted in at leastfour replicates.

2.4. DNA extraction, PCR amplification, cloning, and sequencing

Total soil DNA was extracted from duplicate 0.5 g subsamples(freeze-dried soil) from each sample using the E.Z.N.A.® soil DNA Kit(Omega Bio-tek, Norcross, GA, USA) according to manufacturer’sprotocols. Following extraction, the DNA samples were pooled.

PCR amplification, cloning, and sequencing of bacterial and fungalrRNA genes were performed by the Majorbio Company in Shanghai,China according to Amato et al. (2013). Briefly, bacterial 16S rRNAgene fragments were amplified (95 °C for 2 min, followed by 25 cyclesat 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extensionat 72 °C for 5 min) in triplicate from total soil DNA using primers 338Fand 806R; and fungal 18S rRNA gene fragments were amplified (assame as the bacterial 16S rRNA) using primers 817F and 1196R.Amplicons were subjected to electrophoresis with 2% agarose gels;bands were extracted, dissolved, and purified using the AxyPrep DNAGel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according

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to the manufacturer’s instructions and quantified using QuantiFluor™−ST (Promega, USA). Purified amplicons were pooled in equimolarand paired-end sequenced (2 × 250) on an Illumina MiSeq platform(PE250/PE300) according to the standard protocols.

2.5. Data analysis

Raw fastq files were demultiplexed and quality-filtered using QIIME1.90 with the following criteria. (1) The 300 bp reads were truncated atany site receiving an average quality score< 20 over a 50 bp slidingwindow, discarding the truncated reads that were shorter than 50 bp.(2) Exact barcode matching was performed, only two nucleotidemismatches in primers were allowed, and any reads containingambiguous characters were removed. (3) Only sequences with overlapslonger than 10 bp were assembled based on their overlapping sequence.Reads which could not be assembled were discarded. Operationaltaxonomic units (OTUs) were clustered with a 97% identity cutoffusing UPARSE 7.1, and chimeric sequences were identified andremoved using UCHIME. The taxonomy of each 16S rRNA and 18SrRNA gene sequence was analyzed by RDP Classifier against the SILVA(SSU115) 16S rRNA and 18S rRNA databases, respectively, using aconfidence threshold of 70%. The total number of bacterial and fungalOTUs of each sample were estimated from the rarefaction curvesthrough extrapolation.

According to the experimental design, we had three treatments andeach treatment had four replicates (the fours sampling sites wereregarded as replicates). The experimental data were analyzed basedon the descriptive statistics in SPSS Statistics 22. The final results of

each treatment were expressed as means of the replicates and expressedas the mean ± standard error. The number of OTUs and relativeabundance (percentage of OTUs) of each phylum and genus were usedto represent the bacterial/fungal community composition. The simila-rities of bacterial/fungal community of moss biocrusts vs. fixed sandand moss biocrusts vs. mobile sand were determined by the Sorenson’ssimilarity coefficient (SC) (Osem et al., 2006).

SC c a b= 2 /( + ) (1)

In this equation, a and b represent the numbers of OTUs in the twosamples, respectively, and c represents the numbers of same OTUs ofthe two samples. The differences of similarity indexes among thesamples, including Sorenson’s similarity coefficient, were determinedby the NPAR1WAY in SAS 8.01. The differences among the sampleswere statistically evaluated at the 5% probability level by the paired-samples t-test or one-way ANOVA in SAS 8.01. According to ourexperimental design, the paired-samples t-test was mainly used toevaluate the differences between the paired treatments (i.e., mossbiocrusts vs. fixed sand, moss biocrusts vs. mobile sand) to reduce oreliminate the site effects (e.g., the possible differences in soil properties,geomorphology, micro-climate, and surrounding environments amongthe four sampling sites) in this study. The correlation and regressionanalysis were further conducted to offer more insights into the relation-ships between the relative abundance/community diversity of bacter-ial/fungal communities and the content of soil nutrients. The repre-sentation and graphical fits of experimental data were obtained usingOriginPro 9.2.

Fig. 1. Photos of (a) a representative sampling site with sparse artificially planted shrub-patches and (b) fixed sand with moss biocrusts (∼30-year-old), (c) fixed sand without biocrusts,(d) mobile sand during sampling on the Loess Plateau of China.

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3. Results

3.1. Characteristics of moss biocrusts

We identified seven moss species from the moss biocrusts, includingBryum argenteum Hedw., B. arcticum (R. Brown) B.S.G., B. caepiticiumHedw., Didymodon vinealis (Brid.) Zander, D. nigrescens (Mitt.) Saito,Barbula vinealis Brid., and B. perobtusa (Broth.) Chen. Dominant mossspecies were only B. arcticum (R. Brown) B.S.G. and D. vinealis (Brid.)Zander. The moss biocrusts almost completely (> 95%) covered theland surface in the open interspaces between the sparse artificiallyplanted shrubs. The moss thickness, density, and biomass were18.95 mm, 55.9 gametophyte cm−2, and 152.99 g m−2, respectively.The total chlorophyll content of the moss biocrusts ranged from 0.49 to0.71 mg g−1 (dry moss), with an average of 0.58 ± 0.03 mg g−1.

3.2. Effects of moss biocrusts on soil nutrients and microbial densities

As listed in Table 1, the moss biocrusts had 4.98 and 11.11 timeshigher of organic matter content as compared with the fixed sand andmobile sand. The content of total N, available N, and available P of themoss biocrusts were 2.89, 7.61, and 5.24 times (F≥ 4.64, P≤ 0.032)as high as that of the fixed sand, while they were 5.20, 10.38, and 7.96times as high as that of the mobile sand. The moss biocrusts also had3.03 and 4.09 times higher of microbial C and N as compared with thefixed sand, and they had 5.23 and 6.03 times higher of microbial C andN as compared with the mobile sand. Moreover, the bacterial, fungal,and actinobacterial densities of the moss biocrusts were 2.29, 2.76, and1.80 times (F ≥ 6.06, P ≤ 0.015) as high as that of the fixed sand; andthey were 4.41, 6.14, and 3.48 times as high as that of the mobile sand.Furthermore, the densities of bacteria, fungi, and actinobacteria werelinearly (R2 ≥ 0.954, P≤ 0.097) and positively correlated with eachother.

3.3. Effects of moss biocrusts on soil bacterial community

The moss biocrusts had 56.9% higher OTUs and 7.4% higherShannon diversity index in bacterial community as compared withthe fixed sand (Table 2). Similarly, they had 142% higher OTUs and62.9% higher Shannon index in bacterial community as compared withthe mobile sand (Table 2). The moss biocrusts shared 842 (accountingfor 62.3%) bacterial OTUs with the fixed sand and 409 (accounting for30.6%) bacterial OTUs with the mobile sand. Namely, 64.1% of thebacterial OTUs in the moss biocrusts were same with the fixed sand,while only 31.2% of the bacterial OTUs in the moss biocrusts were samewith the mobile sand. The Sorenson’s similarity coefficient of bacterialcommunity of the moss biocrusts vs. fixed sand and moss biocrusts vs.mobile sand were 0.768 and 0.468, respectively.

The bacterial community of all three treatments was mainly

composed of Proteobacteria, Chloroflexi, Actinobacteria,Acidobacteria, Bacteroidetes, Cyanobacteria, and Gemmatimonadetes(Fig. 2a). However, the moss biocrusts consistently had 97, 69, 50, 24,and 23 more OTUs than the fixed sand in Proteobacteria, Chloroflexi,Bacteroidetes, Cyanobacteria, and Actinobacteria, respectively. Corre-spondingly, they consistently had 165, 106, 73, 43, 35, and 28 moreOTUs than the mobile sand in Proteobacteria, Chloroflexi, Bacteroi-detes, Cyanobacteria, Actinobacteria, and Gemmatimonadetes, respec-tively. At genus level (Fig. 2b), all the three treatments had fewer than31 OTUs in each bacterial genus. The most common genera wereRoseiflexus, Bryobacter, Haliangium, Leptolyngbya, uncultured Anaeroli-neaceae, and unclassified Rhizobiales.

According to the relative abundance of fungal phyla (Fig. 2c), thebacterial community of the moss biocrusts was dominated by Acid-obacteria (24.3%), Proteobacteria (23.8%), Chloroflexi (15.8%), andActinobacteria (14.5%). However, the bacterial community of the fixedsand was dominated by Actinobacteria, Proteobacteria, and Acidobac-teria; and that of the mobile sand was dominated by Actinobacteria,Cyanobacteria, and Proteobacteria. The moss biocrusts had lowerrelative abundance of Actinobacteria (17.2%; t= 6.72, P = 0.001) ascompared with the fixed sand. Similarly, they had lower relativeabundance of Actinobacteria (33.0%; t= 21.76, P< 0.001) andCyanobacteria (14.8%; t = 11.80, P< 0.001) but higher relativeabundance of Acidobacteria (21.9%; t= 18.45, P< 0.001) and Chlor-oflexi (11.3%; t= 10.64, P< 0.001) as compared with the mobilesand. At the genus level (Fig. 2d), the bacterial community of both themoss biocrusts and fixed sand were dominated by RB41; and that of themobile sand was dominated by Arthrobacter and Microcoleus. At thegenus level, there was almost no difference (≤ 5.6%) in the relativeabundance of bacterial community between the moss biocrusts andfixed sand. However, the moss biocrusts had lower relative abundanceof Arthrobacter (31.2%; t= 71.94, P< 0.001) and Microcoleus (21.2%;t= 51.68, P< 0.001) but higher relative abundance of RB41 (14.4%;t= 7.12, P = 0.001) and uncultured Anaerolineaceae (7.4%; t = 6.46,P = 0.001) as compared with the mobile sand.

3.4. Effects of moss biocrusts on soil fungal community

As listed in Table 2, the moss biocrusts had similar number of OTUs,17.9% higher Shannon index, and 19.5% higher Simpson index infungal community as compared with the fixed sand. Correspondingly,they had 26.1% higher OTUs, 42.2% higher Shannon index, and 64.3%higher Simpson index in fungal community as compared with themobile sand (F ≥ 23.11, P< 0.001). The moss biocrusts and fixed sandhad 34 (accounting for 42.5%) fungal OTUs in common; and the mossbiocrusts and mobile sand had 29 (accounting for 39.7%) fungal OTUsin common. Namely, 58.6% of the fungal OTUs in the moss biocrustswere same with the fixed sand; and 50.0% of the fungal OTUs in themoss biocrusts were same with the mobile sand. The Sorenson’s

Table 1General characteristics of the moss biocrusts, fixed sand, and mobile sand measured through conventional methods.

Measurementsa Moss biocrusts Fixed sand Mobile sand F value P value

Organic matter content (%) 1.460 ± 0.114 ab 0.293 ± 0.046 b 0.131 ± 0.014 c 102.61 < 0.001Total N content (%) 0.130 ± 0.026 a 0.045 ± 0.004 b 0.025 ± 0.005 b 12.65 0.001Total P content (%) 0.050 ± 0.002 a 0.048 ± 0.002 a 0.044 ± 0.002 a 2.08 0.167Available N content (mg kg−1) 114.82 ± 9.23 a 15.08 ± 1.19 b 11.06 ± 1.53 b 16.95 < 0.001Available P content (mg kg−1) 4.14 ± 0.78 a 0.79 ± 0.25 b 0.52 ± 0.08 b 11.56 0.002Microbial C content (mg kg−1) 303.90 ± 18.33 a 100.20 ± 19.75 b 58.11 ± 17.71 b 4.64 0.032Microbial N content (mg kg−1) 23.58 ± 6.95 a 5.76 ± 0.95 b 3.91 ± 1.26 b 7.78 0.007Bacterial density ( × 105 CFU g−1) 4.63 ± 0.90 a 2.02 ± 0.42 b 1.05 ± 0.10 b 6.06 0.015Fungal density ( × 102 CFU g−1) 7.24 ± 0.75 a 2.62 ± 0.60 b 1.18 ± 0.08 b 42.72 < 0.001Actinobacterial density ( × 104 CFU g−1) 3.62 ± 0.48 a 2.01 ± 0.16 b 1.04 ± 0.13 c 30.95 < 0.001

a The thickness of the moss biocrusts and uncrusted soil were equally 20 mm because all the samples were taken from top 20 mm soil by petri dishes (90 mm diameter × 20 mmheight).

b Values with in a row followed by the same letter are not significantly different at P ≤ 0.05.

B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177

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similarity coefficient of fungal community of the moss biocrusts vs.fixed sand and moss biocrusts vs. mobile sand were 0.596 and 0.558,respectively.

The fungal community of all the three treatments was mainlycomposed of Ascomycota and Basidiomycota (Fig. 3a). The mossbiocrusts had very similar number of OTUs with the fixed sand, whilethey had 5 and 6 more OTUs in Ascomycota and Basidiomycota,respectively, as compared with the mobile sand. All the three treat-ments had fewer than 6 OTUs in each fungal genus (Fig. 3b), and theirdifferences in each fungal genus were fewer than 2 OTUs. The mostcommon genera were unclassified Ascomycota, Dothideomycetes, andPleosporales.

As shown by relative abundance in Fig. 3c, the fungal community ofthe moss biocrusts, fixed sand, and mobile sand at the phylum level wasconsistently dominated by Ascomycota and Basidiomycota. The mossbiocrusts had very similar relative abundance of Ascomycota and

Basidiomycota with the fixed sand. However, they had lower relativeabundance of Ascomycota (6.8%; t= 7.56, P = 0.001) but higherrelative abundance of Basidiomycota (7.3%; t = 6.48, P = 0.001) ascompared with the mobile sand. At the genus level (Fig. 3d), the mossbiocrusts had a slightly lower (1.4%) relative abundance of Acrosper-mum and unclassified Saccharomycetales but a higher (≤ 2.5%)relative abundance of unclassified Ascomycota, Dothideomycetes,Pleosporales, and Lichinaceae as compared with the fixed sand. Ascompared with the mobile sand, they had a lower (2.1%) relativeabundance of unclassified Ascomycota and a higher (2.1%) relativeabundance of unclassified Lichinaceae.

3.5. Relationships between bacterial/fungal community and soil nutrients

The Pearson’s correlation coefficients between the content of soilnutrients and the number of bacterial OTUs were mostly significant

Table 2Estimated parameters of bacterial and fungal community of the moss biocrusts, fixed sand, and mobile sand using high-throughput DNA sequencing technique.

Treatments OTUsa Ace Chao Coverage Shannon Simpson

Bacterial communityMoss biocrusts 1048 ± 8 ab 1155 ± 11 a 1157 ± 13 a 0.990 ± 0.001 a 5.56 ± 0.07 a 0.011 ± 0.001 aFixed sand 668 ± 21 b 867 ± 8 b 859 ± 11 b 0.964 ± 0.009 b 5.18 ± 0.01 b 0.015 ± 0.001 aMobile sand 433 ± 22 c 559 ± 28 c 557 ± 28 c 0.987 ± 0.001 a 3.41 ± 0.05 c 0.016 ± 0.001 aFungal communityMoss biocrusts 58 ± 3 a 62 ± 3 a 61 ± 2 a 1.000 ± 0.001 a 1.65 ± 0.03 a 0.092 ± 0.001 aFixed sand 56 ± 1 a 58 ± 2 b 57 ± 1 b 1.000 ± 0.001 a 1.40 ± 0.02 b 0.077 ± 0.001 aMobile sand 46 ± 1 b 55 ± 2 b 56 ± 3 b 1.000 ± 0.001 a 1.16 ± 0.02 c 0.056 ± 0.001 a

a Total number of OTUs estimated from the rarefaction curves through extrapolation.b Values with in a row followed by the same letter are not significantly different at P ≤ 0.05.

Fig. 2. Bacterial OTUs (average value of the replicates) of moss biocrusts, fixed sand, and mobile sand. (a) Number of bacterial OTUs at the phylum level; (b) Number of bacterial OTUs atthe genus level; (c) Relative abundance of bacterial OTUs at the phylum level; (d) Relative abundance of bacterial OTUs at the genus level.

B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177

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(r ≥ 0.589), except for total P (Table 3). In contrast to that, thePearson’s correlation coefficients between the content of soil nutrientsand the number of fungal OTUs were insignificant. The results showedthat the content of soil nutrients was significantly and positivelycorrelated with the number of bacterial OTUs, but they were notcorrelated with the number of fungal OTUs.

As listed in Table 4, the content of soil nutrients were positivelycorrelated with the relative abundance of Proteobacteria, Chloroflexi,Acidobacteria, Bacteroidetes, and Gemmatimonadetes. However, theywere negatively correlated with the relative abundance of Actinobac-teria and Cyanobacteria. Similarly, they were positively correlated withthe relative abundance of Basidiomycota but were negatively correlatedwith the relative abundance of Ascomycota. Except for total P, thePearson’s correlation coefficients were significant (r ≥ 0.632) between

the content of soil nutrients and bacterial OTUs in phyla of Chloroflexi,Actinobacteria, and Bacteroidetes. These Pearson’s correlation coeffi-cients were also significant (r ≥ 0.748) between the content of soilnutrients and fungal OTUs in phyla of Ascomycota and Basidiomycota.

4. Discussion

4.1. Characteristics of bacterial/fungal community in biocrusts

Our study showed that the moss biocrusts in semi-arid climate onthe Loess Plateau of China had the highest diversity with 1048 bacterialOTUs and 58 fungal OTUs. Their bacterial community was dominatedby Acidobacteria, Proteobacteria, Chloroflexi, and Actinobacteria; andtheir fungal community was dominated by Ascomycota and

Fig. 3. Fungal OTUs (average value of the replicates) of moss biocrusts, fixed sand, and mobile sand. (a) Number of fungal OTUs at the phylum level; (b) Number of fungal OTUs at thegenus level; (c) Relative abundance of fungal OTUs at the phylum level; (d) Relative abundance of fungal OTUs at the genus level.

Table 3Pearson's correlation coefficients between the OTU numbers of soil bacteria/fungi and the content of soil nutrients.

Number of OTUs Organic matter Total N Total P Available N Available P Microbial C Microbial N

Bacterial community Proteobacteria 0.920** 0.924** 0.198 0.938** 0.850** 0.713* 0.782*

Chloroflexi 0.906** 0.898** 0.131 0.906** 0.835** 0.709* 0.786*

Actinobacteria 0.864** 0.837** 0.222 0.858** 0.761* 0.706 0.788*

Acidobacteria 0.865** 0.917** 0.134 0.853** 0.854** 0.634 0.705Bacteroidetes 0.897** 0.891** 0.232 0.932** 0.875** 0.640 0.736*

Cyanobacteria 0.873** 0.885** 0.086 0.866** 0.822* 0.679 0.745*

Gemmatimonadetes 0.810* 0.827* 0.143 0.846** 0.805* 0.589 0.644Fungal community Ascomycota 0.579 0.572 0.555 0.536 0.456 0.447 0.585

Basidiomycota 0.352 0.258 0.017 0.386 0.509 0.013 0.200

* Significant at the 0.05 probability level (two tailed).** Significant at the 0.01 probability level (two tailed).

B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177

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Basidiomycota. Over the past decade, a number of studies have beenconducted to investigate the microbial community diversity of differenttypes of biocrusts in different drylands, including the Colorado Plateau(Redfield et al., 2002; Gundlapally and Garcia-Pichel, 2006), GreatBasin (Soule et al., 2009), Chihuahuan Desert (Soule et al., 2009; Bateset al., 2012), Sonoran Desert (Soule et al., 2009; Bates et al., 2012),Mojave Desert (Steven et al., 2014; Mueller et al., 2015), and coldsteppe ecosystems in southwestern Idaho (Blay et al., 2017) in USA; theTengger Desert (Zhang et al., 2012; Grishkan et al., 2015), Gurban-tunggut Desert (Zhang et al., 2009, 2011), Mu Us Desert (Zhao et al.,2011), and Horqin Sandy Land (Zhang et al., 2014) in China; theTabernas Desert (Maier et al., 2014) and Sax (Maestre et al., 2015) inSpain; the Negev Desert in Israel (Zaady et al., 2010); and theSantiniketan (Kumar and Adhikary, 2015) in India. Although differentmethods (conventional method or PCR-based DNA-sequencing) wereemployed in these studies, the comparative results (Table 5) indicatethat the bacterial/fungal community are greatly different in varioustypes of biocrusts under different climatic conditions (represented byannual precipitation). For example, Moquin et al. (2012) found that thebacterial communities of moss biocrusts greatly differed from thatreported for cyanobacterial crusts. Grishkan et al. (2015) also believedthat the differences in fungal community of biocrusts between theTengger and Negev deserts were caused by the principal differences intheir precipitation regimes, associated with different rainy seasons withwinter rainfall in the Negev and summer rainfall in the Tengger. Thus,according to the different bacterial/fungal community of biocrustslisted in Table 5, we conclude that the bacterial/fungal community ofbiocrusts are closely related to the climate characteristics at least.

Besides the effects of biocrusts, it has been reported that the soilmicrobial community is very sensitive and could be significantlyaffected by various factors. For example, both Zhang et al. (2012)and Meadow and Zabinski (2012) reported that the composition of soilmicrobial community had high spatial heterogeneity in deserts anddrylands. Moreover, Steven et al. (2013) found that soil microbialcommunities displayed spatial biogeographic patterns associated soilparent material. Additionally, Kuske et al. (2012) and Ferrenberg et al.(2015) affirmed that climate changes, such as the increase of tempera-ture and precipitation, strongly influenced soil microbial communitycomposition. Lastly, soil microbial community was also greatly changedby N addition (Mueller et al., 2015) and physical disturbances fromtrampling (Kuske et al., 2012; Ferrenberg et al., 2015). In a word, soilbacterial/fungal community is originally different across differentclimate regions. However, the presence of biocrusts further intensifiestheir differences and make them become more significant.

In addition, the determination of microbial community diversity inbiocrusts by conventional techniques requires specialized skills, is timeconsuming, and is very poor in repeatability and accuracy (Hill et al.,2000; Redfield et al., 2002). Compared with conventional techniques,DNA-based techniques are capable of providing a more comprehensiveand accurate measure of microbial community diversity in biocrusts,

where soil microbial communities are extreme diverse and maybecontain abundant non-cultured representatives of novel divisions(Kuske et al., 2002; Ansorge, 2009; Steven et al., 2014). In this study,we recorded many OTUs in unclassified phyla and genera as presentedin Figs. 2–3. These OTUs are likely new taxa and have not yet beenreported before (possibly also because that there are generally anumber of OTUs who could not be assigned to certain taxa based onlyon a partial sequence of 16S/18S rRNA gene, especially for fungi).However, it has been reported that significant errors possibly exist inthe investigation of soil microbial community with high-throughputDNA sequencing technique, because that the different communities ofsoil microorganisms maybe have different responses to the conditionsof DNA extraction and PCR amplification (Krsek and Wellington, 1999;Martin-Laurent et al., 2001). Thus, in our opinion, the combination ofconventional techniques and high-throughput DNA sequencing techni-que should be used in the determination of microbial community ofbiocrusts in future.

4.2. Differences of bacterial/fungal community between biocrusts anduncrusted soil

This study showed that the moss biocrusts had very differentbacterial and fungal community diversity with the fixed sand andmobile sand. In dryland ecosystems, particularly desert ecosystem, thesurvival of microorganism in surface soil is greatly restricted by thelong-term extreme low moisture (Pointing and Belnap, 2012), hightemperature (up to 80 °C) (Johnson et al., 2012), lack of C and N(Pointing and Belnap, 2012), and strong solar radiation (Belnap et al.,2008). These microorganisms are also very sensitive to the movementof surface soil particles and sand burial caused by strong wind blowing.However, it has been reported that moss biocrusts could significantlyincrease soil moisture by up to 7.6% at upper 5 cm depth in semi-aridclimates on the Loess Plateau of China (Xiao et al., 2016) (sometimesthey deteriorate deep soil water conditions in hyper-arid regions (Liet al., 2004)). They could also decrease soil surface temperature by upto 11.8 °C under wet and hot conditions in summer, and significantlyincrease soil surface temperature by up to 8.0 °C under dry and coldconditions in winter (Xiao et al., 2013, 2016) (the increasing effectsfrom cyanobacteria or lichen biocrusts on soil temperature were alsoconfirmed by a few studies (George et al., 2003; Kidron and Tal, 2012)).Moreover, moss biocrusts could completely stabilize surface soilparticles and protect soil microorganisms from the destroy of sandmovement and burial (Jia et al., 2012; Tisdall et al., 2012). Addition-ally, moss biocrusts could significantly improve soil fertility throughfixing atmospheric C and N (Zhao et al., 2010; Kidron et al., 2015a),producing more extracellular polymeric substances (Mager andThomas, 2011; Chen et al., 2014), and accumulating soil nutrientsagainst runoff and erosion (Barger et al., 2006; Li et al., 2013). Forthese causes, we reasonably attribute the high relative abundance andcommunity diversity of bacteria/fungi in the moss biocrusts to the

Table 4Pearson's correlation coefficients between the relative abundance of soil bacteria/fungi and the content of soil nutrients.

Relative abundance Organic matter Total N Total P Available N Available P Microbial C Microbial N

Bacterial community Proteobacteria 0.522 0.539 0.472 0.563 0.502 0.392 0.455Chloroflexi 0.850** 0.886** 0.175 0.838** 0.839** 0.632 0.716*

Actinobacteria −0.875** −0.909* −0.102 −0.873* −0.846* −0.665 −0.728*

Acidobacteria 0.686 0.758* 0.173 0.667 0.610 0.612 0.611Bacteroidetes 0.902** 0.929** 0.452 0.842* 0.649 0.861* 0.919**

Cyanobacteria −0.510 −0.575 −0.547 −0.501 −0.375 −0.534 −0.533Gemmatimonadetes 0.723* 0.790* 0.253 0.682 0.583 0.697 0.697

Fungal community Ascomycota −0.883** −0.926** −0.209 –0.883** −0.882** −0.871** −0.898**

Basidiomycota 0.793* 0.838** 0.114 0.794* 0.768* 0.748* 0.769*

* Significant at the 0.05 probability level (two tailed).** Significant at the 0.01 probability level (two tailed).

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Table5

Reg

iona

ldifferen

cesof

theba

cterialan

dfung

alco

mmun

ityof

bioc

rustsin

differen

tdrylan

dsof

North

America,

China

,MiddleEa

st,a

ndSo

uthe

rnEu

rope

.

Reg

ions

Ann

ualprecipitation

(mm)

Bioc

rust

type

sBa

cterialco

mmun

ity

Fung

alco

mmun

ity

Sono

ranDesert,USA

75–2

55Lich

enSh

anno

ninde

x=

2.03

–3.02;

dominated

byCya

noba

cteria

(54.8%

),Actinob

acteria(15.1%

),Proteo

bacteria

(13.8%

),an

dAcido

bacteria

(11.1%

)(N

agyet

al.,20

05)

Shan

noninde

x=

1.67

–2.22;

had5–

19speciesin

10classes,15

orde

rs,a

nd3ph

yla;

dominated

byAscom

ycota(82%

),Ba

sidiom

ycota(8%),an

dZy

gomycota(5%)(Bates

etal.,20

12)

Mojav

eDesert,USA

140

Unk

nown

Shan

noninde

x=

4.7;

96OTU

s;do

minated

byCya

noba

cteria

(42%

),Proteo

bacteria

(∼25

%),an

dActinob

acteria(∼

20%)(Steve

net

al.,20

14)

Shan

noninde

x=

2.9;

41OTU

s;do

minated

byAscom

ycota(∼

75%)an

dBa

sidiom

ycota(∼

21%)(Steve

net

al.,20

14)

Chihu

ahua

nDesert,USA

235

Lich

en–

Shan

noninde

x=

1.58

–1.93;

had3–

17speciesin

10classes,15

orde

rs,a

nd3ph

yla;

dominated

byAscom

ycota(82%

),Ba

sidiom

ycota(8%),an

dZy

gomycota(5%)(Bates

etal.,20

12)

Colorad

oPlateau,

USA

241

Cya

noba

cteria

Shan

noninde

x=

2.1–

3.3;

dominated

byCya

noba

cteria

(38.4%

),Actinob

acteria(11.8%

),β-Proteo

bacteria

(11.5%

),an

dBa

cteriode

tes

(10.6%

)(G

undlap

ally

andGarcia-Pich

el,2

006)

Shan

noninde

x=

1.87

;dom

inated

byAscom

ycota(87%

)(Bates

etal.,

2010

)

Coldstep

peecosystemslocatedin

southw

estern

Idah

o,USA

235–

803

Possibly

liche

nan

dmoss

Shan

noninde

x=

1.48

–1.73;

dominated

byActinob

acteria(36–

51%),

Bacteroide

tes(6–1

7%),Proteo

bacteria

(9–1

2%),an

dCya

noba

cteria

(<11

%);Th

eab

unda

nceof

Actinob

acteriaan

dFirm

icutes

was

high

erat

elev

ations

expe

rien

cing

cooler,wetterclim

ates,w

hile

theab

unda

nceof

Cya

noba

cteria,Proteo

bacteria,an

dChlorofl

exide

creased(201

7)

–

Gurba

ntun

ggut

Desert,China

<15

0Algae,liche

n,an

dmoss

Cya

noba

cteria

dominated

indifferen

tsuccession

alstag

esan

dthenu

mbe

rof

cyan

obacterial

speciesiden

tified

was

25,2

8,an

d31

inmicroalga

e,lic

hen,

andmossbioc

rusts,

respective

ly(Zha

nget

al.,20

09)

–

Teng

gerDesert,China

191

Cya

noba

cteria

and

moss

Had

33sequ

encesin

8major

taxo

nomic

grou

ps;d

ominated

byBa

cterioidetes

(23%

),Proteo

bacteria

(32%

),an

dAcido

bacteria

(12%

)(Zha

nget

al.,20

12)

Cya

noba

cteria

bioc

rusts:Sh

anno

ninde

x=

1.75

–1.94,

21–2

3species;Moss

bioc

rusts:

Shan

noninde

x=

1.95

–2.41,

19–2

4species;

Both

thebioc

rusts

weredo

minated

byAscom

ycota(95.5%

)(G

rishka

net

al.,20

15)

HorqinSa

ndyLa

nd,C

hina

341

Unk

nown

Had

72clon

esin

7ph

yla;

dominated

byProteo

bacteria

(52%

),Acido

bacteria

(19.2%

),Ba

cteroide

tes(9.6%),an

dActinob

acteria(8.2%)

(Zha

nget

al.,20

14)

–

MuUsDesert,China

300–

350

Moss

–Sh

anno

ninde

x=

2.38

;16OTU

s;do

minated

byAscom

ycota(58%

)an

dBa

sidiom

ycota(42%

)(Zha

oet

al.,20

11)

LoessPlateau,

China

290–

505

Moss

Shan

noninde

x=

5.56

;102

9–10

69OTU

s;do

minated

byAcido

bacteria

(24.3%

),Proteo

bacteria

(23.8%

),Chlorofl

exi(15

.8%),an

dActinob

acteria

(14.5%

)(Thisstud

y);T

hecyan

obacteriaha

d54

speciesbe

long

ingto

10ge

nera

and4families

withfilamen

tous

cyan

obacteriado

minan

t(Yan

get

al.,20

13)

Shan

noninde

x=

5.63

;50–

68OTU

s;do

minated

byAscom

ycota(68.0%

),an

dBa

sidiom

ycota(23.8%

)(Thisstud

y)

Arabian

Deserts,Oman

86–3

18Cya

noba

cteria

and

liche

n–

Cultiva

tion

:Sh

anno

ninde

x=

2.43

–3.77,

101speciesin

44ge

nera;

dominated

byAscom

ycota(98%

).Py

rosequ

encing

:142

–341

OTU

sin

6ph

yla;

dominated

byAscom

ycota,

Basidiom

ycota,

andChy

tridiomycota

(Abe

det

al.,20

13)

Tabe

rnas

Desert,Sp

ain

220

Lich

enSh

anno

ninde

x=

7.5,

387OTU

s;do

minated

byProteo

bacteria

(29%

),Actinob

acteria(27%

),Ba

cteroide

tes(12%

),an

dCya

noba

cteria

(7%)

(Maier

etal.,20

14)

–

SanNicolas

Totolapa

n,Mexico

1341

Mossan

dlic

hen-moss

Shan

noninde

x=

4.8–

5.0,

280OTU

sin

7ph

yla;

dominated

byAcido

bacteria

(39.8–

51.3%)an

dα-Proteo

bacteria

(36.9–

39.1%)(N

avarro-

Noy

aet

al.,20

14)

–

B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177

173

favorable microhabitats (with sufficient moisture, moderate tempera-ture, and abundant living nutrition) which is exactly established by themoss biocrusts in harsh environments. Actually, similar results forbiocrusts and uncrusted soil have been reported many times (Garcia-Pichel et al., 2003; Castillo-Monroy et al., 2011a; Steven et al., 2014).For example, Steven et al. (2013) reported that Cyanobacteria andProteobacteria demonstrated significantly higher relative abundance inbiocrusts, and Chloroflexi and Archaea were significantly enriched inuncrusted soil. Gundlapally and Garcia-Pichel (2006) also found thatthe bacterial community of biocrusts consistently displayed fewerrichness and Shannon diversity than typical soil communities, withapparent dominance by few members. On the other hand, it has beenreported that the richness and community diversity of bacteria/fungiincreased with the age and successional stages of biocrusts (in order ofcyanobacteria, algae, lichen, and moss) (Redfield et al., 2002; Zhanget al., 2009; Bates et al., 2010; Grishkan et al., 2015). This result isreasonable because that biocrusts at high successional stages (i.e., soillichen and moss) are capable of providing greater protection againstenvironmental stresses in harsh environments. Subsequently, theycould provide a more suitable microhabitat for soil microorganisms ascompared with biocrusts at early successional stages (i.e., cyanobacter-ia and green-algae). Finally, besides the influences of biocrusts, the soilmicrobial communities are extremely sensitive and could be signifi-cantly affected by many factors including locations, soil properties,geomorphology, micro-climate, and surrounding environments. Thus,an ordination-based approach would possibly give more explanations tothe differences of microbial communities among samples of differenttypes of biocrusts and uncrusted soil, or from different sites. In otherwords, an ordination-based approach should be employed in theinvestigation of microbial communities of biocrusts in further study.

In our study, the moss biocrusts and uncrusted soil shared more than30.6% bacterial OTUs and 39.7% fungal OTUs in their communitycomposition. Their Sorenson’s similarity coefficients of bacterial andfungal community were more than 0.468 and 0.558, respectively.Similar results were also reported by Steven et al. (2014), who foundthat biocrusts and uncrusted soil shared 36.7% of bacterial OTUs and25.7% of fungal OTUs. They also suggested that the differences ofbacterial and fungal communities between biocrusts and uncrusted soilwere largely due to the differences of relative abundances of species incommunity rather than the presence or absence of particular OTUs(Steven et al., 2014). Thus, abundant OTUs found in one habitat arelikely to be co-located in the other, although the relative abundance ofthat OTU may vary widely (Steven et al., 2014). We would like to givesuch explanations to the results obtained from our study: the differencesof bacterial/fungal community between biocrusts and uncrusted soil onthe Loess Plateau of China were mostly attribute to their differences ofrelative abundances of species in community rather than the presenceor absence of particular OTUs. This is exactly why we designed andevaluated the differences of bacterial/fungal community between themoss biocrusts and uncrusted soil by both their species richness andrelative abundance in this study.

4.3. Relationships between soil fertility and bacterial/fungal community ofbiocrusts

In this study, the contents of soil nutrients in the moss biocrustswere significantly correlated with the OTU numbers of bacteria and therelative abundances of bacteria/fungi, implying that the characteristicsof bacterial and fungal community in moss biocrusts play importantroles in C and N cycling (Brankatschk et al., 2013) and improving soilfertility in semi-arid climates. It has been reported that the majority ofN-fixing, ammonia-oxidizing, and denitrifying bacteria are affiliatedwithin the Proteobacteria (Zhang et al., 2014; Paul, 2015). Also, avariety of genera in Proteobacteria and Chloroflexi could fix C andconvert energy from light through photosynthesis (Paul, 2015). There-fore, the Proteobacteria and Chloroflexi maybe play important roles in

nutrient cycling (Zhang et al., 2014). Furthermore, Actinobacteria aremajor contributors to biological buffering of soils and have roles inorganic matter decomposition because they are recognized as theproducers of many bioactive metabolites (Chaudhary et al., 2013). Inaddition, Cyanobacteria species in biocrusts possessed scytonemin inhigher proportion than chlorophyll a, suggesting its role in protectionfrom high solar irradiance and UV (Kumar and Adhikary, 2015). Anumber of cyanobacteria with distinct sheath layer survived within thebiocrusts in desiccated state; however, they revived their metabolicactivity soon after rainfall and immediately played important roles insoil C and N fixation as well as soil nutrient mobilization (Kumar andAdhikary, 2015). Also, Steven et al. (2014) reported that the functionalcategories related to photosynthesis, circadian clock proteins, andheterocyst-associated genes were enriched in biocrusts, where popula-tions of Cyanobacteria were larger. The genes related to potassiummetabolism were also more abundant in biocrusts, suggesting differ-ences in nutrient cycling between biocrusts and uncrusted soil (Stevenet al., 2014). At last, since the large majority of Acidobacteria have notbeen cultured, the function and ecology of these bacteria is not wellunderstood. However, these bacteria may be an important contributorto ecosystems, since they are particularly abundant within soils(Naether et al., 2012). Besides the content of soil nutrients, severalstudies have been carried out to understand the relationships betweenmicrobial community and soil fertility. For example, Grishkan et al.(2015) found that the density of microfungal isolates was linearly andpositively correlated with chlorophyll content. Zhang et al. (2012)showed that the bacterial community abundance was closely correlatedwith soil enzyme activities in different soils. They reported that thepresence of Cyanobacteria was correlated with significant increases inprotease, catalase, and sucrase in the biocrusts and increased urease inthe rhizosphere soil of Artemisia ordosica (Zhang et al., 2012). More-over, the occurrence of Acidobacteria was associated with significantincreases in urease, dehydrogenase, and sucrose in the rhizosphere soilof Caragana korshinski (Zhang et al., 2012). In addition, the presence ofγ-Proteobacteria was correlated with a significant increase in poly-phenol oxidase in the rhizosphere soil of A. ordosica (Zhang et al.,2012).

Even that we gained novel insides from the biocrusts, it is still hardto fully understand the potential implications of the differences inbacterial/fungal community between the moss biocrusts and uncrustedsoil at the present stage of investigations. However, these differencesare definitely closely related to soil C and N cycling according to abovementioned references. Compared with the uncrusted soil, the high levelof soil fertility in the moss biocrusts of our study could be attributed totheir high abundance and diversity of bacterial/fungal community.Similar conclusions were also made by Blay et al. (2017). They foundthat the bacterial communities from rolling biocrusts (possibly lichen-moss) in cold steppe ecosystems were affected by climate regime anddiffered substantially from other cold desert ecosystems, resulting inpotential differences in nutrient cycling and ecosystem dynamics (Blayet al., 2017).

As we shown in this study, the bacterial/fungal community inbiocrusts plays important roles in soil C and N cycling (Brankatschket al., 2013), and therefore in improving soil fertility. However, in turn,the accumulation of soil nutrients probably changed the microbialcomposition of biocrusts (Zhang et al., 2009). For example, Schulz et al.(2016) pointed out that the soil parameters including pH, electricalconductivity, carbonate content, total contents of C, N, P, and thebioavailable P-fraction, might have an influence on the species compo-sition of biocrusts, even not significant. In our opinion, for a betterunderstanding the complicated relationships between the microbialcommunity of biocrusts and soil fertility, more comparative studiesshould be conducted for different types of biocrusts in different climateregions in future. These studies would provide more experimental datafor resolving the causality, and contribute a lot for understanding thecomplicated relationships between bacterial/fungal community and

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soil fertility.Although the roles in C and N cycling and improving soil fertility of

biocrusts are mostly attributed to the abundance and diversity ofmicrobial community (cyanobacteria, bacteria, and fungi) throughtheir photosynthesis and respiration, C and N fixation, and effects onsoil enzyme activities (Belnap et al., 2003b; Barger et al., 2016; Sanchoet al., 2016), the mosses and other cryptogams (i.e., soil lichens andgreen-algae) in biocrusts also contribute a lot to soil nutrients andfertility through capturing depositional N and dust and decreasingnutrients losses via dissolved, gaseous (N), and erosional loss (Bargeret al., 2016). However, it is hard to separate and estimate thecontribution of each component in biocrusts to soil fertility due toour limited knowledge at present, especially for different types ofbiocrusts (e.g., moss, lichen, or cyanobacteria biocrust). For example,moss biocrusts are certainly capable of accumulating more soil nu-trients through increasing dust capture and decreasing nutrient losswith runoff and sediment by their larger biomass as compared withcyanobacteria or lichen biocrusts (Xiao et al., 2016). However, theypossibly have no advantages in photosynthesis and respiration, C and Nfixation, and improving soil enzyme activities, which is mainlyattributed to the abundance and diversity of bacterial and fungalcommunity. Conversely, cyanobacteria or lichen biocrusts probablyare more efficient in photosynthesis and respiration, C and N fixationand transformation processes but less efficient in accumulating soilnutrients owing to its lower biomass as compared with moss biocrusts.

5. Conclusions

From this study, we concluded that moss biocrusts harbor a largenumber and high diversity of bacteria and fungi in semi-arid climateson the Loess Plateau of China. The microbial communities of mossbiocrusts greatly differ from both that of uncrusted soil and that ofbiocrusts from the other climate regions around the world. The mossbiocrusts on the Loess Plateau of China are dominated byAcidobacteria, Proteobacteria, Chloroflexi, and Actinobacteria in bac-terial community, and they are dominated by Ascomycota andBasidiomycota in fungal community. More importantly, the diversifiedbacteria and fungi in the moss biocrusts possibly play important roles inthe cycling of soil C and N and improving the fertility of semi-aridclimate soil, because that the contents of soil nutrients were signifi-cantly correlated with the OTU numbers of bacteria and the relativeabundances of bacteria/fungi. Our results contribute to a better under-standing the microbial composition and corresponding ecologicalfunctions of biocrusts in semi-arid climates on the Loess Plateau ofChina, and similar climate regions in other parts of the world.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (grant number 41671221); the FundamentalResearch Funds for the Central Universities (grant numbers2017QC048, 2017QC126); and the Open Fund from the State KeyLaboratory of Soil Erosion and Dryland Farming on the Loess Plateau(grant number A314021402-1513). We give ours thanks to Dr. GC Dingfor his professional help in the analysis of DNA sequences. We alsothank the Shenmu Experimental Station of Soil Erosion andEnvironment, CAS for its logistical support.

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