Applied Soil Ecology 117–118 (2017) 165–177

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Applied Soil Ecology

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Moss-dominated biocrusts increase soil microbial abundance and MARK community diversity and improve soil fertility in semi-arid climates on the Loess Plateau of China ⁎ Bo Xiaoa,b, , Maik Vestec,d a Department of Soil and Water Sciences, China Agricultural University, Beijing, 100193, China b 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, China c University of Hohenheim, Institute of Botany (210), Garbenstrasse 30, Stuttgart, 70599, Germany d Brandenburg University of Technology Cottbus-Senftenberg, Soil Protection and Recultivation, Konrad-Wachsmann-Allee 6, Cottbus, 03046, Germany

ARTICLE INFO ABSTRACT

Keywords: Various ecological functions of biocrusts are mostly determined by their bacterial and fungal abundance and Biological soil crust community diversity, which has not yet been fully investigated. To provide more insights into this issue, we Microbiotic crust collected samples of moss biocrusts, fixed sand, and mobile sand from a watershed with semi-arid climate on the Microbial community composition Loess Plateau of China. The relative abundances and community diversities of soil bacteria and fungi of the Microbial community diversity samples were determined using high-throughput DNA sequencing. Finally, we analyzed the characteristics of Relative abundance of species bacterial and fungal community of the moss biocrusts and their relationships to the content of soil nutrients. Our High-throughput sequencing results showed that the moss biocrusts had 1048 bacterial OTUs (operational taxonomic units) and 58 fungal OTUs, and their Shannon diversity indexes were 5.56 and 1.65, respectively. The bacterial community of the moss biocrusts was dominated by Acidobacteria (24.3%), Proteobacteria (23.8%), Chloroflexi (15.8%), and Actinobacteria (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 as compared with the uncrusted soil, and their Sorenson’s similarity coefficients of bacterial and fungal communities 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 and fungi. 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 soil fertility.

1. Introduction (Porada et al., 2014; Lenhart et al., 2015; Belnap et al., 2016). The dominant components of biocrusts and their small-scale dis- Biocrusts (also named biological soil crusts) of dry environments are tribution depend on topography, soil characteristics, climates, plant formed by a highly specialized communities of moss and living communities, microhabitats, successional stages, and disturbance re- microorganisms (including soil lichens, green algae, cyanobacteria, gimes (Kidron et al., 2010; Bowker et al., 2016; Bu et al., 2016), but are fungi, and bacteria) as well by excretion of biopolymers (Belnap et al., mostly determined by the local water regimes which is regulated by soil 2016; Xiao et al., 2016). According to the dominating components and texture, microclimatic conditions, and precipitation (Bowker et al., successional development, biocrusts are usually classified as cyanobac- 2016). Generally, biocrusts are dominated by cyanobacteria and soil teria (blue-green algae)-, green algae-, soil lichen-, or moss-dominated lichens in super-arid and arid climates with less than 250 mm of annual biocrusts (Belnap et al., 2016). It has been reported that biocrusts are precipitation (e.g., the Gurbantunggut Desert of China (Zhang et al., widespread in arid and semi-arid climates throughout the world with 2011)), while they are mostly dominated by mosses in semi-arid climate varying species composition and coverage (Belnap et al., 2003a; with 250–500 mm of annual precipitation (e.g., the Loess Plateau of Bowker et al., 2016). Thus, they are considered as an important China (Xiao and Hu, 2017)). In the Negev Desert of Israel, cyanobacter- component of vegetation and land cover in dryland ecosystems ia and green-algae are characteristic for biocrusts in areas with less than

⁎ Corresponding author at: Department of Soil and Water Sciences, China Agricultural University, Beijing, 100193, China. E-mail addresses: [email protected], [email protected] (B. Xiao). http://dx.doi.org/10.1016/j.apsoil.2017.05.005 Received 20 December 2016; Received in revised form 2 May 2017; Accepted 5 May 2017 0929-1393/ © 2017 Elsevier B.V. All rights reserved. B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177

170 mm of annual precipitation (Kidron et al., 2010), while moss cover particularly important in nutrient-limited dryland ecosystems because and thickness increase with increasing annual rainfall along the of its usual low level, susceptibility to depletion, and difficulties of climatic gradient (Yair et al., 2011). replenishment (Ravi et al., 2010). Previous studies confirmed that Although the negative effects of biocrusts have been reported biocrusts play a significant role in N cycling of dryland ecosystems, as several times (for example, they smooth the soil surface and prevent they contribute major N inputs via biological fixation (Zhao et al., 2010; plant seeds from penetrating the soil (Deines et al., 2007; Su et al., Su et al., 2011) and capture of dust (Williams and Eldridge, 2011) and 2007; Langhans et al., 2009)), many studies have confirmed that depositional N, harbor intense internal N transformation processes (Hu biocrusts mostly perform positive roles in various ecological processes et al., 2015; Kidron et al., 2015b), and direct N losses via dissolved, such as preventing water and wind erosion (Bowker et al., 2008), gaseous (Lenhart et al., 2015), and erosional loss processes (Li et al., enhancing soil water retention (Zhang et al., 2008), increasing soil C 2013; Barger et al., 2016). Similarly, soil C cycling is also significantly and N (Green et al., 2008), facilitating vascular plant establishment and changed by biocrusts through photosynthetic activity (Hui et al., 2014; growth (Godínez-Alvarez et al., 2012), and promoting soil biodiversity Kidron et al., 2015a) and soil respiration (Castillo-Monroy et al., 2011b; (Castillo-Monroy et al., 2011a). On the other hand, they give strong Yu et al., 2014). On the other side, the soil microorganisms in biocrusts influences on hydrological processes through enhancing or weakening accelerate the decomposition of organic matter, mainly due to the soil infiltration and runoff production depending from the species increasing soil enzyme activities (e.g., urease, alkaline phosphatase, composition (Belnap, 2006; Yair et al., 2011). Particularly, moss invertase, and protease) (Zhang et al., 2012; Liu et al., 2014). In other biocrusts attract more attention because they usually generate much words, the mosses and other cryptogams (i.e., lichens and green-algae) stronger influences on various ecological processes than cyanobacteria, in biocrusts are mainly responsible for the effects on soil formation and green-algae or soil lichen biocrusts due to their greater biomass water conservation (soil physical processes) through their functions in − (> 10 mg cm 2) and larger thickness (> 15 mm vs. ∼3 mm), espe- stabilizing soil surface and holding soil water (Kidron and Tal, 2012; cially in stabilizing soil surface and changing soil water regimes (Xiao Xiao et al., 2016). Their roles in C and N cycling and improving soil et al., 2016; Xiao and Hu, 2017). In general, it is believed that biocrusts fertility (soil chemical and biological processes) are mostly attributed to are important communities for the soil processes and ecosystem the cyanobacteria, green-algae, bacteria, and fungi through their functioning (Bowker et al., 2010), and their rehabilitation are impor- photosynthesis, nitrogen fixation, and effects on soil enzyme activities tant measures for combating land degradation and desertification (Xiao (Belnap et al., 2003b). It is well known that both soil bacteria and fungi et al., 2015). are responsible for important processes (Paul, 2015) even in biocrusts Through stabilizing the soil surface (Zhang et al., 2006), conserving (Maier et al., 2014; Steven et al., 2014; Mueller et al., 2015). For these soil water (Langhans et al., 2009; Xiao et al., 2016), and accumulating reasons, the bacterial and fungal abundance and community diversity nutrients (Li et al., 2008), biocrusts create a favorable microhabitat for of biocrusts are of very high concern owing to their significant roles in other soil microorganisms in dry environments. Therefore, they usually maintaining and improving soil fertility, which are crucial for the harbor a large number and high diversity of soil microorganisms as restoration of degraded lands and vegetation in arid and semi-arid compared with uncrusted soil (Garcia-Pichel et al., 2003; Steven et al., climates (Abed et al., 2013; Steven et al., 2014; Zhang et al., 2014; 2014). These diversified soil microorganisms fundamentally determine Mueller et al., 2015). In addition, the bacterial and fungal community the various important ecological functions of biocrusts (Castillo-Monroy diversity of biocrusts are usually used to identify the successional stages et al., 2011a), but till now we still have no detailed information about of biocrusts in natural development or restoration processes (Lan et al., them. According to the common theories of biodiversity and ecosystem 2013; Steven et al., 2015), and to evaluate their responses to dis- functioning, species diversity gives positive short-term effects on turbance or climate change (Ferrenberg et al., 2015; Mueller et al., ecosystem processes (such as primary productivity and nutrient reten- 2015). tion) through functional niche complementarity (the complementarity The Loess Plateau in China covers an area of 640,000 km2 and has effect) and selection of extreme trait values (the selection effect), and it the world's highest soil erosion rates (including water erosion in contributes to the stability and maintenance of ecosystem processes in summer as well as wind erosion in winter and early spring) across the face of perturbations (long-term effects of biodiversity) (Loreau, the world (Xin et al., 2008). This is due to the fact that vascular plants 2000; Loreau et al., 2001; Delgado-Baquerizo et al., 2016). In this are heavily degraded and cover less than 5% in some areas owing to context, it is well believed that soil microbial community diversity climate changes and human activities (e.g., agricultural overexploita- provides the cornerstone for support of soil ecosystem services by key tion) (Wang et al., 2008; Xin et al., 2008). In order to combat land roles in soil organic matter turnover, C sequestration, and even water degradation and desertification, the Grain for Green Project has been cycling (Nannipieri et al., 2003; Brussaard et al., 2007). In other words, implemented to restore vegetation in recent decades (Cao et al., 2009). various soil processes (especially C, N cycling) would possibly benefita During the project, the agricultural activities have ceased over a large lot from the abundant and diversified soil microorganisms inhabited area and a large number of native shrubs have been artificially planted biocrusts (Blay et al., 2017). Thus, their characteristics, particularly to conserve soil and water (Cao et al., 2009). Owing to stabilization of abundance and community diversity, are of great importance for a land surface resulted from the decreasing disturbances of agricultural better understanding the functions of biocrusts in relation to soil activities and increasing protection of the artificially planted shrubs, development in drylands and other ecosystems (Gundlapally and biocrusts spontaneously recolonized the soil surface and gradually Garcia-Pichel, 2006; Castillo-Monroy et al., 2011a). However, up to developed from cyanobacteria to mosses (or directly developed from now only a few studies have been conducted to investigate the bare land to moss biocrusts in the regions with abundant precipitation) microbial community composition of different types of biocrusts in over several years (Zhao et al., 2014a; Bu et al., 2016). Nowadays, moss different climate regions around the world (e.g., Soule et al., 2009; biocrusts are extensively developed and are widely distributed with a Zhang et al., 2011, 2012; Bates et al., 2012). It seems that the microbial coverage reaching 70% in most areas of semi-arid climates on the Loess community of biocrusts could be affected by many environmental Plateau (Wang et al., 2016; Xiao et al., 2016). These moss biocrusts are factors, but it mostly depends on the development of biocrusts and able to store water and stabilize the soil surface and are an important the site-specific microclimatic conditions (Moquin et al., 2012). How- contribution to combat desertification, which are serious problems in ever, most of the research has been restricted to arid climate regions, this region (Xiao et al., 2014, 2015). Moss biocrusts have been while the microbial community of biocrusts in semi-arid climate regions intensively investigated on the Loess Plateau of China, especially for (e.g., the Loess Plateau of China) is less known. soil water cycling (Xiao et al., 2010, 2011b, 2016; Wei et al., 2014), soil Soil fertility (especially C and N) is essential for sustaining fertility (Zhao et al., 2010, 2014b), and wind and water erosion (Wang cryptogams and vascular plants in terrestrial ecosystems, and it is et al., 2013; Zhao and Xu, 2013; Zhao et al., 2014a). However, their

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

− microbial community composition has not yet been fully investigated, were 12.6%, 0.8%, and 10.5 cm h 1, respectively (Xiao et al., 2016). which is important to understand the functions of biocrusts for Although the soil on the Loess Plateau is dominated by loess soil ecosystems functioning. (ustochnept in USDA soil taxonomy or cambisols in FAO soil classifica- In this study, we hypothesized that the moss biocrusts on the Loess tion) with clayey or loamy texture, the aeolian sandy soil covers an area Plateau of China harbor a large number and high diversity of bacteria of 79,200 km2 and accounts for 12.2% of the total area (Yamamoto and and fungi as compared with uncrusted soil, and these diversified Endo, 2014). Especially in the northern Loess Plateau along the Great bacteria and fungi possibly play important roles in improving soil Wall, the aeolian sandy soil covers up to 64.6% of the area and the loess fertility including soil C, N, P. Based on these hypotheses, we collected soil covers 15.2% only (Yamamoto and Endo, 2014). samples of moss biocrusts, fixed sand, and mobile sand from a In each sampling site, the top 20 mm of the fixed sand with moss representative semi-arid climate on the Loess Plateau of China. biocrusts (moss biocrusts which was ∼30 years old with > 95% moss Afterwards, the relative abundance and diversity of bacterial and coverage and naturally developed on fixed sand after the plantation of fungal communities of these samples, at both phylum and genus levels, artificially planted shrubs; see Fig. 1b), fixed sand without biocrusts were determined using high-throughput DNA sequencing technique in (hereafter fixed sand which was constituted of abundant stabilized sand laboratory. We also measured the characteristics of biocrusts, the particles, small pieces of organic materials (possibly the litters of content of soil nutrients, and microbial densities of the samples by surrounding vascular plants), and few moss stems (< 5%); see conventional methods. The analysis of soil nutrient content allowed us Fig. 1c), and mobile sand (aeolian sand with frequent disturbances of to explore relationships between the composition of bacterial/fungal the surface; see Fig. 1d) were randomly sampled from 12 sampling community of biocrusts and soil fertility. The objective of this study was points by petri dishes (90 mm diameter × 20 mm height) on Sep. 23, to determine the bacterial and fungal community diversity of moss 2014. The sub-samples collected from the 12 sampling points at each biocrusts and its implications for soil fertility on the Loess Plateau of sampling site were mixed together for each treatment, and finally there China. The results will provide a better understanding of the microbial were 12 samples in total (3 treatments × 4 sampling sites). The three composition and corresponding ecological functions of moss biocrusts treatments were sampled in nearby locations with similar soil texture in in semi-arid climates on the Loess Plateau of China, and comparable each sampling site, and they were mostly resulted by the different ecoregions of the world. degree of protections from shrubs rather than their locations. In other words, we set three treatments and each treatment had four replicates 2. Materials and methods in this study. The samples were homogenized and sieved (< 2 mm) to remove any root material or pebbles. They were packed on dry ice and 2.1. Study area stored at −20 °C until processing.

The study area located at the Liudaogou watershed 2.3. Characterization of moss biocrusts and soil nutrients (38°46′–38°51′ N and 110°21′–110°23′ E; an area of 6.89 km2 with elevation of 1081–1274 m a.s.l.) on the northern Loess Plateau of The biocrust thickness was measured through a digital caliper (CD- China. The mean annual precipitation is 409 mm (with ∼80% occur- 6′’-ASX, Mitutoyo, Japan). Moss species in the biocrusts were visually ring during summer) and potential evaporation is 1337 mm, respec- identified (e.g., shape of stems and leaves) and analyzed for size, color, tively (Xiao et al., 2011a). The mean annual temperature is 8.4 °C and and habitats; moss density was calculated from the total moss game- the mean monthly temperature ranges from −9.7 °C in winter (Dec.–- tophytes in a 20-mm square sample; and moss plants were washed out Feb.) to 23.7 °C in summer (Jun.–Aug.) (Cha and Tang, 2000). Due to with a 2-mm screen and dried at 65 °C for 24 h before measuring their the serious degradation of natural vegetation as a result of inappropri- biomass. The total chlorophyll content was also recorded through ate land use practices in combination with high rainfall intensities and laboratory analysis with a UV–vis spectrophotometer (DR 5000, complex landforms, the region is highly affected by severe soil loss. As a Hach, USA) to indicate the cryptogam biomass of biocrusts. result of water erosion in summer as well as wind erosion in winter and Moreover, the bacterial, fungal, and actinobacterial densities were early spring, the observed soil erosion rate varies between 15,000 and measured by the plate counts as described by Schinner et al. (1995). − − 20,000 t km 2 a 1 (Cha and Tang, 2000). Additionally, the content of soil nutrients, including organic matter, For restoration of the landscape, native shrubs including Artemisia total N and P (that reflect the biocrust establishment as well as biocrust ordosica Krasch. (Asteraceae) and Caragana korshinskii Kom. biomass), available N and P (that can explain the biocrust establish- (Leguminosae) were planted in the watershed about 30 years ago ment), and microbial C and N, were measured according to Carter and (Xiao and Hu, 2017). Currently, the planted shrubs are distributed in Gregorich (2006). All above measurements were conducted in at least patches and cover 20–30% of the watershed. Owing the restriction of four replicates. limited precipitation (409 mm per year in average) and soil moisture, the artificially planted shrubs are finally distributed in sparse patches 2.4. DNA extraction, PCR amplification, cloning, and sequencing after about 30 years (Xiao and Hu, 2017). In the study area, moss biocrusts are extensively developed (they were initially recorded Total soil DNA was extracted from duplicate 0.5 g subsamples ® around 30 years ago) on fallow lands, shrub lands, and grasslands (freeze-dried soil) from each sample using the E.Z.N.A. soil DNA Kit and now cover approximately 70–80% of the soil surface (Xiao et al., (Omega Bio-tek, Norcross, GA, USA) according to manufacturer’s 2010). protocols. Following extraction, the DNA samples were pooled. PCR amplification, cloning, and sequencing of bacterial and fungal 2.2. Experimental design and sample collection rRNA genes were performed by the Majorbio Company in Shanghai, China according to Amato et al. (2013). Briefly, bacterial 16S rRNA In the watershed, we selected four sampling sites (see Fig. 1a) with gene fragments were amplified (95 °C for 2 min, followed by 25 cycles representative moss biocrusts and very sparse shrub lands, composed of at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension artificially planted A. ordosica Krasch., C. korshinskii Kom., and Salix at 72 °C for 5 min) in triplicate from total soil DNA using primers 338F psammophila C. Wang et Chang Y. Yang (Salicaceae). The soil on the and 806R; and fungal 18S rRNA gene fragments were amplified (as sampling sites was an aeolian sandy soil (entisols in USDA soil same as the bacterial 16S rRNA) using primers 817F and 1196R. taxonomy or arenosols in FAO soil classification), and its texture was Amplicons were subjected to electrophoresis with 2% agarose gels; loamy sand (USDA) with 81% sand, 14% silt, and 5% clay (Xiao et al., bands were extracted, dissolved, and purified using the AxyPrep DNA 2016). It’s field capacity, wilting point, and steady-state infiltration rate Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according

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

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. to the manufacturer’s instructions and quantified using QuantiFluor™ each treatment were expressed as means of the replicates and expressed −ST (Promega, USA). Purified amplicons were pooled in equimolar as the mean ± standard error. The number of OTUs and relative and paired-end sequenced (2 × 250) on an Illumina MiSeq platform abundance (percentage of OTUs) of each phylum and genus were used (PE250/PE300) according to the standard protocols. to represent the bacterial/fungal community composition. The simila- rities of bacterial/fungal community of moss biocrusts vs. fixed sand 2.5. Data analysis and moss biocrusts vs. mobile sand were determined by the Sorenson’s similarity coefficient (SC)(Osem et al., 2006). Raw fastq files were demultiplexed and quality-filtered using QIIME SC=2 c /( a + b) (1) 1.90 with the following criteria. (1) The 300 bp reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding In this equation, a and b represent the numbers of OTUs in the two window, discarding the truncated reads that were shorter than 50 bp. samples, respectively, and c represents the numbers of same OTUs of (2) Exact barcode matching was performed, only two nucleotide the two samples. The differences of similarity indexes among the mismatches in primers were allowed, and any reads containing samples, including Sorenson’s similarity coefficient, were determined ambiguous characters were removed. (3) Only sequences with overlaps by the NPAR1WAY in SAS 8.01. The differences among the samples longer than 10 bp were assembled based on their overlapping sequence. were statistically evaluated at the 5% probability level by the paired- Reads which could not be assembled were discarded. Operational samples t-test or one-way ANOVA in SAS 8.01. According to our taxonomic units (OTUs) were clustered with a 97% identity cutoff experimental design, the paired-samples t-test was mainly used to using UPARSE 7.1, and chimeric sequences were identified and evaluate the differences between the paired treatments (i.e., moss removed using UCHIME. The taxonomy of each 16S rRNA and 18S biocrusts vs. fixed sand, moss biocrusts vs. mobile sand) to reduce or rRNA gene sequence was analyzed by RDP Classifier against the SILVA eliminate the site effects (e.g., the possible differences in soil properties, (SSU115) 16S rRNA and 18S rRNA databases, respectively, using a geomorphology, micro-climate, and surrounding environments among confidence threshold of 70%. The total number of bacterial and fungal the four sampling sites) in this study. The correlation and regression OTUs of each sample were estimated from the rarefaction curves analysis were further conducted to offer more insights into the relation- through extrapolation. ships between the relative abundance/community diversity of bacter- According to the experimental design, we had three treatments and ial/fungal communities and the content of soil nutrients. The repre- each treatment had four replicates (the fours sampling sites were sentation and graphical fits of experimental data were obtained using regarded as replicates). The experimental data were analyzed based OriginPro 9.2. on the descriptive statistics in SPSS Statistics 22. The final results of

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Table 1 General 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 < 0.001 Total N content (%) 0.130 ± 0.026 a 0.045 ± 0.004 b 0.025 ± 0.005 b 12.65 0.001 Total P content (%) 0.050 ± 0.002 a 0.048 ± 0.002 a 0.044 ± 0.002 a 2.08 0.167 − Available N content (mg kg 1) 114.82 ± 9.23 a 15.08 ± 1.19 b 11.06 ± 1.53 b 16.95 < 0.001 − Available P content (mg kg 1) 4.14 ± 0.78 a 0.79 ± 0.25 b 0.52 ± 0.08 b 11.56 0.002 − Microbial C content (mg kg 1) 303.90 ± 18.33 a 100.20 ± 19.75 b 58.11 ± 17.71 b 4.64 0.032 − Microbial N content (mg kg 1) 23.58 ± 6.95 a 5.76 ± 0.95 b 3.91 ± 1.26 b 7.78 0.007 − Bacterial density ( × 105 CFU g 1) 4.63 ± 0.90 a 2.02 ± 0.42 b 1.05 ± 0.10 b 6.06 0.015 − Fungal density ( × 102 CFU g 1) 7.24 ± 0.75 a 2.62 ± 0.60 b 1.18 ± 0.08 b 42.72 < 0.001 − Actinobacterial 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 mm height). b Values with in a row followed by the same letter are not significantly different at P ≤ 0.05.

3. Results composed of Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, Bacteroidetes, Cyanobacteria, and Gemmatimonadetes 3.1. Characteristics of moss biocrusts (Fig. 2a). However, the moss biocrusts consistently had 97, 69, 50, 24, and 23 more OTUs than the fixed sand in Proteobacteria, Chloroflexi, We identified seven moss species from the moss biocrusts, including Bacteroidetes, Cyanobacteria, and Actinobacteria, respectively. Corre- Bryum argenteum Hedw., B. arcticum (R. Brown) B.S.G., B. caepiticium spondingly, they consistently had 165, 106, 73, 43, 35, and 28 more Hedw., Didymodon vinealis (Brid.) Zander, D. nigrescens (Mitt.) Saito, OTUs than the mobile sand in Proteobacteria, Chloroflexi, Bacteroi- Barbula vinealis Brid., and B. perobtusa (Broth.) Chen. Dominant moss detes, Cyanobacteria, Actinobacteria, and Gemmatimonadetes, respec- species were only B. arcticum (R. Brown) B.S.G. and D. vinealis (Brid.) tively. At genus level (Fig. 2b), all the three treatments had fewer than Zander. The moss biocrusts almost completely (> 95%) covered the 31 OTUs in each bacterial genus. The most common genera were land surface in the open interspaces between the sparse artificially Roseiflexus, Bryobacter, Haliangium, Leptolyngbya, uncultured Anaeroli- planted shrubs. The moss thickness, density, and biomass were neaceae, and unclassified Rhizobiales. − − 18.95 mm, 55.9 gametophyte cm 2, and 152.99 g m 2, respectively. According to the relative abundance of fungal phyla (Fig. 2c), the The total chlorophyll content of the moss biocrusts ranged from 0.49 to bacterial community of the moss biocrusts was dominated by Acid- − − 0.71 mg g 1 (dry moss), with an average of 0.58 ± 0.03 mg g 1. obacteria (24.3%), Proteobacteria (23.8%), Chloroflexi (15.8%), and Actinobacteria (14.5%). However, the bacterial community of the fixed 3.2. Effects of moss biocrusts on soil nutrients and microbial densities sand was dominated by Actinobacteria, Proteobacteria, and Acidobac- teria; and that of the mobile sand was dominated by Actinobacteria, As listed in Table 1, the moss biocrusts had 4.98 and 11.11 times Cyanobacteria, and Proteobacteria. The moss biocrusts had lower higher of organic matter content as compared with the fixed sand and relative abundance of Actinobacteria (17.2%; t = 6.72, P = 0.001) as mobile sand. The content of total N, available N, and available P of the compared with the fixed sand. Similarly, they had lower relative moss biocrusts were 2.89, 7.61, and 5.24 times (F ≥ 4.64, P ≤ 0.032) abundance of Actinobacteria (33.0%; t = 21.76, P< 0.001) and as high as that of the fixed sand, while they were 5.20, 10.38, and 7.96 Cyanobacteria (14.8%; t = 11.80, P< 0.001) but higher relative times as high as that of the mobile sand. The moss biocrusts also had abundance of Acidobacteria (21.9%; t = 18.45, P< 0.001) and Chlor- 3.03 and 4.09 times higher of microbial C and N as compared with the oflexi (11.3%; t = 10.64, P< 0.001) as compared with the mobile fixed sand, and they had 5.23 and 6.03 times higher of microbial C and sand. At the genus level (Fig. 2d), the bacterial community of both the N as compared with the mobile sand. Moreover, the bacterial, fungal, moss biocrusts and fixed sand were dominated by RB41; and that of the and actinobacterial densities of the moss biocrusts were 2.29, 2.76, and mobile sand was dominated by Arthrobacter and Microcoleus. At the 1.80 times (F ≥ 6.06, P ≤ 0.015) as high as that of the fixed sand; and genus level, there was almost no difference (≤ 5.6%) in the relative they were 4.41, 6.14, and 3.48 times as high as that of the mobile sand. abundance of bacterial community between the moss biocrusts and Furthermore, the densities of bacteria, fungi, and actinobacteria were fixed sand. However, the moss biocrusts had lower relative abundance linearly (R2 ≥ 0.954, P ≤ 0.097) and positively correlated with each of Arthrobacter (31.2%; t = 71.94, P< 0.001) and Microcoleus (21.2%; other. 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, 3.3. Effects of moss biocrusts on soil bacterial community P = 0.001) as compared with the mobile sand.

The moss biocrusts had 56.9% higher OTUs and 7.4% higher 3.4. Effects of moss biocrusts on soil fungal community Shannon diversity index in bacterial community as compared with the fixed sand (Table 2). Similarly, they had 142% higher OTUs and As listed in Table 2, the moss biocrusts had similar number of OTUs, 62.9% higher Shannon index in bacterial community as compared with 17.9% higher Shannon index, and 19.5% higher Simpson index in the mobile sand (Table 2). The moss biocrusts shared 842 (accounting fungal community as compared with the fixed sand. Correspondingly, for 62.3%) bacterial OTUs with the fixed sand and 409 (accounting for they had 26.1% higher OTUs, 42.2% higher Shannon index, and 64.3% 30.6%) bacterial OTUs with the mobile sand. Namely, 64.1% of the higher Simpson index in fungal community as compared with the bacterial OTUs in the moss biocrusts were same with the fixed sand, mobile sand (F ≥ 23.11, P< 0.001). The moss biocrusts and fixed sand while only 31.2% of the bacterial OTUs in the moss biocrusts were same had 34 (accounting for 42.5%) fungal OTUs in common; and the moss with the mobile sand. The Sorenson’s similarity coefficient of bacterial biocrusts and mobile sand had 29 (accounting for 39.7%) fungal OTUs community of the moss biocrusts vs. fixed sand and moss biocrusts vs. in common. Namely, 58.6% of the fungal OTUs in the moss biocrusts mobile sand were 0.768 and 0.468, respectively. were same with the fixed sand; and 50.0% of the fungal OTUs in the The bacterial community of all three treatments was mainly moss biocrusts were same with the mobile sand. The Sorenson’s

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

Table 2 Estimated 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 community Moss biocrusts 1048 ± 8 ab 1155 ± 11 a 1157 ± 13 a 0.990 ± 0.001 a 5.56 ± 0.07 a 0.011 ± 0.001 a Fixed sand 668 ± 21 b 867 ± 8 b 859 ± 11 b 0.964 ± 0.009 b 5.18 ± 0.01 b 0.015 ± 0.001 a Mobile sand 433 ± 22 c 559 ± 28 c 557 ± 28 c 0.987 ± 0.001 a 3.41 ± 0.05 c 0.016 ± 0.001 a Fungal community Moss biocrusts 58 ± 3 a 62 ± 3 a 61 ± 2 a 1.000 ± 0.001 a 1.65 ± 0.03 a 0.092 ± 0.001 a Fixed sand 56 ± 1 a 58 ± 2 b 57 ± 1 b 1.000 ± 0.001 a 1.40 ± 0.02 b 0.077 ± 0.001 a Mobile 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 at the genus level; (c) Relative abundance of bacterial OTUs at the phylum level; (d) Relative abundance of bacterial OTUs at the genus level. similarity coefficient of fungal community of the moss biocrusts vs. Basidiomycota with the fixed sand. However, they had lower relative fixed sand and moss biocrusts vs. mobile sand were 0.596 and 0.558, abundance of Ascomycota (6.8%; t = 7.56, P = 0.001) but higher respectively. relative abundance of Basidiomycota (7.3%; t = 6.48, P = 0.001) as The fungal community of all the three treatments was mainly compared with the mobile sand. At the genus level (Fig. 3d), the moss composed of Ascomycota and Basidiomycota (Fig. 3a). The moss biocrusts had a slightly lower (1.4%) relative abundance of Acrosper- biocrusts had very similar number of OTUs with the fixed sand, while mum and unclassified Saccharomycetales but a higher (≤ 2.5%) they had 5 and 6 more OTUs in Ascomycota and Basidiomycota, relative abundance of unclassified Ascomycota, Dothideomycetes, respectively, as compared with the mobile sand. All the three treat- Pleosporales, and Lichinaceae as compared with the fixed sand. As ments had fewer than 6 OTUs in each fungal genus (Fig. 3b), and their compared with the mobile sand, they had a lower (2.1%) relative differences in each fungal genus were fewer than 2 OTUs. The most abundance of unclassified Ascomycota and a higher (2.1%) relative common genera were unclassified Ascomycota, Dothideomycetes, and abundance of unclassified Lichinaceae. Pleosporales. As shown by relative abundance in Fig. 3c, the fungal community of 3.5. Relationships between bacterial/fungal community and soil nutrients the moss biocrusts, fixed sand, and mobile sand at the phylum level was consistently dominated by Ascomycota and Basidiomycota. The moss The Pearson’s correlation coefficients between the content of soil biocrusts had very similar relative abundance of Ascomycota and nutrients and the number of bacterial OTUs were mostly significant

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

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 the genus level; (c) Relative abundance of fungal OTUs at the phylum level; (d) Relative abundance of fungal OTUs at the genus level.

Table 3 Pearson'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 0.705 Bacteroidetes 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 0.644 Fungal 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).

(r ≥ 0.589), except for total P (Table 3). In contrast to that, the the content of soil nutrients and bacterial OTUs in phyla of Chloroflexi, Pearson’s correlation coefficients between the content of soil nutrients Actinobacteria, and Bacteroidetes. These Pearson’s correlation coeffi- and the number of fungal OTUs were insignificant. The results showed cients were also significant (r ≥ 0.748) between the content of soil that the content of soil nutrients was significantly and positively nutrients and fungal OTUs in phyla of Ascomycota and Basidiomycota. correlated with the number of bacterial OTUs, but they were not correlated with the number of fungal OTUs. 4. Discussion As listed in Table 4, the content of soil nutrients were positively fl correlated with the relative abundance of Proteobacteria, Chloro exi, 4.1. Characteristics of bacterial/fungal community in biocrusts Acidobacteria, Bacteroidetes, and Gemmatimonadetes. However, they were negatively correlated with the relative abundance of Actinobac- Our study showed that the moss biocrusts in semi-arid climate on teria and Cyanobacteria. Similarly, they were positively correlated with the Loess Plateau of China had the highest diversity with 1048 bacterial the relative abundance of Basidiomycota but were negatively correlated OTUs and 58 fungal OTUs. Their bacterial community was dominated with the relative abundance of Ascomycota. Except for total P, the by Acidobacteria, Proteobacteria, Chloroflexi, and Actinobacteria; and ’ ffi fi ≥ Pearson s correlation coe cients were signi cant (r 0.632) between their fungal community was dominated by Ascomycota and

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

Table 4 Pearson'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 0.455 Chloroflexi 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 0.611 Bacteroidetes 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 −0.533 Gemmatimonadetes 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).

Basidiomycota. Over the past decade, a number of studies have been where soil microbial communities are extreme diverse and maybe conducted to investigate the microbial community diversity of different contain abundant non-cultured representatives of novel divisions types of biocrusts in different drylands, including the Colorado Plateau (Kuske et al., 2002; Ansorge, 2009; Steven et al., 2014). In this study, (Redfield et al., 2002; Gundlapally and Garcia-Pichel, 2006), Great we recorded many OTUs in unclassified phyla and genera as presented Basin (Soule et al., 2009), Chihuahuan Desert (Soule et al., 2009; Bates in Figs. 2–3. These OTUs are likely new taxa and have not yet been et al., 2012), Sonoran Desert (Soule et al., 2009; Bates et al., 2012), reported before (possibly also because that there are generally a Mojave Desert (Steven et al., 2014; Mueller et al., 2015), and cold number of OTUs who could not be assigned to certain taxa based only steppe ecosystems in southwestern Idaho (Blay et al., 2017) in USA; the on a partial sequence of 16S/18S rRNA gene, especially for fungi). Tengger Desert (Zhang et al., 2012; Grishkan et al., 2015), Gurban- However, it has been reported that significant errors possibly exist in tunggut Desert (Zhang et al., 2009, 2011), Mu Us Desert (Zhao et al., the investigation of soil microbial community with high-throughput 2011), and Horqin Sandy Land (Zhang et al., 2014) in China; the DNA sequencing technique, because that the different communities of Tabernas Desert (Maier et al., 2014) and Sax (Maestre et al., 2015)in soil microorganisms maybe have different responses to the conditions Spain; the Negev Desert in Israel (Zaady et al., 2010); and the of DNA extraction and PCR amplification (Krsek and Wellington, 1999; Santiniketan (Kumar and Adhikary, 2015) in India. Although different Martin-Laurent et al., 2001). Thus, in our opinion, the combination of methods (conventional method or PCR-based DNA-sequencing) were conventional techniques and high-throughput DNA sequencing techni- employed in these studies, the comparative results (Table 5) indicate que should be used in the determination of microbial community of that the bacterial/fungal community are greatly different in various biocrusts in future. types of biocrusts under different climatic conditions (represented by annual precipitation). For example, Moquin et al. (2012) found that the bacterial communities of moss biocrusts greatly differed from that 4.2. Differences of bacterial/fungal community between biocrusts and reported for cyanobacterial crusts. Grishkan et al. (2015) also believed uncrusted soil that the differences in fungal community of biocrusts between the Tengger and Negev deserts were caused by the principal differences in This study showed that the moss biocrusts had very different their precipitation regimes, associated with different rainy seasons with bacterial and fungal community diversity with the fixed sand and winter rainfall in the Negev and summer rainfall in the Tengger. Thus, mobile sand. In dryland ecosystems, particularly desert ecosystem, the according to the different bacterial/fungal community of biocrusts survival of microorganism in surface soil is greatly restricted by the listed in Table 5, we conclude that the bacterial/fungal community of long-term extreme low moisture (Pointing and Belnap, 2012), high biocrusts are closely related to the climate characteristics at least. temperature (up to 80 °C) (Johnson et al., 2012), lack of C and N Besides the effects of biocrusts, it has been reported that the soil (Pointing and Belnap, 2012), and strong solar radiation (Belnap et al., microbial community is very sensitive and could be significantly 2008). These microorganisms are also very sensitive to the movement affected by various factors. For example, both Zhang et al. (2012) of surface soil particles and sand burial caused by strong wind blowing. and Meadow and Zabinski (2012) reported that the composition of soil However, it has been reported that moss biocrusts could significantly microbial community had high spatial heterogeneity in deserts and increase soil moisture by up to 7.6% at upper 5 cm depth in semi-arid drylands. Moreover, Steven et al. (2013) found that soil microbial climates on the Loess Plateau of China (Xiao et al., 2016) (sometimes communities displayed spatial biogeographic patterns associated soil they deteriorate deep soil water conditions in hyper-arid regions (Li parent material. Additionally, Kuske et al. (2012) and Ferrenberg et al. et al., 2004)). They could also decrease soil surface temperature by up (2015) affirmed that climate changes, such as the increase of tempera- to 11.8 °C under wet and hot conditions in summer, and significantly ture and precipitation, strongly influenced soil microbial community increase soil surface temperature by up to 8.0 °C under dry and cold composition. Lastly, soil microbial community was also greatly changed conditions in winter (Xiao et al., 2013, 2016) (the increasing effects by N addition (Mueller et al., 2015) and physical disturbances from from cyanobacteria or lichen biocrusts on soil temperature were also trampling (Kuske et al., 2012; Ferrenberg et al., 2015). In a word, soil confirmed by a few studies (George et al., 2003; Kidron and Tal, 2012)). bacterial/fungal community is originally different across different Moreover, moss biocrusts could completely stabilize surface soil climate regions. However, the presence of biocrusts further intensifies particles and protect soil microorganisms from the destroy of sand their differences and make them become more significant. movement and burial (Jia et al., 2012; Tisdall et al., 2012). Addition- In addition, the determination of microbial community diversity in ally, moss biocrusts could significantly improve soil fertility through biocrusts by conventional techniques requires specialized skills, is time fixing atmospheric C and N (Zhao et al., 2010; Kidron et al., 2015a), consuming, and is very poor in repeatability and accuracy (Hill et al., producing more extracellular polymeric substances (Mager and 2000; Redfield et al., 2002). Compared with conventional techniques, Thomas, 2011; Chen et al., 2014), and accumulating soil nutrients DNA-based techniques are capable of providing a more comprehensive against runoff and erosion (Barger et al., 2006; Li et al., 2013). For and accurate measure of microbial community diversity in biocrusts, these causes, we reasonably attribute the high relative abundance and community diversity of bacteria/fungi in the moss biocrusts to the

172 .Xa,M Veste M. Xiao, B.

Table 5 Regional differences of the bacterial and fungal community of biocrusts in different drylands of North America, China, Middle East, and Southern Europe.

Regions Annual precipitation Biocrust types Bacterial community Fungal community (mm)

Sonoran Desert, USA 75–255 Lichen Shannon index = 2.03–3.02; dominated by Cyanobacteria (54.8%), Shannon index = 1.67–2.22; had 5–19 species in 10 classes, 15 orders, and Actinobacteria (15.1%), Proteobacteria (13.8%), and Acidobacteria 3 phyla; dominated by Ascomycota (82%), Basidiomycota (8%), and (11.1%) (Nagy et al., 2005) Zygomycota (5%) (Bates et al., 2012) Mojave Desert, USA 140 Unknown Shannon index = 4.7; 96 OTUs; dominated by Cyanobacteria (42%), Shannon index = 2.9; 41 OTUs; dominated by Ascomycota (∼75%) and Proteobacteria (∼25%), and Actinobacteria (∼20%) (Steven et al., 2014) Basidiomycota (∼21%) (Steven et al., 2014) Chihuahuan Desert, USA 235 Lichen – Shannon index = 1.58–1.93; had 3–17 species in 10 classes, 15 orders, and 3 phyla; dominated by Ascomycota (82%), Basidiomycota (8%), and Zygomycota (5%) (Bates et al., 2012) Colorado Plateau, USA 241 Cyanobacteria Shannon index = 2.1–3.3; dominated by Cyanobacteria (38.4%), Shannon index = 1.87; dominated by Ascomycota (87%) (Bates et al., Actinobacteria (11.8%), β-Proteobacteria (11.5%), and Bacteriodetes 2010) (10.6%) (Gundlapally and Garcia-Pichel, 2006) Cold steppe ecosystems located in 235–803 Possibly lichen and Shannon index = 1.48–1.73; dominated by Actinobacteria (36–51%), – southwestern Idaho, USA moss Bacteroidetes (6–17%), Proteobacteria (9–12%), and Cyanobacteria (< 11%); The abundance of Actinobacteria and Firmicutes was higher at elevations experiencing cooler, wetter climates, while the abundance of Cyanobacteria, Proteobacteria, and Chloroflexi decreased (2017) Gurbantunggut Desert, China < 150 Algae, lichen, and Cyanobacteria dominated in different successional stages and the number – moss of cyanobacterial species identified was 25, 28, and 31 in microalgae, 173 lichen, and moss biocrusts, respectively (Zhang et al., 2009) Tengger Desert, China 191 Cyanobacteria and Had 33 sequences in 8 major taxonomic groups; dominated by Cyanobacteria biocrusts: Shannon index = 1.75–1.94, 21–23 species; Moss moss Bacterioidetes (23%), Proteobacteria (32%), and Acidobacteria (12%) biocrusts: Shannon index = 1.95–2.41, 19–24 species; Both the biocrusts (Zhang et al., 2012) were dominated by Ascomycota (95.5%) (Grishkan et al., 2015) Horqin Sandy Land, China 341 Unknown Had 72 clones in 7 phyla; dominated by Proteobacteria (52%), – Acidobacteria (19.2%), Bacteroidetes (9.6%), and Actinobacteria (8.2%) (Zhang et al., 2014) Mu Us Desert, China 300–350 Moss – Shannon index = 2.38; 16 OTUs; dominated by Ascomycota (58%) and Basidiomycota (42%) (Zhao et al., 2011) Loess Plateau, China 290–505 Moss Shannon index = 5.56; 1029–1069 OTUs; dominated by Acidobacteria Shannon index = 5.63; 50–68 OTUs; dominated by Ascomycota (68.0%), (24.3%), Proteobacteria (23.8%), Chloroflexi (15.8%), and Actinobacteria and Basidiomycota (23.8%) (This study) (14.5%) (This study); The cyanobacteria had 54 species belonging to 10 genera and 4 families with filamentous cyanobacteria dominant (Yang et al., 2013) Arabian Deserts, Oman 86–318 Cyanobacteria and – Cultivation: Shannon index = 2.43–3.77, 101 species in 44 genera; lichen dominated by Ascomycota (98%). Pyrosequencing: 142–341 OTUs in 6 phyla; dominated by Ascomycota, Basidiomycota, and Chytridiomycota Applied S (Abed et al., 2013) Tabernas Desert, Spain 220 Lichen Shannon index = 7.5, 387 OTUs; dominated by Proteobacteria (29%), – Actinobacteria (27%), Bacteroidetes (12%), and Cyanobacteria (7%) oil Ecology117–118(2017)165–177 (Maier et al., 2014) San Nicolas Totolapan, Mexico 1341 Moss and lichen-moss Shannon index = 4.8–5.0, 280 OTUs in 7 phyla; dominated by – Acidobacteria (39.8–51.3%) and α-Proteobacteria (36.9–39.1%) (Navarro- Noya et al., 2014) B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177 favorable microhabitats (with sufficient moisture, moderate tempera- nutrient cycling (Zhang et al., 2014). Furthermore, Actinobacteria are ture, and abundant living nutrition) which is exactly established by the major contributors to biological buffering of soils and have roles in moss biocrusts in harsh environments. Actually, similar results for organic matter decomposition because they are recognized as the biocrusts and uncrusted soil have been reported many times (Garcia- producers of many bioactive metabolites (Chaudhary et al., 2013). In Pichel et al., 2003; Castillo-Monroy et al., 2011a; Steven et al., 2014). addition, Cyanobacteria species in biocrusts possessed scytonemin in For example, Steven et al. (2013) reported that Cyanobacteria and higher proportion than chlorophyll a, suggesting its role in protection Proteobacteria demonstrated significantly higher relative abundance in from high solar irradiance and UV (Kumar and Adhikary, 2015). A biocrusts, and Chloroflexi and Archaea were significantly enriched in number of cyanobacteria with distinct sheath layer survived within the uncrusted soil. Gundlapally and Garcia-Pichel (2006) also found that biocrusts in desiccated state; however, they revived their metabolic the bacterial community of biocrusts consistently displayed fewer activity soon after rainfall and immediately played important roles in richness and Shannon diversity than typical soil communities, with soil C and N fixation as well as soil nutrient mobilization (Kumar and apparent dominance by few members. On the other hand, it has been Adhikary, 2015). Also, Steven et al. (2014) reported that the functional reported that the richness and community diversity of bacteria/fungi categories related to photosynthesis, circadian clock proteins, and increased with the age and successional stages of biocrusts (in order of heterocyst-associated genes were enriched in biocrusts, where popula- cyanobacteria, algae, lichen, and moss) (Redfield et al., 2002; Zhang tions of Cyanobacteria were larger. The genes related to potassium et al., 2009; Bates et al., 2010; Grishkan et al., 2015). This result is metabolism were also more abundant in biocrusts, suggesting differ- reasonable because that biocrusts at high successional stages (i.e., soil ences in nutrient cycling between biocrusts and uncrusted soil (Steven lichen and moss) are capable of providing greater protection against et al., 2014). At last, since the large majority of Acidobacteria have not environmental stresses in harsh environments. Subsequently, they been cultured, the function and ecology of these bacteria is not well could provide a more suitable microhabitat for soil microorganisms as understood. However, these bacteria may be an important contributor compared with biocrusts at early successional stages (i.e., cyanobacter- to ecosystems, since they are particularly abundant within soils ia and green-algae). Finally, besides the influences of biocrusts, the soil (Naether et al., 2012). Besides the content of soil nutrients, several microbial communities are extremely sensitive and could be signifi- studies have been carried out to understand the relationships between cantly affected by many factors including locations, soil properties, microbial community and soil fertility. For example, Grishkan et al. geomorphology, micro-climate, and surrounding environments. Thus, (2015) found that the density of microfungal isolates was linearly and an ordination-based approach would possibly give more explanations to positively correlated with chlorophyll content. Zhang et al. (2012) the differences of microbial communities among samples of different showed that the bacterial community abundance was closely correlated types of biocrusts and uncrusted soil, or from different sites. In other with soil enzyme activities in different soils. They reported that the words, an ordination-based approach should be employed in the presence of Cyanobacteria was correlated with significant increases in investigation of microbial communities of biocrusts in further study. protease, catalase, and sucrase in the biocrusts and increased urease in In our study, the moss biocrusts and uncrusted soil shared more than the rhizosphere soil of Artemisia ordosica (Zhang et al., 2012). More- 30.6% bacterial OTUs and 39.7% fungal OTUs in their community over, the occurrence of Acidobacteria was associated with significant composition. Their Sorenson’s similarity coefficients of bacterial and increases in urease, dehydrogenase, and sucrose in the rhizosphere soil fungal community were more than 0.468 and 0.558, respectively. of Caragana korshinski (Zhang et al., 2012). In addition, the presence of Similar results were also reported by Steven et al. (2014), who found γ-Proteobacteria was correlated with a significant increase in poly- that biocrusts and uncrusted soil shared 36.7% of bacterial OTUs and phenol oxidase in the rhizosphere soil of A. ordosica (Zhang et al., 25.7% of fungal OTUs. They also suggested that the differences of 2012). bacterial and fungal communities between biocrusts and uncrusted soil Even that we gained novel insides from the biocrusts, it is still hard were largely due to the differences of relative abundances of species in to fully understand the potential implications of the differences in community rather than the presence or absence of particular OTUs bacterial/fungal community between the moss biocrusts and uncrusted (Steven et al., 2014). Thus, abundant OTUs found in one habitat are soil at the present stage of investigations. However, these differences likely to be co-located in the other, although the relative abundance of are definitely closely related to soil C and N cycling according to above that OTU may vary widely (Steven et al., 2014). We would like to give mentioned references. Compared with the uncrusted soil, the high level such explanations to the results obtained from our study: the differences of soil fertility in the moss biocrusts of our study could be attributed to of bacterial/fungal community between biocrusts and uncrusted soil on their high abundance and diversity of bacterial/fungal community. the Loess Plateau of China were mostly attribute to their differences of Similar conclusions were also made by Blay et al. (2017). They found relative abundances of species in community rather than the presence that the bacterial communities from rolling biocrusts (possibly lichen- or absence of particular OTUs. This is exactly why we designed and moss) in cold steppe ecosystems were affected by climate regime and evaluated the differences of bacterial/fungal community between the differed substantially from other cold desert ecosystems, resulting in moss biocrusts and uncrusted soil by both their species richness and potential differences in nutrient cycling and ecosystem dynamics (Blay relative abundance in this study. et al., 2017). As we shown in this study, the bacterial/fungal community in 4.3. Relationships between soil fertility and bacterial/fungal community of biocrusts plays important roles in soil C and N cycling (Brankatschk biocrusts et al., 2013), and therefore in improving soil fertility. However, in turn, the accumulation of soil nutrients probably changed the microbial In this study, the contents of soil nutrients in the moss biocrusts composition of biocrusts (Zhang et al., 2009). For example, Schulz et al. were significantly correlated with the OTU numbers of bacteria and the (2016) pointed out that the soil parameters including pH, electrical relative abundances of bacteria/fungi, implying that the characteristics conductivity, carbonate content, total contents of C, N, P, and the of bacterial and fungal community in moss biocrusts play important bioavailable P-fraction, might have an influence on the species compo- roles in C and N cycling (Brankatschk et al., 2013) and improving soil sition of biocrusts, even not significant. In our opinion, for a better fertility in semi-arid climates. It has been reported that the majority of understanding the complicated relationships between the microbial N-fixing, ammonia-oxidizing, and denitrifying bacteria are affiliated community of biocrusts and soil fertility, more comparative studies within the Proteobacteria (Zhang et al., 2014; Paul, 2015). Also, a should be conducted for different types of biocrusts in different climate variety of genera in Proteobacteria and Chloroflexi could fix C and regions in future. These studies would provide more experimental data convert energy from light through photosynthesis (Paul, 2015). There- for resolving the causality, and contribute a lot for understanding the fore, the Proteobacteria and Chloroflexi maybe play important roles in complicated relationships between bacterial/fungal community and

174 B. Xiao, M. Veste Applied Soil Ecology 117–118 (2017) 165–177 soil fertility. Ansorge, W.J., 2009. Next-generation DNA sequencing techniques. New Biotechnol. 25, 195–203. Although the roles in C and N cycling and improving soil fertility of Barger, N.N., Herrick, J.E., Van Zee, J., Belnap, J., 2006. Impacts of biological soil crust biocrusts are mostly attributed to the abundance and diversity of disturbance and composition on C and N loss from water erosion. Biogeochemistry microbial community (cyanobacteria, bacteria, and fungi) through 77, 247–263. fi ff Barger, N.N., Weber, B., Garcia-Pichel, F., Zaady, E., Belnap, J., 2016. Patterns and their photosynthesis and respiration, C and N xation, and e ects on controls on nitrogen cycling of biological soil crusts. In: Weber, B., Büdel, B., Belnap, soil enzyme activities (Belnap et al., 2003b; Barger et al., 2016; Sancho J. (Eds.), Biological Soil Crusts: An Organizing Principle in Drylands. Springer et al., 2016), the mosses and other cryptogams (i.e., soil lichens and International Publishing, Switzerland, pp. 257–285. green-algae) in biocrusts also contribute a lot to soil nutrients and Bates, S.T., Nash, T.H., Sweat, K.G., Garcia-Pichel, F., 2010. Fungal communities of lichen-dominated biological soil crusts: diversity, relative microbial biomass, and fertility through capturing depositional N and dust and decreasing their relationship to disturbance and crust cover. J. Arid Environ. 74, 1192–1199. nutrients losses via dissolved, gaseous (N), and erosional loss (Barger Bates, S.T., Nash, T.H., Garcia-Pichel, F., 2012. Patterns of diversity for fungal et al., 2016). However, it is hard to separate and estimate the assemblages of biological soil crusts from the southwestern United States. Mycologia 104, 353–361. contribution of each component in biocrusts to soil fertility due to Belnap, J., Büdel, B., Lange, O.L., 2003a. Biological soil crusts: characteristics and our limited knowledge at present, especially for different types of distribution. In: Belnap, J., Lange, O.L. (Eds.), Biological Soil Crusts: Structure, biocrusts (e.g., moss, lichen, or cyanobacteria biocrust). For example, Function, and Management. Springer Berlin & Heidelberg, pp. 3–30. Belnap, J., Phillips, S., Duniway, M., 2003b. In: Alsharhan, W.W., Goudie, A.S., Fowler, moss biocrusts are certainly capable of accumulating more soil nu- A., Abdellatif, E.M. (Eds.), Soil Fertility in Deserts: a Review on the Influence of trients through increasing dust capture and decreasing nutrient loss Biological Soil Crusts and the Effect of Soil Surface Disturbance on Nutrient Inputs with runoff and sediment by their larger biomass as compared with and Losses. Desertification in the Third Millennium. Swets & Zeitlinger Publishers, Lisse, The Netherlands, pp. 245–252. cyanobacteria or lichen biocrusts (Xiao et al., 2016). However, they Belnap, J., Phillips, S.L., Flint, S., Money, J., Caldwell, M., 2008. Global change and possibly have no advantages in photosynthesis and respiration, C and N biological soil crusts: effects of ultraviolet augmentation under altered precipitation fixation, and improving soil enzyme activities, which is mainly regimes and nitrogen additions. Glob. Change Biol. 14, 670–686. Belnap, J., Weber, B., Büdel, B., 2016. Biological soil crusts as an organizing principle in attributed to the abundance and diversity of bacterial and fungal drylands. In: Weber, B., Büdel, B., Belnap, J. (Eds.), Biological Soil Crusts: An community. Conversely, cyanobacteria or lichen biocrusts probably Organizing Principle in Drylands. Springer International Publishing, Switzerland, pp. are more efficient in photosynthesis and respiration, C and N fixation 3–13. and transformation processes but less efficient in accumulating soil Belnap, J., 2006. The potential roles of biological soil crusts in dryland hydrologic cycles. Hydrol. Process. 20, 3159–3178. nutrients owing to its lower biomass as compared with moss biocrusts. Blay, E.S., Schwabedissen, S.G., Magnuson, T.S., Aho, K.A., Sheridan, P.P., Lohse, K.A., 2017. Variation in biological soil crust bacterial abundance and diversity as a 5. Conclusions function of climate in cold steppe ecosystems in the Intermountain West, USA. Microb. Ecol. 1–10. http://dx.doi.org/10.1007/s00248-00017-00981-00243. (in press). From this study, we concluded that moss biocrusts harbor a large Bowker, M.A., Belnap, J., Bala Chaudhary, V., Johnson, N.C., 2008. Revisiting classic number and high diversity of bacteria and fungi in semi-arid climates water erosion models in drylands: the strong impact of biological soil crusts. Soil Biol. Biochem. 40, 2309–2316. on the Loess Plateau of China. The microbial communities of moss Bowker, M.A., Maestre, F.T., Escolar, C., 2010. Biological crusts as a model system for biocrusts greatly differ from both that of uncrusted soil and that of examining the biodiversity-ecosystem function relationship in soils. Soil Biol. biocrusts from the other climate regions around the world. The moss Biochem. 42, 405–417. Bowker, M.A., Belnap, J., Büdel, B., Sannier, C., Pietrasiak, N., Eldridge, D., Aguilar- biocrusts on the Loess Plateau of China are dominated by Rivera, V., 2016. Controls on distribution patterns of biological soil crusts at micro- to Acidobacteria, Proteobacteria, Chloroflexi, and Actinobacteria in bac- global scales. In: Weber, B., Büdel, B., Belnap, J. (Eds.), Biological Soil Crusts: An terial community, and they are dominated by Ascomycota and Organizing Principle in Drylands. Springer International Publishing, Switzerland, pp. fi 173–197. Basidiomycota in fungal community. More importantly, the diversi ed Brankatschk, R., Fischer, T., Veste, M., Zeyer, J., 2013. Succession of N cycling processes bacteria and fungi in the moss biocrusts possibly play important roles in in biological soil crusts on a Central European inland dune. FEMS Microbiol. Ecol. 83, the cycling of soil C and N and improving the fertility of semi-arid 149–160. climate soil, because that the contents of soil nutrients were signifi- Brussaard, L., de Ruiter, P.C., Brown, G.G., 2007. Soil biodiversity for agricultural sustainability. Agr. Ecosyst. Environ. 121, 233–244. cantly correlated with the OTU numbers of bacteria and the relative Bu, C.F., Zhang, P., Wang, C., Yang, Y.S., Shao, H.B., Wu, S.F., 2016. Spatial distribution abundances of bacteria/fungi. Our results contribute to a better under- of biological soil crusts on the slope of the Chinese Loess Plateau based on canonical – standing the microbial composition and corresponding ecological correspondence analysis. Catena 137, 373 381. Cao, S.X., Chen, L., Yu, X.X., 2009. Impact of China's Grain for Green Project on the functions of biocrusts in semi-arid climates on the Loess Plateau of landscape of vulnerable arid and semi-arid agricultural regions: a case study in China, and similar climate regions in other parts of the world. northern Shaanxi Province. J. Appl. Ecol. 46, 536–543. Carter, M.R., Gregorich, E.G., 2006. Soil Sampling and Methods of Analysis, second ed. CRC Press, Boca Raton, FL, USA. Acknowledgments Castillo-Monroy, A.P., Bowker, M.A., Maestre, F.T., Rodríguez-Echeverría, S., Martinez, I., Barraza-Zepeda, C.E., Escolar, C., 2011a. Relationships between biological soil crusts, This work was supported by the National Natural Science bacterial diversity and abundance, and ecosystem functioning: insights from a semi- arid Mediterranean environment. J. Veg. Sci. 22, 165–174. Foundation of China (grant number 41671221); the Fundamental Castillo-Monroy, A.P., Maestre, F.T., Rey, A., Soliveres, S., Garcia-Palacios, P., 2011b. Research Funds for the Central Universities (grant numbers Biological soil crust microsites are the main contributor to soil respiration in a 2017QC048, 2017QC126); and the Open Fund from the State Key semiarid ecosystem. Ecosystems 14, 835–847. Cha, X., Tang, K.L., 2000. Study on comprehensive control model of small watershed eco- Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau environment in water and wind crisscrossed erosion zone (in Chinese with English (grant number A314021402-1513). We give ours thanks to Dr. GC Ding abstract). J. Nat. Resour. 15, 97–100. for his professional help in the analysis of DNA sequences. We also Chaudhary, H.S., Soni, B., Shrivastava, A.R., Shrivastava, S., 2013. Diversity and thank the Shenmu Experimental Station of Soil Erosion and versatility of Actinomycetes and its role in antibiotic production. J. Appl. Pharm. Sci. 3, 83–94. Environment, CAS for its logistical support. 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