INTRODUCTION

Biological soil crusts(BSCs)are the organic complex in the surface layer of soil and are colonized by cyanobacteria primarily(Mazor et al.,1996);afterwards,bacteria,eukaryotic algae,fungi,lichens,and mosses may grow(Belnap and Lange,2001;Caba′la and Rahmonov,2004).Biological soil crusts are widely distributed in arid and semiarid regions(Belnap and Lange,2001).They have important functions in the stability of the surface soil,reduction of wind and water erosion,and increase of soil fertility(Eldridge and Greene,1994;Belnap and Lange,2001;Yeager et al.,2004;Bowker et al.,2006).The process of the formation,development,and maturation of BSCs under the driving of various ecological factors and time is BSC succession,and during the succession,abundant life forms change(Lange et al.,1992;Zaady et al.,2000;Lan et al.,2013).According to the different successional stages of BSCs,they can be divided into algal crusts,lichen crusts,and moss crusts which represent the early,middle,and final successional stages,respectively(Belnap and Lange,2001;Hu et al.,2003;Chamizo et al.,2012).Lichen crusts can also be divided into cyanolichen and green alga-lichen crusts according to the symbiotic algae in the structure of lichen(Wu et al.,2013).As sensitive ecosystems in an extreme environment,BSCs are highly susceptible to disturbances and environmental changes(Belnap and Lange,2003;Bowker et al.,2010;Steven et al.,2015)and thus can be used as a model of an ecological system to study the general mechanism of ecosystem succession and to explore the ecological factors impacting the microbial community structure(Bowker et al.,2014).

During the development and succession of BSCs,morphological features and mineralogical components change(Chen et al.,2009);what is more important is that composition of microorganisms will also vary(Belnap and Lange,2001).Microorganisms are the important participants and drivers during BSC formation and development.The autotrophic and heterotrophic microorganisms participate in and dominate the biogeochemical cycle and energy flow of BSCs as the primary producers and consumers/disintegrators in the ecosystem,respectively(Garcia-Pichel et al.,2003;Nagy et al.,2005;Abed et al.,2010;Angel and Conrad,2013).Microorganisms are also an important constituent of BSCs,in terms of the fine physical structure of BSCs.Algal filaments and hyphae can twine soil particles and fix the sand(Hu et al.,2003;Chamizo et al.,2012).Thus,microorganisms are the basis of the formation,development,and execution of ecological functions of BSCs.To understand the mechanism of BSC succession and ecological functions,it is necessary to get the detailed and reliable information about microbial community composition(Garcia-Pichel et al.,2001;Bowker et al.,2008).So far,there have been many studies that have investigated the microbial community composition and diversity in BSCs worldwide.Among them,due to the significant colonization status,cyanobacteria in BSCs were studied most frequently(Hu et al.,2000;Garcia-Pichel et al.,2001;Red field et al.,2002;Hu and Liu,2003;Yeager et al.,2004;Langhans et al.,2009;Steven et al.,2012;Dojani et al.,2014;Kumar and Adhikary,2015;Pushkareva et al.,2015).At the same time,heterotrophic bacteria(Garcia-Pichel et al.,2003;Smith et al.,2004;Nagy et al.,2005;Gundlapally and Garcia-Pichel,2006;Zaady et al.,2010;Navarro-Noya et al.,2014),archaea(Soule et al.,2009),fungi(Grishkan et al.,2006;Bates and Garcia-Pichel,2009;Abed et al.,2013),actinomycetes(Gonz´alez et al.,2005),and eukaryotic algae(Lewis and Lewis,2005;Nyati et al.,2007;Cardon et al.,2008)have also been studied.However,almost all these studies focused on the diversity,whereas the community composition was studied rarely.E-specially with the succession of BSCs,little is known about the differences in the overall microbial components,and the rules governing the variation are also poorly understood.

As for the environmental factors driving BSC succession,the existing studies showed that the main factors in fluencing cyanobacteria diversity and community structure are the aridity level and precipitation gradient(Hagemann et al.,2015;Zhang et al.,2015),salinity(Li et al.,2013),soil texture(Garcia-Pichel et al.,2001),and so on.The main environmental factors affecting the heterotrophic bacteria in BSCs may be the vertical gradients of physicochemical differences(Garcia-Pichel et al.,2003).Moisture,temperature,and organic matter may affect the fungi distribution(Grishkan et al.,2006).However,the main environmental factors affecting the overall microbial community composition of BSCs at the phylum level are not clear.In recent years,due to the development of the high-throughput sequencing technology,accuracy of the microbial component surveys has greatly improved.This technology has been widely used in thefield of microbial diversity in soil environment(Abed et al.,2012,2013;Meadow and Zabinski,2012;Steven et al.,2012,2015;Maier et al.,2014;Pushkareva et al.,2015;Zhang et al.,2015).With application of the high-throughput sequencing technology,we may synthetically study prokaryotic and eukaryotic microorganisms at the same time,and this approach makes for a more precise analysis of the shift of microorganisms at different successional stages of BSCs.This approach also makes analyses of the main ecological driving factors possible.

In this study,we compared the prokaryotic and eukaryotic microorganisms of BSCs at different successional stages in the Tengger Desert of China with the pyrosequencing analysis,and we also investigated the main ecological factors affecting microbial components of BSCs.In short,this study aimed to explore the succession mechanism of BSCs and changes in ecological functions of BSCs.

MATERIALS AND METHODS

Study area and sampling

Biological soil crusts were sampled in April 2013 in Shapotou,Ningxia Hui Autonomous Region,China(37°27′N,104°57′E),and the district is located on the southeast edge of the Tengger Desert,China.The study area has a typical continental climate,and the soil type is the semiarid brown calcic soil.Average air temperature is 24.3 °C in July and −6.9 °C in January,and soil surface temperature reaches to 74°C in summer and −25 °C in winter.Mean annual precipitation is approximately 203 mm,and the rainy season is mainly between June and September(Lan et al.,2013).The sampling was conducted following the methods described by Wu et al.(2013).Specifically,sterilized petri dishes were buttoned and pressed into the BSCs,and then gently inverted,and the samples were stripped with a mini shovel.Under dry conditi-ons,samples were transported quickly to the laboratory and stored in a dryer.Alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts–four types of BSCs representing different successional stages–were sampled.Each of the BSC types was randomly collected with multiple replicates for subsequent analyses.

Determination of morphological and physicochemical characteristics

The morphological characteristics of BSCs were observed under a dissecting microscope,and abundant organisms,coverage of abundant organisms,and surface features were identified.Physicochemical properties of BSCs,thickness,moisture,total nitrogen(TN)content,organic carbon(OC)content,electrical conductivity(EC),pH,and mechanical composition of soil particles,were analyzed.The thickness was determined by a vernier caliper.Moisture was determined by the drying method(105± 2°C).Content of TN was determined by the Kjeldahl method(Bremner,1960),and OC content was determined by the ignition method(550± 5°C,6 h)in a muffle furnace(Mingorance et al.,2007).Electrical conductivity and pH were determined in a 1∶5(weight∶volume)BSC-solution suspension using calibrated YSI instruments(Abed et al.,2010).The mechanical composition was analyzed by the Robinson pipette method(Gee and Bauder,1986).Each measurement of physicochemical characteristics was carried out in triplicate.

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DNA extraction,polymerase chain reaction(PCR)amplification,and pyrosequencing

As shown in Fig.3a,the abundant prokaryotic phyla across all samples were Proteobacteria,Cyanobacteria,Actinobacteria,Bacteroidetes,Acidobacteria,and Chloro flexi,and all these phyla accounted for 85.2%of total prokaryotic sequences.In AL crusts,Cyanobacteria sequences were the most abundant and accounted for 51.9%of prokaryotic sequences in this type.In the rest of samples,the most abundant phylum was Proteobacteria,accounting for 23.3%,30.6%,and 32.1%of own respective prokaryotic sequences in CY,GR,and MO crusts.In addition,the low-abundance phyla Planctomycetes,Armatimonadetes,Firmicutes,Gemmatimonadetes,and Nitrospirae were all detected in each type.After comparison of the prokaryotic community composition among all samples,the results showed that with the succession of BSCs,Cyanobacteria abundance declined.The abundance of Proteobacteria and Actinobacteria had a tendency to increase with the succession.The abundance levels of the phyla Acidobacteria,Armatimonadetes,Bacteroidetes,and Chloro flexi in lichen crusts were higher than those in AL and MO crusts.As shown in Fig.3b,the most abundant eukaryotic microorganism in all samples was Ascomycota,accounting for 65.7%of the total detected eukaryotic sequences.Besides,the unclassified eukaryotic microorganisms could also be found in each BSC type and accounted for about 27.8%of the total sequences.Two types of lichen crusts had extremely high Ascomycota abundance∶in GR crusts,Ascomycota accounted for 77.0%of the eukaryotic sequences,and its abundance in CY crusts reached 93.0%.In AL and MO crusts,Ascomycota accounted for 48.4%and 47.4%,respectively.In addition,in all the samples,the phyla Chlorophyta,Chytridiomycota,Bacillariophyta,and Basidiomycota were detected at low abundance levels.By contrast,the phylum Ascomycota reached the highest abundance at the middle successional stage(lichen).Along the succession,the abundance level of Chlorophyta in the respective samples had a tendency to decrease.Bacillariophyta,which also belong to eukaryotic algae,had the highest abundance at the early succession stage of AL crusts,but the abundance in the two types of lichen crusts differed widely.Green alga-lichen crusts had a much higher Bacillariophyta abundance than CY crusts.In contrast,the phyla Chytridiomycota and Basidiomycota had the lowest abundance in lichen crusts and the highest abundance in MO crusts.

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The DNA concentration of each PCR product was determined using the PicoGreen dsDNA quantitation reagent(Invitrogen,Carlsbad,USA)and was qualitycontrolled on QuantiFluorTM-ST(Promega,Madison,USA).After quantification,they were pooled in a proportion according to the amount of each sample in terms of sequencing requirements.Emulsion-based clonal amplification(EmPCR)products were prepared using the Roche-specified Roche emPCRAmp-Lib L kit(Roche,Branford,USA),and then the pyrosequencing was performed on the Roche genome sequencer FLX+(Roche,Branford,USA).The sequences were uploaded to the National Center for BiotechnologyInformation(NCBI)sequenceread archive database,and the accession numbers are SRP-063082 and SRP063545.

Data processing and analysis

The significance of differences in each of the abovementioned physicochemical characteristics of the data BSCs was analyzed using one-way analysis of variance(ANOVA)at P＜0.05 in the SPSS 13.0 software.The analyses of the sequences were performed by the Mothur project(http∶//www.mothur.org/wiki/Mothur manual).According to the barcode sequences,we distinguished sequencing data from different samples;we discarded low-quality sequences with ambiguous base pairs and/or wrong length to get valid sequences and then removed primers and adaptors to obtain trimmed sequences.After that,the trimmed se-quences were aligned against the Silva database to get taxonomy information,and operational taxonomic units(OTUs)were clustered at a 97%sequence similarity level.The diversity indexes(ACE,Chao,and Shannon indexes)and rarefaction curves were obtained through the Mothur project based on the OTU cluster results.Venn diagrams were made using online tools Venny(http∶//bioinfogp.cnb.csic.es/tools/venny/)according to the OTU distribution tables.The community composition was determined by means of the taxonomy analysis results;the chloroplast and mitochondrial genes representing plants and animals were discarded,and the no-rank,unclassified sequences,or those with abundance lower than 1%were grouped into“Others”.Heatmaps and clusters at the class level were constructed using the HemI 1.0 software,and the Pearson distance was used as a similarity metric.Sorting analyses of microbial community structure at the phylum level were conducted with the Canoco 4.5 ecology software.

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RESULTS

Morphological and physicochemical characteristics

Judging by the surface features of the BSCs,AL crusts had a flat surface,and their color was light and close to the sand and soil color;the abundant organisms cyanobacteria were mainly distributed at 20–1000 µm depth in the surface,and AL crusts had nearly 100%coverage relative to bare sand.Cyanolichen crusts had a deep black color and colloidal thallus;the coverage of the thallus was about 68%in the dry condition,and the symbionts were cyanobacteria and fungi.Surface of GR crusts had a brown color,and their thallus was a circular plate;their thallus coverage was above 80%in the dry condition,and the symbionts were green algae and fungi.Moss crusts were light green and had a blanket shape,covered by thick bryophyta and their sporophyte,and the coverage of bryophyta was more than 95%in the dry condition.In addition,various physicochemical characteristics of four types of BSCs showed significant di-fferences(Table I).Thickness and OC and TN contents were all the highest in MO crusts and the lowest in AL crusts,and there was no significant difference between the two lichen crusts.Alga crusts had the significantly lower moisture than the rest of BSCs,and there were no significant differences among the rest of BSCs.Two lichen crusts had the lowest EC,and there was no significant difference between them.There were no significant differences in pH among all the crusts.As for the mechanical composition,AL crusts had the highest sand content and the lowest silt and clay contents;two lichens had the highest clay content,and the contents of silt and sand were intermediate;MO crusts had the highest silt content and the lowest sand content.

Dataset and diversity

There have been many studies that use various approaches to explore prokaryotic community composition of BSCs;for example,there have been studies involving PCR cloning and sequencing(Li et al.,2013;Navarro-Noya et al.,2014),DGGE fingerprinting(Nagy et al.,2005;Gundlapally and Garcia-Pichel,2006),high-throughput sequencing(Steven et al.,2013;Maier et al.,2014),etc.Our results also showed that Proteobacteria,Actinobacteria,Cyanobacteria,Bacteroidetes,and Acidobacteria are the most abundant phyla in all samples.We also observed that the different successional stages of BSCs had obviously different prokaryotic community composition and,in particular,Proteobacteria and Actinobacteria had the relatively high abundance in MO crusts,Cyanobacteria in AL crusts,and Bacteroidetes and Acidobacteria in lichen crusts(Figs.3a and 6a).At the class level,the cluster analysis results showed that AL crusts was the most different from the rest crust types in terms of prokaryotic microorganisms,and two lichen types were relatively close(Fig.4).These results further proved that AL crusts,lichen crusts,and MO crusts had di-fferent successional status,but CY and GR crusts may have relatively similar successional status.

TABLE I Physicochemical characteristics of four types of biological soil crusts,alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts

a)TN=total N;OC=organic C;EC=electrical conductivity. b)Means±stand deviations(n=3). c)Values with different letters in the same row are significantly different at P ＜ 0.05.

Characteristica) AL CY GR MO Abundant species Cyanobacteria Lichen Lichen Moss Coverage(%) ca.100 ca.68 ca.80 ca.99 Surface color Ashen Black Brown Light green Thickness(mm) 3.37±0.08b)cc) 10.05±0.74b 10.91±0.51b 19.55±0.77a Moisture(%) 0.51±0.11b 0.68±0.04a 0.66±0.11a 0.74±0.05a TN(g kg−1) 0.64± 0.09c 1.26± 0.33b 1.24± 0.12b 1.72± 0.13a OC(g kg−1) 10.37±0.76c 14.80± 1.02b 16.36± 2.42b 25.87±1.10a EC(µS cm−1) 192.7± 26.1a 106.0± 15.5b 101.7± 16.1b 165.7± 13.7a pH 7.85±0.05 7.77±0.15 7.64±0.17 7.60±0.20 Sand(%) 85.5±2.32a 62.95±2.46c 68.84±1.41b 56.12±1.99d Silt(%) 13.77±2.24d 35.41±2.84b 29.82±1.56c 42.82±2.17a Clay(%) 0.72±0.08b 1.64±0.63a 1.34±0.32ab 0.74±0.05ab

Rarefaction curves(Fig.1)showed that at the 97%similarity level,the prokaryotic sequence libraries of all samples were far from saturated(the slope does not go to zero),while eukaryotic sequence libraries of MO crusts were close to saturation.The rarefaction curves of AL and MO crusts were obviously different,and the curve slopes of the two types of lichen crusts were close to each other or even overlapping.As shown in Table III,the Chao and ACE indexes,which represent OTU richness,were the highest in MO crusts,followed by GR crusts,and the lowest in AL crusts in the prokaryotic sequence results.As to the eukaryotic sequence results,GR crusts had the highest Chao and ACE indexes,followed by CY crusts,whereas MO crusts had the lowest indexes.The Shannon index,which represents diversity,was the highest in GR crusts,with CY crusts being the second and AL crusts the lowest in terms of prokaryotic sequence results.The eukaryotic sequence results showed that CY crusts had the highest Shannon index,with GR crusts being the second andMO crusts the lowest.In general,at a similar read number,higher diversity and more OTUs were detected in prokaryotic sequences than in eukaryotic sequences.Among the different successional BSC samples,differences in OTU richness and diversity of both prokaryotic and eukaryotic sequences were observed.In particular,there were clear differences between AL and MO crusts,but two different types of lichen crusts were close in the OTU number and diversity.

TABLE II Numbers of valid sequences and trimmed sequences of the prokaryotes and eukaryotes in four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts,based on pyrosequencing data

BSCsProkaryote Eukaryote Valid TrimmedPercen-Valid TrimmedPercentage tage AL 2329616996 72.96% 2575622384 86.91%CY 2500120091 80.36% 2335419004 81.37%GR 2909522945 78.86% 2571721724 84.47%MO 2719121851 80.36% 2703722450 83.03%

TABLE III Comparison of the estimated reads,operational taxonomic units(OTUs),and diversity indexes(Shannon,Chao,and ACE)of the prokaryotes and eukaryotes for clustering at 97%sequence similarity based on pyrosequencing data of four types of biological soil crusts(BSCs),algal(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts

BSCs Prokaryotes Eukaryotes Reads OTUs Shannon index Chao index ACE index Reads OTUs Shannon index Chao index ACE index AL 14740 2907 6.37 5405 6772 22217 1057 3.15 1682 2100 CY 17927 3978 7.22 6645 8208 18639 1168 3.87 2025 2591 GR 20556 4327 7.33 7741 9542 21440 1252 3.66 2131 2718 MO 17311 4321 7.17 8187 10803 22411 853 3.01 1443 1800

Fig.1 Rarefaction curves of the number of operational taxonomic units(OTUs)vs.the number of prokaryotic and eukaryotic reads based on pyrosequencing data at the 97%sequence similarity level of four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts.

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Taxonomic composition

Through a taxonomic analysis of sequences,it was found that all samples contained a large number of chloroplast and mitochondrial sequences;especially,the sequences of chloroplasts accounted for 76.2%of the total eukaryotic sequences.The chloroplast-related sequences mainly came from the phylum Streptophyta,which were identified as bryophyta environmental samples at the species level,and there were also a few algal chloroplasts.The mitochondrial sequences were mainly from the Metazoa.The sequences that represented plants and animals were eliminated from the microbial community structure analysis.

Total DNA was extracted from 0.5-g BSC samples following the protocol of a commercial soil DNA isolation kit(Mag-Bind Soil DNA Kit Inc.,Norcross,USA).Each isolated DNA was eluted with 60µL of double-distilled H2O and then stored at−80°C.Each type of the BSCs was subjected to DNA extraction in triplicate and then pooled as a template for PCR amplification.

Fig.2 Venn diagrams showing the numbers of unique prokaryotic(a)and eukaryotic(b)operational taxonomic units(OTUs)at the 97%sequence similarity level as well as occurrence of shared OTUs across four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),or moss(MO)crists.

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PCR amplifications and 454 pyrosequencing were conducted in Majorbio Biotech Company in Shanghai,China.The PCR amplification primers 27F(5′-AGAGTTTGATCCTGGCTCAG-3′)and 533R(5′-TTACCGCGGCTGCTGGCAC-3′)containing the A and B sequencing adaptors(454 Life Sciences,Roche,Branford,USA)were used to amplify a ca.500-bp fragment of the 16S rRNA gene V1–V3 region of prokaryotic microorganism.Eukaryotic microorganism amplification primers which also contained the A and B adaptors were 3NDF(5′-GGCAAGTCTGGTGCCAG-3′)and V4euk R2(5′-ACGGTATCT(AG)ATC(AG)TCTTCG-3′),and the target fragment was the V4 region of 18S ribosomal DNA(rDNA),the length of which was about 450 bp.ABI GeneAmp®9700 PCR(Life Technologies,Waltham,USA)was used for PCRs.All the PCRs were carried out in triplicate in 20-µL volume with 0.4 µL of each primer(2.5 mmol L−1),10 ng of template DNA,4 µL of 5 × FastP-fu buffer,2 µL of dNTPs(2.5 mmol L−1),and 0.4µL of TransStart Fastpfu DNA polymerase(MBI Fermentas,Waltham,USA).The amplification program consisted of an initial denaturation step at 95°C for 2 min,followed by 25 cycles of 94 °C for 30 s,55 °C for 30 s,and 72°C for 30 s,and a final extension of 72°C for 5 min.Replicate PCR products of the same crust sample were pooled within a PCR tube.Then,they were checked on 2%(weight∶volume)agarose gel in Tris-borate-EDTA buffer,and purified with a DNA gel extraction kit(Axygen Bio,San Francisco,USA).

The shared and unique OTUs among the BSC samples are illustrated by a Venn diagram(Fig.2).As shown in Fig.2a,prokaryotic sequence analysis showed that MO crusts had the largest number of own unique OTUs,accounting for 24.0%of the total number(11146)of prokaryotic OTUs.Between CY and GR crusts,the shared OTUs accounted for 14.1%of the total number,and the shared OTUs among all the BSC samples accounted for only 3.3%.As shown in Fig.2b,the unique OTU number of MO crusts was also the highest,and reached 21.6%of the total number(2931)of eukaryotic OTUs;the shared OTUs between CY and GR crusts accounted for 16.4%of the total number,and the shared OTUs among all the samples accounted for only 2.7%.

At the class level,the main prokaryotic sequences belong to α-Proteobacteria,Cyanobacteria,Actinobacteria,Acidobacteria,and Cytophagia,accounting for 64.9%of total prokaryotic sequences.A hierarchical heatmap based on the prokaryotic community at the class level is shown in Fig.4.The sample clustering results showed that MO crusts had the furthest Pearson distance from the others,and the distance between CY and GR crust samples was the smallest.The AL crusts were closer to two lichen crust types in the Pearson distance.Prokaryotic microorganisms could be clustered into multiple groups according to the abundance differences among different crust samples.Bacilli and Cyanobacteria clustered together because their abundance levels were relatively higher in AL crusts than in the others.The abundance levels of Actinobacteria,β-Proteobacteria,and Caldilineae were higher in MO crusts than in others,and they got together as a cluster.The rest of prokaryotes at the class level were more abundant in lichen crusts than in AL and MO crusts.The most abundant sequences of eukaryotic microorganisms belong to classes Sordariomycetes,Dothideomycetes,Eurotiomycetes,Leotiomycetes,and Pezizomycetes,accounting for 68.5%of total eukaryotic sequences.As shown in Fig.5,AL crusts had the most different eukaryotic community composition(at the class level)from that of the others,the two types of lichen crusts also were close to each other,and MO crusts were closer to lichen crusts than to AL crusts.The cluster results on eukaryotic microorganisms showed that Sordariomycetes became a lone cluster because their abundance was far higher in CY crusts than in the others.Besides,the abundance levels of Chlorophyceae,Mucoromycotina,Kickxellomycotina,Bacillariophyceae,and Saccharomycetes were relatively higher in AL crusts than in the others and got together to form a group.Eurotiomycetes and Agaricomycetes had the highest abundance in MO crusts,so they got together to form a cluster.In GR crusts,Dothideomycetes,Orbiliomycetes,Leotiomycetes,and Trebouxiophyceae were the most abundant and clustered together.

Fig.3 Relative abundance of the abundant prokaryotic(a)and eukaryotic(b)phyla revealed by pyrosequencing of four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts.

Fig.4 Heatmap and cluster analyses of prokaryotic microbial communities at the class level in four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts,based on pyrosequencing data.

Fig.5 Heatmap and cluster analyses of eukaryotic microbial communities at the class level in four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts,based on pyrosequencing data.

Relationships between microbial community composition and environmental factors

The structure of prokaryotic and eukaryotic comm-unities at the phylum level was evaluated by detrended correspondence analysis using Canoco 4.5 software.The results showed that the length of the gradient is＜3 standard deviations(SD)and,therefore,the linear model was chosen for the subsequent constrained ordination analyses;that is,redundancy analysis(RDA)was performed to study the effects of environmental factors on abundant phyla.As shown in Fig.6a,the first two axes explained 90.3%and 8.6%,respectively,of the total variation in the relationship of phyla with environmental factors.Only CY and GR crusts were located in the same quadrant,showing that abundant phyla of the lichen crusts were similar,and those of AL and MO crusts were more different.The phylum Cyanobacteria had the highest correlation coefficient with Axis 1;the differences among all crust samples mainly were reflected in Cyanobacteria abundance.Specifically,AL crusts had the most abundant Cyanobacteria,and prokaryotic communities of AL crusts formed a separate group associated with higher sand content.The abundant phyla Proteobacteria and Actinobacteria in MO crusts were closely related to OC,thickness,and TN.The abundance levels of Firmicutes and “Others” were higher in CY crusts than in the rest crust types and were closely related to higher clay content and low EC.Compared with other BSC types,GR crusts had average prokaryotic microorganism and environmental factor gradients.In addition,RDA results demonstrated that moisture(P=1.16,Monte Carlo test)accounted for the greatest amount of variability in the prokaryotic community,followed by sand and silt contents.

标准环境下养护28 d后的试验混凝土，用SCQ-U自动切石机进行切割制备渗透性测试样本。其中，每个试件切出6片分别进行其水和气体渗透性测试的试件(3片用于水渗透测试，3片用于气体渗透测试)。因此，每个规格的混凝土的水和气体渗透测试的试件数量均为6片，以此消除混凝土材料随机性对测试结果的影响。如图2为混凝土水和气体渗透性的样本切割示意图。

The RDA results of eukaryotic phyla and environmental factors(Fig.6b)showed that the first two axes accounted for 98.2%and 1.8%of the total variation in data,respectively.Although CY and GR crusts tended to group together,they were located in different quadrants.Ascomycota and Axis 1 had the highest correlation coefficient;namely,the eukaryotic community composition differences among all crusts were mainly reflected in the Ascomycota abundance.Specifically,AL crusts contained the highest abundance of Chlorophyta and Bacillariphyta relatively,which was closely related to high sand content and low moisture.The phylum Ascomycota enriched in CY crusts was positively correlated with clay content,but negatively with EC.Similarly,the eukaryotic microorganism abundance of GR crusts was also relatively average.The relative abundance of Basidiomycota was the highest in MO crusts and had a relationship with high OC,thickness,and TN.Analysis of RDA further con firmed that clay content(P=0.1740,Monte Carlo test)had the greatest in fluence on eukaryotic community composition,followed by EC.

但迄今对超千米深井不同岩层倾角条件下巷道围岩变形破坏的时空规律鲜见报道[15]，为进一步探讨千米深井高应力巷道围岩的变形演化规律，弄清巷道围岩各部分之间的内在联系，本文以新汶矿区华丰煤矿千米深井地质力学条件为研究背景，利用相似模拟的方法，探讨超千米深井巷道围岩各部位之间变形破坏从时间和空间上的内在联系，以期为超千米深井巷道围岩控制提供理论依据。

DISCUSSION

Fig.6 Redundancy analysis of abundant prokaryotic(a)and eukaryotic(b)phyla and the main environmental factors for individual samples of four types of biological soil crusts(BSCs),alga(AL),cyanolichen(CY),green alga-lichen(GR),and moss(MO)crusts.M=moisture;TN=total N;T=thickness;OC=organic C;EC=electrical conductivity.

The AL,lichen(CY and GR crusts),and MO crusts represent the different stages of succession of the BSC ecosystem(Belnap and Lange,2001).This work used the 454 pyrosequencing approach to compare microbial community composition of BSCs at different successional stages.Compared with the traditional molecular biological approaches(clone library,denaturing gradient gel electrophoresis(DGGE),etc.),the highthroughput sequencing technology can provide more sequence information because of its greater sequencing depth(Roesch et al.,2007;Li et al.,2015).We obtained a total of 81883 valid 16S rRNA gene sequences and 85562 valid 18S rRNA gene sequences;the total sequence number is much greater than that in most other existing small subunit ribosome(SSU)partial fragment high-throughput sequencing studies on BSCs(Meadow and Zabinski,2012;Steven et al.,2012;Abed et al.,2013;Maier et al.,2014;Zhang et al.,2015).Through the comparison of diversity based on OTUs,we found that the middle stage of BSC succession had the highest microbial diversity.Although there were no existing studies that showed a similar pattern of the integrity of microbial diversity during BSC succession,one report(Red field et al.,2002)found that the middle succession of BSCs has the highest diversity of cyanobacteria.We speculated that in early succession,the environmental conditions are harsh,and only some microorganisms with high stress resistance can grow.At the final stage,moss occupied the most space of BSCs,and some part of BSCs turned into an anaerobic environment;these conditions may affect most microbes.

In this study,a total of 104583 valid prokaryotic sequences and 101864 valid eukaryotic sequences were obtained by 454 pyrosequencing,with the average length of 410 and 419 bp,respectively.After screening and optimization,we got 81883 of trimmed(optimized)16S rDNA sequence fragments with the average length of 449 bp(20470.8 ± 2597.8 reads sample−1)and 85562 of trimmed 18S rDNA sequence fragmentswith the average length of 449 bp(21390.5±1624.4 reads sample−1).The detailed valid and trimmed sequence numbers in each crust type can be found in Table II.After the OTU clustering of the trimmed sequences at the 97%similarity level,the prokaryotic sequences were divided into 11146 OTUs(3883.3±671.0 OTUs sample−1),and the eukaryotic sequences were divided into 2931 OTUs(1082.5±172.6 OTUs sample−1).As shown in Table III,the OTU number of 16S rDNA fragment sequences ranged from 2907 to 4327 among samples,and MO crusts had the greatest,while AL crusts the smallest number.The OTU numbers of 18S rDNA fragment sequences were also different among the samples,of which MO crusts had the lowest OTU number,and the GR crusts the largest.

Most of the Proteobacteria are copiotrophic bacteria and are closely related to the existence of a large number of available nutrients(Fierer et al.,2007;Zhao et al.,2014).Results of RDA also showed that Proteobacteria were positively correlated with OC and TN contents.As the final stage of BSC succession,MO crusts accumulated the most nutrients;these findings explained well why Proteobacteria themselves were enriched in MO crusts.Meanwhile,Proteobacteria themselves are effective in maintaining the stability of the BSCs and biogeochemical cycle;for example,some of Proteobacteria can secrete a sticky protein,promote the adhesion of soil particles,and some are capable of heterotrophic photosynthesis,including Methylobacterium,Belnapia,and Sphingomonas(Maier et al.,2014),and they were also detected in this work.Consistent with our results,previous studies also found that Actinobacteria are common and diverse in soil(Zhao et al.,2014)and play an important role in the formation and stability of BSCs(Gonz´alez et al.,2005;Gundlapally and Garcia-Pichel,2006).

As the important oxygen-producing photosynthetic organism in BSCs,cyanobacteria played the role of primary producers,and its abundance was the most different among different successional stages of BSCs(Fig.6a).With the development of BSCs,the abundance of cyanobacteria tended to decrease,which was in accord with previous research results(Red field et al.,2002;Nagy et al.,2005;Rivera-Aguilar et al.,2006).The reasons may be related to the improvement of environmental conditions;consequently,other heterotrophic microorganisms can multiply remarkably and squeeze the living space of cyanobacteria(Lan et al.,2013).Results of RDA revealed that the abundance of cyanobacteria was positively correlated with sand content and negatively correlated with moisture.We speculated that the high sand content and soil porosity at the early successional stage of BSCs were more advantageous for mobile filamentous Cyanobacteria(e.g.,Microcoleus)for obtaining light,water,and other resources(Hu et al.,2003).However,at the end of the succession of BSCs,the clay content increased,porosity declined,and thickness increased;thus,it became harder for Cyanobacteria to move,and therefore Cyanobacteria could not effectively utilize light and water within a short period,such as after rain,and their survival advantage was also certainly in fluenced.In addition,Zaady et al.(2010)suggested that different aridity levels correspond to different BSC successional stages.Therefore,moisture may mainly affect Cyanobacteria abundance by reflecting the development direction of BSCs.However,the water type that really affected cyanobacteria was mainly the episodic water,like condensate water,and this type of water was hard to measure accurately.

Bacteroidetes have been reported commonly in previous studies on BSC microorganisms;therefore,some of them can secrete large amounts of extracellular polysaccharides and are helpful for the formation of BSCs(Nagy et al.,2005;Gundlapally and Garcia-Pichel,2006).Relatively,the abundance of Bacteroidetes was the highest at the middle successional stage and positively correlated with clay content.Thus,we speculated that in lichen crusts,clay content was higher and Bacteroidetes could adhere to soil more tightly,which might be helpful for the stable structure of lichens.Acidobacteria are heterotrophic bacteria with highly diverse functions,but only a small percentage of Acidobacteria have been cultured(Ward et al.,2009),and Eichorst et al.(2011)suggested that their abundance correlates with plant polymers in soil.As acidophilic bacteria,the relative abundance of Acidobacteria is also related to pH(Lauber et al.,2009).However,because the pH value showed no significant differences among our samples,the in fluence of pH on Acidobacteria was not obvious.In addition,our study did not detect archaea,but Soule et al.(2009)found that although the diversity of archaea in BSCs is limited,Archaea themselves are very common in BSCs.Bates et al.(2011)found that the most common phylum of archaea is Crenarchaeota.We speculated that the remarkable discrepancy may be due to the differences in primers,and it may be difficult to amplify the archaea DNA with the primers we used.

As to the existing studies on the eukaryotic community composition of BSCs,most focus on the diversity and composition of fungi and eukaryotic algae.As for the composition and diversity of fungi,Aebed et al.(2013)found that more than 96%of the sequences belong to Ascomycota,Basidiomycota,and Chytridiomycota in BSCs using fungi-specific primers and pyrosequencing.Our results were similar,though we used prokaryotic universal primers.Furthermore,the research on the eukaryotic algae in BSCs mainly involves traditional culture methods(Rivera-Aguilar et al.,2006;Langhans et al.,2009;Zhang et al.,2009,2011).Very few researchers have surveyed the whole eukaryotic microbial components synchronously.For example,Meadow and Zabinski(2012)via the pyrosequencing approach found that the eukaryotic microorganisms have spatial heterogeneity and the main eukaryotes are diatoms and fungi.Because of the different study sites,their results are different from those of our work.Our results mainly showed that only fungi were the most abundant eukaryotic microorganism,especially in lichen crusts.In lichen crusts,the formation of a symbiont structure that was composed of fungi and algae may be the main reason why fungi increased in abundance obviously(Wu et al.,2013).Although the eukaryotic algae were not abundant in our BSCs,Chlorophyta and Bcillariophyta had the highest abundance at the early successional stage,which has not been reported before.This is probably because there was no sufficient organic matter at the early stage and,therefore,the heterotrophic eukaryotic microorganisms were deficient.We also found that on average,about 20%of the eukaryotic sequences shared a low similarity with sequences in public databases,indicating that there were a large number of new unknown eukaryotic microorganisms in our samples.The RDA results showed that the differences among crust samples were mainly reflected in Ascomycota,Chytridiomycota,and “Others” abundance levels.The abundance of Ascomycota and clay content were positively correlated,and Chytridiomycota and “Others”abundance levels had a positive correlation with EC.Clay content may affect the fungi abundance by in fluencing the soil gap and available nutrients.Electrical conductivity revealed the ion concentration differences and may have in fluences on the osmotic regulation of microorganisms.Because the in fluenced “Others” are mainly unclassified eukaryotic microorganisms,their acclimatization mechanisms are not clear;therefore further indepth studies are needed.

CONCLUSIONS

Using the 454 pyrosequencing analysis of four BSC types,we obtained a detailed structure of the microbial community and found that the microbial community composition shifted remarkably during the process of development and succession of BSCs,but the composition was relatively close in two types of lichen crusts with the different dominant symbiotic algae.We also found that moisture and mechanical composition had the greatest effects on the prokaryotic community structure,whereas the in fluences of EC and mechanical composition were the greatest on eukaryotic community composition.This work also con firmed that the high-throughput sequencing technology is suitable for surveying microbial community composition of BSCs rapidly and comprehensively.

ACKNOWLEDGEMENT

This work was supported by the National Natural Science Foundation of China(Nos.31170464,41573111,and 31300322).

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