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Bacterial Communities in Soil Under Moss and Lichen-Moss CrustsYendi E. Navarro-Noya a , Angélica Jiménez-Aguilar a , César Valenzuela-Encinas b , RocioJ. Alcántara-Hernández c , Víctor M. Ruíz-Valdiviezo a , Alejandro Ponce-Mendoza d , MarcoLuna-Guido a , Rodolfo Marsch a & Luc Dendooven aa Laboratory of Soil Ecology , ABACUS , Cinvestav , México D. F. , Méxicob Department of Chemistry , Unidad Profesional Interdisciplinaria de Biotecnología-IPN(UPIBI) , México D. F. , Méxicoc Laboratorio de Ecología Bacteriana y Epigenética Molecular , Instituto de Ecología , UNAM ,México D. F. , Méxicod Laboratorio de Ecología Microbiana, Departamento El Hombre y su Ambiente , UniversidadAutónoma Metropolitana-Xochimilco , México D. F. , MéxicoAccepted author version posted online: 15 Jul 2013.Published online: 02 Dec 2013.

To cite this article: Yendi E. Navarro-Noya , Angélica Jiménez-Aguilar , César Valenzuela-Encinas , Rocio J. Alcántara-Hernández , Víctor M. Ruíz-Valdiviezo , Alejandro Ponce-Mendoza , Marco Luna-Guido , Rodolfo Marsch & Luc Dendooven(2014) Bacterial Communities in Soil Under Moss and Lichen-Moss Crusts, Geomicrobiology Journal, 31:2, 152-160, DOI:10.1080/01490451.2013.820236

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Geomicrobiology Journal (2014) 31, 152–160Copyright C© Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490451.2013.820236

Bacterial Communities in Soil Under Mossand Lichen-Moss Crusts

YENDI E. NAVARRO-NOYA1, ANGELICA JIMENEZ-AGUILAR1, CESAR VALENZUELA-ENCINAS2, ROCIO J.ALCANTARA-HERNANDEZ3, VICTOR M. RUIZ-VALDIVIEZO1, ALEJANDRO PONCE-MENDOZA4, MARCOLUNA-GUIDO1, RODOLFO MARSCH1, and LUC DENDOOVEN1∗

1Laboratory of Soil Ecology, ABACUS, Cinvestav, Mexico D. F., Mexico2Department of Chemistry, Unidad Profesional Interdisciplinaria de Biotecnologıa-IPN (UPIBI), Mexico D. F., Mexico3Laboratorio de Ecologıa Bacteriana y Epigenetica Molecular, Instituto de Ecologıa, UNAM, Mexico D. F., Mexico4Laboratorio de Ecologıa Microbiana, Departamento El Hombre y su Ambiente, Universidad Autonoma Metropolitana-Xochimilco,Mexico D. F., Mexico

Received March 2013, Accepted June 2013

Biological soil crusts are symbiotic microbial communities formed by green algae, mosses, fungi, lichens, cyanobacteria and bacteriain different proportions. Crusts contribute to soil fertility and favour water retention and infiltration. However, little is known aboutthe bacterial community structure in soil under the crusts. Soil was sampled under a moss crust (considered the MOSS group),lichen plus moss (considered the LICHEN group) and bare soil (considered the BARE group) and the microbial communitiesdetermined using nearly full 16S rRNA gene libraries. Bacteria belonging to seven different phyla were found and the Acidobacteriaand Alphaproteobacteria were the dominant in each group. The crusts affected negatively the abundance of the Burkholderiales. Thephylogenetic diversity and bacterial community membership were different in the LICHEN group compared to the BARE and MOSSgroups, but not species richness and community structure. The beta diversity analysis also revealed a different bacterial communitystructure beneath the LICHEN and MOSS crusts, suggesting species-specific influence. This is a first insight into the effect of abiological soil crust on the bacterial community structure in an organic matter rich soil of a high altitude mountain forest.

Keywords: 16S rRNA gene libraries, forest soil, high altitude mountain, symbiotic relations, UniFrac analysis

Introduction

The upper layer of soil favors symbiotic microbial communi-ties in what is named biological soil crusts (BSC). The BSC areformed by green algae, mosses, fungi, lichens, cyanobacteriaand bacteria in different proportions (Marsh et al. 2006). It iswell documented that BSC play an important role in ecosys-tem functioning and nutrient cycling (Bowker et al. 2005).Soil crusts contribute to soil fertility by fixing atmospheric N2(principally cyanolichens and associated free-living bacteria)and CO2, thereby increasing soil organic matter (Abed et al.2010; Belnap 2002b; Zhao et al. 2010).

Many of the studies of the biology, functionality and bio-geography of the BSC focus on arid and semi-arid environ-ments (Belnap 2002a; Bowker et al. 2005; Castillo-Monroyet al. 2011; Eldrich 2001; Housman et al. 2006) as the crustsare related to a first successional stage in extreme conditionsthereby increasing the inorganic and organic nutrient contentin soil (Veluci et al. 2006). However, the distribution of BSC

∗Address correspondence to Luc Dendooven, Laboratory ofSoil Ecology, ABACUS, Cinvestav, Av. I.P.N. 2508, C.P. 07360,Mexico D. F., Mexico; Email: dendooven@me.com

can occur in a wide range of environments and can be foundin shrub-steppe grasslands, wet polar areas, tropical moun-tains and alpine ecosystems (Cardinale et al. 2012). In someenvironments, crust cover all bare soil providing 70% of theground cover (Evans and Johansen 1999).

Different types of soil crusts have been defined, mostlybased on their composition. Depending on the dominant or-ganism group, e.g., cyanobacteria, lichen or moss, differenttypes of BSC can be differentiated (Root and McCune 2012).The contribution of the crusts to C and N cycling varies greatlydepending on species composition (Housman et al. 2006), theassociation between them and the lichen and/or mosses (Bateset al. 2011), rainfall (Eldridge 2001), and soil texture and pH(Belnap 2002a). Crusts also favor water retention and infiltra-tion, provide microhabitats for other species, modify the pH ofthe soil underneath and protect the soil from wind and watererosion and temperature fluctuations during the day (Jimenez-Aguilar et al. 2009; Eldridge et al. 2010). The bacterial popu-lations composing the microbial community of the BSC havebeen studied intensively (Abed et al. 2010; Castillo-Monroyet al. 2011; Jimenez-Aguilar et al. 2009; Marsh et al. 2006).However, the effect of the BSC on the soil bacterial commu-nities underneath the crust is still unknown. Our hypothesis isthat the changes generated by the BSC could have an effect in

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the bacterial community structure in the soil underneath thecrust, similarly to the rhizospheric effect of vascular plants onsoil microbial communities.

The ‘Pico del Aguila’ can be found to the southwest ofMexico City and reaches 3894 m. This high altitude naturalreserve is one of the last remaining green areas in Mexico Cityand important for capturing water and replenishing aquifers ofthis megapolis. The vegetation at the natural reserve is mostlygrass with some trees and different types of soil crusts can befound (Garcia-Aguirre et al. 2007). The microbial compositionof different crusts, e.g. lichen crust (Castillo-Monroy et al.2011), lichen crust thalli of rock outcrops (Bates et al. 2011)and squamous lichen crust (Placidium spp.) (Nagy et al. 2005)have been studied. As such, the objectives of this study wereto i) describe the bacterial populations in the soil under thecrusts and ii) compare them with the bacterial communities inthe bare soil.

Materials and Methods

Site Description and Sampling Characteristics

The study area was located on the Pico del Aguila, San NicolasTotolapan (D.F., Mexico) (latitude 19◦ 12′ 46.8” N, longitude99◦ 15′ 25.2” W, 3894 m.a.s.l.). Major relief shaping factorsof this site were volcanic activity and glacial phenomena. Theeast slope is defined as volcanic rocks of the Mid Tertiary(Schlaapfer 1968). The oldest rocks are known as andesitic se-ries (Mooser 1957). The vegetation is dominated by the grassesFestuca amplissima Rupr. and Muhlenbergia macroura (Kunth)Hitchc. (Garcia-Aguirre et al. 2007). At the nearest climatol-ogy station, i.e., “Monte Alegre” at 2420 m.a.s.l., the climateis temperate and humid. The average annual temperature is8.1◦C and the average annual precipitation is 1341 mm with1145 mm between May and October.

In the Pico del Aguila, the soil bacterial communities wasdetermined under two different morphotypes of BSC natu-rally found in the study area. Since the main objective of thisstudy was to determine the soil bacterial community in soilunder two types of biological soil crusts, only morphotypeswith high abundance and with a minimum surface area of10 cm × 10 cm were selected to assure that bacterial commu-nities under BSC were well developed. The first morphotypewas composed of mosses only: Polytrichum sp. and Breuteliasp. (considered the MOSS group), and the second morpho-type composed by lichen (Cladonia sp.) and moss (of the orderBryales) (considered the LICHEN group). Also, soil visuallybarren of soil crust was referred to as the BARE group. Crustsamples were collected at three locations within an area of 2ha. At each site, three composite samples were collected frompoints within 50 m of one another. As such, a total of ninesoil samples were obtained. Each composite sample consistedof five subsamples taken from beneath five areas covered byeach BSC. To collect each subsample, the soil crust was sep-arated from the soil and discarded. A 1-cm deep soil cubewas taken. Each subsample was put in sterile bags. The soilsamples were placed on ice and taken to the laboratory. In thelaboratory, the nine soil samples were 1-mm sieved separately.This field-based replication was maintained in the study.

Soil Characterization

Water content, water holding capacity (WHC), electrolyticconductivity (EC), soil particle size distribution, pH, organicC and total N of the different samples were determined asdescribed in Franco-Hernandez et al. (2003).

Metagenomic DNA Extraction and Amplification of Bacterial16S rRNA Genes

The DNA was directly extracted from soil associated to thecrusts. The technique used was based on the method de-scribed by Ceja-Navarro et al. (2010). The universal bacterialprimers (Relman 1993) were used for amplification of nearlyfull-length, ca. 1500 bp-long, 16S rRNA gene segments. Am-plification conditions included a denaturation step at 94◦Cfor 10 min followed by 20 cycles at 93◦C for 1 min, at 57◦Cfor 1 min, at 72◦C for 2 min, and a final step at 72◦C for10 min. The amplification was done using Touchgene Gradi-ent (Techne, Cambridge United Kingdom).

Construction of the Ribosomal RNA Libraries and Sequencing

PCR amplicons from five reactions were pooled, purified withthe UltraCleanTM PCR Clean-up (MoBio, Carlsbad, CA,USA) and used to clone in the pCR2.1 vector of the TOPO R©TA cloning kit (Invitrogen, Carlsbad, CA). The constructionswere transformed in chemo-competent cells of Escherichiacoli DH5α (Invitrogen). Colonies were grown and selectedin 96 well plates in Luria-Bertani broth supplemented withKanamycin 50 μg/L. They were incubated at 37◦C all night,40% glycerol was added to each well, the plates were shakenand centrifuged at 500 rpm at 4◦C for 1 min. The plates werestored at –60◦C until send to LANGEBIO (Laboratorio Na-cional de Genomica para la Biodiversidad, Irapuato, Mexico)for sequencing.

Phylogenetic Analyses and Statistical Analysis

All the sequences obtained from the nine soil samples, i.e., atotal of 1549 sequences, were aligned using the NAST toolfrom Greengenes (DeSantis et al. 2006) and chimeras weredetected using Bellerophon v. 3.0. The screened sequences(1062) were imported to the QIIME software pipeline ver-sion 1.5.0 (Caporaso et al. 2010b). Operational taxonomicunits (OTU) were determined by using the Uclust OTUpicker version 1.2.21q at a similarity threshold of 97% (Edgar2010). The representative sequence for each of the clus-ters were aligned to the Greengenes core–set–aligned avail-able at http://greengenes.lbl.gov/ using PyNast version 1.1(Caporaso et al. 2010a). The minimum percent sequenceidentity to include a sequence in the alignment was set at75%. Taxonomy assignation was done by using the naıveBayesian rRNA classifier from the Ribosomal Data Project(http://rdp.cme.msu.edu/classifier/classifier.jsp) (Wang et al.2007) at a confidence threshold of 80%. Rarefaction, richnessestimators and diversity indices were calculated by consecu-tive sub–sampling the population of bacterial occurrences foreach soil sample.

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Fig. 1. Alpha diversity rarefaction of the bacterial communities in the bare soil (BARE) and soil sampled under lichen plus moss(LICHEN) and moss crust (MOSS) as revealed by 16S rRNA libraries. Error bars represent intervals of confidence.

The phylogenetic community analysis was done using FastUniFrac (Hamady et al. 2010) within the QIIME pipeline.The representative sequence set alignment was used to con-struct a bacterial phylogeny using FastTree with a maximumlikelihood approximation method (Price et al. 2009). This treedata served as input for the UniFrac analysis, resulting in apairwise distance matrix of soil samples. Principal coordinateanalyses (PCoA) were done using the pairwise distance matrixof soil samples with weighted and unweighted distances. Per-mutational multivariate analysis of variance (perMANOVA)was done to test significant differences between bacterial com-munities from the different groups (n = 999) with the scriptcompare categories.py within QIIME. As only three replicatesfor each soil crust were used, the rejection of the null hypoth-esis of no difference was based on an adjusted alpha value of0.1.

A UniFrac analysis was done to describe the dissimilar-ity among soil crust samples and measure the fraction of the

branch length in a phylogenetic tree that is unique to any com-munity (Lozupone and Knight 2005). Evolutionary distancecould be more powerful for understanding differences in com-munities, because distant branches of the microbial phylogenyon average have more dissimilar community functions (func-tional diversity) than closely related bacterial lineages. Theunweighted approach calculates the ratio of branch lengthunique to any community to the total branch length in thetree. The weighted approach divides the total branch length ofa tree among the different communities and the frequency ofoccurrences of bacterial phylotypes observed among samples(Lozupone et al. 2007).

Nucleotide Sequence Accession Numbers

The sequences were deposited in the GenBank database andassigned the accession numbers JN023098-JN024099.

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Bacterial Communities under Biological Soil Crusts 155

Results

Soil Characteristics

The soil characteristics were not significantly different betweenthe samples (ANOVA test). The soil had an acid pH (5.46 ±0.23) with electrolytic conductivity 0.11 ± 0.02 dS/m. The wa-ter content was 402 ± 49 g/kg and the water holding capacity1440 ± 246 g/kg. The particle size distribution of the silt loamsoil was 40 g clay/kg soil, silt 550 g/kg soil and sand 410 g/kgsoil.

The soil organic C content and total N content was similarfor the different groups. The organic C of the BARE soil was166 ± 40 g C/kg and the total N content 6.90 ± 1.70 g N/kgdry soil, while that of the MOSS group was 179 ± 31 g C/kgand 7.39 ± 0.35 g N/kg, and of the LICHEN group 171 ±50 g C/kg and 7.39 ± 0.60 g N/kg.

Alpha Diversity Analysis

From a total of 1064 sequences with an average of 1460 pblong, 280 different OTUs were detected at similarity thresh-old of 97%. Rarefaction analysis of different alpha diversityparameters showed that similar species richness and hetero-geneity was found in the groups, as revealed by the Chao1estimator and Shannon index (H’) (Figure 1). However, sig-nificant difference in terms of phylogenetic diversity (PD) wasfound (p < 0.05). In general, the LICHEN group showed lowerdiversity compared with the BARE and MOSS groups. H’ andPD were similar for BARE and MOSS groups.

Bacterial Community Composition

OTUs belonging to six different phyla were found at the sam-pling sites, i.e., Acidobacteria, Actinobacteria, Chloroflexi,Firmicutes, Gemmatimonadetes and Proteobacteria (Fig-ure 2A). Of all the OTUs, 2.3% remained unclassified. OTUsclassified into the Acidobacteria phylum (between 39.8 ± 12.8and 51.3 ± 8.3%) were the most abundant followed by thosebelonging to the Proteobacteria (between 36.9 ± 6.8 and39.1 ± 18.3%). Among Proteobacteria, OTUs classified intothe class of the Alphaproteobacteria were the most abundantand Betaproteobacteria the least (Figure 2A). Most OTUsthat belonged to the Acidobacteria were identified within theorder Gp1 and the least within the orders Gp4 and Gp7 (Fig-ure 2B). The order of Burkholderiales differed significantlyamong LICHEN, MOSS and BARE (F = 6.05, p < 0.05).The most abundant order within the Alphaproteobacteriawas Sphingomonadales that also included the most abundantgenus, i.e., 11.76% of the overall OTUs belonged to the genusSphingomonas (Table 1). The second most abundant genuswas Steroidobacter (Xanthomonadales, Gammaproteobacte-ria) with an average of 4.88% of all the sequences.

Among the OTUs identified at genus level (Table 1), 65.64%of the genera, i.e. Iamia, Ktedonobacter, Rhodoplanes, Sphin-gomonas, Steroidobacter and the Acidobacteria GP1, GP3,GP6 and GP17, were found in the three crust groups. OTUsbelonging to the Pseudomonas, Caulobacter and Cellvibriogenera were detected uniquely in the soil under the crusts.

Table 1. Percentages of clones belonging to different genera andAcidobacteria orders based on the ribosomal data project in baresoil (BARE), soil under lichen and moss (LICHEN) and undermoss (MOSS)

BARE LICHEN MOSS TotalSequences 341 370 351 1062Taxon (%)

Gp1a 35.02 33.78 30.94 33.25Sphingomonas 4.81 11.61 18.85 11.76Gp3 5.59 7.43 6.50 6.51Steroidobacter 6.62 2.59 5.44 4.88Gp6 8.19 3.81 2.15 4.72Iamia 1.99 2.55 0.54 1.69Rhodoplanes 1.11 1.68 1.35 1.38Pseudolabrys 2.77 1.05 0.00 1.28Dokdonella 2.40 0.86 0.00 1.09Gp17 0.94 1.05 0.27 0.75Pseudomonas 0.00 1.98 0.27 0.75Gemmatimonas 0.55 0.00 1.61 0.72Ktedonobacter 0.89 0.95 0.27 0.70Aquicella 0.84 0.95 0.00 0.60Novosphingobium 0.55 0.00 1.10 0.55Gp5 0.95 0.35 0.00 0.43Gp2 0.61 0.64 0.00 0.42Methylibium 1.17 0.00 0.00 0.39Stella 0.89 0.00 0.27 0.39Anaeromyxobacter 0.00 0.00 1.07 0.36Caulobacter 0.00 0.49 0.27 0.25Bradyrhizobium 0.00 0.74 0.00 0.25Massilia 0.67 0.00 0.00 0.22Cellvibrio 0.00 0.25 0.27 0.17Rhodanobacter 0.00 0.49 0.00 0.16Gp7 0.00 0.35 0.00 0.12Mesorhizobium 0.33 0.00 0.00 0.11Dyella 0.33 0.00 0.00 0.11Rhodoferax 0.28 0.00 0.00 0.09Rudaea 0.28 0.00 0.00 0.09Hyphomicrobium 0.00 0.25 0.00 0.08

aGp1, Gp3, Gp6, Gp17, Gp5, Gp2, Gp7: Acidobacteria groups.

Comparisons of the Bacterial Communities

When the membership of the phylogenetic branches constitut-ing the bacterial communities were compared, i.e., unweightedUniFrac distances, the bacterial communities of the LICHENgroup were separated from the BARE and MOSS groups (Fig-ure 3). However, when the weighted data were considered noclear pattern was found. Less variation was found in the BAREand MOSS groups than in the LICHEN group. Significant dif-ferences were found between the groups considering the un-weighted distances (Table 2) and the grouping of the BAREand MOSS groups was also significantly different from theLICHEN group (p = 0.008).

Discussion

Bacterial Composition at Phylum and Class Level

It was found that bacterial communities in soil under baresoil or BSCs with moss only differed from those under

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Fig. 2. Taxonomic distribution of A) phyla and four classes of Proteobacteria and B) order level, of the bacterial communities in baresoil (BARE) and soil sampled under lichen plus moss (LICHEN) and moss crust (MOSS) as revealed by 16S rRNA libraries. GP1:Acidobacteria Gp1. Error bars represent the standard deviation.

BSCs composed of moss and lichen. Acidobacteria andProteobacteria were the most dominant phyla in the differentsoil samples. The dominance of these two phyla has beenreported for different soils (Janssen 2006). For instance, Linet al. (2011) studying soil bacterial communities in nativeand regenerated perhumid montane forests found a similardominance of these two phyla as did Zhang et al. (2011)while studying the rhizosphere of cotton. In the bacterialcommunities associated with lichen, however, Acidobacteriarepresented between 10–20% of the OTUs (Bates et al. 2011).

Acidobacteria are characterized as versatile heterotrophs,among the most abundant bacteria in soil and their abun-dance is increased by the plant polymers (Eichorst et al. 2011).Only a few of the known Acidobacteria have been isolated(Kishimoto et al. 1991; Hiraishi et al. 1995; Ward et al. 2009),while the majority remain uncultured although they are abun-dant in soil. Low percentages of Firmicutes have generallybeen reported in soil. Lin et al. (2011) reported percentagesof Firmicutes ranging from 0.3 to 2.7% and Nacke et al.(2011) reported that members of this phylum represented

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Fig. 3. Principal coordinate analysis using weighted and unweighted UniFrac with the different sequences found in the three replicatesof bare soil (BARE), in the soil under moss (MOSS) and under moss plus lichen (LICHEN). The unweighted approach calculatesthe ratio of branch length unique to any community to the total branch length in the tree. The weighted approach divides the totalbranch length of a tree among the different communities and the frequency of occurrences of bacterial phylotypes observed amongsamples (Lozupone and Knight 2005).

only 1.2% of the total sequences, while Will et al. (2010)reported 3.2%.

However, in Colorado plateau soil crusts (5.2%) and hyper-arid deserts soil gypsum crusts (12% to 18%) percentages ofFirmicutes were high (Gundlapally and Garcıa-Pichel 2006).In this study, Firmicutes comprised between 5.52 ± 2.87 and13.74 ± 2.32% of all sequences. However, using nearly-full16S rRNA gene sequence the OTUs were identified up tophylum level, this means that the sequences did not cluster

Table 2. PERMANOVA analyses testing the effect of biologi-cal soil crusts on the soil bacterial community structure usingUniFrac pairwise distances

UniFrac Distances

Unweighted Weighted

Comparison F value p value F value p value

BARE versus LICHENversus MOSS

1.37 0.028 1.08 0.377

Bare versus Crusts 1.22 0.132 1.30 0.243LICHEN versus MOSS 1.27 0.208 0.76 0.602BARE versus LICHEN 1.70 0.090 1.68 0.098BARE versus MOSS 1.13 0.200 0.77 0.587BARE + MOSS versus

LICHEN1.52 0.008 1.74 0.107

with knowing orders or even classes among Firmicutes in thepublic databases. This suggested that novel Firmicutes weredetected in this poorly investigated environment. The same oc-curred with Sphingomonadaceae members in the mycosphereof Laccaria proxima (Scop.) Cooke and Russula exalbicans(Pers.) Melzer and Zvara (Boersma et al. 2009).

Alphaproteobacteria (19.7 ± 8.0 to 28.0 ± 15.5%) were themost abundant class within the Proteobacteria and Betapro-teobacteria the least (0 to 2.3 ± 0.4%). A similar distributionwas reported previously in different forest soils (Lin et al. 2011;Nacke et al. 2011). Alphaproteobacteria were also found to bethe most abundant group of lichen-associated bacteria (Bateset al. 2011; Cardinale et al. 2006; Cardinale et al. 2012; Grubeet al. 2009).

Bacterial Composition at Order and Genus Level

The Sphingomonadales with their abundance ranging from5.6 ± 3.0 to 20.5 ± 11.3% were the most dominant Al-phaproteobacteria. Within the Sphingomonadales, the genusSphingomonas was the most abundant in the three groups.Sphingomonas is a well studied genus of bacteria and oftenabundant in soil. OTUs belonging to this genus have beenfound in soil crusts (Csotonyi et al. 2010), they have thecapacity to degrade cellulose and posses several plant growthpromoting traits (Kang et al. 2007).

The relative abundance of Burkholderiales was signif-icantly lower in the two types of crust. Generally, large

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proportions of Burkholderia spp. have been detected in lichen-associated bacterial communities by different techniques, e.g.,culture-dependent and FISH (fluorescence in situ hybridiza-tion) (Cardinale et al. 2006; Grube et al. 2009). Pseudomonas,Caulobacter and Cellvibrio were detected uniquely in the soilunder the crusts. Pseudomonas and Caulobacter were foundfrequently associated with BSCs (Bates et al. 2011; Liba et al.2006). Culture-dependent approaches have revealed that theproduction of growth promoting substances and nutrientacquisition, e.g. solubilize phosphate and release amino acidsand phytohormones, could be involved in the promotion ofthese organisms (Liba et al. 2006).

Differences in the Bacterial Diversity and CommunityStructure

In general, the diversity in terms of H’ and PD was lower in theLICHEN group than in the MOSS and BARE groups, whilespecies richness was similar. Phylogenetic diversity is a morepowerful indicator of diversity than other ecological statis-tics based on frequency and occurrence, because distant ordeep phylogenetic branches of the microbial phylogeny surelyhave more different functions than those closely related (Faith1992). It is probable that a selective effect is being exerted onthe bacterial community in the soil under the lichen crust.An enlargement of the species richness and diversity could beexpected due to the organic molecules exuded by the plants,as is found in the rhizosphere of plants. However, it was alsoshown that roots of vascular plants, select for protective bac-terial populations against phytopathogens (Berendsen et al.2012). In the mycosphere of fruiting bodies of the ectomyc-orrhizal fungi L. proxima and R. exalbicans growing in for-est soil, also favoured members of the Sphingomonadaceae(Boersma et al. 2009), consequently, reducing the speciesrichness.

The beta diversity analysis revealed that the bacterial com-munity composition of the LICHEN group was different fromthe MOSS group, and that the bacterial community structuresof the BARE and MOSS soils were similar. There exists alsostrong evidence that the bacteria associated with lichen sym-biosis are selected specifically from the surrounding soil by thedifferent species in the lichen (Bates et al. 2011; Grube et al.2009). Besides, several soil factors, such as pH, organic-C con-tent, that are known to affect the bacterial community struc-ture were similar in the three groups studied. No differencesin the BARE and MOSS bacterial communities were found,presumably due to similar nutrient availability. The moss crusthas been found to have lower nitrogen fixation rates comparedwith lichen or cyanobacterial-algal crusts (Su et al. 2011).

It is clear that replication is the fundamental way to quan-tify random and systematic variation in a environmental sys-tem (Lennon 2011). There is risk of drawing weak or invalidconclusions when unreplicated data is used in an experimen-tal design. This has been addressed by taking samples ofthe three groups of crusts at three different locations. Al-though this is still a limited number of samples, our studyprovides a first insight on the bacterial communities under soilcrusts.

Conclusions

The dominant taxa in all groups were Acidobacteria andAlphaproteobacteria. However, an effect on the phylogeneticdiversity and bacterial community membership, but not in thespecies richness and community structure, was observed inthe LICHEN crust compared to the BARE and MOSS soils.The beta diversity analysis also revealed different structureon the soil bacterial communities beneath the LICHEN andMOSS crusts. It was found that bacterial communities in soilunder bare soil or BSCs with moss differed only from thoseunder BSCs composed of moss and lichen.

Acknowledgments

This work was funded by ‘Instituto de Ciencia y Tecnologıa delDistrito Federal’ (ICyTDF) and Cinvestav (Mexico). Y.E.N.-N. received grant-aided support from ABACUS and ‘ConsejoNacional de Ciencia y Tecnologıa’ (CONACyT) and A. J.-A.from ICyTDF.Yendi E. Navarro-Noya and Angelica Jimenez-Aguilar con-tributed equally to this study

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