Fungal communities of lichen-dominated biological soil crusts: Diversity, relative microbial biomass, and their relationship to disturbance and crust cover

Journal of Arid Environments 74 (2010) 1192e1199

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Fungal communities of lichen-dominated biological soil crusts: Diversity, relative microbial biomass, and their relationship to disturbance and crust cover Scott T. Bates a, b, *, Thomas H. Nash III a, Ken G. Sweat a, Ferran Garcia-Pichel a a b

School of Life Sciences (SoLS), Arizona State University, Tempe, AZ 85287-4501, USA Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, CO 80309, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 August 2009 Received in revised form 21 April 2010 Accepted 31 May 2010 Available online 25 June 2010

Molecular methodologies were used to characterize fungal communities associated with lichen-dominated biological soil crusts (BSCs) at two sites on the Colorado Plateau (USA) in order to investigate their diversity and abundance, in relation to that of bacteria, as well as how these parameters corresponded to overall soil crust cover and the presence of anthropogenic disturbance. Fungal community diversity and composition were assessed with denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR amplified ribosomal genes and by sequencing. Quantitative PCR, specific for fungi as well as bacteria, was used to evaluate relative microbial densities. Two sites with similar soil characteristics, both of which contained well developed BSCs dominated by lichens, were studied. Results indicated that while a considerable diversity of fungi is present within these BSCs, much higher than what has previously been determined for cyanobacteria-dominated crusts, fungi contribute less biomass and are less diverse than their bacterial counterparts. Fungal diversity in lichen-dominated BSCs was negatively correlated with disturbance and positively correlated with crust cover. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Bacteria Biological soil crust Colorado Plateau DGGE Fungal diversity Lichens

1. Introduction Biological soil crusts (BSCs) are formed by communities of microorganisms that bind together the upper strata of soil (Belnap and Gardner, 1993). Known alternatively as cryptogamic, cryptobiotic, cyanobacterial, or microbiotic soils, BSCs are common in arid lands, such as those found in the southwestern United States. These communities are comprised of varying assemblages of cyanobacteria, eukaryotic microalgae, fungi, lichens, mosses, as well as other bacteria, and archaeal populations have also been documented recently from BSCs (Soule et al., 2009). Of these organisms, it is cyanobacteria that have a central position in establishing and maintaining the crust community (Garcia-Pichel and Wojciechowski, 2009). The many functional roles BSCs play in ecosystems (e.g., soil hydrology and soil stabilization) have been documented previously (Belnap and Lange, 2001; Evans and Johansen, 1999), and arguably the most important of these is nutrient cycling. Within arid environments, BSCs have even been termed ‘mantles of fertility’ as they are considered hotspots of biogeochemical inputs (Garcia-Pichel et al., 2003), fixing 0.7e100 kg N ha
1 year
1, and from 4 to 370 kg C ha
1 year
1 * Corresponding author at: Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO 80309, USA. Tel.: þ1 602 481 9013. E-mail address: [email protected] (S.T. Bates). 0140-1963/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2010.05.033

(Evans and Lange, 2001). These substantial numbers point to the relevance of BSC mediated C and N cycling on a global scale, especially considering approximately one-third of the Earth’s terrestrial surface is arid or semi-arid land where BSC cover can be as high as 70% in some areas (Belnap et al., 2001). Fungi typically form networks of highly branched mycelia comprised of filamentous hyphae, and those associated with BSCs may play several important roles within arid land ecosystems. For example, it has been suggested that fungal mycelia links BSCs with patches of arid land vegetation, mediating nutrient exchange between these systems (Collins et al., 2008; Green et al., 2008). The critical function of stabilizing arid land soils against erosion has generally been relegated to BSCs (Belnap and Gillette, 1998; Guo et al., 2008). Although cyanobacteria play a critical role in this process (Belnap and Gardner, 1993; Garcia-Pichel et al., 2001; Garcia-Pichel and Wojciechowski, 2009), the mycelium of freeliving fungi can significantly increase the stability of soils in general (Meadows et al., 1994) and are also known to aid in soil particle aggregation associated with BSCs (Barger et al., 2006). Fungi are frequently cited as crust components (Belnap et al., 2001; Galun and Garty, 2001; Garcia-Pichel et al., 2001), and despite their potential to provide valuable ecosystem services, few studies (Bates and Garcia-Pichel, 2009; Green et al., 2008; Grishkan et al., 2006; States and Christensen, 2001) have specifically examined fungal diversity in BSCs or how it varies between different crust types.

S.T. Bates et al. / Journal of Arid Environments 74 (2010) 1192e1199

Studies examining the influence of small-scale disturbance (e.g., footprints, hoof prints, and vehicle tracks) and overall crust cover on the diversity of free-living arid land BSC fungi are also lacking. Anthropogenic disturbance is known to have a negative impact on BSCs and their functional capacity to acquire and retain nutrients (Belnap, 1995, 2002; Barger et al., 2006). As BSC fungal communities are tightly linked to crusts that they inhabit, being noticeably different and more diverse than those of the surrounding soils (Bates and Garcia-Pichel, 2009), disturbance, even at a small scale, and loss of crust cover may be detrimental to these communities, their diversity, and the ecosystem services that they provide. Interestingly, Zak (1992) and Morris et al. (2007) have suggested small-scale disturbance may actually increase fungal diversity in soils by creating greater spatial heterogeneity that provides new areas for fungal colonization; however, these authors also acknowledge the dynamics of small-scale disturbance are poorly understood and that there is a need for more study in this area. In this study, we assessed the fungal diversity and microbial population densities (fungi vs. bacteria) from well developed, lichen-dominated crusts, and compared our findings to those of a previous study that investigated crusts dominated by cyanobacteria using the same methods. The sites (both representing the same soil class) selected for this investigation experienced moderate but contrasting levels of disturbance, which allowed us to examine the relationship that crust cover and anthropogenic disturbance may have to fungal diversity and microbial abundance. In addressing these topics we ask the following questions: (i) What fungi are prevalent in lichen crusts, and how does fungal abundance compare to that of bacteria in these systems? (ii) How does fungal diversity and relative abundance, in relation to crustdwelling bacteria, vary between our sites as well as between crust types in general? (iii) How do fungal diversity and microbial abundance correspond to small-scale disturbance and overall crust cover? 2. Methods

1193

absence based on visual evidence, such as foot/hoof imprints (humans, cattle, or horses) or vehicular travel (motorcycles or allterrain-vehicles), of relatively recent disruption events. For sampling, the crust was wetted with sterile ultra-pure laboratory grade (Milli-Q) water to soften the upper layer of crust. The bottoms of 55 mm diameter Petri plates were inserted into the surface to excise a circular-shaped portion of the crust, each sampled to a depth of approximately 1 cm without disturbing the upper layer. Samples were then given unique identifier numbers, allowed to dry, sealed, and transported to the laboratory. Samples were stored dry, a method that has been suggested for preserving desert soil samples (Campbell et al., 2009), in the lab repository until DNA extractions were carried out. 2.2. Isolation of soil DNA Approximately 1.0 g of crusted soil from each sample was transferred aseptically into micro-centrifuge vials. As initial PCR runs were inhibited by the soluble salts within these soils, each sample was washed 3 times sequentially by adding w1 ml of sterile Milli-Q water, centrifuged for 10 min, and the supernatant was then discarded. The samples were then transferred to a vial containing the bead solution of the commercially available Ultra Clean Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). These vials were then subjected to 3 freeze-thaw treatment cycles (each consisting of 15 min in a 65  C water bath, followed by immersion in a liquid N2 bath for a period of 5 min) to optimize the yield off fungal DNA. After a final heating step (15 min in a 65  C water bath), the manufacturer’s protocol was followed. The extracted DNA was eluted into 50 ml of the kit’s ‘EB’ elution buffer and assessed for quality against a standard (EZ Molecular Weight Marker, Bio-Rad Laboratories, Hercules, CA, USA) in a 1% agarose gel after ethidium bromide staining and visualization under a Fluor-S Multi-Imager system (Bio-Rad Laboratories). Community genomic DNA concentration was quantified using a NanoDrop ND1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

2.1. Collection sites and sampling 2.3. PCR-amplification of fungal rRNA genes and sequencing Two sites in northwestern Arizona, both consisting of gypsic soil covered by well developed BSCs dominated by lichen cover (lichen crusts), were examined. The first site, ‘Ft. Pearce’ (36 590 N, 113 220 W), is located in the Fort Pearce Area of Critical Environmental Concern (ACEC) managed by the Arizona Strip field office of the Bureau of Land Management (BLM). The second site, ‘Gyp Hills’ (36170 N, 113 550 W), is located in Grand Canyon-Parashant National Monument that is jointly managed by the National Parks Service (NPS) and the BLM. The soil of both sites has been classified as a fine sandy loam of the Gypill series (DeWall, 2004), and the measured pH at both sites is in the 8.0e8.5 range. Both sites were characterized by the presence of following lichen species: Acarospora nodulosa (Dufour) Hue, Diploschistes diacapsis (Ach.) Lettau, Fulgensia bracteata (Hoffm.) Räsänen, Gypsoplaca macrophylla (Zahlbr.) Timdal, Peltula obscurans var. hassei (Zahlbr.) Wetmore, and Psora decipiens (Hedw.) Hoffm. Squamarina lentigera (Weber) Poelt was prevalent at Ft. Pearce, but apparently lacking at the Gyp Hills site. At each site, three 500 m-long transects were sampled. Each consisted of 10 duplicate samples taken every 50 m. For each transect, data on the degree of crust cover, the presence or absence of anthropogenic disturbance (both every 10 m, within a m2 frame), and ‘on point’ presence or absence of crust (every 5 m) were collected. Crust cover was visually estimated using the following categories: low (below 35%), medium (35e65%), or high (above 65%). Anthropogenic disturbance was scored by presence or

For use in denaturing gradient gel electrophoresis (DGGE), PCR amplicons of approximately 250 bp in length were generated from community DNA templates, targeting the variable D1eD2 domain of LSU rDNA using the fungi-specific primers NL1f (GC) of O’Donnell (1993), 50 -ATATCAATAAGCGGAGGAAAAG-30 , and LS2r of Cocolin et al. (2001), 50 -ATTCCCAAACAACTCGACTC-30 . A GC-rich clamp (CGCCCGCCGCGCGCGGCGGGGGGGCGGGGGCC) was added to the 50 end of NL1f to improve subsequent band separation in DGGE (Muyzer et al., 1993). PCR was performed in 50 ml reactions using Takara Ex Taq DNA polymerase premix PCR kits (Takara Bio, Madison, WI, USA) with the commercially obtained primers (Operon Biotechnologies, Huntsville, AL, USA) described above. Each reaction contained: 25 pmol of each primer, 4 ml dNTPs (2.5 mM each), 1 ml 10% bovine serum albumin, 5 ml 10 reaction buffer (containing 20 mM MgCl2), 1.25 units Ex Taq polymerase, w50e100 ng of template DNA, and sterile Milli-Q water to volume. PCR was performed in a Bio-Rad iCycler thermal cycler with the following cycling parameters: 5 min at 94  C, followed by 30 cycles of 30 s at 94  C, 30 s at 55  C and 60 s at 72  C, with a final extension for 5 min at 72  C. Products were checked for quality on 1% agarose gels as above. For sequencing, eluted DGGE bands (see below) were first reamplified using the same protocol and primer pair [NL1f (unclamped)/LS2r] as above, and approximately 50 ng of purified PCR product was sequenced commercially using the unclamped

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forward primer NL1f. When possible, multiple bands putatively representing a single phylotype (e.g., band at the same position horizontally across the gel) were excised from different lanes and sequenced in order to confirm their identity. Retrieved sequences exhibiting 98% similarity or greater were considered to be unique ‘phylotypes’, and representative sequences of the phylotypes obtained were submitted to GenBank (GQ4111020eGQ411055). The sequences were also subjected to similarity searches, performed using the BLASTn (Altschul et al., 1997) function available from GenBank (http://www.ncbi.nlm.nih.gov). 2.4. Quantitative PCR (qPCR) for relative microbial abundance To assess the abundance of microbial populations, fungal 25e28S rRNA and bacterial 16S rRNA gene copy numbers were determined using qPCR (Ferre, 1992; Filion et al., 2003; Haugland et al., 2004; Morrison et al., 1998; Nagy et al., 2005), a method shown to be effective in approximating the biomass of BSC fungi (Bates and Garcia-Pichel, 2009). In this analysis, the ABI Prism 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used employing the fluorescent dye SYBR Green to detect dsDNA. Each reaction mixture (20 ml) contained: 10 ml of SYBR GreenER qPCR Supermix (Invitrogen, Carlsbad, CA, USA), 25 pmol of each primer (Operon), 1 or 5 ml diluted genomic community DNA (bracketed 500-/1000-fold dilutions for fungi and 10-/100-fold for bacteria, respectively), and sterile Milli-Q water to volume. The fungal qPCR analysis used the primer pair NL1f (unclamped)/LS2r (see above), and the bacterial quantification used the primer pair GM5f (50 -TACGGGAGGCAGCAG-30 )/907r (50 -CCGTCAATTCCTTTRAGTTT-30 ), which yields amplicons of approximately 590 bp (Muyzer et al., 1995). Each run contained sample triplicates at the two different dilutions (six analytical replicates per sample in total) using the following thermal cycle conditions: 10 min at 95  C (100% ramp), followed by 40 cycles of 15 s at 95  C (100% ramp) and 1 min at 57 /55  C (fungal and bacterial cycles respectively; both at 100% ramp). Cycle threshold (Ct) values were determined using the SDS software package (Applied Biosystems) based on fluorescence data recorded during each qPCR run. Melting curve profiles were obtained after each 40-cycle reaction to verify the source of the fluorescence signal as being produced from dsDNA rather than artifacts such as primer dimmers. These profiles used the following cycle parameters: 15 s at 95  C, 15 s at 57/55  C (fungal and bacterial cycles, respectively) and 15 s at 95  C (all 100% ramp). All ribosomal gene copy numbers (fungal or bacterial) were determined from values that could be interpolated within a calibration curve obtained from known quantities of standards that were included in each qPCR run. These standards used either genomic DNA isolated from Neurospora crassa (188 rDNA copies per 40 Mb genome) (Galagan et al., 2003) or Escherichia coli (seven rDNA copies per 4.6 Mb genome) (Fogel et al., 1999) as template. For these the template mass was determined spectrophotometrically and transformed to the number of rRNA gene copies using the figures given in parentheses above. Log-linear correlation coefficients between the number of copies and Ct values were all >0.92. Failed reactions were not considered in the analyses; therefore, values for each sample represented an average of three to six successful replicate determinations. All valid analytical replicates from both dilutions were then averaged in calculating the values of rDNA copy numbers per gram of soil. 2.5. Denaturing gradient gel electrophoresis (DGGE) DGGE was run on 6% polyacrylamide gels containing a denaturing gradient (30e60% of urea/formamide) to separate PCR

products based primarily on sequence differences in the gene fragments (Muyzer et al., 1993; van Elsas et al., 2000). All gels were loaded with approximately 300 ng of DNA per lane and run for 6 h at 180 V and 60  C in a DCode Universal Mutation Detection System (Bio-Rad). An internal standard was also run on each gel to serve as anchors for gel-to-gel comparisons and to ensure gel fidelity. The standard contained a mixture of three 16S rRNA alleles from known cultivated bacterial isolates. Gels were stained for 30 min with a solution of 3 ml concentrated SYBR Gold Nucleic Acid Gel Stain (Invitrogen) diluted in 15 ml of TAE buffer, de-stained in TAE buffer for approximately 5 min, and then imaged using the Fluor-S MultiImager system (Bio-Rad). Bands of interest were excised with a sterile scalpel, placed in 1.5 ml vials with 50 ml of 10% Tris buffer, and allowed to elute for 4 days at 4  C prior to preparation for sequencing. Each lane in the gel contained the PCR amplified products from a single sample, and all 10 samples representing a single 500 m transect were run in different lanes of a single DGGE gel (e.g., see Fig. 1). Some of the eluted bands that did not yield clean sequences (e.g., separate bands that were in close proximity on the gel) were re-amplified, run again on DGGE gels under a more narrow gradient (e.g., 40e50%) to further separate bands corresponding to distinct phylotypes, and the resultant bands were excised, re-amplified, and sequenced using the same protocol cited above. 2.6. Statistics, quantification, and analyses of DGGE fingerprints In order to quantify diversity, digitized images of DGGE fingerprints were analyzed using QuantityOne software (Bio-Rad). The procedure to obtain biodiversity estimates from DGGE gels has been described in detail by Nübel et al. (1999) and has been used previously in quantifying diversity of BSC fungi (Bates and GarciaPichel, 2009). Briefly, a threshold of intensity is set which facilitates “band” detection, and it is assumed that each band in a lane represents a single unique phylotype (although it is possible that two or more phylotypes may exist in the same band, it is not highly probable). For each lane, the relative density of DNA for individual bands (based on cumulative pixel intensities) is then reported by the software. From these data, richness is estimated simply as the number of bands detected in a single lane, and the Shannon index is calculated using the relative abundances of all phylotypes in a lane. The QuantityOne software (Bio-Rad) also allowed us to match bands across the DGGE (lane-to-lane) by position, thus providing a method to determine whole-transect frequencies for a given phylotype and to estimate richness values for the total transect. These data (presence or absence, and abundance of phylotypes across DGGE gels), as well as those from a previous study of fungal diversity associated with cyanobacteria-dominated BSCs (Bates and Garcia-Pichel, 2009), were then used to construct community matrices for use in the rarefaction analyses. In comparing diversity measures between sites or among transects, the Student’s t-test for independent samples or ANOVAs were used to assess the significance of differences in measured or calculated parameters, after confirming homoschedasticity in the data set by the Levene’s test for equal variance. In a few cases where the assumption of homoschedasticity was rejected, the non-parametric two-sample Wilcoxon test was used in place of the Student’s t-test. To assess correlation between our estimates of crust cover or the presence of anthropogenic disturbance and diversity measures (richness and Shannon index), abundance values (fungal 25e28S or bacteria 16S rRNA copy numbers quantified by qPCR), or relative microbial biomass estimate ratios (bacterial 16S to fungal 25e28S rRNA copy numbers), the non-parametric Kendall tau rank correlation coefficient was calculated. In this analysis, 5 data points (3 from the Ft. Pearce and 2 from the Gyp Hills site) were removed

S.T. Bates et al. / Journal of Arid Environments 74 (2010) 1192e1199

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Fig. 1. A representative DGGE fungal community fingerprint (10 samples, labeled 1e10, along a 500 m transect e lanes labeled ‘S’ are standards) of lichen-dominated biological soil crust from the Gyp Hills site within the Grand Canyon-Parashant National Monument. Sequences from an excised band later determined to be allied with Alternaria (arrow ‘a’) is indicated.

from the correlation analysis where natural phenomena (extreme water erosion on steep slopes) prevented accurate estimation of crust cover or anthropogenic disturbance. All analyses (statistical and rarefaction) were performed using BiodiversityR and R statistical software (http://www.r-project.org/). 3. Results 3.1. Fungal diversity: community fingerprints, molecular survey, and rarefaction In the molecular survey, we were able to successfully recover and sequence over one-hundred DGGE bands. Fungal sequences closely allied with free-living hyphal and yeast forms were present, as were some attributable to lichenized fungi. The Ascomycota were the dominant phylum (87% of all sequences), with a majority of these (67%) belonging to the Pleosporales, an order containing numerous species with darkly pigmented hyphae or spores. In assessing the identity of our phylotypes by similarity to sequences in public databases, the high diversity of these lichen crusts was reflected in the number of fungal taxa detected (3 phyla and 11 orders). These included: Ascomycota: Hypocreales, Lecanorales, Pezizales, Pleosporales, Sordariales, Thelebolales, and Verrucariales; Basidiomycota: Agaricales, Corticiales, and Tremellales; Zygomycota: Mortierellales. The dominant (those most abundant and frequent across transects) phylotypes are given in Table 1. Fungal diversity parameters, quantified from the number of distinct 25e28S phylotypes (bands) detected in DGGE gels, a technique that may not capture the microbial diversity with the detail of exhaustive clone libraries or pyrosequencing (Bent and Forney, 2008) but allows for reliable comparisons of patterns in beta diversity (Gundlapally and Garcia-Pichel, 2006; Muyzer et al., 1993; Nagy et al., 2005; Wang et al., 2008), are reported in Table 2. We statically compared these diversity measures among transects (n ¼ 3) to test if these parameters were consistent at each site.

ANOVAs revealed significant differences among site transects for both diversity estimates of richness (P ¼ 0.003, P ¼ 0.0002) and Shannon index (P ¼ 0.0003, P ¼ 3.9  10
5) at both the Gyp Hills and Ft. Pearce sites, respectively, with one transect at each site accounting for the variance. Cumulative richness values determined along whole 500 m-long transects at both sites were much higher than those of individual samples, suggesting a high degree of patchiness in fungal community structure at the meter-scale. This same patchiness was obvious from inspection of community fingerprints as well (e.g., see Fig. 1). Significant differences were Table 1 Abundant and/or frequently occurring molecular phylotypes recovered from DGGE fingerprints of lichen-dominated biological soil crusts, their closest match (at 98e100% query coverage) to known fungal sequences in GenBank, and the locality in which they were recovered (Ft. Pearce ¼ FP; Gyp Hills ¼ GH; transects 1e3 at either site). Max. Identity

Closest match in database

99% 100% 98% 98%

Acremonium spp. Alternaria spp. Camarosporium sp. Coniothyrium palmarum; Leptosphaeria maculans Cryptococcus spp. Deflexula nana; Pterula spp. Diplodina coloradensis Endocarpon petrolepideum Laetisaria fuciformis Lophiostoma crenatum Marcelleina persoonii Mortierella alpina Omphalina velutipes Preussia spp. Pseudeurotium zonatum Sporormia lignicola Thelebolus spp. Phoma spp.

100% 99% 100% 97% 99% 95% 99% 100% 96% 100% 99% 100% 100% 98%

FP1

FP2

FP3

GH1

GH2

GH3

x

x x

x x x

x

x

x

x

x

x

x

x

x

x x x x x x x

x x

x x x

x x

x x

x

x x

x x

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Table 2 Diversity calculated from DGGE fungal community fingerprints of lichen-dominated biological soil crusts from Ft. Pearce Area of Critical Environmental Concern and Grand Canyon-Parashant National Monument. Site

Ft. Pearcea Gyp Hillsb a b

Richness

Shannon index

Sample range

Sample average  SD

Transect range

Transect average  SD

Sample range

Sample average  SD

3e15 5e28

7.71  3.49 14.24  5.69

22e58 53e70

41  15 59  7.6

0.81e2.41 0.96e3.06

1.64  0.41 2.10  0.50

Samples, n ¼ 28; transects, n ¼ 3. Samples, n ¼ 29; transects, n ¼ 3.

also detected between sites for total fungal diversity (richness, P ¼ 3.3  10
5, two-sample Wilcoxon test; Shannon index, P ¼ 0.0005, Student’s t-test), confirming that these sites, which varied in degree of crust cover and disturbance, also differed in their diversity. We also statistically compared fungal diversity measures from our lichen crusts to those previously determined (using identical methods) for cyanobacterial BSCs on the Colorado Plateau of Utah by Bates and Garcia-Pichel (2009). Lichen- and cyanobacteriadominated crust sites were significantly different for both richness (two-sample Wilcoxon test P ¼ 5.9  10
10) and Shannon index (Student’s t-test P ¼ 6.7  10
13), with lichen crusts typically displaying higher diversity. Similar results were obtained in the rarefaction analyses: lichen crusts were nearly always much more diverse than those of cyanobacterial crusts. With the same sampling effort, rarefaction curves (Fig. 2) revealed only one case where a cyanobacterial crust transect (the Sunday Churt site) exceeded the diversity of a single lichen crust transect from Ft. Pearce. 3.2. Microbial population density assessed by rRNA genes The microbial abundances and the relative ratio of bacterial-tofungal ribosomal RNA genes, quantified by qPCR, are reported in

Table 3. Fungal abundance averaged around 107e108 rDNA copies (25e28S) per gram of soil; whereas, bacterial abundance was in the range of 109e1011 copies (16S) per gram of soil range. Across all samples from both sites, we found bacterial abundance to be significantly different (two-sample Wilcoxon test P ¼ 2.3  10
9) from, and much higher than, that of fungi. Between the sites, Ft. Pearce and Gyp Hills, bacteria-to-fungi rDNA copy ratios were not significantly different (two-sample Wilcoxon test P ¼ 0.61). No significant differences were detected between the cyanobacterial BSCs of Bates and Garcia-Pichel (2009) and our lichen crusts for bacteria-to-fungi rDNA copy ratios (two-sample Wilcoxon test P ¼ 0.11). 3.3. Anthropogenic disturbance, crust cover, and correlation analysis The presence of crusted soil was detected more often in the Gyp Hills site (287 “on-point” BSC occurrences in 300 transect points; 96%) than in the Ft. Pearce site (206 “on-point” BSC occurrences in 300 transect points; 69%). Crust cover scores were, on average, “high” at the Gyp Hills site and “medium” at the Ft. Pearce site. The frequency of disturbance was moderate (present in 44% of the points examined) at the Ft. Pearce site, and much lower (present in 24% of the points examined) at the Gyp Hills site. Fungal diversity, as measured by richness and Shannon index, was negatively correlated with anthropogenic disturbance at Ft. Pearce (respectively, P ¼ 3.2  10
4, s ¼
0.62; P ¼ 3.4  10
4, s ¼
0.60; n ¼ 27), Gyp Hill (respectively, P ¼ 1.0  10
4, s ¼
0.64; P ¼ 3.8  10
4, s ¼
0.58; n ¼ 28), and across both sites (respectively, P ¼ 2.5  10
6, s ¼
0.55; P ¼ 2.4  10
6, s ¼
0.54; n ¼ 55). Conversely, crust cover exhibited a positive correspondence with richness and Shannon index at Ft. Pearce (respectively, P ¼ 3.4  10
5, s ¼ 0.68; P ¼ 5.5  10
5, s ¼ 0.64; n ¼ 27), Gyp Hill (respectively, P ¼ 0.02, s ¼ 0.38; P ¼ 0.04, s ¼ 0.33; n ¼ 28), and across both sites (respectively, P ¼ 3.0  10
7, s ¼ 0.57; P ¼ 1.1  10
6, s ¼ 0.53; n ¼ 55). Bacterial abundance (16S rRNA gene copy number), fungal abundance (25e28S rRNA gene copy number), and the bacteria-to-fungi rDNA copy ratios were not correlated with diversity measures (richness or Shannon index; P > 0.05; n ¼ 17), nor did they correspond to anthropogenic disturbance (P > 0.05; n ¼ 17) or crust cover (P > 0.05; n ¼ 17). 4. Discussion 4.1. Fungal diversity of lichen-dominated BSCs

Fig. 2. Rarefaction curves depicting the richness (as number of unique phylotypes) determined at an equal sampling effort (as the number of sampled individuals) of fungal communities associated with lichen- and cyanobacteria-dominated biological soil crusts from sites on the Colorado Plateau.

In addition to the ascomycetous lichens that dominate crusts at our sites, we found that free-living fungi from the Ascomycota are also conspicuous components of these BSCs; a finding that mirrors all surveys of BSC fungi to date (Bates et al., in press). Ascomycetous species from the anamorphic genus Alternaria have also proven to be prominent crust fungi. In this study a phylotype corresponding to Alternaria overwhelmingly dominated these lichen crusts at both

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Table 3 Absolute abundance of bacteria and fungi (copies of ribosomal genes per gram of soil) in lichen-dominated biological soil crusts from Ft. Pearce area of Critical Environmental Concern and Grand Canyon-Parashant National Monument quantified by qPCR and their paired ratios. Site

Ft. Pearce Gyp Hills a b c d

Bacterial abundancea,b

Fungal abundancec,d

Ratio (16S/25e28S)d

Range

Average  SD

Range

Average  SD

Range

Average  SD

4.16  109e3.87  1011 1.06  109e6.37  1010

1.00  1.45  1011 2.20  1.83  1010

2.56  107e3.41  108 1.69  107e4.36  107

1.01  1.07  108 2.36  0.95  107

1.63  100e2.09  103 3.25  101e3.03  103

7.91  102 1.07  103

16S rDNA copies per gram of soil. Samples, n ¼ 10. 25e28S rDNA copies per gram of soil. Samples, n ¼ 9 and n ¼ 8, Ft. Pearce and Gyp Hills, respectively.

sites, being highly abundant in nearly every sample. Species in this genus have also been cited in nearly all published molecular and culture-dependant arid land BSC fungal surveys (Bates et al., in press). Alternaria, as well as Acremonium, were also recovered and dominant in nearby cyanobacterial crusts on the Colorado plateau (Bates and Garcia-Pichel, 2009). Acremonium was also present at both of our sites; however, it was only among the principal phylotypes at the Ft. Pearce site (within one transect; see Table 1). These data suggest that Alternaria is a conspicuous crust fungus over a very broad geographic range and over varying classes of soils, while the importance of Acremonium may be relegated more specifically to cyanobacteria-dominated BSC. We also found that phylotypes closely matched to yeast species, Cryptococcus (Basidiomycota) and Thelebolus (Ascomycota), were among the more dominant members of the fungal crust community. Interestingly, many of the closely matching sequences in these genera originate from psychrophilic species; for example, Cryptococcus adeliensis Scorzetti et al. (2000) (GenBank AF137603) isolated from decayed algae in Antarctica and Thelebolus globosus Brumm. and de Hoog (GenBank FJ176905) from “biomats” in ultraoligotrophic Antarctic lakes (de Hoog et al., 2005). The presence of phylotypes in BSCs related to these species may represent fungi that are associated with decaying crust components or those that are adapted to survive in soil microbial communities were only a low level of organic carbon is available, as is typical for crusts (Bates and Garcia-Pichel, 2009). Although yeast have been reported in other crust surveys (States and Christensen, 2001), this study is the first to suggest that they may be among the more prominent members of some arid land BSC fungal communities. A novel crustdwelling black yeast, Exophiala crusticola, has also been isolated from two geographically distant western BSC sites (Bates et al., 2006). E. crusticola has the ability to survive on cyanobacterial exudates, which likewise points to the fact that yeasts may be an integral component of BSCs.

4.2. Variation in fungal diversity between crust types The fungal communities recovered from these lichen-dominated BSCs are clearly more diverse, by nearly every measure, than those of cyanobacteria crusts from sandy calcareous soil sites on the Colorado Plateau. The sequences that we were able to obtain from the DGGE fingerprints revealed the presence of more higher-level taxa (3 phyla and 11 orders) than those (2 phyla and 5 orders) detected in cyanobacteria-dominated BSCs by Bates and GarciaPichel (2009), even with a similar survey effort. Rarefaction curves as well as diversity measures (richness and Shannon’s index) all revealed notably higher diversity, on average, than what had been reported previously for fungi from cyanobacteria crusts (Bates and Garcia-Pichel, 2009), which were effectively lichen-free. In fact, our diversity measures approached those reported for bacteria (Gundlapally and Garcia-Pichel, 2006; Nagy et al., 2005) inhabiting other arid land BSCs.

As BSCs develop, additional macroscopic organisms (e.g., lichens and mosses) can be established in the crusts community (GarciaPichel et al., 2001) that can potentially harbor a considerable diversity of microbes, such as bryophilous (moss-associated), endolichenic (growing within the cells of the lichen mycobiont), and lichenicolous (lichen-associated) fungi (Arnold et al., 2009; Diederich, 2004; Döbbeler, 1997; Lawrey and Diederich, 2003; States and Christensen, 2001; Suryanarayanan et al., 2005). Our results are congruent with this concept, as on average there was a 2-to-3 fold increase (as high as 14 fold) in fungal diversity within these lichen crusts over that of less developed cyanobacterial crusts (Bates and Garcia-Pichel, 2009), which cannot be explained by the addition of the lichen species alone. It is also possible that endemic fungal species in these gypsic soils, known to support high endemism for some groups of organisms (e.g., lichens and vascular plants) (Evans and Johansen, 1999; Meyer, 1986), contributed to the higher levels of fungal diversity observed at these sites. 4.3. Fungal diversity of lichen crusts between and within sites Overall the distribution of fungal diversity at these lichendominated BSC sites appears to be patchy at the meter-scale, as has been noted before for BSC microbial communities (Bates and Garcia-Pichel, 2009; Gundlapally and Garcia-Pichel, 2006). Statistical analysis of diversity measures across transects at each or our sites revealed fungal diversity is also somewhat patchy at the landscape scales. Despite this patchiness, however, a few principal phylotypes (Table 1), were present among transects from both sites. One pleosporalean phylotype (e.g., see band labeled ‘a’, Fig. 1), corresponding to Alternaria spp. (100% identity), was widespread (present in all transects, and in 70e100% of the samples within each of these) and frequently among the more dense phylotypes (represented as the brightest bands) within individual samples. Interestingly, a phylotype outside of the Ascomycota corresponding to Mortierella alpina (100% identity; Zygomycota) was present in 4 of 6 transects (2 from each site) and also among the more dense phylotypes. Additional widespread and dominant phylotypes that were present at both sites included two, Thelebolus spp. (100% identity; Thelebolales) and Cryptococcus (100% identity; Tremellales), that were closely allied with the genera of yeasts. An Acremonium (Hypocreales) related phylotype (99% identity) was widespread and dominant in two transects at the Ft. Pearce site. Other dominant phylotypes were not widespread across the sites, being present in only one transect. 4.4. The relation of small-scale disturbance and crust cover to diversity of BSC fungi By our metrics, the Gyp Hill site was clearly more pristine than the Ft. Pearce site, where anthropogenic disturbance occurred more frequently and crust cover was lower overall. Fungal diversity estimates at the Gyp Hills site, with its more extensive BSC cover, were likewise significantly different from and on average much higher

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than those of the Ft. Pearce site. This finding was suggestive of one correspondence revealed by our analysis: fungal diversity was positively correlated with crust cover in these well developed lichen crusts. In contrast, fungal diversity was negatively correlated to disturbance and, likewise, significantly lower at the Ft. Pearce site. The decrease in fungal diversity observed in the relatively more disturbed crusts of the Ft. Pearce site may simply be attributed to a diminished crust cover. On the other hand, it is possible that particular BSC fungi are sensitive to disturbance and do not recover after disruption events such as trampling by cattle or compaction from off-road vehicles. Disruption events may also contribute to the loss of disturbance sensitive, macroscopic crust-dwelling organisms (e.g., lichens and mosses) (Evans and Johansen, 1999) that can act as hosts supporting an abundance of fungal diversity (e.g., bryophilous, endolichenic, and lichenicolous fungi). Regardless of the mechanisms that control fungal diversity in BSCs, our findings also speak against the assumption that small-scale disturbance can enhance fungal diversity in soils. The site with the highest frequency of disturbance, Ft. Pearce, also exhibited significantly lower fungal diversity by every measure. Furthermore, the rarefaction analysis revealed that fungal diversity in one Ft. Pearce transect was diminished to such a degree that it was within the range determined for cyanobacterial crusts, even falling below the level of one prokaryotic dominated BSCs site. 4.5. Relative microbial abundance ratios for BSCs Bacteria-to-fungi ratios of rRNA gene copy numbers for our lichen-dominated BSCs were not significantly different between our sites, and averaged at roughly 1000:1 for both sites. These numbers are consistent with, and not significantly different from, previously determined estimates from cyanobacteria-dominated crusts on the Colorado Plateau (Bates and Garcia-Pichel, 2009). In addition to suggesting the general tendency of bacterial biomass to dominate over that of fungi in BSCs, these statistics also imply that there is an overall stability to the allocation of microbial biomass in crusted soils. As autotrophic and diazatrophic bacteria of BSCs are responsible for the majority of nutrient inputs in these systems, this stability can perhaps be attributed to stoichiometric controls over fungal growth exerted by these crust-dwelling bacteria. Similar phenomena are known from other systems (Danger et al., 2007). 4.6. Conclusions We have demonstrated that fungal communities of lichendominated BSC are structurally similar to those of other crusted soils examined in earlier studies, yet they also contain sizeable populations of particular fungal taxa (e.g., yeasts species) that have not been previously reported. Fungi associated with these lichen crusts are also considerably more diverse than those of cyanobacteria-dominated BSCs. Furthermore, this fungal diversity has a positive correspondence with the degree of crust cover and an inverse relationship with disturbance. As disturbance can be detrimental to crust cover, we cannot determine unequivocally the degree to which each of these factors influenced fungal diversity in these BSCs; however, our study clearly demonstrates that the diversity of crust fungi is intimately connected with the properties of the BSCs that they inhabit. As such, the well documented sensitivity of BSCs to human activities (Barger et al., 2006; Belnap, 2002; Belnap and Eldridge, 2001; Belnap et al., 2007; Bowker, 2007; Evans and Belnap, 1999; Neff et al., 2005) and climate change (Schwinning et al., 2008) also has implications for diversity of arid land soil fungi as well as the ecosystem services that they may provide. Management practices that strive to conserve biological soil crusts are likewise those that ensure the maintenance of

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