Influence of sand burial on cultivable micro-fungi inhabiting biological soil crusts

Pedobiologia 58 (2015) 89–96

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Influence of sand burial on cultivable micro-fungi inhabiting biological soil crusts Isabella Grishkan a,∗ , Rong Liang Jia b , Xin Rong Li b a

Institute of Evolution, University of Haifa, 199 Aba Khoushy Ave, Mount Carmel, Haifa 3498838, Israel Shapotou Desert Research and Experiment Station, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, 320 Donggang West Road, Lanzhou 730000, China b

a r t i c l e

i n f o

Article history: Received 19 October 2014 Received in revised form 26 March 2015 Accepted 26 March 2015 Keywords: Arenosols Below-crust sandy layers Melanin-containing fungi Aspergillus fumigatus Species composition Diversity

a b s t r a c t We examined the influence of sand burial of 0.5-mm and 10-mm depths on micro-fungal communities inhabiting moss-dominated and mixed biocrusts in the vicinity of the Shapotou Research Station in the Tengger Desert, China. The buried communities were compared with those in unburied crusts, as well as with the sandy below-crust and topsoil communities. We isolated 65 fungal species belonging to 43 genera using the soil dilution plate method. Compared to the unburied communities, the buried crust communities were characterized by lower abundance of melanin containing species, especially those with large many-celled conidia, but higher abundance of species producing small light-colored and onecelled conidia, mainly mesophilic Penicillium spp. Sand deposition also caused reduction of isolate density that was more pronounced in the mixed crusts. Diversity characteristics of micro-fungal communities and isolate densities varied in the two crust types in response to the same level of sand burial. This difference between the mixed and moss-dominated crusts was less expressed when the communities had been subjected to the deep sand burial. Below-crust sandy communities showed a more significant decrease both in isolate density and species richness compared to buried crust communities, which was accompanied by the substitution of dominant species – the thermotolerant Aspergillus fumigatus in the subcrust sandy layers instead of melanized species with large multicellular conidia in the crusts. On the whole, the influence of sand burial on crust micro-fungal communities was likely associated with the shielding effect of sand layer which might protect the crust layer from evaporation and UV-radiation. Whereas sand deposition in the crusts partly changed the substrate quality and microclimatic conditions for soil micro-fungi, transition from the crust to the below-crust sandy layer much more substantially altered the environmental situation (mainly in nutrient status, from the comparatively rich organic crust layer to the poor mineral sandy layer) thus leading to the more significant changes in the micro-fungal communities. © 2015 Elsevier GmbH. All rights reserved.

Introduction In arid and semi-arid lands, sand burial is considered a noticeable environmental disturbance (e.g., Littmann, 1997; Bristow and Lancaster, 2004; Clemmensen and Murray, 2005; Rao et al., 2012). It affects soil temperature and moisture regime, as well as the availability of light, various nutrients, and oxygen (e.g., Harris and Davy, 1988; Maun, 1994, 2004; Williams and Eldridge, 2011). Sand burial is known to lower and slow the germination of seeds and seedling emergence (e.g., Ren et al., 2002). This kind of disturbance may be a source of severe stress for cyanobacterial crusts causing

∗ Corresponding author. Tel.: +972 4 8249697. E-mail address: [email protected] (I. Grishkan). http://dx.doi.org/10.1016/j.pedobi.2015.03.003 0031-4056/© 2015 Elsevier GmbH. All rights reserved.

the reduction of chlorophyll content and biomass, including a decrease in total carbohydrate reserve, especially polysaccharides (Wang et al., 2007; Rao et al., 2012). Sand deposition may also influence the communities of soil microorganisms changing the ratio between aerobic and anaerobic microbes and reducing the amount of mycorrhizal fungi (Maun, 2004). In the Shapotou revegetated region of the Tengger Desert, mobile sand dunes have been successfully transformed into stable, productive ecosystems, thereby displaying an example of human reversal of desertification in China (Li et al., 2004). Over the past 50 years, the structure and function of the stabilized and revegetated zone have changed considerably, and the development of biological soil crusts became an important factor in the restoration. The development of crust communities has substantially changed the physical and chemical soil properties, intercepting precipitation

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and modifying the vegetation dynamics of these ecosystems (Li et al., 2002, 2003; Duan et al., 2004). Crust communities play an important role in the maintenance of the vegetation function, especially in areas where the sand binding role of previously planted shrubs has weakened with time (Li et al., 2004). However, these crusts every year are inevitably exposed to repeated sand or dust burial of various depths. This burial is caused by two different processes: by wind blowing, which mostly happens in spring when wind speed is usually the highest and precipitation is low; and by animal activity (burrows of ants, lizards, and rabbits) occurring mainly in summer and autumn when precipitation is relatively high. Sand burial has resulted in multiple organic horizons of ‘fossilized crusts” in areas where they have survived this stress and barren spaces where they have not; both “fossilized crusts” and barren spaces weaken the protective function of the crusts. Hence, it is considered an important task to conserve these crusts in order to prevent or reduce the hazard associated with sandstorms and desertification in the region (Li et al., 2003). To address this, the effect of sand burial of various depths under different water availability conditions on ecophysiological and morphological parameters of moss- and lichen-dominated crusts was studied in the revegetated area of the Tengger Desert (Jia et al., 2008). The study revealed a significant decrease in crust respiration rate and a significant increase of moss shoot elongation as a result of burial. Both of these responses might have acted as compensatory mechanisms that favored the recovery of crusts after sand burial. Free-living micro-fungi are known to be an essential part of biological soil crusts. Together with heterotrophic bacteria, cyanobacteria, green algae, lichens, and mosses, they play a remarkable role in crust composition and functioning (e.g., States et al., 2001). In the revegetated area of the Tengger Desert, our pioneer mycological study revealed a diverse crust mycobiota composed of 134 species (Grishkan et al., 2015). In the present research, we examined the influence of sand burial on culturable microfungal communities inhabiting different crust types in the area. We hypothesized, based on our previous findings, that the composition and structure of these communities (reflected in the abundance of groupings with different life-history strategies), their diversity level, and the amount of micro-fungi would be affected by sand deposition. To test this hypothesis, the following characteristics of the communities were analyzed in the course of the study: species composition; contribution of the major taxonomic and ecological groupings to community structure; the dominant groups of species, density of isolates, and the diversity level (species richness, heterogeneity, and equitability).

Fig. 1. Experimental plots covered by moss-dominated crusts (surrounded by solid line) and mixed crusts (surrounded by dashed line), with tubes for control (1) and for simulation of shallow (2) and deep (3) sand burial.

content of the crusts during dry periods is very low and does not exceed 2%. The vegetation in the region is dominated by psammophytes, such as Hedysarum scoparium Fisch., Agriophyllum squarrosum Moq., Stilpnolepis centiflora Krasch and Pugionium calcaratum Kom., etc., covering about one percent of the area (Li et al., 2004). The region consists of huge, dense and continuous reticulate barchan dunes with loose, impoverished, and mobile blown sand. The non-crusted dunes (Arenosols, according to the FAO-UNESCO system, 1974) consist of 99.7% of sand and only 0.3% of finer particles (silt and clay). However, the biological soil crusts (BSCs) contain up to 30–35% of finer particles (Li et al., 2006). The non-irrigating vegetation system was initially established in 1956 to protect the Baotou–Lanzhou railway line from sand burial. This system was further expanded in 1964, 1981, and 1987. The stabilization method used included a combination of windbreaks, straw checkerboard barriers, and planted xerophytic shrubs (Duan et al., 2004). Following surface stabilization, biological (cyanobacterial) soil crusts began to colonize the dune surfaces. These crusts were then gradually converted to moss and lichen crusts (Li et al., 2003). Currently, the BSC cover more than 80% of the total revegetated desert sections. Sampling design

Materials and methods Site description The study was conducted in the Shapotou research station (SRS) of the Cold and Arid Regions and Environmental and Engineering Research Institute, CAS, located in Zhongwei County in the Ningxia Hui Autonomous Region at the southeastern edge of the Tengger Desert (37◦ 32 N, 105◦ 02 E, elevation of 1340 m). It is an ecotone between steppified desert and desertified steppe, being also a transitional zone between sandy and re-vegetated deserts (Li et al., 1998). The mean, absolute minimum and maximum annual air temperatures reach 10.0 ◦ C, −25.1 ◦ C, and 38.1 ◦ C, respectively. The mean, absolute maximum, and minimum annual precipitation is approximately 186, 304 and 88 mm, respectively, 80% of which falls between May and September (Jia et al., 2008). The temperature of the crust surface can reach 54 ◦ C (the south-exposed cyanobacterial crusts at midday hours in July 2011). Moisture

Crust and below-crust samples were collected in September 2014, from two experimental plots covered by two crust types: moss-dominated (Bryum argenteum Hedw.) and mixed (lichens, green algae, cyanobacteria, and mosses in similar proportions). In these plots, located on the windward slope of the area with revegetation started at 1964, cylindrical polyvinyl chloride (PVC) tubes (104 mm diameter, 20 cm depth) were randomly placed into the soil in March 2013 (Fig. 1). Nine tubes were placed in each plot. Three tubes, with 6.5 g of air-dried drifting sand were gently and evenly distributed over a crust forming the 0.5-mm layer (hereafter – shallow sand burial). In another three tubes, 130 g of sand were distributed over a crust forming the 10-mm layer (hereafter – deep sand burial) and the remaining three tubes stayed intact and served as controls. The uppermost edges of the tubes were kept 0.5 cm above the lower flat surface to avoid the sand from blowing out of the tubes. In each tube, the below-crust sandy samples were also collected at depths of 0.5–1 cm (under the mixed crusts) and

I. Grishkan et al. / Pedobiologia 58 (2015) 89–96

1–1.5 cm (under the moss-dominated crusts). To avoid edge effects, all samples were taken from the middle of the tubes using sterilized cylindrical PVC tubes of 5 cm in diameter. For additional, sandy surface control, three samples were taken as well from the nearby, non-crusted sandy dunes stabilized by the straw checkerboard barriers. The samples were from the middle of the dunes and near the border of the barriers, at depths of 0–0.5 cm. Altogether, 42 samples were collected and examined. The samples were processed in the laboratory of the Institute of Evolution, University of Haifa, Israel, within 4–5 weeks after sampling. Isolation of micro-fungi Micro-fungi were isolated by means of the soil dilution plate method (Davet and Rouxel, 2000). Despite certain limitations and biases, such as possible overestimation of heavily-sporulating species, loss of rare fungi and fungi that cannot grow on culture media, this method remains a useful and valid approach for the initial characterization of the ecology of fungal communities (e.g., Bills et al., 2004). It is especially relevant for desert soils where fungi may exist for a long period in a dormant (spore) state. Ten grams of each soil sample were used in a dilution series. Two culture media with different carbon and nitrogen sources were employed: Malt Extract Agar (MEA) and Czapek’s Agar (CzA) (Sigma–Aldrich Inc., St. Louis, USA). Streptomycin (Spectrum Chemical Mfg. Corp, Gardena, CA, USA) was added to each medium (100 ␮g/ml) to suppress bacterial growth. One mL of soil suspension from the dilutions 1:10–1:100 (soil:sterile water) was mixed with the agar medium at 40 ◦ C in Petri dishes (90-mm diameter). The plates were incubated at 25 ◦ C in darkness for 10–15 days (3 plates for each medium).

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To analyze burial-dependent and depth-dependent variations in the micro-fungal community structure, three major groupings were chosen: Penicillium spp. consisting mainly of mesic fungi, Aspergillus spp., consisting mainly of thermotolerant and thermophilic fungi, and the major desert group of melanin-containing fungi. In the latter grouping, melanized species with large multicellular spores were examined separately. The contribution of each group was estimated as an average of its density (number of isolates of a particular group in the sample/total number of all isolates in the sample, i.e., its relative abundance in total isolate number in the sample) and its contribution (percentage) in the Shannon index (Grishkan et al., 2008). We used the latter characteristic together with direct density because (i) it is logarithmic, thus preventing an overestimation of heavily-sporulating species, and (ii) it takes into account not only the number of isolates but also the number of species comprising the aforementioned groupings. Statistical analysis was conducted using XLSTAT (http://www.xlstat.com). We employed a one-way ANOVA followed by multiple-comparison tests to compare data from different habitats on diversity characteristics, contribution of micro-fungal groupings, and isolate density. A two-way unbalanced ANOVA with interactions was used to test the effect of different environmental factors (locality type, burial depth, and soil depth), separately and in interaction, on the above mycological parameters. To evaluate similarity between micro-fungal communities from different habitats, the clustering of the communities based on species relative abundances was made by the Unweighed pair-group average method with Chi squared distance as the distance coefficient. Results

Identification

Composition and diversity of micro-fungal communities

After incubation, the emerging fungal colonies were transferred to MEA and CzA for purification and further taxonomic identification. In an attempt to induce sporulation, all non-sporulating isolates were also grown on Oatmeal Agar (Sigma–Aldrich Inc, St. Louis, USA), as recommended by Bills et al. (2004), and on Water Agar (agar – 20 g, water – 1000 ml). Taxonomic identification was based on morphological characteristics of fungal isolates. All names of species are cited according to the database of Kirk et al. (2014).

Altogether, 65 species were isolated from Mucoromycotina (4 species), teleomorphic (morphologically sexual) Ascomycota (19), and anamorphic (asexual) Ascomycota (42). The species belonged to 43 genera of which the most common were Chaetomium (9), Aspergillus (4), and Penicillium (3). Eight types of micro-fungal strains remained non-sporulating in culture. In the mixed crusts, the lowest and the highest numbers of micro-fungal species were isolated from the unburied and the deeply buried crusts, respectively, while in the moss-dominated crusts, species richness varied in the opposite way (Table 1). The micro-fungal communities from the deeply buried mixed crusts were also significantly more heterogeneous and even compared to the shallow buried and unburied crusts, whereas the equitability of the communities from the buried and unburied moss-dominated crusts was similar (Table 1). The topsoil sandy micro-fungal communities were the most even in comparison to the other crust communities (Table 1).

Data analyses Density of fungal isolates was expressed as “colony forming of biodiversity was units” (CFU) per gram dry substrate. Analysis based on the Shannon–Wiener index (H = − pi ln(pi ), where pi is the proportion of species i in a sampling plot) and evenness (J = H/Hmax where Hmax is the maximum value of H for the number, S, of species present; Hmax = lnS) (Krebs, 1999).

Table 1 Diversity characteristics of micro-fungal communities (S – number of species, including sterile strains, H – Shannon index, J – evenness) in unburied crusts, buried crusts, sandy surfaces, and sandy below-crust layers at SRS. Means with the same letters are not significantly different (comparisons made within each of the three characteristics across all habitats; a one-way ANOVA, the Tukey (HSD) test, at the 5% level). Locality

Mixed crusts under deep sand burial Mixed crusts under shallow sand burial Unburied mixed crusts Moss crusts under deep sand burial Moss crusts under shallow sand burial Unburied moss crusts Sand from the middle of straw checkerboard Sand from the border of straw checkerboard

Below crust

Crust/sand S

H

J

S

H

J

28ab 25abc 18cde 20bcd 23abc 30a 26ab 18bcd

2.20ab 1.29cde 1.28cde 1.79bcd 1.90abc 2.07ab 2.36a 2.14ab

0.66abc 0.40de 0.44cde 0.60bcd 0.61bcd 0.61bcd 0.72abc 0.74ab

17cde 9e 8e 14de 8e 16de – –

1.68bcd 0.84f 1.39cde 2.03abc 0.73f 0.98def – –

0.59bcd 0.38ef 0.67abc 0.77a 0.35f 0.35f – –

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Fig. 2. Differences in contribution of main micro-fungal groupings in unburied crusts, buried crusts, sandy surfaces, and sandy below-crust layers at SRS: – Penicillium spp.; – Aspergillus spp.; – melanin-containing spp. The area below the white line on the bars of melanin-containing micro-fungi indicates contributions of species with large multicellular spores. Means with the same letters are not significantly different (comparisons made within each of the four groupings, Penicillium spp., Aspergillus spp., melanin-containing spp., and melanincontaining spp. with large multicellular spores, across all habitats; a one-way ANOVA, the Tukey (HSD) test, at the 5% level).

The differences between crust and sandy below-crust microfungal communities in diversity characteristics in most pair-wise comparisons were more pronounced than the differences between the buried and unburied crust communities. In all cases, species

richness was significantly higher in the crust localities. Moreover, the sandy communities below unburied mixed crusts and deeply buried moss-dominated crusts, were significantly more heterogeneous and even compared to the respective crust communities (Table 1). Melanin-containing micro-fungi dominated all topsoil communities studied and also prevailed in the sandy layers below deeply buried crusts (Table 2). Melanized species comprised 42.4–95.3% and 28–80.5% of the contribution index in the topsoil and below-crust communities, respectively (Fig. 2). The main core of melanin-containing fungi in all crust communities (except for the deeply buried mixed crust) was represented by species with large (>20 ␮m) multicellular spores such as Embellisia phragmospora, Alternaria alternata, and Ulocladium atrum (Table 2) (31.4–82.3% of the contribution index – Fig. 2). In the topsoil sandy localities, the contribution of these species was significantly lower (Fig. 2). Abundance of melanized fungi (especially with large many-celled spores) was lower in the buried compared to the unburied crusts and more significantly – in the below-crust as compared to the crust communities (Fig. 2). The abundance of Aspergillus spp. (mostly A. fumigatus) accounted for the most pronounced differences between the crust and below-crust habitats being nearly 30-fold higher in the below-crust sandy micro-fungal communities. The topsoil sandy communities as well the shallow buried crust communities were characterized by higher contribution of aspergilli in comparison to the unburied crust communities but these differences were much less expressed than the depth-wise differences (Fig. 2). The contribution of Penicillium spp. (mainly P. aurantiogriseum) to community composition was the highest (24.4% and 26.4%) in the shallow and deeply buried moss-dominated crusts. It was nearly two-fold lower in the deeply buried mixed crusts and in the sandy edge habitats, while in other micro-fungal communities, the abundance of penicillii was very low, 0–1.6% (Fig. 2). Cluster analysis based on relative abundances of species separated the micro-fungal communities into few groups with different levels of similarity (Fig. 3). One of the groups contained

Fig. 3. Clustering the micro-fungal communities from unburied crusts, buried crusts, sandy surfaces, and sandy below-crust layers at SRS.

Table 2 Common micro-fungal species from unburied crusts, buried crusts, sandy below-crust layers, and sandy surfaces at SRS, with their average relative abundance (%). Species in bold are melanin-containing. Species

Moss – deep burial

Mixed – shallow burial

Mixed – deep burial

Moss – unburied

Mixed – unburied

Sand

Crusts

Crusts

Below

Crusts

Below

Crusts

Middle

– – –

– 2.9 0.4

– – 0.9

– 0.8 – – 0.4 – –

– 0.2 – – 0.2 – –

– 78 – 0.4 0.4 – – – – 6.6 1.6 2 – 0.4 0.4 3.7 0.4 – 0.4 – –

Crusts

Below

Crusts

Mucoromycotina Absidia corymbifera Mortierella humilis Rhizopus oryzae

2.4 – 2.2

– 1.1 2.2

0.9 – 0.3

0 – 16.4

0.4 0.7 0.7

– 0.4 16.2

– – 1.4

– 5.4 3.3

Teleomorphic Ascomycota Aspergillus nidulans Chaetomium strumarium Ch. succineum Chaetomium sp. Pleospora tarda Sordaria fumicola Sporormiella minima

– – – 0.2 – – –

– – – – – – –

– 2 – – – 0.9 0.9

– 1.4 – 5.5 – – 4.1

– 0.7 0.4 – – 1.1 –

– 0.9 0.4 – – – –

0.8 1.1 25.9 – – 0.3 –

0.3 – 38.3 – – – –

1.3 2.1 1 – 0.3 0.5 –

3.2 2.3 – – – 0.9 – – 0.3 44.8 0.6 2.6 – – 28.8 7.7 0.6 0.3 1.1 0.9 –

1.4 5.5 – 1.4 – – – – – 12.3 – 5.5 – 2.7 – 38.3 – – – – –

4 3.6 – 0.7 – 2.8 – 0.4 – 71.1 – 1.4 – 0.4 – – 0.7 – – 3.2 2.2

– 75 – – – – – – – 5.5 – – – 0.85 0.4 – – – – – –

3.8 1.1 – 0.5 – 7.2 4 – 0.3 26.3 0.8 2.7 1.1 – 10 0.8 – – 0.3 0.8 2.2

0.9 33.3 8.6 0.3 – 0.3 – – – 3.9 – 2.4 0.3 0.9 0.3 – – – – – –

9.4 0.5 – 7.3 – 3.4 – – 0.8 50.8 0.25 3 1.6 1 1 2.6 1.3 1.6 – 5.2 0.3

– – – –

– – – –

– – 1.1 0.8

– 0.3 1.2 –

Anamorphic Ascomycota Alternaria alternata Aspergillus fumigatus A. ustus Boeremia exigua Cladosporium cladosporioides Coleophoma empetri Coniothyrium olivaceum Curvularia inaequalis C. spicifera Embellisia phragmospora Fusarium oxysporum Giberella acuminata Myrothecium verrucaria Papulaspor apannosa Penicilium aurantiogriseum Pyrenochaeta cava Setosphaeriar ostrata Stachybotrys chartarum Trichoderma koningii Ulocladium atrum Westerdykella capitulum Mycelia sterilia Dark Dark 1 Dark 2 Light

4.8 23.5 – – 0.3 0.5 – – 0.2 20.4 0.2 1 – 1 33.4 5.8 0.7 0.2 – 0.3 1.2

– 93.9 – – – – – – – 0.3 – 0.5 – – – – 0.2 – – 0.2 –

0.2 – – –

– – – 0.6

Below

– 1.8 0.7 0.4

Below

– – – 0.4

1.3 – – –

– 0.8 – –

Below – – 15

Border

– – 0.5

– – –

1.9 – – – – – –

– 0.4 – 0.7 0.2 – –

– – – – – – –

14.9 0.7 – 0.9 – – – 6.5 0.2 47.5 – 0.9 – – 0.4 19.4 1.7 – – 5.4 –

– 64.3 – – 0.5 – – – – – – 5.6 – – – 12.7 – – – – –

24.4 16.4 – 10.6 9.2 0.4 – – – 6 0.2 3.1 7.8 – 0.5 0.5 0.2 10.8 – 3.1 1.2

– – – –

– – – –

1.5 – – –

5.4 21.8 – 5.1 1.1 0.2 – – 0.2 – 14.3 – 11.4 – 8.4 – 0.3 19.9 0.1 8 –

I. Grishkan et al. / Pedobiologia 58 (2015) 89–96

Moss – shallow burial

– – – –

93

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Fig. 4. Isolate density in unburied crusts, buried crusts, sandy surfaces, and sandy below-crust layers at SRS: – crusts/sandy surface; – below-crust sandy layers. Vertical bars represent standard deviations (n = 3). Means with the same letters are not significantly different (comparison made across all habitats; a one-way ANOVA, the Tukey (HSD) test, at the 5% level).

the communities from the crust habitats (except for the deep buried mixed crusts) that were clustered together due to the dominance of the above-mentioned melanin-containing species with large many-celled conidia. Another group consisted of the sandy communities from the habitats below unburied and shallow buried crusts. These communities were highly similar to each other because of the overwhelming dominance of A. fumigatus (Table 2). The topsoil sandy communities also grouped together with a comparatively high level of similarity, as well as the communities from the deeply buried mixed crust and below-crust habitats (due to high abundance of Chaetomium succineum). The communities from the sand below the deeply buried moss-dominated crusts clustered apart from other communities because of the high abundance of Pyrenochaeta cava (Table 2). Density of micro-fungal isolates In the mixed crusts, the CFU number was significantly (1.7–2.3fold) higher in the unburied crusts compared to the buried crusts, while in the moss-dominated crusts, the shallow buried and the deeply buried crusts contained the highest and the lowest isolate density, respectively (Fig. 4). Both in the topsoil and below crust sandy habitats, the CFU numbers were significantly lower than in the crusts (Fig. 4). The lowest numbers of CFU were counted below the deeply buried moss-dominated crusts (Fig. 4). Effect of locality type, burial depth, and soil depth on mycobiotic characteristics Among the mycobiotic parameters studied, diversity characteristics, especially species richness, were less affected by the above environmental factors. However, the contributions of different micro-fungal groupings as well as isolate density were very significantly influenced both by each factor separately and their interactions (Table 3). Among the environmental factors, soil depth affected the characteristics of micro-fungal communities most markedly, followed by the depth of burial (Table 3). Discussion As already mentioned, sand burial is known as a remarkable environmental factor, which alters various physical and chemical soil properties in the arid and semiarid areas. Extended periods of

burial could cause the increase of nitrogen bioavailability in the cyanobacterial crusts (Williams and Eldridge, 2011) likely due to the death of crust organisms, their autolysis, and the liberation of extracellular N. Sand deposition was shown to slightly increase pH and moisture content of the cyanobacterial crusts probably because of the alkaline sand substances penetrating into the crust layers and the shielding effect of a sand layer on the water evaporation in the crusts (Rao et al., 2012). Given that it is a very well established fact that composition and activity of soil fungal communities are closely related to physical, chemical, and structural properties of the soil (e.g., Ritz and Young, 2004), sand burial was expected to influence diversity and distribution of micro-fungi in the biological soil crusts of the Tengger Desert. The influence of sand burial was reflected in species composition of the micro-fungal communities. All crust communities were characterized by dominance of melanin-containing species, which is considered a typical feature of almost all mycologicallystudied arid soils (e.g., Ranzoni, 1968; Christensen, 1981; Halwagy et al., 1982; Skujins, 1984; Abdullah et al., 1986; Hashem, 1991; Ciccarone and Rambelli, 1998; Zak, 2005, Grishkan and Nevo, 2010) and particularly of biological soil crusts (Bates and Garcia-Pichel, 2009; Bates et al., 2010; Porras-Alfaro et al., 2011; Bates et al., 2012; Grishkan and Kidron, 2013). Nevertheless, the unburied crust micro-fungal communities were remarkably similar to each (Fig. 3) and significantly differed from the buried crust communities both in total abundance of melanized fungi and abundance of melanized species with large thick-walled and many-celled conidia. The latter group can be considered the most resistant to UV- and drought stresses due to their protective spore morphology. Sand deposition also increased the contribution of mesophilic Penicillium spp. (except for the shallow buried mixed crusts). The sand layer, protecting to some extent the crust layer from the evaporation and UV-radiation, likely creates more suitable conditions for penicillii to survive and develop. On the other hand, the moss-dominated crusts under shallow sand burial hosted significantly higher abundances of the thermotolerant Aspergillus spp., which was similar to what was observed in the proximate topsoil sandy habitats. In the two crust types studied, diversity characteristics of microfungal communities varied with the depth of burial. In the mixed crusts, sand burial increased both species richness and evenness of the communities, while in the moss-dominated crusts, species richness varied in the opposite way, and community evenness was not sensitive to sand deposition. Similarly, different trends were found in the variation of isolate densities. In the mixed crusts, communities characterized by maximum isolate densities and consisting of 80% melanized species with large many-celled conidia were isolated from the unburied habitats. At the same time, in the moss-dominated crusts, the shallow buried communities with comparatively high contributions of light-colored Penicillium and Aspergillus species with small one-celled conidia contained the highest CFU numbers (Fig. 4). Notably, the deeply buried microfungal communities in both crust types were similar to each other in isolate densities. The moss-dominated crusts located on the north-facing slopes are characterized by lower midday surface temperatures, longer wetness duration, and consequently, by greater thickness and higher chlorophyll content, compared to the mixed crusts confined to the intermediate positions between the north-facing and south-facing slopes (Grishkan et al., 2015). Such microclimatic differences between the crust types may explain not only differences between their unburied micro-fungal communities but also the dissimilar responses of the communities to the same level of sand burial. Interestingly, this dissimilarity was less expressed when the communities had been subjected to deep sand burial. All analyses performed indicated that the depth-dependent variations in the community parameters were more pronounced

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Table 3 Data of two-way unbalanced ANOVA analysis for the effect of locality type, burial depth, soil depth, and the interactions among them on different parameters of micro-fungal communities at SRS. Parameter

Locality

Burial

Depth

Locality × burial

Locality × depth

Burial × depth

Locality × burial × depth

Species richness Shannon index Evenness Melanin-containing spp. Melanized spp. with multi-cellular spores Penicillium spp. Aspergillus spp. Isolate density

8.5** 4.1@ NS 42.4**** 6.8** 25.6**** NS 6**

NS 19.3**** 28.2**** 38 **** 14.6**** 31.5**** 84.3**** 16.2****

117.5**** 54.3**** NS 217.6**** 447.8**** 101 **** 769.5**** 302.1****

17.8**** NS 7** 31.3**** 32.9**** 11.1** 27.9**** 22.5***

NS 4.4@ 6@ 37.9**** 27.4**** 29.9**** 39.3**** 8.7**

NS 7** 8.9*** 81.6**** 47.6**** 23.6**** 37.1**** 13.4****

NS 5.7* 15.3**** 9.9** 8.5*** 16.7**** 43.7**** 13.7****

@ * ** *** ****

≤0.05. ≤0.01. ≤0.005. ≤0.001. ≤0.0001.

than the burial-dependent variations as well as the variations between the crust and sandy surface communities. This fact has a logical explanation: whereas sand deposition in the crust layer partly changed the substrate quality and microclimatic conditions for soil micro-fungi, transition from the crust to the sandy, especially the below-crust layer, substantially altered the environmental situation. This alteration in nutrient status from the comparatively rich organic crust layer to the poor mineral sandy layer expectedly and significantly decreased isolate density, which can be considered an indirect characteristic of fungal biomass. It was also accompanied by the significant reduction in species richness with depth as well as by the remarkable alteration in composition and structure of the micro-fungal communities associated primarily with different dominant species. The dark-colored species with large thick-walled multicellular conidia, which dominated the crust communities, were replaced in the subcrust sandy layers with Aspergillus fumigatus, a thermotolerant light-colored species with small one-celled conidia. A. fumigatus is known as a ubiquitous saprotrophic fungus (e.g., Domsch et al., 2007) with a wide temperature range for growth and with optimum temperatures between 35 ◦ C and 38 ◦ C (e.g. Magan, 2007). It was frequently isolated and in high numbers from the soils of different arid regions (e.g., Ranzoni, 1968; Halwagy et al., 1982; Moubasher et al., 1985; Abdullah et al., 1986; Abdel-Hafez et al., 1989; Bokhary, 1998; Mandeel, 2002; Sharma et al., 2010; Baeshen et al., 2014). In the biological soil crusts of the Negev desert (Israel) and the Tengger Desert, as well as in other Negev soils, A. fumigatus dominated or co-dominated the thermotolerant communities isolated at 37 ◦ C (Grishkan and Nevo, 2010; Grishkan and Kidron, 2013; Grishkan et al., 2015). Recent taxonomic studies revealed that A. fumigatus is a complex composed of several species that are morphologically nearly identical to each other but differ in some gene sequences as well as in their growth temperature regimes (all species being thermotolerant) and the extrolite profile (Hong et al., 2005). Apparently, in sandy substrates, especially in the subcrust, subsurface layers, A. fumigatus obtains the most favorable conditions for development, thus avoiding strong competition with melanin-containing fungi with multicellular conidia, which overwhelmingly dominate the crust micro-fungal communities. Conclusion The present study revealed the influence of sand burial on qualitative and quantitative characteristics of crust micro-fungal communities in the Tengger Desert, China. It was likely associated with the shielding effect of the sand layer even of shallow depth, which might protect the crust layer from evaporation and UV-radiation. The influence was expressed in the decreasing abundance of melanin containing species, especially with large

many-celled conidia, and the simultaneous increase in the abundance of species producing small light-colored and one-celled conidia, mainly mesophilic Penicillium spp. Sand deposition also caused the reduction in isolate density, which was more pronounced in the mixed crusts. Transition from the crust to the subcrust sandy layer was accompanied by more significant changes in the micro-fungal communities compared to the sand burial. Substantial alteration in edaphic conditions (mainly in nutrient status, from the comparatively rich organic crust layer to the poor mineral sandy layer) led to the significant decrease both in isolate density and species richness, and to the substitution of dominant species – the thermotolerant A. fumigatus sensu lato in the subcrust sandy layers instead of melanized species with large multicellular conidia in the crust layers. Thus, continued and deep sand burial of biological soil crusts in the area may disrupt the stability and functioning of crust micro-fungal communities, which could reduce the amount of micro-fungi, their diversity, and change their species composition, especially the group of dominant and frequently occurring species. Acknowledgments We thank the Chinese Academy of Sciences (visiting professorship for senior international scientists, Grant No. 2011T1Z16), the National Natural Science Foundation of China (Grant No. 41371099), and the Israeli Ministry of Absorption for financial support of this research. References Abdel-Hafez, S.I.I., Mohawed, S.M., El-Said, A.H.M., 1989. Seasonal fluctuations of soil fungi of Wadi Qena at Eastern desert of Egypt. Acta Mycologica XXV, 113–125. Abdullah, S.K., Al-Khesraji, T.O., Al-Edany, T.Y., 1986. Soil mycoflora of the Southern Desert of Iraq. Sydowia 39, 8–16. Baeshen, N.A., Sabir, J.S., Zainy, M.M., Baeshen, M.N., Abo-Aba, S.E.M., Moussa, T.A.A., Ramadan, H.A.I., 2014. Biodiversity and DNA barcoding of soil fungal flora associated with Rhazya stricta in Saudi Arabia. Bothalia J. 44, 301–314. Bates, S.T., Garcia-Pichel, F., 2009. A culture-independent study of free-living fungi in biological soil crusts of the Colorado Plateau: their diversity and relative contribution to microbial biomass. Environ. Microbiol. 11, 56–67. Bates, S.H., Nash, T.H., Sweat, K.G., Garcia-Picel, F., 2010. Fungal communities of lichen-dominated biological soil crusts: diversity, relative microbial biomass and ther relationship to disturbance and crust cover. J. Arid Environ. 74, 1192–1199. Bates, S.H., Nash, T.H., Garcia-Pichel, F., 2012. Patterns of diversity for fungal assemblages of biological soil crusts from the southwestern United States. Mycologia 104, 353–361. Bills, G.F., Christensen, M., Powell, M., Thorn, G., 2004. Saprobic soil fungi. In: Mueller, G.M., et al. (Eds.), Biodiversity of Fungi. Inventory and Monitoring Methods. Elsevier Academic Press, California, pp. 271–302. Bokhary, H.A., 1998. Mycoflora of desert sand dunes of Riyadh region, Saudi Arabia. J. King. Saud. Univ. 10, 15–29. Bristow, S., Lancaster, N., 2004. Movement of a small slipfaceless dome dune in the Namib Sand Sea, Namibia. Geomorphology 59, 189–196. Christensen, M., 1981. Species diversity and dominance in fungal community. In: Carroll, G.W., Wicklow, D.T. (Eds.), The Fungal Community, its Organization and Role in the Ecosystem. Marcell Dekker, New York, pp. 201–232.

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