Biocrust-inhabiting cultured microfungi along a dune catena in the western Negev Desert, Israel

European Journal of Soil Biology 56 (2013) 107e114

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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Biocrust-inhabiting cultured microfungi along a dune catena in the western Negev Desert, Israel Isabella Grishkan a, *, Giora J. Kidron b a b

Institute of Evolution, University of Haifa, Mount Carmel, Haifa 310905, Israel Institute of Earth Sciences, The Hebrew University of Jerusalem, Givat Ram Campus, Jerusalem 91904, Israel

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 November 2012 Received in revised form 11 March 2013 Accepted 22 March 2013 Available online 3 April 2013 Handling editor: Bryan Griffiths

We examined the variations in microfungal communities inhabiting 5 biocrust types (4 cyanobacterial, A eD, and one moss-dominated, E) along a north-facing dune catena at the Nizzana research station (NRS) in the western Negev Desert, Israel. The crusts characterizing by variable abiotic conditions showed an increase in chlorophyll content (A < B < C < D < E) correlated positively and negatively with surface wetness duration and irradiance intensity, respectively. A total of 78 microfungal species from 48 genera was isolated using the soil dilution plate method. Similar to other Negev regions, the NRS microfungal communities were dominated by melanin-containing species with large, multi-celled conidia. Abundance of this xeric group increased toward the more xeric crusts, while mesic Penicillium spp. and Mucoromycotina displayed the opposite trend. Density of microfungal isolates positively correlated with chlorophyll content indicating possible significant influence of organic matter content and wetness duration on fungal biomass. The moss dominated crust differed markedly from the cyanobacterial crusts on species relative abundances, diversity level, and isolate density. The study showed a similarity between the variations in crust microfungal communities within a dune catena at NRS and along the precipitation gradient in Negev, implying that microclimatic differences and regional climatic variability may have a comparable effect on microfungi. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Microfungal communities Dune catena Biological soil crusts Chlorophyll content

1. Introduction In arid and semiarid landscapes, widely distributed biocrusts (known also as biological soil crusts or microbiotic crusts) play an important role in soil surface stabilization, hydrology, and nutrient assimilation (e.g., [1e6]). Biocrusts are considered an appropriate model system to study the relationship between biodiversity and its function [7]. Free-living microfungi, together with bacteria, cyanobacteria, green algae, lichens, and mosses contribute to the composition and functioning of the biocrusts. Yet, only little is known thus far about fungal diversity in crusts. In the studies conducted by States and Cristensen [8] and States et al. [9], a total of 92 and 106 taxa from different categories of crust-inhabiting fungi were isolated in the desert grasslands of Wyoming and Utah, respectively, with 43 taxa being prevalent and characteristic for the microfungal communities. Recent studies of crust free-living fungi from the Colorado plateau [10,11] and the semiarid grassland in central New Mexico, USA [12], which were based on culture-independent molecular methodologies, revealed * Corresponding author. Tel.: þ972 4 8249697; fax: þ972 4 8246554. E-mail address: [email protected] (I. Grishkan). 1164-5563/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejsobi.2013.03.005

comparatively rich fungal diversity belonging mainly to the phylum Ascomycota. In the Negev Desert of Israel, mycobiota in biocrusts and in the non-crusted soil under dominant shrubs was examined in 10 locations along a northesouth rainfall gradient. The mycobiota of the biocrusts (80 species) was characterized by dominance of melanin-containing fungi and the remarkable contribution of morphologically sexual ascomycete species [13]. In the Nizzana research station (NRS) located in the western Negev Desert, Israel, five types of biocrusts (referred hereafter as A, B, C, D, and E) were defined, four cyanobacterial crusts (AeD) and one moss-dominated crust (E). The crusts occupy the mid and bottom slopes of the westeeast trending longitudinal dunes and the interdunes, serving as a protective shield against wind erosion. The crusts differ in various physical and biological parameters including thickness, biomass, and photoautotrophic species composition, with north-facing crusts exhibiting higher biomass and more diverse species composition [14]. While the highbiomass crusts, crusts BeE, extend along the north-facing catena of the dune, the low-biomass crust, crust A, spreads over the southfacing slopes and the interdune. It also covers the top of low dunes (which are therefore called stabilized) where wind velocity is low enough to allow for its establishment.

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Among a number of abiotic factors (such as parent material, dust accumulation, rain, water-holding capacity, and daylight wetness duration) examined in order to evaluate their effect on the crust chlorophyll content, only surface wetness duration yielded significant positive relationship with the chlorophyll content [15]; at the same time, significant negative correlation was found between crust chlorophyll content and radiation intensity [16]. The data thus implied that differences in evaporation in accordance with irradiance determined differences in surface wetness duration and hence in crust biomass, and that variations in crust chlorophyll content might reflect differences in the above edaphic parameters. Following the close link between surface wetness duration, irradiance, and composition of autotrophic species and their biomass [15], we hypothesized that the different crust types at NRS might differ also on their microfungal communities. Based on our previous findings [13,17], we further hypothesized that the above abiotic variables would shape the crust’s microfungal communities influencing the abundance of their xeric (melanin-containing fungi) and mesic (Penicillium spp. and mucoromycotouse species) components. The current research was therefore designed to study the variation in the microfungal communities within the different crust types of NRS. In the course of the study, the following characteristics of the communities were analyzed: species composition; contribution of major taxonomic and ecological groupings to community structure; dominant groups of species, density of isolates, and diversity level-species richness, the ShannoneWiener and the evenness indices. The effect of chlorophyll content on the above characteristics was also estimated. 2. Material and methods 2.1. Site description The research site is located in the Nizzana research station, NRS (34 23’E, 30 56’N), within the Hallamish dune field, at the western Negev Desert, Israel. Mean annual precipitation is 95 mm, occurring primarily between November and April [18]. Being the eastern extension of the Sinai dune fields, the Hallamish dune field is comprised of longitudinal dunes (up to 15e 20 m high), trending westeeast, and separated by wide (50e 200 m) interdunes. The dunes are characterized by relatively long north-facing slopes and by relatively short south-facing slopes. Most of the dunes have a mobile crest and are therefore considered active. The mobile crest lacks biocrusts and is covered by very sparse vegetation (<5%) while the lower flanks of the dunes and the sandy interdunes have 10e20% vegetation cover [19] and are almost entirely covered by biocrusts. Low dunes, below 6e8 m, have a complete crust cover and are therefore considered stabilized.

Five crust types (AeE) were defined at NRS, four (AeD) cyanobacterial crusts and one (E) moss-dominated crust (Fig. 1). The main crust properties are shown in Table 1. Topographically-induced surface wetness duration determined the crust chlorophyll content [15] as well as the crust type [14]; the chlorophyll content significantly negatively correlated with radiation intensity and positively e with surface wetness duration ([16]; Fig. 2). While the most xeric crust A is located on the south-facing slope and the interdune (termed herein as Ab), and at the top of the stabilized dune (termed herein as At), the high-biomass crusts BeE occupy the north-facing slopes, from gently sloping surfaces (crust B) to steeper slope sections (crust C and especially crust D). Crust E is located at the interface between the north-facing slope and the interdune and was divided herein to an inclined section (termed herein as Ei) and a horizontal section (termed herein as Eh). In addition to extended shading (and thus low evaporation rate), crust E receives extra amounts of water by runoff and subsurface flow, all substantially contributing to a relatively long surface wetness duration [20]. While both Ei and Eh showed similar surface wetness duration [3], Eh exhibited more xeric characteristics owing to its more sun-exposed position. 2.2. Sampling Crust samples were collected during the summer of 2009 (August) and 2011 (June); some climatic characteristics during these periods are shown in Table 2. The samples were taken from the 50  50 cm plots which were demarcated along two transects, w10 m apart, which extended along a north-facing catena. The transects extended from the upper top of a low (w8 m high) crusted (and hence stabilized) dune to the interdune. In this way, sampling of the non-crusted mobile sand was avoided. All together, 7 points (At, Ab, B, C, D, Ei, and Eh) covering all 5 crust types defined within the research site, were included in the analysis. Two crust samples (one sample, 20  20  0.3 cm, from each transect) were collected from each plot at each sampling period. The samples were taken from the uppermost crust layer that hosts all photoautotrophic components. The crust samples, placed in sterile paper bags, were stored in dry conditions (22e27% of relative humidity) until processing. 2.3. Chlorophyll content Concentration of chlorophyll a (hereafter chlorophyll) was measured in 24 crust cores of 1.2 cm in diameter that were taken from each plot at the end of the growing period. Chlorophyll of each crust core was extracted by hot methanol (70  C, 20 min) in the presence of MgCO3 (0.1% w v1) in sealed test tubes and assayed according to Wetzel and Westlake [21]. 2.4. Characterization of fungal communities

Fig. 1. Schematic cross section (from top to bottom) along a 8 m-high stabilized dune (At e crust A located at the top of the stabilized dune; Ab e crust A located on the south-facing slope and the interdune; Ei e inclined section of crust E; Eh e horizontal section of crust E).

For isolation of microfungi, the soil dilution plate method [22] was employed. Despite certain limitations and biases such as overestimation of highly sporulating species, loss of rare fungi and the inability of some fungi to grow on culture media [23], this method remains a useful approach for the initial characterization of the ecology of fungal communities (e.g., [24,25]). It is especially applicable to 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 were employed: Malt Extract Agar (MEA) and Czapek’s Agar (CzA) (SigmaeAldrich Inc, St. Louis, USA). Streptomycine (Spectrum Chemical Mfg. Corp, Gardens, USA) was added to each medium (100 mg ml1) to suppress bacterial growth. One mL of soil

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Table 1 Physical and chemical properties of crusts determined during long-term monitoring at the Nizzana research station. Number in parenthesis indicates one SD; N indicates number of samples. Crust thickness, strength, and chlorophyll, protein, and carbohydrate content exhibited significant between-type differences (P < 0.05) (data after[14,15]). Crust Color (according to the type Munsell color chart)

A B C D E

Dry

Wet

10YR6/4 10YR6/4 10YR6/4 10YR6/4 10YR6/3 10YR6/3

10YR5/4 10YR5/3 10YR4/4 10YR4/3 10YR3/3 10YR3/3

Thickness (mm)

1.1 1.5 2.0 2.8

(0.3) (0.3) (0.4) (0.3)

N N N N

¼ ¼ ¼ ¼

24 12 12 18

Crust strength (g cm2)

Chlorophyll (mg m2)

175.4 (78.8) N ¼ 24 95.5 (31.2) N ¼ 12 293.7 (85.1) N ¼ 12 438.7 (209.1) N ¼ 14

16.7 20.7 28.5 43.4

(9.9) N ¼ 196 (10.5) N ¼ 196 (16.7) N ¼ 196 (27.6) N ¼ 196

Protein (g m2)

Carbohydrate (g m2)

4.17 (1.36) N ¼ 97 5.65 (2.19) N ¼ 84 6.34 (2.69) N ¼ 95 10.42 (4.62) N ¼ 98

5.28 (2.27) N ¼ 92 7.65 (3.28) N ¼ 84 9.15 (4.34) N ¼ 98 15.92 (5.97) N ¼ 98

Main photoautotrophic speciesa

MIC; MIC; MIC; MIC; OSC; 10.3 (1.4) N ¼ 12 782.0 (290.4) N ¼ 12 53.2 (25.8) N ¼ 196 31.55 (13.28) N ¼ 79 33.16 (14.71) N ¼ 89 BRY; OSC;

Moss cover (%)

PHO <0.1 SCY 0.1 SCY 1 SCY; 10 SCH; NOS TOR; MIC; 80 SCH; NOS

a Main species: cyanobacteria: MIC ¼ Microcoleues sp; PHO ¼ Phormidium sp; Scy ¼ Scytonema sp; OSC ¼ Oscillatoria sp; NOS ¼ Nostoc sp; SCH ¼ Schizothrix sp, mosses: BRY ¼ Bryum dunense; TOR ¼ Tortula brevissima.

suspension from the dilutions 1:10e1:100 (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 and 37  C in darkness for 10e15 days (3 plates for each medium and temperature). 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 Water Agar (agar e 20 g, water e 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. [26]. 2.5. Data analyses Density of fungal isolates was expressed as “colony forming units” (CFU) per g dry substrate. Analysis of biodiversity was based

on the ShannoneWiener index (H) and evenness (J ¼ H/Hmax) [27]. To test similarity between the microfungal communities of 2009 and 2011, the percentage similarity coefficient [27] was used. To analyze spatiotemporal variations in the microfungal community structure, five major groupings were chosen: Penicillium spp., Aspergillus spp., teleomorphic Ascomycota (species producing morphologically expressed sexual state in culture), Mucoromycotina, and melanin-containing microfungi. These groupings were used in all of our previous studies on soil microfungi in Israel [28]. The contribution of each group to mycobiota structure 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 relative abundance (percentage) in the Shannon index [29]. 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 the non-parametric Wilcoxon matched pair test and a one-way ANOVA followed by multiple comparisons tests to compare data from different crust types on chlorophyll content, diversity characteristics, relative abundance of microfungal groupings, and isolate densities. The relationship of these data with chlorophyll content was estimated by linear and nonlinear regression analyses. To evaluate similarity between microfungal communities from different crust types, the clustering of the communities based on species relative abundances was performed by the Unweighed pair-group average method with Euclidian distance as the distance coefficient. 3. Results 3.1. Rainfall and chlorophyll content The years of the investigation were extremely dry with rain amounts during 2008/09 and 2010/11 to only 31.3 and 30.6 mm, respectively, which is much less than the long-term annual average of 95 mm. In both years the highest daily rain events were

Table 2 Climatic variables (T e temperature at 50 cm above the ground; RH e relative humidity; Rad e irradiance) measured during August, 2009 and June, 2011 at the Kadesh Barnea meteorological station (http://www.meteo-tech.co.il/negev/negev_ weekly.asp) located near 5 km south of NRS. Fig. 2. Relationships of crust chlorophyll content with irradiance intensity (a) and daylight surface wetness duration (b). Bars indicate one SE (modified from Refs. [15,16]).

Period

Tmax,  C

Tmin,  C

RHmax, %

RHmin, %

Radmax, W m2

August, 2009 June, 2011

39.7 40.1

17.4 16.1

90 100

13 16

918 1101

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up to 4e6 mm and therefore surface wetness duration was relatively short and in most days the surface dried during midday. Chlorophyll content showed a clear gradient with A < B < C < D < E. This parameter was w2.5-fold higher in the most mesic crust, crust E, than that in the most xeric crust A (Fig. 3).

Table 3 Diversity characteristics of microfungal communities (S e number of species, including sterile strains, H e Shannon index, J e evenness) in the crusts of NRS. Means with the same letter are not significantly different (a one-way ANOVA, at the 5% level). Crust type

2009

At Ab B C D Ei Eh

26 27 26 32 31 23 25

S

3.2. Composition and diversity of microfungal communities The crust mycobiota of NRS is composed of 78 species from Mucoromycotina (2 species), teleomorphic Ascomycota (20), anamorphic (asexual) Ascomycota (55), and Basidiomycota (1). The species belonged to 48 genera; the most common were Chaetomium (7), Phoma (5), Aspergillus, and Alternaria (4 each). We isolated 56 and 52 species from the crusts sampled during the summer of 2009 and 2011, respectively. Notably, the communities of the predominant inclined section of moss-dominated crust (Ei) were characterized by significantly lower diversity characteristics than the communities from the cyanobacterial crusts (Wilcoxon test and a one-way ANOVA for both Shannon and evenness indexes, p < 0.05) (Table 3). As Fig. 4 shows, melanin-containing microfungi predominated in all communities e they comprised 61% of the total species number and 57.3e99.8% of the contribution index (the species are in bold in Table 4). The main core of melanin-containing species was represented by species with large (>20 m) multicellular spores (Ulocladium atrum, Alternaria alternata, Embellisia phragmospora, Sporormiella minima). All other microfungal groupings contributed much less to the communities’ structure: Aspergillus spp. e 0e3.5%, Penicillium spp. e 0e18.8%, Mucoromycotina e 0e14.3%, teleomorphic Ascomycota e 0.6e16.5%; the last grouping was mainly composed of melanin-containing species. A complex of the most abundant and/or frequently occurring species in the crust soil involved: U. atrum, A. alternata (frequency of occurrence 100%), Cladosporium cladosporioides, Stachybotrys chartarum, E. phragmospora, Stemphyium state of Pleospora tarda, Mortierella humilis, S. minima, Thielavia terricola, Pyrenochaeta cava, Fusarium oxysporum, Penicillium aurantiogriseum, and three thermotolerant species isolated mainly at 37  C e Aspergillus fumigatus, Canaryomyces notabilis, and Chaetomium strumarium. 3.3. Spatial variations in microfungal communities

2011 H

a a a a a a a

1.78 1.95 1.90 2.38 2.14 1.25 2.03

J a a a a a b a

0.58 0.59 0.58 0.69 0.62 0.40 0.63

S b ab ab a ab c ab

26 26 26 28 27 22 22

H a a a a a a a

2.35 2.31 2.25 2.23 2.50 1.82 2.16

J a a a a a b a

0.72 0.71 0.69 0.67 0.76 0.59 0.70

a a a a a b a

Notes: At e crust A located at the top of the stabilized dune; Ab e crust A located on the south-facing slope and the interdune; Ei e inclined section of crust E; Eh e horizontal section of crust E.

percentage similarity in the different crust types). This enabled us to group the results of both years. Cluster analysis based on relative abundances of species indicated that the moss-dominated crust E differed markedly from the cyanobacterial crusts AeD (Fig. 5). In the studied crusts, the CFU number varied from 2130 to 19,360 g1 dry soil. In each sampling set, the moss-dominated crust was significantly richer in microfungal isolates than the cyanobacterial crusts (p < 0.01) while the cyanobacterial crusts AeD did not differ remarkably from each other on this characteristic (2130e 3620 CFU g1 dry soil). In this regard one should note the difference in isolate density between the horizontal and inclined sections of crust E, with the inclined section having 1.5-fold higher CFU number. 3.4. Relationships of mycobiotic characteristics with chlorophyll content Strongly significant (p < 0.01) positive linear relationship was found between isolate density and chlorophyll content (Fig. 6a). Abundance of teleomorphic Ascomycota also displayed significant (p < 0.05) but negative linear correlation with chlorophyll content (Fig. 6b); notably, this microfungal grouping consisted almost only of melanin-containing species. More than half of the variability in abundance of Mucoromycotina could be explained by the variation in chlorophyll content when employing the non-linear regression

Composition of the microfungal communities sampled in 2009 and 2011 was highly similar to each other (63.4e78.6% of 100

contribution index, %

90 80 70 60 50 40 30 20 10 0

At

Fig. 3. Average chlorophyll content in different crust types monitored during the end of growing period (At e crust A located at the top of the stabilized dune; Ab e crust A located on the south-facing slope and the interdune; Ei e inclined section of crust E; Eh e horizontal section of crust E). Bars indicate one SE. Means with the same letter are not significantly different (a one-way ANOVA, at the 5% level).

Ab

B

C

D

Ei

Eh

Fig. 4. Spatial dynamics of relative abundance of main microfungal groupings in different crust types of NRS: e Mucoromycotina; e teleomorphic Ascomycota; e Penicillium spp.; e Aspergillus spp.; e melanin-containing spp. The area below the white line on the bars of melanin-containing microfungi indicates contributions of species with large multicellular spores (At e crust A located at the top of the stabilized dune; Ab e crust A located on the south-facing slope and the interdune; Ei e inclined section of crust E; Eh e horizontal section of crust E).

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Table 4 Common microfungal species in the crusts of NRS, with their average relative abundance (%). Bolded species are melanin-containing. Species

Mucoromycotina Mortierella humilis Teleomorphic Ascomycota Canaryomyces notabilis Chaetomium cochlioides Ch. globosum Ch. nigricolor Ch. strumarium Ch. succineum Sporormiella minima Thielavia terricola Ascomycetes (immature) Anamorphic Ascomycota Alternaria alternata Aphanocladium album Aspergillus fumigatus A. niger Botryotrichum piluliferum Cladosporium cladosporioides Drechslera australiensis Embellisia chlamydospora E. phragmospora Fusarium oxysporum F. equiseti Geotrichum candidum Lecanicillium psalliotae Papulaspora pannosa Penicillium aurantiogriseum Phoma exigua Pyrenochaeta cava Stachybotrys chartarum Stemphylium state of Pleospora tarda Ulocladium atrum Basidiomycota Sporotrichum sp. Mycelia sterilia, light dark

Crust type At

Ab

e

e

0.3 e e e e 0.1 1.1 e e

B

C

D

Ei

Eh e

3.2

3.4

6.6

3.5

0.4 e e e e e 6.2 2.8 0.15

0.5 0.8 e e 0.1 0.8 2.8 3.8 0.8

0.4 0.07 1.0 0.2 0.07 e 1.7 5.5 e

0.6 e e 0.2 0.1 0.08 1.5 e 4.2

e 0.4 0.4 0.02 0.3 0.4 0.02 0.09

0.2 0.2 e 0.06 0.06 e 5.8 e 0.1

16.5 e 0.3 e e 0.6 e 7.6 e e 1.6 e e e 1.8 0.6 1.6 2.5 9.5 38.6

18.5 e 0.15 0.05 0.5 1.5 e e 3.2 1.3 e 2.0 e e 1.6 3.5 0.5 1.5 3.8 46.5

20.1 e 0.2 e e 1.2 e e 1.4 0.5 1.6 e 0.1 e e 3.4 7.8 7.6 42.0

14.2 0.7 0.07 e e 2.8 0.2 e 1.6 2.3 e e 3.2 0.07 1.7 0.2 6.3 2.5 8.4 34.5

14.3 0.4 0.8 0.8 e 1.8 e 1.9 5.8 0.4 0.4 e 0.8 0.8 2.5 e 3.5 0.8 7.4 39.2

1.8 e 0.02 1.0 0.6 0.1 0.05 e 5.6 1.3 e e e 0.2 2.1 13.6 0.1 0.7 2.7 58.8

8.4 e e e e 0.1 e 2.3 23.5 1.6 e 0.2 e e e 13.8 0.06 2.7 9.6 32.3

e 5.8 1.1

0.5 0.8 2.5

e 0.6 2.4

0.1 1.6 6.3

1.9 1.5 e

0.9 0.7 3.2

e e e

Notes: At e crust A located at the top of the stabilized dune; Ab e crust A located on the south-facing slope and the interdune; Ei e inclined section of crust E; Eh e horizontal section of crust E.

analysis (Fig. 6c). Species richness also displayed the non-linear relationship with chlorophyll content (Fig. 6d), with the highest number of species found in the crusts C and D which are characterized by intermediate values of chlorophyll content.

Fig. 5. Clustering the microfungal communities from different crust types of NRS based on species relative abundances (At e crust A located at the top of the stabilized dune; Ab e crust A located on the south-facing slope and the interdune; Ei e inclined section of crust E; Eh e horizontal section of crust E).

4. Discussion The present study highlights the role of topographicallyinduced irradiance and moisture variability in structuring the microfungal communities. Variations in structure of the communities through different crust types at NRS accompanied the variations in crust composition (dominance of cyanobacteria e dominance of mosses). Cluster analysis, which was based on species relative abundance, showed that the moss-dominated crust E differed markedly from the cyanobacterial crusts AeD. Similar variations in fungal diversity were found at the Colorado plateau between cyanobacterial and lichen-dominated crusts [11]. In both cases, the high-biomass crusts (either moss or lichen) differed from the low-biomass (cyanobacterial) crusts. In our study, the mycological parameter that sharply differentiated the cyanobacterial and mosses-dominated crusts was associated with the density of the fungal isolates which can be considered an indirect characteristic of fungal biomass. The isolate density was 3.6e9.0-fold higher in crust E than in crusts AeD. Furthermore, this parameter displayed significant positive linear relationship with the chlorophyll content which showed a clear gradient with the crusts A < B < C < D < E in agreement with our previous findings [15,30]. Following the close link between chlorophyll content and organic matter of the crusts [14] and between the chlorophyll content and surface wetness duration [15], one may conclude that surface wetness duration not only dictates the biomass of the autotrophic components of the crusts (as deduced

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Fig. 6. Relationships of characteristics of microfungal communities with crust chlorophyll content.

from their chlorophyll content) or the organic matter of the crust but, expectedly, also influences the fungal biomass. Similar patterns were observed in the central (Makhtesh Ramon area) and southern (Nahal Shaharut) Negev. In both cases, relatively small spatiotemporal variation in microfungal community structure was found. At the same time, there were drastic fluctuations in the isolate densities exhibited high-positive dependence on the organic matter content [17,31]. Yet, while having much higher isolate density, crust E (its predominant inclined section) was poorer in species richness and diversity level of the microfungal communities in comparison with crusts AeD. Similarly, in the Makhtesh Ramon area the habitats under shrub canopies were characterized by lower diversity indices (Shannon, evenness) compared to the sunny open habitats [17]. The negative correlation between the diversity level and the CFU number was indeed expected. Often, an increase in the isolate number is caused by an abundant development of one or two species, which decrease both species richness and diversity level of the microfungal communities. Expectedly, the mycobiota of the uppermost crust layer at NRS displayed similar features to that of other mycobiotas in the Negev Desert [13,17,31]. It was expressed in the predominance of melanincontaining species both in number and abundance which is considered a typical trait for almost all mycologically studied desert soils (e.g., [32e40]). The prevalence of melanized fungi with large multicellular spores found in most localities of the Negev Desert

was also characteristic for the NRS crusts. These fungi, belonging to the order Pleosporales, were also found to predominate at the Colorado plateau and in the semiarid grassland in central New Mexico, USA [10e12]. These studies along with our survey at NRS suggest that dark pigmentation as well as multicellular spore morphology are very important for dispersal and resting functions in climatically stressful desert habitats. However, these characteristics may not be as critical for temperate biocrusts, such as in central Europe, where the abundance of melanized Pleosporales in the crusts was relatively low [41]. Being overwhelmingly prevailing in all habitats, melanized species with large manycelled spores displayed however notable spatial variations in their abundance which increased from Ei to At. To a lesser extent, the opposite trend was characteristic for the mesic groupings of Penicillium spp and Mucoromycotina. Spatial variation of these microfungal groupings was not significantly influenced by crust chlorophyll content, yet we can interpret this variation as a result of increasing irradiance intensity and, subsequently, as a result of increasing xeric conditions. While penetrating only up to 100 microns into soil depth [42], UV irradiance nevertheless plays an important role in the organization and distributional pattern of microfungal communities (e.g., [43,44], and references therein). Therefore, the dominance of darkcolored microfungi was characteristic for all crust types in NRS. On a regional scale, melanized fungi with large thick-walled multicelled conidia increased their abundance southward in the central

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and southern Negev Desert [13], overwhelmingly dominating all microfungal communities in the southern Negev [31]. At NRS, increasing abundance of this microfungal group from the mossdominated crust Ei to the cyanobacterial crust A resembles the above regional pattern. The effect of irradiance can also be seen in the more sun-exposed horizontal section of crust E, Eh, which in comparison with its inclined section, Ei, was characterized by remarkably higher amount of melanized fungi. Our study revealed the same dominant and frequent microfungal species at the crusted dune slopes of NRS and in the noncrusted locations along a northesouth gradient in the Negev Desert. Similar absence of crust-specific mycobiota was also found in the crusts of the Colorado plateau following the use of the culture independent molecular fingerprint method [9]. Nevertheless, for a decisive conclusion to be made, further mycological analysis of crust-covered desert areas is needed which will employ both the culture-based isolation technique and the metagenomic approach. This can certainly improve our understanding of fungal diversity and the role of environmental factors in shaping the crust fungal communities. The present study showed that the mycobiota of the uppermost crust layer in NRS, similar to other mycobiota in the Negev Desert, was dominated by melanin-containing species with large multicelled conidia. Abundance of these xeric species increased spatially, with increasing xeric conditions, while mesic Penicillium spp. and Mucoromycotina displayed the opposite trend. The variations in crust composition (dominance of cyanobacteria e dominance of mosses) were accompanied by the variations in microfungal community structure. The moss-dominated crust differed notably from the cyanobacterial crusts on species relative abundances, diversity level, and isolate density. Following the similar pattern found at NRS along a 20 m transect containing xeric and mesic habitats, and along a precipitation gradient of w100 km in the Negev Desert [13], we would like to argue for the advantage of the drainage basin approach for study of abioticebiotic relations. Contrary to a regional research during which the inevitable involvement of additional variables such as cloudiness, air temperatures, relative humidity or soil type cannot be controlled, a local research within a single drainage basin or catena ensures, as much as it is possible under field conditions, maximum control of the variables examined. Acknowledgments We thank the Israeli Ministry of Absorption for financial support of this research. References [1] S.D. Warren, Ecological role of microphytic soil crusts in arid ecosystems, in: D. Allsopp, D.L. Hawksworth, R.R. Colwell (Eds.), Microbial Diversity and Ecosystem Function, Cab International, London, 1995, pp. 199e209. [2] G.J. Kidron, A. Yair, Rainfallerunoff relationships over encrusted dune surfaces, Nizzana, western Negev, Israel, Earth Surf. Process. Landf. 22 (1997) 1169e1184. [3] G.J. Kidron, Differential water distribution over dune slopes as affected by slope position and microbiotic crust, Negev Desert, Israel, Hydrol. Process. 13 (1999) 1665e1682. [4] J. Belnap, O. Lange, Preface, in: J. Belnap, O. Lange (Eds.), Biological Soil Crusts: Structure, Function, and Management, Springer-Verlag, Berlin, New York, 2001, pp. VeIX. [5] T.M. Langhans, C. Storm, A. Schwabe, Community assembly of biological soil crusts of different successional stages in a temperate sand ecosystem, as assessed by direct determination and enrichment techniques, Microbiol. Ecol. 58 (2009) 394e407. [6] Y. Bashan, L.E. de-Bashan, Microbial populations of arid lands and their potential for restoration of deserts, in: P. Dion (Ed.), Soil Biology and Agriculture in the Tropics. Soil Biology Series 21, Springer, Berlin, Heidelberg, 2010, pp. 109e137 Chapter 6.

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