Bacterial diversity and fatty acid composition of hypersaline cyanobacterial mats from an inland desert wadi

Journal of Arid Environments 115 (2015) 81e89

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Journal of Arid Environments journal homepage: www.elsevier.com/locate/jaridenv

Bacterial diversity and fatty acid composition of hypersaline cyanobacterial mats from an inland desert wadi b 
b, Peter Gajdos b, Milan Certík Raeid M.M. Abed a, *, Tatiana Klempova a

Sultan Qaboos University, College of Science, Biology Department, P.O. Box 36, Postal Code 123, Al Khoud, Oman Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinsk
eho 9, 812 37, Bratislava, Slovak Republic

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2014 Received in revised form 8 December 2014 Accepted 14 January 2015 Available online 19 January 2015

Hypersaline cyanobacterial mats are infrequently reported in desert streams and information on such mats is very scarce. We investigated bacterial diversity and fatty acid composition in hypersaline cyanobacterial mats from Wadi Muqshin, located inland near the Empty Quarter desert in Oman. Most of the detected cyanobacteria belonged to known halotolerant, thermotolerant and UV resistant types that were typically reported in other hypersaline mats. A total of 84,834 ribosomal sequences were obtained, with 62e79% of the sequences affiliated to Cyanobacteria, Proteobacteria, Bacteroidetes, Clostridia and Chloroflexi. Cluster analysis showed that Mat 5 with the highest salinity was profoundly different from the other mats and shared species were
72% between all mats. While most Deltaproteobacteria in the wadi mats belonged to sulfate-reducing bacteria, a number of sequences related to purple sulfur, purple non-sulfur as well as green no-sulfur bacteria were also detected. Different saturated, branched and mono- and di-unsaturated fatty acids were detected in all mats, with the saturated 16:0 and 18:0 and the monounsaturated 16:1 and 18:1 fatty acids accounting for relative amounts of 70e77% of total fatty acids. We conclude that microbial diversity and fatty acids composition in the desert wadi hypersaline cyanobacterial mats resemble their counterparts from other hypersaline environments. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Desert Wadi Cyanobacterial mats Fatty acids Pyrosequencing Cyanobacteria

1. Introduction Microbial mats are accretionary, cohesive, macroscopic accumulations of microbial communities, which are often laminated and grow mostly on submerged or moist surfaces (Pierson, 1992). They are distributed worldwide in a surprisingly wide range of environments (Pierson, 1992). Under hypersaline conditions, microbial mats develop well, mainly because of the restricted abundance and activity of animal grazers and the absence of competition from macrophytes (Farmer, 1992). Most studied hypersaline mats originated from intertidal flats, closed basins (e.g. solar salterns and evaporation ponds), hypersaline lakes and hot springs, but few from arid and semiarid inland saline lakes. Hypersaline mats from inland saline lakes were mainly reported from Australia, Spain, lakes of the Great Rift Valley (Africa) and the deserts of America (Bauld, 1981, 1986; Guerrero and de Wit, 1992;

* Corresponding author. Sultan Qaboos University, College of Science, Biology Department, P.O. Box 36, Al Khoud, Postal Code 123, Muscat, Oman. E-mail address: [email protected] (R.M.M. Abed). http://dx.doi.org/10.1016/j.jaridenv.2015.01.010 0140-1963/© 2015 Elsevier Ltd. All rights reserved.

Jonkers et al., 2003). So far, there have been very few reports on hypersaline microbial mats from inland saline lakes or streams in the arid deserts of the Arabian Peninsula (Jupp et al., 2008), although such mats were described in intertidal flats of the region (Abed et al., 2007, 2008). It is of interest to find out whether hypersaline mats from arid deserts harbor similar/different microbial communities to their counterparts in other hypersaline environments. The tropical Arabian Desert is the fourth largest desert in the world and it occupies most of the Arabian Peninsula. It is characterized by extreme environmental conditions, with intense temperatures reaching as high as 60  C in hot summers, high humidity and a rainfall average less than 4 inches (100 mm) a year throughout the desert. During torrential rains, drainage basins become flooded and wadis are formed in mountain valleys. Wadis, which is an Arabic term used to describe corridors for fluvial run-off that may contain perennial, intermittent or ephemeral surface flow, are widespread in the Arabian region. Many wadis can be found across Oman, due to its special geology, and large areas of these wadis are covered by laminated cyanobacterial mats. These mats experience extreme conditions of temperature, desiccation and UV

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and light intensity, thus are expected to attract well-adapted extremophilic microorganisms. Unusually, few wadis in Oman were found to contain hypersaline cyanobacterial mats, instead of the typical freshwater mats, due to the high evaporation rates and the recharge regimes from deep groundwater aquifers (Jupp et al., 2008). An example is Wadi Muqshin, located inland, ca. 200 km from the Arabian Sea coast, close to the Empty Quarter, and consists of a series of pools with different salinities ranging from 5 to 25% (Jupp et al., 2008). Hypersaline cyanobacterial mats in desert wadis have been infrequently reported and rarely examined in any detail. In this study, we investigated bacterial communities (using direct microscopy and pyrosequencing) and fatty acid composition in five cyanobacterial mats from Wadi Muqshin, Oman. In particular, we asked the question, “how comparable are the hypersaline mats from wadi ecosystems to their counterparts from other habitats?”. This study will provide the first detailed insights into microbial communities and lipid biomarkers in hypersaline mats from inland saline streams in the Arabian Desert. 2. Materials and methods 2.1. Sampling sites and nutrient analysis Five cyanobacterial mats were sampled from Wadi Muqshin (48 km long) in the southwest of the Sultanate of Oman, bordering the dunes of the Empty Quarter desert. Detailed description of the site and information on its hydrogeology and mineralogy are given in Jupp et al. (2008). The wadi contains pools that are flooded during raining season, but then evaporated through intense summer (temperature can reach as high as 55  C), resulting in extremely hypersaline conditions (salinity is between 5% and 25%). Large areas of these pools are covered with benthic, well developed, laminated cyanobacterial mats. Five mat samples were collected from different pools of the wadi; two from a downstream pool (Mat 1 and 2), two from a middle stream pool (Mat 3 and 4) and one sample from an upstream pool (Mat 5). The pools were ca. 100e200 m apart from each other and had the salinities 6.5%, 7.3 and 12% at the time of sampling, respectively. The collected mats differed in appearance, texture and salinity. The mats were cut carefully using a sterile scalpel and stored in Petri plates. A map of the sampling location, photographs of the mats and the wadi pools and detailed physical and chemical parameters of the sampling sites can be found in Jupp et al. (2008) and Abed et al. (2011a). 2.2. Microscopy and morphotype quantification of cyanobacteria The depth of the oxygenic photosynthetic layer in each mat was determined by microsensor measurements (not shown). The top cyanobacterial layer of the mats (1e3 mm) was excised under a dissecting microscope with a clean scalpel blade and sterile forceps. Samples were torn apart, mounted in water on a microscope glass slide and observed using transmitted light, phase contrast and fluorescence microscopy. Different morphotypes were identified and photographed. Three cores from each mat sample were observed microscopically to ensure a good overall representation of resident morphotypes. Morphological identification was carried
rek and out in accordance with traditional phycological (Koma Anagnostidis, 2005) and bacteriological (Castenholz et al., 2001) systems. 2.3. Pyrosequencing and sequence analyses The mat samples (ca. 300e500 mg each) were subjected to DNA extraction using the Power Biofilm DNA isolation kit (MOBIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer's

instructions. Purified DNA extracts were submitted to Molecular Research MR DNA Laboratory (Shallowater, TX, USA) for tagpyrosequencing. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed as described before (Dowd et al., 2008) using the GS FLX titanium sequencing kit XLR70. One-step PCR was performed using a mixture of hot start and hot start high fidelity taq polymerases resulting in amplicons that extend 350e450 bp from the 27F region (Escherichia coli rRNA numbering). The bTEFAP sequencing was performed according to the MR DNA protocols (www.mrdnalab.com). All sequences were analyzed and taxonomically classified using the NGS analysis pipeline of the SILVA rRNA gene database project (SILVAngs) (Quast et al., 2013). Each read was aligned using the SILVA Incremental Aligner (SINA) (Pruesse et al., 2012) against the SILVA SSU rRNA SEED and quality controlled (Quast et al., 2013). Reads shorter than 50 aligned nucleotides and reads with more than 2% of ambiguities, or 2% of homopolymers, respectively, were excluded from further processing. Identical reads were then identified (dereplication), the unique reads were clustered (OTUs), and the reference read of each OTU was classified using cd-hit-est (version 3.1.2; http://www.bioinformatics.org/cd-hit). The classification was performed by a local nucleotide BLAST search against the non-redundant version of the SILVA SSU Ref dataset (release 111; http://www.arb-silva.de) using blastn (version 2.2.22þ; http:// blast.ncbi.nlm.nih.gov/Blast.cgi) with standard settings. The classification of each OTU reference read was mapped onto all reads that were assigned to the respective OTU, yielding the number of individual reads per taxonomic path. Reads without any BLAST hits or reads with weak BLAST hits were assigned to the meta group “No Relative” in the SILVAngs fingerprint.

2.4. Fatty acid analysis Mat materials were gently dried at 65  C for 10 h and weighed. Lipids were extracted from dry homogenized mat two times using 100 ml chloroform/methanol (2:1, v/v) for 3 h at laboratory tem perature with occasional stirring (Certík et al., 1996). After extraction, the mixture was filtered and the extracts were collected. 0.9% KCl (1.2-fold of total extract volume) was then added, the mixture was stirred vigorously for 1 min and centrifuged to effect phase separation. The chloroformelipid containing layer was filtered through anhydrous Na2SO4 and evaporated under vacuum. Total lipids were determined gravimetrically (triplicate standards of dry yeast cells were used to assess reproducibility) and used for further analysis. Fatty acids from total lipids were converted to their methylesters by methanolic solution of sodium methoxide and methanolic HCl and analyzed by gas chromatography (GC-6890 N, Agilent Technologies) using a capillary column DB-23 (60 m  0.25 mm, film thickness 0.25 mm, Agilent Technologies) and a FID detector (constant flow, hydrogen 40 ml/min, air 450 ml/ min, 250  C) under a temperature gradient (150  C held for 3 min; 150e175  C at a program rate 7.0  C/min; 175  C held for 5 min; 175e195  C at a program rate 5.0  C/min; 195e225  C at a program rate 4.5  C/min; 225  C held for 0.5 min; 225e215  C at a program rate 10  C/min; 215  C held for 7 min; 215e240  C at a program rate 10  C/min; 240  C held for 7 min) with hydrogen as carrier gas (flow 2.5 ml/min, velocity 57 cm/s, pressure 220 kPa) and a split ratio of 1/20 (Inlets: heater 230  C; hydrogen flow 51 ml/min for 2 min, then hydrogen flow 20 ml/min; pressure  220 kPa) (Certík et al., 1996). The fatty acid methylester peaks were identified by authentic standards of C4eC24 fatty acid methylesters mixture (Supelco, USA) and evaluated by ChemStation B 01 03 (Agilent Technologies).

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3. Results 3.1. Cyanobacterial morphotypes as revealed by direct microscopy The mats harbored a variety of morphologically recognizable filamentous and coccoid cyanobacterial types, differing in their distribution and abundance in every mat (Fig. 1AeN, Table 1). The most dominant cyanobacterial morphotype was the filamentous Microcoleus chthonoplastes (Fig. 1A), which is characterized by closely bundled trichomes (2e5 mm diameter) in a common sheath, and by bullet-shaped apical cells. This cyanobacterium was very common in Mats 1e4, which experience fluctuating salinities, but was not observed in Mat 5 with the highest salinity. The chroococcalean cyanobacterium Johannesbaptistia pellucida (Fig. 1B) was encountered in all mats except Mat 2. This cyanobacterium has cells that are much shorter than wide forming pseudofilaments. The nitrogen fixing cyanobacterium Scytonema was only detected in Mat 3. Scytonema possess intercalary heterocytes and form filaments that are 9e13 mm in diameter, usually yellow brown due to the presence of the UV sunscreen pigment scytonemin (Fig. 1C). Filaments of Schizothrix with a one to two trichomes surrounded by wide hyaline sheaths often with disconnected cells were observed in 3 out of 5 mats (Fig. 1D). Very thin cyanobacteria (1e3 mm), classified as Leptolyngbya, with filaments containing consistently single trichomes within thin but firm sheaths occurred within colonies of other cyanobacteria or within common gelatinous matter secreted by the entire microbial community (Fig. 1EeF). Several types of unicellular cyanobacteria belonging to the genera Gloeocapsa, Chroococcus, Aphanocapsa and Synechococcus were detected in the mats but with different distribution (Fig. 1GeK). Gloeocapsa was only observed in Mat 4, and this cyanobacterium is composed of small groups of irregularly arranged cells, enveloped by wide, usually stratified individual gelatinous envelopes, joined together into a formless mass (Fig. 1G). Other coccoid cyanobacterium, which divides in two or three alternating dimensions by successive cell cleavage, was represented in most mats by species of Chroococcus (Fig. 1H). Aphanocapsa with more or less spherical cells was detected in all mats whereas Synechococcus was only detected

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in three mats (Table 1). Minor microscopically conspicuous components in the mat samples included filamentous morphotypes with tightly coiled trichomes corresponding to the botanical species Spirulina labyrinthiformis (trichome diameter ca.1.5e2 mm) and Spirulina subsalsa (trichome diameter ca 2.5e4 mm) (Fig. 1LeN). 3.2. Bacterial diversity as revealed by pyrosequencing A total of 84,834 sequences of 16S rRNA gene were generated from the five mat samples, with 11e19% of the total number of sequences without any close relatives (Table 2). The highest bacterial richness, as determined by the number of OTUs and Chao1 index based on a 97% sequence similarity threshold to define OTU, was highest in Mat 1 and lowest in Mat 5, which had the highest salinity (Table 2). All sequence libraries were far from saturation as indicated by the rarefaction curves that did not level off at the 97% cut-off (Fig. 2A) and higher number of sequences is apparently required to cover the whole community diversity. Cluster analysis showed that Mat 5 was different from other mat samples and formed a separate branch (Fig. 2B). Pairwise comparison of presence/absence of species showed
72% shared species between all mats (Fig. 2C). The microbial community of Mat 5 was profoundly different and shared less than 65% of its species with the other mats. The majority of sequences (62e79%) were affiliated to Cyanobacteria, Proteobacteria, Bacteroidetes, Clostridia and Chloroflexi (Fig. 3A and B). Cyanobacteria constituted 8e29% of the total sequences with the highest abundance in Mat 2 and lowest in Mat 1 (Fig. 3A). Sequences belonging to the cyanobacterial genera Synechococcus, Leptolyngbya, Lyngbya, Microcoleus and Spirulina were detected in all mats (Fig. 4). In contrast, sequences related to known halophilic and halotolerant as well as UV resistant cyanobacteria such as Cyanothece, Chroococcidiopsis, Euhalothece, Dactylococcopsis, Haloleptolyngbya, Halomicronema were encountered in some mats but not others (Fig. 4). Nitrogen fixing heterocystous cyanobacteria of the genera Nostoc and Scytonema were only detected in Mat 4 and 5 but Stigonema was only detected in Mat 1 (Fig. 4).

Fig. 1. Photomicrographs of identified filamentous and unicellular cyanobacterial morphotypes in the wadi mats. The identification and distribution of these morphotypes among the five mats are shown in Table 1. Scale bar is 10 mm for all slides.

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Table 1 Comparison of the cyanobacterial communities among the five wadi mats as determined by epifluorescent microscopy. Morphotype

A B C D EeF G H I K LeN

Probable taxon

Cells

Microcoleus chthonoplastes Johannesbaptistia sp. Scytonema sp. Schizothrix splendida Leptolyngbya spp. Gloeocapsa sp. Chroococcus sp. Aphanocapsa sp Synechococcus sp. Spirulina spp.

Colony color

Width (mm)

Shape

2e5 2e6 9e13 1e4 1e3 2e3 5e8 5e7 1e2 1e3

Isodiametric Discoid Isodiametric Isodiametric Isodiametric Spherical Spherical oval Spherical Spherical Isodiametric

Table 2 Pyrosequencing and bacterial diversity estimators for the studied mat samples. Sample Total number of % of sequences with % of Number of ID sequences no relative OTUs0.03a OTUs0.03a b Singletons

Chao1

Mat Mat Mat Mat Mat

4845 3620 4466 4198 3300

a b

1 2 3 4 5

19,231 15,364 18,753 18,042 13,444

11.1 15.9 19.11 12.92 18.11

6.6 6.3 6.3 6.2 6.7

Operational taxonomic unit at 3% sequence dissimilarity. Singletons are sequences that were observed once.

3085 2260 2809 2580 2209

Dark green Olive/Yellowish green Greyish green Pale green Yellowish green Yellowish brown Dark brown Colorless Yellowish green Pale green

Microbial mat sample Mat 1

Mat 2

Mat 3

Mat 4

Mat 5

þþ þþ e e þþ e þ þ e e

þþ e e þ e e þþ þ þ þþ

þþ þ þ þ þþ e e þ e þ

þþ þ e e þþ þþ þþ þ þ þ

e þþ e þ e e þþ þ þþ e

Among the proteobacterial classes, Alpha-, Gamma-, and Deltaproteobacteria were most frequently encountered with few sequences from Betaand Epsilonproteobacteria (
3%). Alphaproteobacteria was the most dominant class among all proteobacterial classes and made up between 13 and 32% of total sequences (Fig. 3A). Most of the sequences (i.e. 20e29% of alphaproteobacterial sequences) in all mats were phylogenetically affiliated to the genera Dichotomicrobium and Parvularcula while the remaining sequences belonged to the genera Roseovarius, Erythrobacter, Roseibacterium, Paracoccus, Sediminimonas and Skermanella. Gammaproteobacteria occurred at comparable frequencies

Fig. 2. A) Calculated rarefaction curves of observed OTUs (sequences that have 97% similarity are defined as one OTU) richness in the five wadi mats; B) Cluster analysis showing similarities among the five mats' microbial communities based on pyrosequencing data and C) Percentage of species that are shared between the different mats.

Fig. 3. A) Taxonomic affiliation and relative abundance (% of total sequences) of the most common bacterial groups in the five studied wadi microbial mats encountered by pyrosequencing and B) The absolute number of sequences (represented by shape of the symbol) and the number of OTUs (represented by the size of the symbol) in each taxonomic path.

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Fig. 4. Sequence frequency in the investigated wadi mat samples showing the major encountered bacterial genera. The shape of the symbol represents the number of sequences in each taxonomic bath, the size of the symbol represents the number of OTUs at deeper taxonomic levels with that taxonomic path and the color of the symbol indicates the relative frequency of the taxonomic path within the sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in all mats (3e5% of total sequences) and sequences belonging to the same genera were detected in all mats (Fig. 4). Deltaproteobacteria exhibited its higher abundance in Mat 1 (i.e. 12% of total sequences) and lowest abundance in Mat 2 (1.5%) and most of the detected sequences belonged to well-known sulfate-reducing bacterial genera (Figs. 3 and 4). While sequences belonging to the genera Desulfovibrio, Desulfomonile and Desulfonema, were encountered in all mats, sequences belonging to the genera Desulfobacterium, Desulfobacillum, Desulfarculus, Desulfosalsimonas and Desulfopila were encountered in some mats but not others (Fig. 4). Sequences belonging to the phylum Bacteroidetes were abundant in all mats (9e22% of all sequences). The highest abundance of this group was detected in Mat 5 and made up 22% of total sequences (Fig. 3A). Most of the sequences belonged to the genera Lewinella, Croceitalea and Saprospira, and these genera were detected in all mats (Fig. 4). Chloroflexi sequences composed 6e21% of total sequences in all mats, with the highest abundance in Mat 3 (Fig. 3A and B). More than 90% of the sequences of the Chloroflexi group belonged to uncultured Anaerolineaceae, uncultured Caldilineaceae and Candidatus chlorothrix (Fig. 4), a filamentous anoxygenic photoautotroph isolated from Guerrero Negro hypersaline mats in Mexico. The classes Planctomycetes and Spirochaetae were

encountered in all mats and each made up 2e9% of total sequences (Fig. 3). Most sequences of the class Planctomycetes and Spirochaetae belonged to the genus Phycisphaera and Spirochaeta, respectively. The remaining less dominant groups (
3% of total sequences) were distributed among several other groups (Fig. 5A) including Verrucomicrobia, Acidobacteria, Actinobacteria, Chlorobi, Firmcutes and Deferribacteres (Fig. 3). 3.3. Fatty acid composition Different saturated, branched and mono- and di-unsaturated fatty acids in the range of C14eC24 were detected in all mats, with minor quantitative differences (Fig. 5A and B). The saturated straight-chain fatty acids 16:0 and 18:0 and the monounsaturated fatty acids 16:1 and 18:1 dominated all mat samples and accounted for relative amounts of 70e77% of total fatty acids (Fig. 5B). The most dominant fatty acids were 16:0 and made up ca. 26% of total fatty acids in Mat 2, 3 and 4 and >28% Mat 1 and 5 (Fig. 5B). The amount of the fatty acid 18:0 ranged between 10% and 24% with the lowest in Mat 4 and highest in Mat 2, respectively. Several monounsaturated fatty acids were detected including 16:1-9c (4e9% of total fatty acids), 18:1-11t (<1%), 18:1-9c (6e11%) and 18:1-11c (5e15%). The highest abundance of these fatty acids was found in

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Fig. 5. A) A representative total ion current chromatogram showing the peaks of the identified fatty acids; B) Relative abundance (% of total fatty acids) and distribution of fatty acids in the wadi microbial mats.

Mat 1 and 4 (Fig. 5B). The concentrations of the cis configuration of the fatty acid 18:1-11 were much higher (10e75%) than the trans configuration in all mats. The only detected di-saturated fatty acids 18:2-9c, 12c displayed a relative abundance of <3% in all mats (Fig. 5B). Branched saturated fatty acids were found in amounts between 5% (in Mat 1 and 5) and 10% (in Mat 3) and consisted mainly of two iso (i) and one anteiso (ai) fatty acids (i.e. 14:0 iso-13me, 15:0 iso 14-me and 14:0 anteiso 12-me, respectively). 4. Discussion 4.1. Cyanobacteria in the wadi mats Most identified cyanobacteria in the wadi microbial mats belonged to extremophilic types such as Microcoleus, Spirulina, Chroococcidiopsis, Euhalothece, Dactylococcopsis, Haloleptolyngbya and Halomicronema, which were typically detected in other hypersaline mats around the world (Campbell and Golubic, 1985; Abed and Garcia-Pichel, 2001; Jonkers et al., 2003; Richert et al., 2006). The 16S rRNA phylogenetic affiliation of detected cyanobacteria was in overall good agreement with their microscopic identification, especially for the most dominant types such as Microcoleus, Leptolyngbya, Spirulina, Scytonema and Gloeocapsa. Pyrosequencing provided a more comprehensive overview of the diversity and distribution of cyanobacteria in the mats than direct microscopy. Similar to other hypersaline mats in other parts of the world, M. chthonoplastes was also detected as the chief matforming cyanobacterium in the wadi mats. The cosmopolitan distribution of this cyanobacterium points out to its importance in the formation and stabilization of hypersaline cyanobacterial mats (Garcia-Pichel et al., 1996). Recently, this cyanobacterium has been shown to possess a special tactic movement, known as halotaxis, in order to cope with elevated salinities in intertidal flats (Kohls et al., 2010). Additionally, M. chthonoplastes was shown to possess a complete nif-gene cluster and to express these genes under natural

conditions, although this cyanobacterium was not previously assigned as a diazotroph (Bolhuis et al., 2010). Several types of unicellular cyanobacteria were detected in the wadi mats including Johannesbaptistia, Gloeocapsa, Chroococcus, Aphanocapsa, Synechococcus, Cyanothece, Euhalothece and Dactylococcopsis, however with different distributions. All these cyanobacteria are known for their tolerance to desiccation and elevated salinities and have been reported from hypersaline marine mats, lagoons and inland evaporitic lakes (Campbell and Golubic, 1985; Grilli Gaiola et al., 1996; Richert et al., 2006; Abed et al., 2008). Examples of the environments where such cyanobacteria were frequently encountered includes the hypersaline mats from the intertidal flats of Abu Dhabi, coastal ponds of the Rangiroa Atoll in French Polynesia, the Great Salt Lake in the United States, Chiprana lake in Spain and the Solar Lake in Egypt (Grilli Gaiola et al., 1996; Jonkers et al., 2003; Richert et al., 2006; Abed et al., 2008). Previous studies demonstrated that extremely halotolerant unicellular cyanobacteria of similar morphology formed a diverse but monophyletic cluster based on the analysis of 16S rRNA genes (GarciaPichel et al., 1998). While unicellular cyanobacteria can tolerate high salinities through the production and accumulation of compatible solutes (Galinsky, 1995), they developed several strategies to cope with desiccation including production of polyhydroxyl carbohydrates, efficient repair of the DNA damage and the production of the UV sunscreen pigment scytonemin (Potts, 1994; Dillon et al., 2002). Indeed, scytonemin pigment was observed by microscopy around cells of the unicellular cyanobacteria Chroococcus and Chroococcidiopsis, as well as the filamentous cyanobacteria Scytonema and Nostoc. Scytonemin production was shown to be stimulated by temperature, UV-A irradiation and periodic desiccation (Dillon et al., 2002). The detection of thin filamentous cyanobacteria in the wadi mats is in agreement with their widespread distribution in almost every mat system. This group of cyanobacteria was found to dominate field samples, especially following environmental

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perturbations and to overgrow other cyanobacteria in cultures
ska et al., 2012). The detection of cyanobacteria belonging to (Palin Halomicronema in the wadi mats is consistent with the high salinity and temperature in Wadi Muqshin since this genus was introduced to describe moderately halophilic, moderately thermophilic, very thin filamentous cyanobacteria isolated from hypersaline microbial mats (Abed et al., 2002). The tightly coiled trichomes belonging to Spirulina are also typical for hypersaline mats (Abed and GarciaPichel, 2001; Jonkers et al., 2003; Sørensen et al., 2005). Similar Spirulina morphotypes from hypersaline waters have been shown to cluster together, justifying the establishment of a new genus Halospirulina, based on morphology, high salt tolerance and 16S rRNA signatures (Nübel et al., 2000). Although few sequences related to known heterocystous nitrogen-fixing genera such as Nostoc, Scytonema and Stigonema were detected in the mats, these cyanobacteria may play a role in enriching the mat system with nutrients. 4.2. Bacterial diversity in the wadi mats The wadi mats harbored a high diversity of microorganisms, as indicated by OTU richness and Chao index estimates. Although similar bacterial groups were detected in all mats, clear differences in the distribution of species were still observable. This is clearly reflected by the percentage of shared species among the mats, which was between 57% and 72%. Mat 5 showed the lowest similarity with the other mats, which could be attributed to the slightly higher salinity in this mat. The mats had a large proportion of novel types since
18% of the total sequences had no close match in publicly available 16S rRNA gene databases. The uniqueness of these bacteria is probably a result of the harsh environmental conditions of the desert. Most of the acquired sequences from Alphaproteobacteria were phylogenetically related to halotolerant and thermotolerant species, isolated previously from hypersaline environments. For instance, Dichotomicrobium, which is the most dominant genus in the mats includes thermophilic and halotolerant species isolated from the Solar Lake, Egypt and the coastal saline lake in Brazil (Hirsch and Hoffmann, 1989). Most of the detected alphaproteobacterial genera contained strictly aerobic and bacteriochlorophyll a-containing species. Thus, these bacteria are likely residing in the top layer of the mats, in close association with cyanobacteria. Indeed, bacteria belonging to Rhodobacteraceae were previously isolated from the oxic layer of hypersaline mats and were shown to grow on cyanobacterial exudates (Jonkers and Abed, 2003). The presence of bacteriochlorophyll a in the detected Alphaproteobacteria points out to their ability to perform anoxygenic photosynthesis, besides their chemotrophic mode of life. Alphaproteobacteria such as Paracoccus denitrificans may play a role in the nitrogen cycle through the use of nitrate as an electron acceptor (Baker et al., 1998). Interestingly, this organism is able to perform denitrification, even under oxic conditions (Baker et al., 1998). This makes this organism suitable to survive in microbial mats and to cope with the fluctuating conditions of high oxygen concentration due to cyanobacterial photosynthesis during the day and anoxia in the dark. Deltaproteobacteria in the wadi mats belonged mainly to sulfatereducing bacteria, thus indicating an active sulfur cycle in these mats. The detection of phototrophic purple sulfur bacteria (e.g. Halochromatium and Marichromatium) as well as green no-sulfur bacteria (i.e. Chloroflexi) further supports this assumption. Most of the acquired genera of sulfate-reducing bacteria in the wadi mats were previously reported in other pristine as well as polluted hypersaline microbial mats (Teske et al., 1998; Abed et al., 2011b). Increased temperatures have been experimentally shown to stimulate the activity of sulfate-reducing activity in hypersaline mats

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(Abed et al., 2006). Hence, the elevated temperatures in the deserts of Oman, which exceeds 50  C in hot summers, could have a direct effect on the activity of sulfate-reducing bacteria in the wadi mats. Chloroflexi, which constituted between 6 and 21% of total sequences in the wadi mats, have also been reported to be conspicuously present in other hypersaline microbial mats (Pierson et al., 1994; Klappenbach and Pierson, 2004). This group of bacteria accommodates species that have the ability to perform anoxygenic photosynthesis. Candidatus chlorothrix, which was found dominant in all wadi mats, was also detected in hypersaline mats from Guerrero Negro, Mexico and intertidal flats in Abu Dhabi (Klappenbach and Pierson, 2004; Abed et al., 2007). The high abundance of Bacteroidetes group in all mats points out to the importance of this group in the mats. This group is known to include species that are specialized in the degradation of extrapolymeric substances (EPS). EPS production by phototrophs was shown to be stimulated under high salt stress (Liu and Buskey, 2000). This could be the reason why Bacteroidetes displayed their highest abundance in Mat 5, where salinity is highest. 4.3. Fatty acids and environmental adaptation Fatty acids gain their importance not only because of their use as biomarkers for different bacterial groups in the mats but also because they reflect the adaptation of bacteria to environmental € tter, stress (Grimalt et al., 1992; Abed et al., 2008; Scherf and Rullko 2009). Fatty acids in the wadi mats resembled those reported in other hypersaline mats from coastal and marine environments (Grimalt et al., 1992; Wieland et al., 2003; Abed et al., 2008; Scherf €tter, 2009). The detection of 70e77% of total fatty acids and Rullko belonging to saturated straight-chain fatty acids 16:0 and 18:0 and the monosaturated fatty acids 16:1 and 18:1 is indicative of the dominance of cyanobacteria (Grimalt et al., 1992; Scherf and € tter, 2009). Even the detected di-saturated fatty acids 18:2Rullko 9c, 12c were also reported in cyanobacteria (Grimalt et al., 1992; Abed et al., 2008). While the monosaturated fatty acids 16:0 and 18:0 accounted for 36e50% of the total detected fatty acids in all wadi mats, the mono-unsaturated fatty acids 16:1 and 18:1 accounted for 24e33%. Desaturation of fatty acids is carried out by acyl-lipid desaturases as a post-biosynthesis modification (Chintalapati et al., 2004) and is a well-established mechanism in cyanobacteria to increase membrane fluidity in order to tolerate extreme conditions of salinity, temperature and desiccation (Hufleijt et al., 1990; Singh et al., 2002). For instance, an increase in the amounts of unsaturated fatty acids was observed within the first hour after a sudden elevation of NaCl concentration in the cyanobacterium Synechococcus 6311 (Hufleijt et al., 1990). Similarly, around 60% of the total phospholipids of the desiccation tolerant cyanobacterium Nostoc commune UTEX584 were found to belong to the unsaturated 20:3u3 fatty acid (Olie and Potts, 1986). Unsaturation of fatty acids in membrane lipids stimulates the synthesis of the Naþ/Hþ antiport(s) and/or Hþ ATPase(s), which results in a decrease in the concentration of Naþ in the cytosol, thus leading to the protection of PSI and PSII against NaCl-induced inactivation (Singh et al., 2002). The fatty acids iso- and anteiso C14 and iso-C15 were detected in all wadi mats in significant proportions (i.e. 5e10% of total lipids). These branched fatty acids have been found abundant in Gram positive bacteria as well as in sulfate-reducing bacteria and sulfuroxidizing bacteria (Grimalt et al., 1992; Wieland et al., 2003), which were detected in the wadi mats by pyrosequencing. Significant amounts of cis/trans unsaturated fatty acids were detected, indicating the importance of these lipids in the wadi mats. Cisetrans isomerization has been shown to be part of a general stresseresponse adaptation mechanism of microorganisms

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allowing them to withstand exposure to organic solvent, heavy metals and high temperature and salinity (Pedrotta and Witholt, 1999; Heipieper et al., 2003; Heipieper and Fischer, 2010). This process has been detected in several gammaproteobacterial strains (Heipieper et al., 2003). Interestingly, 94% of these fatty acids belonged to the cis form in the wadi mats, although nearly equal amounts of both configurations were detected in other hypersaline mats (Abed et al., 2008). Since the increase in trans-unsaturated fatty acids has been shown to decrease membrane fluidity (Pedrotta and Witholt 1999), it is postulated that the mats' microorganisms use the isomerization of unsaturated lipids to accumulate more of the cis form and to increase the fluidity of membranes. The cis/trans conversion has been shown to be quantitatively less effective than the desaturation of fatty acids, but still substantially influences the fluidity/rigidity of membranes (Heipieper and Fischer, 2010 and references therein). In conclusion, the hypersaline cyanobacterial mats in the studied desert wadi harbor similar bacterial communities to other hypersaline mats from intertidal flats, evaporation ponds and hypersaline lakes. These microorganisms belonged to extremophilic types that are tolerant to the hypersalinity of the wadi as well as to the elevated temperature and UV and light intensity of the desert. Acknowledgment The authors would like to thank the Hansewissenschaftskolleg, Institute for Advanced Studied in Delmenhorst, Germany for supporting RA. This research was funded by Sultan Qaboos University (grant No. IG SCI/BIOL/11/01) and by grant VEGA 1/0975/12 from the Grant Agency of the Ministry of Education, Slovak Republic. References Abed, R.M.M., Dobrestov, S., Al-Kharusi, S., Schramm, A., Jupp, B., Golubic, S., 2011a. Cyanobacterial diversity and bioactivity of inland hypersaline microbial mats from a desert stream in the Sultanate of Oman. Fottea 11, 215e224. Abed, R.M.M., Garcia-Pichel, F., 2001. Changes after long-term transplant in microbial mat cyanobacterial community composition revealed with a polyphasic approach. Environ. Microbiol. 3, 53e62. Abed, R.M.M., Garcia-Pichel, F., Hernandez-Marine, M., 2002. Polyphasic characterization of benthic, moderately halophilic, moderately thermophilic cyanobacteria with very thin trichomes and the proposal of Halomicronema excentricum gen. nov., sp. nov. Arch. Microbiol. 177, 361e370. Abed, R.M.M., Kohls, K., de Beer, D., 2007. Effect of salinity changes on the bacterial diversity, photosynthesis and oxygen consumption of cyanobacterial mats from an intertidal flat of the Arabian Gulf. Environ. Microbiol. 9, 1384e1392. Abed, R.M.M., Kohls, K., Schoon, R., Scherf, A.-K., Schacht, M., Palinska, K.A., €tter, J., Golubic, S., 2008. Lipid biomarkers, Hassani, H.A., Hamza, W., Rullko pigments and cyanobacterial diversity of microbial mats across intertidal flats of the arid coast of the Arabian Gulf (Abu Dhabi, UAE). FEMS Microbiol. Ecol. 65, 449e462. Abed, R.M.M., Musat, N., Musat, F., Mussmann, M., 2011b. Structure of microbial communities and hydrocarbon-dependent sulfate reduction in the anoxic layer of a polluted microbial mat. Mar. Pollut. Bull. 62, 539e546. Abed, R.M.M., Polerecky, L., Al-Najjar, M.A., de Beer, D., 2006. Effect of temperature on photosynthesis, oxygen consumption and sulfide production in an extremely hypersaline cyanobacterial mat. Aquat. Microb. Ecol. 44, 21e30. Baker, S.C., Ferguson, S.J., Ludwig, B., Page, M.D., Richter, O.M., Spanning, R.J., 1998. Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility. Microbiol. Mol. Biol. Rev. 62, 1046e1078. Bauld, J., 1981. Occurrence of benthic microbial mats in saline lakes. Hydrobiologia 81, 87e111. Bauld, J., 1986. Benthic microbial communities of Australian saline lakes. In: de Deckker, P., Williams, W.D. (Eds.), Limnology in Australia. Dr. W. Junk Pub., Boston, pp. 95e111. Bolhuis, H., Severin, I., Confurius-Guns, V., Wollenzien, U.I.A., Stal, L., 2010. Horizontal transfer of the nitrogen fixation gene cluster in the cyanobacterium Microcoleus chthonoplastes. ISME J. 4, 121e130. Campbell, S.E., Golubic, S., 1985. Benthic cyanophytes (cyanobacteria) of Solar Lake (Sinai). Arch. Hydrobiol./Suppl. 71 Algol. Stud. 38/39, 311e329. Castenholz, R.W., Rippka, R., Herdman, M., Wilmotte, A., 2001. Subsection III. (Formerly Oscillatoriales Elenkin 1934). In: Garrity, G. (Ed.), Bergey's Manual of Systematic Bacteriology, second ed. Springer-Verlag, New York, pp. 539e562.

 
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