Geomicrobiology Journal

ISSN: 0149-0451 (Print) 1521-0529 (Online) Journal homepage: http://www.tandfonline.com/loi/ugmb20

Biodiversity of isolated cyanobacteria from desert

soils in Iran

Atefeh Etemadi-Khah, Ahmad Ali Pourbabaee, Mostafa Noroozi, Hossein Ali

Alikhani & Laura Bruno

To cite this article: Atefeh Etemadi-Khah, Ahmad Ali Pourbabaee, Mostafa Noroozi, Hossein
Ali Alikhani & Laura Bruno (2017): Biodiversity of isolated cyanobacteria from desert soils in Iran,
Geomicrobiology Journal, DOI: 10.1080/01490451.2016.1271064

To link to this article: http://dx.doi.org/10.1080/01490451.2016.1271064

Accepted author version posted online: 07

Feb 2017.

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Biodiversity of isolated cyanobacteria from desert soils in Iran

Atefeh Etemadi-Khah1*, Ahmad Ali Pourbabaee1, Mostafa Noroozi2, Hossein Ali Alikhani1,

Laura Bruno3

1
Department of Soil Science, University College of Agriculture and Natural Resources,

University of Tehran, Tehran, Iran

2
Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

3
Department of Biology, University of Rome “Tor Vergata”, Italy

*
Corresponding author: Atefeh Etemadi-Khah (etemadia@ut.ac.ir)

Abstract

Cyanobacteria are among the most ubiquitous, ecologically important photo--autotrophs in the

earth. They play important roles in terrestrial environments, especially in arid and semi--arid

regions. In this study, morphological and genetic diversity of the cyanobacteria inhabiting desert

soil in Iran was investigated for the first time. The samples were collected at 40 different sites in

the Kavir National Park. After cultivation and morphological identification, the 16S rRNA gene

was sequenced from the cyanobacterial cultures. Twenty-seven different isolates were

genetically and morphologically identified in 23 sites. Morphotypes fitting the description of five

genera Phormidium, Trichocoleus, Leptolyngbya, Microcoleus and Tychonema with an

abundance 44.45%, 37.04%, 11.11%, 3.7% and 3.7%, respectively. Sequence comparisons of

samples on the National Center for Biotechnology Information and morphological data showed

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that 48et and 50et strains had 94% similarity to Phormidium animale and 97% similarity to

Microcoleus sp. respectively. These strains formed a separate and longer branch in the

phylogenetic tree, suggesting relatively distant phylogenetic relationships with other sequences

in this study. It seems that these sequences are new strains and it is needed another markers for

further investigation. Soil analysis showed salinity ranged from 0.23-87.8 dS/m, the highest

salinity tolerance and the most commonly isolated genus was Phormidium. The presence of

cyanobacterial strains in the Kavir National Park showed that, despite the harsh conditions of this

place, it is however biologically active.

Keywords

morphology, phylogeny, soil cyanobacteria, 16S rRNA gene, Kavir National Park

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1. Introduction

Cyanobacteria, also known as blue-green algae include a genetically highly diverse group of

Gram-negative photosynthetic prokaryotes (Kulasooriya, 2011; Heidari et al., 2012). It is

believed that they appeared on the Earth 3.4 billion years ago as witnessed in the fossilized

stromatolites (Kulasooriya, 2011). They are found in almost every aquatic and terrestrial

environment (Castenholz and Waterbury, 1989) due to their broad ecological tolerances (Whitton

and Potts, 2000; Waterbury, 2006). Soil is the most important non-aqueous habitat for

cyanobacteria where (Zenova et al., 1995) cyanobacteria play important roles, especially in arid

and semi--arid regions (Cameron, 1966; Belnap, 2003; Yeager et al., 2007). They can improve

the physico-chemical parameters of the terrestrial environment (Nayak and Prasanna, 2007).

The analysis of microbial diversity in natural communities needs a polyphasic approach that

combines the use of traditional and molecular techniques of community diversity

characterization (Garcia-Pichel et al., 2001; Bruno et al., 2009). Identification of cyanobacteria

using the traditional classification is primarily based on features such as morphology of the

filaments, vegetative cells, heterocysts and akinetes (Komarek and Anagonistidis, 1989). The

form of the colony, shape of the terminal cells, presence or absence of sheath and gas vesicles, as

well as life cycle are additional features used for identification of some genera (Rajaniemi et al.,

2005). Sequence analysis of genes encoding small-subunit rRNA (16S rRNA gene) is currently

the most promising approach for the phylogenetic classification of cyanobacteria, but alone it is

not appropriate for resolving species relationships (Garcia-Pichel et al., 2001; Deshmukh et al.,

2010; Casamatta et al. 2005).

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The diversity of terrestrial cyanobacteria from various regions across the world was widely

investigated. For example, Garcia-Pichel et al. (2001) investigated cyanobacteria in soil desert

crusts from the Colorado Plateau by using phylogenetic and morphological approaches,

Alwathnani and Johansen (2011) described the cyanobacteria present in soils from a Mojave

Desert Ecosystem only by means of morphological characterization. Muhlsteinova et al. (2014)

characterized 24 strains isolated from the Atacama, Colorado and Mojave Deserts using

morphological and molecular approaches. They carried out phylogenetic analyses by sequencing

the 16S rRNA gene and the 16S--23S internally transcribed spacer (ITS) region. Dojani et al.

(2014) applied the combined approach to determine the cyanobacterial diversity in biological soil

crusts of the Succulent Karoo and Nama Karoo of southern Africa. At the same time the

knowledge of the cyanobacteria present in desert soils of Iran is particularly weak. About 61% of

the 164 million hectares of Iran has arid and semi-arid conditions, and the annual rainfall in these

regions is low and between 50 to 250 mm (Modarres, 2006; Feizi et al., 2014). We will focus on

the fact that probably this large area present biological soil crusts formed mostly by

cyanobacteria that were never been characterized. This study was carried out in Kavir National

Park that is a protected ecological zone in northern Iran with an area of 4,000 square kilometers

(1,500 mile²). The park is located 120 kilometers south of Tehran and 100 kilometers east of

Qom, and it sits on the western end of one of Iran’s two major deserts, the Dasht-e Kavir (Great

Salt Desert). Siahkuh (Black Mountain), a large, semi-circular rock outcropping sits in roughly

the park’s center.

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The aim of this study was the taxonomic evaluation of the cyanobacteria isolated in culture from

soil of Kavir National Park using both morphological observations with light microscope and

molecular data from 16S rRNA gene sequences.

2. Materials and methods

2.1 Site Descriptions and Sampling

Kavir National Park is located between 34o 17′ N and 51o 25′ E to 35o 11′ N and 53o 03′ E (Fig.

1). Typically, the area receives around 150 millimeters (6 inches) of rain each year, most of

which falls between November and May. The humidity is at the rate of 41%. For isolation of

cyanobacteria, soil samples were randomly collected up to at 0-5 cm depth from 40 different

sites. Samples were collected on 16th to 18th April 2014. After collection, the samples were

maintained in sterile plastic bags and brought in the laboratory of the Department of Soil

Science, College of Agriculture and Natural Resources University of Tehran. The samples were

then divided into two portions one for physico-chemical analysis and the other for

microbiological study. One portion of soil samples was air dried, ground and passed through a 2

mm sieve. After that, the samples were analyzed for soil pH and electrical conductivity. The

physico-chemical parameters of samples were analyzed using the standard methods (Strickland

and Parson, 1972).

2.2 Isolation, purification and cultivation of cyanobacteria

One g of the soil samples were added on both BG11 and BG110 (Rippka et al., 1979) mediums

(with/without nitrogen). Three dilutions of soil 102, 103 and 104 were prepared. One ml from

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each dilution was added to BG11 plates. Inoculated plates were incubated at 22-25 ˚C under cool

white fluorescent light (40 μE m−2 s−1) with 12 h light/12 h dark photoperiod until good growth

was obtained (3--6 weeks). Standard plating/streaking techniques were used for isolation and

purification of cyanobacterial strains (Nayak and Prasanna, 2007). The cultures were made pure

by repeated and frequent subculturing in agarized medium (1.5%) (Deshmukh et al., 2010;

Nongbri and Syiem, 2012). For purification of some samples the stock solution of imipenem

antibiotics, and cycloheximide (1.25% [wt/vol]) were prepared fresh, sterilized by filtration, and

added to the medium (Ferris and Hirsch, 1991).

2.3 Morphological study

Cyanobacterial specimens were observed by using light microscope (Olympus BX51) with 40X

and 100X magnifications. Identification of cyanobacteria was carried out according to Komarek

and Anagnostidis (1998, 2005). For all the strains examined in this study, morphological

characteristics were noted, such as the shape and size of vegetative cells (cell shape, width, and

length of the cells), the nature of filaments, presence of sheaths, trichome width, shape of end

cells, heterocysts and akinetes, presence or absence of false branching, presence or absence of

necridia, constrictions at the cross-walls (Garcia-Pichel et al., 2001; Alwathnani and Johansen,

2011; Martineau et al., 2013).

2.4 DNA Extraction, PCR and cloning

DNA of cyanobacteria culture isolates was extracted by genomic DNA extraction Gene

MATRIX Plant & Fungi DNA Purification kit (EURx Ltd., Gdańsk, Poland). Genomic DNAs

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were extracted from cells after six cycles of freezing and thawing as previously described (Bruno

et al., 2005).

For DNA amplification, the 16S rRNA gene, approximately 1000 bp in length, were amplified

by PCR (Applied Biosystem, Foster City, CA) using the CYA359F (5´

GGGGAATYTTCCGCAATGGG 3´) and PcR (5´ACGGGCGGTGTGTAC3´) primers. The

PCR amplification was performed in an eppendorf by using a 50 μL solution mixture consisting

of 5.0 μL of 10X PCR buffer, 4.0 μL of 2.0 mM MgCl2, 2.0 μL of each primer (0.4 mM), 1.0 μL

of dNTP, 0.5 μL of Taq DNA polymerase, 31.5 μL of H2O, and 4.0 μL (10 ng) of genomic DNA.

The thermal PCR profile was as follows: initial denaturation for 7 min at 95 ˚C, followed by 33

incubation cycles each consisting of 40 sec at 95˚C, 1 min at 52 ˚C, 1:30 min at 72 ˚C and a final

elongation step at 72 ˚C for 10 min. PCR products were separated by electrophoresis in 1.5%

agarose gels in TAE buffer (40 mM Tris--acetate,1 mM EDTA, pH 8.0) and were purified from

agarose gels with the Wizard® SV Gel and PCR Clean-Up System and subsequently cloned by

using the pGEM-T Easy vector system (Promega) according to the manufacturer’s instructions.

The cloning was performed to insure high quality sequencing from a single product. Four

colonies were picked for each sample and grown overnight, and plasmids were purified with

Wizard®Plus SV Minipreps DNA Purification System (Promega).

2.5 Phylogenetic analyses

Partial sequencing was performed by C.R.I.B.I. sequencing service (Padua, Italy). The primers

used were CYA359F and PcR that allowed the sequencing of about 1000 bp of the 16S rRNA

gene. Ambiguous bases were checked and altered using the BioEdit program. Sequences were

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compared to known samples and environmental samples using the BLAST search tool on the

National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov).

The alignments were generated using Clustal W (MEGA) and phylogenetic tree was obtained

with MEGA (version 6) by neighbor-joining (NJ) (Kumar et al., 2004; Studier and Keppler,

1988), maximum-parsimony (MP) and maximum-likelihood (ML) (Saitou and Imanishi, 1989).

The bootstrap proportions were calculated using 1000 replicates (Felsenstein, 1985). The

Escherichia coli sequence was designated as an outgroup.

2.6 Nucleotide sequence accession numbers

The sequences determined in this study were deposited in GenBank and their accession numbers

are listed in Table 1.

3. Results

3.1 Diversity of morphotypes

The microscopy observations of the 40 samples showed that 23 sampling sites were

characterized by the presence of cyanobacteria (Table 1). Other sites did not have any

cyanobacteria, although a few small diatoms and green algae were observed, but these are not

discussed here. We identified 27 morphotypes in five genera (Fig. 2-28) based on the

classification scheme of Komarek and Anagnostidis (1989, 1998, 2005). There was little

difference in the number of species that occurred in each site. The highest diversity of

cyanobacteria were found in the sites 10, 16, 27 and 29, each with two species. The

cyanobacteria genera were identified as Phormidium, Microcoleus, Leptolyngbya, Trichocoleus

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and Tychonema. The dominant genus was Phormidium (44.45%) followed by the Trichocoleus

(37.04%), Leptolyngbya (11.11%), Microcoleus (3.7%) and Tychonema (3.7%), respectively.

Strain designation is as follows:

The strain 2et most closely resembled Phormidium autumnale with solitary trichomes in thin

open sheaths; the trichomes were straight or curved, 5--7 μm wide, tapering. Not constricted at

the granular cross-walls, with no evident necridia. Cells green or dirty green in color, shorter

than width. End cells attenuated, rounded, conical. Calyptra present (Fig. 2). The strain 44et was

identified as Phormidium autumnale. Filaments with solitary trichomes, black green or olive

green, mostly straight and parallel, 6-7 µm width. Cells shorter than wide, 3-4 µm long. Thin and

colorless sheaths, opened at the ends. Not constricted at the granular cross-walls, sometimes with

biconcave necridic cells. Apical cell rounded, conical with calyptra (Fig. 3). The strain 54et was

identified as Phormidium autumnale, with solitary, straight or slightly flexuous trichomes in firm

and thick open sheaths, tapering, not constricted at the cross-walls, not granular, without

necridia, cells 4.5-5.5 μm wide, shorter than width, green to dark blue green in color. End cells

attenuated, rounded, conical. Calyptra present (Fig. 4). The strain 79et fitted most closely the

description of Phormidium autumnale, with solitary trichomes, in fairly thick open sheaths,

trichomes straight, flexuous or rarely curved, 6.5-7.5 μm wide. Cells shorter than wide, 3-4 µm

long. Tapering, granular content, not constricted at the cross-walls, with no evident necridia.

Cells brownish green or yellowish in color. End cells rounded, conical, without calyptra (Fig. 5).

The microscopic observation of the morphological study of strain 60et confirmed its

classification as Phormidium autumnale. Filaments with solitary and long trichomes, straight or

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curvy, 5.5-6.5 µm width, green to blue green. Cells shorter than wide. Thin and colorless

sheaths, opened at the ends. Not constricted or slightly constricted at the cross-walls, sometimes

with biconcave necridic cells. Apical cell attenuated, rounded, conical, with calyptra (Fig. 6).

The features of Phormidium autumnale, was also observed in strain 61et. Filaments with solitary

trichomes in fairly thick, colorless and opened sheaths, 6.5-7.5 μm wide. Trichomes straight,

rarely curvy, tapering. Granular content, not constricted at the cross-walls, without necridia.

Cells green to brownish green in color, shorter than width, 5-6 μm long. End cells rounded,

conical, calyptra absent (Fig. 7). The strain 65et was similar to Phormidium autumnale, with

solitary and long trichomes in very thin, colorless open sheath, trichomes usually straight or

rarely curved at the ends, 5-5.5 μm wide. Not constricted or slightly constricted at the cross-

walls, often with biconcave necridic cells. Cells blue green or olive green in color, shorter than

width. End cells attenuated, rounded, conical (Fig. 8). Calyptra present. The strain 73et most

closely represented Phormidium autumnale, filaments with solitary and long trichomes, straight

or rarely curvy, 7.5-8.5 µm width, dark green or grayish green, cells shorter than wide, 2.5-3 µm

long. Firm and thick, colorless sheaths, opened at the ends. Untapering to slightly tapering. Not

constricted at the granular cross-walls, rarely with biconcave necridic cells. End cells, rounded,

conical, with calyptra (Fig. 9). The features of strain 76et was similar to Phormidium autumnale,

with solitary trichomes in fairly thick open sheaths, trichomes straight or rarely curved, 5--6 μm

wide, untapering to slightly tapering. Not constricted at the granular cross-walls, with no evident

necridia. Cells green or blue green in color, longer than width, 6.5-7.5 μm long. End cells

attenuated, rounded, conical. Calyptra present (Fig. 10). The strain 40et fitted most closely the

description of Phormidium autumnale, with one trichome per sheath. Sheath firm, thick,

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colorless and unlamellated. Trichomes straight, rarely curvy, motile, green to brownish green,

tapering toward the ends, not constricted at the cross-walls, 3.5-4 µm wide, shorter than wide.

Often granular content. End cells attenuated, rounded, conical, with a capitate calyptra (Fig. 11).

A typical feature of Phormidium autumnale, fasciculate filaments, was consistently observed in

strain 74et. Filaments sometimes solitary, straight, variably curved, dirty green to dark blue

green, attenuated at the ends. Sheaths fairly thick, usually colorless, not lamellated. Not

constricted at the often granular cross-walls. Cells shorter than wide, 4.5-5 µm width, 3-3.5 µm

long. Rarely with biconcave necridic cells. End cells, rounded, conical, with calyptra (Fig. 12).

The strain 48et most closely resembled Phormidium animale, with solitary trichomes in thin,

colorless open sheath, trichomes straight or rarely curved, 4-5 μm wide. Not constricted or

slightly constricted at the mostly ungranulated cross-walls, with no evident necridia. Cells

brownish green to brown in color, shorter than width. End cells attenuated, rounded, conical.

Calyptra absent (Fig. 13). The strain 50et was identified as a member of the genus Microcoleus,

filaments with few arranged trichomes per sheath or solitary. Sheath thin, colorless,

unlamellated. Trichomes straight, rarely curvy, 4-5 µm width, tapering toward the ends. Not

constricted at the cross-walls. Cells usually granular at the cross-walls, dark green to blue green,

shorter than wide. Apical cells attenuated, rounded, conical, with calyptra. Rarely with necridic

cells. Reproduction by disintegration and by hormogonia (Fig. 14). The microscopic observation

of the morphological study of strain 56et confirmed its classification as Leptolyngbya scottii.

Filaments occasionally coiled, rarely solitary, forming colonies, without false branching, not

attenuated at the ends. Trichomes 2-2.5 μm wide, thin and colorless sheaths, with no evident

necridia. Cells isodiametric, green to yellowish in color, non-granular, constricted at the cross-

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walls. End cells acute-conical, calyptra absent (Fig. 15). The strain 57et was identified as

belonging to the Leptolyngbya scottii, with single trichome per sheath, rarely forming colonies,

with no false branching. Sheaths fairly thick, open and colorless. Trichomes slightly flexuous,

not attenuated at the ends, constricted at the cross-walls, without necridia. Cells shorter than

wide, 2-2.5 μm wide, 1.5-2 μm long, green to blue green, non-granular or with central granule.

Apical cells acute-conical, without calyptra (Fig. 16). The strain 53et was identified as

Leptolyngbya subtilissma. Filaments strongly coiled together, with thin and colorless sheath.

Trichomes rarely solitary or forming compact, gelatinous, green or olive green mats, 1-1.5 μm

wide, more or less isodiametric, slightly constricted at the cross-walls. Apical cells rounded,

without calyptra (Fig. 17). The strain 35et was identified as Trichocoleus desertorum, containing

one to several trichomes entangled together in very thin and colorless sheaths. Trichomes slightly

flexuous, arranged in parallel fascicles, mostly pale green or blue-green, slightly tapering,

constricted at the cross-walls, lacking calyptra, without necridia, 1.5-2 μm wide. Cells non-

granular, longer than wide. Apical cells slightly longer than wide, 1.7-2 µm wide in middle part

and 5-6 µm long, conical to pointed (Fig. 18). The strain 41et was similar to Trichocoleus

desertorum. Filaments with solitary trichome in thin and colorless sheaths. Trichomes straight,

slightly flexuous, tapering, strongly constricted at the cross-walls, rarely with necridia, cells 2-

2.5 μm wide. Hormogonia short and numerous. Cells green to blue green in color, not granular,

isodiametric or longer than width, 2.5-3 μm long. Apical cells longer than wide, 1.8-2.1 µm wide

in middle part and 7-7.5 µm long, conical to sharply pointed, lacking calyptra (Fig. 19). The

strain 43et was identified as belonging to the Trichocoleus desertorum, with solitary trichome in

thin and colorless sheath. Trichomes straight or slightly curved and entangled together, slightly

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attenuated at the ends, constricted at the cross-walls, with no evident necridia, 2-2.5 μm wide.

Cells green to blue green in color, isodiametric or longer than width, not granular. Apical cells

longer than wide, conical to sharply pointed, 2.2-2.4 µm wide in middle part and 7.5-8 µm long,

calyptra absent (Fig. 20). 46et most closely simulated Trichocoleus desertorum, with solitary

trichome in thin and colorless sheath. Trichomes motile, mostly straight and slightly entangled

together, 2-2.5 µm width, mostly dirty green or dark blue green, constricted at the cross-walls,

tapering trichomes with pointed apical cell, cells shorter than wide, 1.5-2 µm long. Hormogonia

short and little. Apical cells conical to sharply pointed, longer than wide, 2.3-2.5 µm wide in

middle part and 7.3-7.5 µm long, lacking calyptra, without necridia (Fig. 21). The features of

strain 51et was similar to Trichocoleus desertorum. Filaments with one trichome per sheath.

Sheaths very thin, open, colorless. Trichomes straight, slightly flexuous and entangled together,

strongly constricted at the cross-walls, lacking calyptra, without necridia, non-granular, 2.5-3 μm

wide. With evident involution cells. Cells pale green to yellowish green, isodiametric or shorter

than wide. Apical cells longer than wide, 2.8-3 µm wide in middle part and 5.5-6.5 µm long,

conical to pointed (Fig. 22). The strain 52et was identified as belonging to the Trichocoleus

desertorum, with solitary trichome in very thin and colorless sheaths. Trichomes motile, straight

or curvy, 1.5-2 µm wide, blue green to olive green, constricted at the cross-walls, cells

isodiametric or longer than wide. Hormogonia short and numerous. With no evident necridia.

Occasionally with one to a few small granules in cell center. Apical cells conical to pointed,

longer than wide, 1.7-2 µm wide in middle part and 4.5-5 µm long, calyptra absent (Fig. 23).

Strain 58et was similar to Trichocoleus desertorum, with trichomes in fairly thick and colorless

sheath. Trichomes mostly straight, rarely flexuous, green to dark blue green, 1.5-2 μm wide,

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slightly attenuated at the ends, strongly constricted at the cross-walls, with some necridic cells,

longer than wide, with some small or large granules in cell center. Apical cells longer than wide,

conical to pointed, 1.8-2 µm wide in middle part and 7.2-7.5 µm long, calyptra absent (Fig. 24).

The features of Trichocoleus desertorum, was also observed in strain 59et. Trichomes motile,

solitary, straight, rarely curvy, somewhat entangled together. Sheaths thin and colorless. Cells

longer than wide, 2-2.5 μm wide, tapering, lacking necridia, green to blue green, strongly

constricted at the cross-walls, with a few small granules in cell center, rarely involution cells.

Hormogonia short and numerous. Apical cells longer than wide, conical to sharply pointed, 2.3-

2.5 µm wide in middle part and 6-6.5 µm long, calyptra absent (Fig. 25). The strain 64et fitted

most closely the description of Trichocoleus desertorum. Filaments with solitary, straight

trichomes. Sheaths absent. Tapering trichomes with pointed apical cell, constricted at the cross-

walls, with no evident necridia. Cells blue green to yellowish green in color, 1.5-2 μm wide,

longer than wide. Rarely with one small granules in the cell center. Hormogonia short and

numerous. Apical cells longer than wide, conical to sharply pointed, 1.8-2.1 µm wide in middle

part and 6.5-8.5 µm long, without absent (Fig. 26). The strain 69et most closely resembled

Trichocoleus desertorum. Filaments with one or few trichomes in very thin and colorless sheath.

Trichomes straight or curvy, sometimes entangled together, tapering, strongly constricted at the

cross-walls, rarely with necridia, cells 1.5-2 μm wide, longer than wide, green to blue green in

color, non-granular. Some short hormogonia. Apical cells longer than wide, 1.8-2 µm wide in

middle part and 6-6.3 µm long, conical to pointed, lacking calyptra (Fig. 27). The strain 39et

most closely represented a Tychonema species, with cylindrical and solitary trichomes, green or

olive green in color. Filaments are straight or occasionally slightly irregularly coiled, more or

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less curved. Trichomes more or less heteropolar, without false branching, sheaths absent, 3-3.5

μm wide, isodiametric or longer than wide, usually not constricted at the cross walls, attenuated

toward ends. End cells rounded or conical. Calyptra present (Fig. 28).

3.2 Phylogenetic diversity of 16S rRNA genes

Morphological taxonomy alone is unable to resolve the evolutionary relationships within

cyanobacteria (Garcia-Pichel et al., 2001). Therefore, a partial 16S rRNA gene sequence (1000

bp) of 27 samples was obtained and compared with closely related taxa retrieved from GenBank

database. The neighbor-joining tree (Fig. 29) is based on a data set of 43 16S rRNA gene

sequences, having the same topology of the trees inferred by the maximum-likelihood and the

maximum-parsimony methods (not shown). In the phylogenetic analysis, using MEGA all of the

cyanobacterial phylotypes were found within three distinct clusters (Fig. 29). In the first cluster

were presented Phormidium animale and two strains of Microcoleus. In the second cluster, our

samples belonged to species of Phormidium autumnale, and only a strain of Tychonema sp.. The

third cluster contained strains of Leptolyngbya scottii, Trichocoleus desertorum and a strain of

Leptolyngbya sutilissima.

Cluster I was formed by 48et strain with 94% similarity to Phormidium animale and 50et strain

with 97% similarity to Microcoleus sp. Considering that their identity with samples in GenBank

were less than 98%, it seems that these sequences are new strains (Stackebrandt and Goebel,

1994) and it is needed another markers for further investigation. These two strains were grouped

with Microcoleus MOA3-1A and had common lineage but their morphological characteristics

were completely different. 50et and 48et strains formed a separate and longer branch, suggesting

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relatively distant phylogenetic relationships with other sequences in this study. 61et, 60et, 79et,

2et, 73et, 44et, 74et, 54et and 40et strains formed a subcluster (A), supported by 60-99% of

bootstrap values, with one Microcoleus sp. and one Oscillatoria sp that were isolated from soil.

In this subcluster, some of the strains of P. autumnale had 100% similarity to each other (data

not shown). For example 44et, 54et and 74et strains had the same phenotype, but their

morphotypes were different. Another subcluster (B) was formed by 76et and 39et. Even though

39et and Tychonema sp. SAG. 23.89 strains 99% were similar to each other but were scattered in

distinct lineage. 39et Tychonema sp. strain was located in the same lineage with Phormidium and

Microcoleus genera but they were quite different morphologically. In subcluster C, 65et with

Phormidium autumnale and Oscillatoria amoena were grouped. A similarity search using the

BLAST software revealed that cluster III contained most of cyanobacterial sequences found from

desert soils (Garcia-Pichel et al., 2001; Boyer et al., 2002; Muhlsteinova et al., 2014). 53et

formed subcluster D with two Leptolyngbya spp. isolates and had a sequence identity of 99%

with the marine isolate of Leptolyngbya sp. LEGE 07314. In cluster III, 51et, 41et, 57et, 56et,

58et, 46et, 64et, 52et, 43et, 35et, 69et and 59et strains formed subcluster E with the most

similarity were between 56et and 57et; 46et (or 52et) and 69et; 35et and 69et, 100%, 99.89% and

99.89%, respectively and the most variation was observed between 58et and 64et with 94.28%

similarity. However 58et and 64et strains had some difference with each other genetically but

were scattered in common lineage. 51et and 41et strains were located in this subgroup with a

longer branch and its evolution was less than other strains.

16
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4. Discussion

In the present work, we investigated the morphological diversity of cyanobacteria from arid soil

in addition to the study of the 16S rRNA gene sequences. Fox et al. (1992) demonstrated that

16S rRNA gene similarity data are not sufficient to absolutely identify species in prokaryotes. It

has been suggested that a single species of a cyanobacterium must be genetically, ecologically

and morphologically uniform (Komarek and Mares 2012). Based on the morphology observed

under the light microscope, a total of 27 morphotypes belonging to five genera were identified

for the samples collected in the Kavir National Park. All the strains studied belonged to the order

of Oscillatoriales being characterized by filamentous trichomes, the lack of heterocysts, akinetes

and false branching. However, a very low diversity was evident in these samples that were

assigned to only five genera: Phormidium, Leptolyngbya, Trichocoleus, Tychonema and

Microcoleus. Most cyanobacterial phenotypes found in this study have been reported for other

arid environments (Garcia-Pichel et al., 2001; Boyer et al., 2002; Alwathnani and Johansen,

2011; Zheng et al., 2011; Muhlsteinova et al., 2014). Four genera including Leptolyngbya,

Trichocoleus, Phormidium and Microcoleus found in the present study also occur in Mojave

Desert soil (Alwathnani and Johansen, 2011). Microcoleus is found in arid land soils worldwide

(Johansen, 1993; Evans and Johansen, 1999; Starkenburg et al., 2011). It builds large biomass on

soil surface or it is a part of microbial crusts (Boyer et al., 2002; Rehakova et al. 2011). Data

collected from other studies indicated that Microcoleus vaginatus, was the most abundant species

in soil desert crusts (Ashley et al., 1985; Metting, 1991; Flechtner et al., 1998; Johansen et al.,

2001; Garcia-Pichel et al., 2001; Zheng et al., 2011), but M. vaginatus was not found M.

vaginatus from Kavir National Park, we have isolated only one strain of Microcoleus that was

17
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consistent with the results of Johansen et al. (2008). The genus Trichocoleus has been

characterized by Muhlsteinova et al. (2014) using molecular, morphological, and ecological

information on isolated strains from the Atacama, Colorado and Mojave deserts. Our

morphological strains 35et, 41et, 43et, 46et, 51et, 52et, 58et, 59et, 64et and 69et were very

similar to T. desertorum from the Atacama, Colorado and Mojave deserts. These strains had

distinct morphology and geography. They were different in some characters like the presence of

the sheath, the apical cell form and the constriction at cross wall.

Considering that in this study, the samples were collected in an environment with soil salinity

ranges from 0.23-87.8 dS/m, similar species that have been isolated from different sites due to

the different morphology, are probably different strains. The highest salinity tolerance were

related to the genera Phormidium and Trichocoleus respectively (data not shown). Four strains of

Phormidium, 79et, 61et, 65et and 48et, in the range of 33.1-87.8 dS/m and three strains of

Trichocoleus, 52et, 58et and 64et, in the range of 33.1-67.7 dS/m were isolated. Generally of the

27 morphotypes, 11 morphotypes were isolated from saline soil (EC>4) (about 40.74%). This

study showed that, despite the hypersalinity and drought in this area, it is biologically active. The

cyanobacterial species found to the present study also occur in other saline and hypersaline

ecosystems (Javor, 1989, Montoya and Golubic, 1991; Garcia-Pichel et al., 2001, Montoya,

2009, Dadheech et al., 2013).

The molecular data obtained, revealed higher cyanobacterial diversity respect that showed by the

observation at the light microscope. Most of the sequences from this study are like those

recovered from other previously studied regions of the Earth (Garcia-Pichel et al., 2001; Boyer et

18
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al., 2002; Muhlsteinova et al., 2014). In the case of 48et P. animale and 50et Microcoleus sp.

molecular and morphological data did not match and their genetic similarity was 99.31%. With

sequence comparisons of 48et and 50et samples on the National Center for Biotechnology

Information they indicated 94% similarity to P. animale CCAP 1459/6 (Gachon, 2013) and 97%

similarity to Microcoleus sp. MOA3-1A (Johansen et al., 2008) respectively. Due to large

genetic similarity of strains 48et to 50et and Microcoleus sp. MOA3-1A, it is likely that with the

sequencing of other genes of 48et, its classification will change in the future. One of the most

problematic cyanobacterial genera in terms of phylogenetic assessment is Phormidium

(Marquardt and Palinska, 2007; Comte et al., 2007; Komarek, 2010). In the cluster II P.

autumnale exhibits phylogenetic closeness to M. vaginatus. This has been reported by Hasler et

al. (2012) from ponds and lakes in the Czech Republic, Austria and Italy. The structure and

morphology of trichomes in both these types are very similar, but M. vaginatus occurs in

vegetative stages mostly with fasciculated trichomes, enveloped by one wide and closed sheath,

while P. autumnale is characterized by solitary trichomes, enveloped each facultative by one

firm, but attached (and at the end mostly open) sheath. Both these species up to now are

distinguished by the morphology of the sheath (number of trichomes in sheaths), structure of

cells and partly also by ecology (Komarek, 2003; Hasler et al., 2012). Because there is a high

similarity in the 16S rRNA gene species definition, to separate them, additional data such as

sequences from other genes or physiological/biochemical characterizations are probably needed

(Muhlsteinova et al. 2014). In the cluster II strain 61et P. autumnale was isolated from a site with

higher salinity, although it was 100% similar to the strains 40et, 73et, 60et, 79et, 54et, 2et and

19
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44et, but none of these strains were resistant to salinity (data not shown). The use of more

markers is need for a proper identification.

T. desertorum cultures investigated in this study demonstrated marked variability within strains.

Because the samples were diverse geographically. According to a BLAST search of GenBank,

these species were 99% similar to strains that were isolated by Muhlsteinova et al. (2014) and

had a broader distribution, occurring commonly in the Atacama, Colorado, and Mojave Deserts

(Muhlsteinova et al. 2014). Dojani and et al. (2014) isolated L. scottii from biological soil crusts

in the Succulent Karoo and Nama Karoo of southern Africa that our strains 56et and 57et had

99% similarity to it. 53et strain was isolated from soil and had close phylogenetic relationship

with the marine strains. There may be due to Tis shallow sea in the Kavir National Park which

dried over time because of warm air. The results showed that one of the isolated strains, 39et

Tychonema sp., while not dominant, is important in the cyanobacterial communities in our desert

soil. Because this genus sequence is different from other strains in the present study and is

located in a common cluster with Phormidium and Microcoleus genera. 39et Tychonema sp. was

99% similar to that species reported by Friedl et al. (2014) in the soil. The results of phylogenetic

tree showed that Phormidium with Microcoleus, Oscillatoria, Tychonema genera and

Leptolyngbya with Trichocoleus genus were close phylogenetically which have evolved during

the time.

5. Conclusion

This is, to our knowledge, the first time that the indigenous cyanobacterial populations in desert

soil in Iran have been investigated by a polyphasic approaches combining isolation in culture,

20
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morphological studies and 16S rRNA gene sequencing. Twenty-seven morphotypes were

assigned to 5 genera; 7 species were isolated from sites of the Kavir National Park that seem two

of them are new strains. Some strains were isolated with a salinity up to 87.8 dS/m, thus, they

can be defined as extremophiles cyanobacteria. The extreme environmental conditions of these

sites are mirrored by the low biodiversity present in the samples investigated and this study

pointed out that cyanobacteria are one of the major group of microorganisms able to grow in

extreme conditions of drought and salinity. The presence of cyanobacterial strains in the Kavir

National Park showed also that, despite the harsh conditions of this place, it is however

biologically active.

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Table 1. Cyanobacterial collection sites, some physico-chemical properties of the soil samples

and the cyanobacteria isolated from soil according to the sequence similarity using BLAST

function of GenBank and accession number

Sequenc GenBan
Conduct
Locat GPS e k
pH ivity Isolated taxon
ion position similarit accessio
(dS/m)
y n no.

34° 55'

57.5142" N
8.1 Phormidium KX7591
Site 1 4.93 99%
2 autumnale 60et 31
52° 1'

16.4238" E

34° 54'

12.114" N
7.6
Site 2 59.6 - - -
4
52° 2'

45.4164" E

34° 52'

38.8446" N
7.7
Site 3 19.07 - - -
1
52° 4'

34.179" E

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34° 50'

41.2044" N
8.2 Trichocoleus KX7591
Site 4 1.25 99%
7 desertorum 35et 14
52° 6'

27.8892" E

34° 48'

19.1586" N
7.5
Site 5 37.7 - - -
4
52° 7'

51.9342" E

34° 48'

6.9798" N
8.1
Site 6 1.72 - - -
2
52° 10'

30.1368" E

34° 49'

11.9238" N
7.9 KX7591
Site 7 2.05 Tychonema sp. 39et 99%
1 12
52° 13'

23.1708" E

34° 48'
8.1 Phormidium KX7591
27.2772" N
Site 8 99%
2 autumnale 40et 15
1.55
52° 16'

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21.1512" E

34° 47'

22.3224" N
8.1 Trichocoleus KX7591
Site 9 99%
3 desertorum 41et 16
52° 19' 1.16

33.9594" E

34° 47' Trichocoleus KX7591

54.801" N desertorum 43et 99% 17

Site 7.8

10 7
52° 23' 2.13 Phormidium 99% KX7591

6.5436" E autumnale 44et 18

34° 44'

3.3066" N
Site 7.8
- - -
11 1
52° 25' 3.61

0.2526" E

34° 42'

37.9764" N
Site 7.6 Trichocoleus KX7591
99%
12 9 desertorum 46et 19
52° 26' 6.74

53.9628" E

34° 40'
Site 7.4 Phormidium animale KX7591
94%
44.1618" N
13 3 48et 20
45.4

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52° 28'

52.6152" E

34° 41'

0.423" N
Site 7.2
- - -
14 6
52° 30' 85.3

56.2098" E

34° 41'

20.7486" N
Site 7.4
- - -
15 2
52° 34' 68.2

4.0764" E

34° 41' KX7591

Microcoleus sp. 50et
12.6168" N 97% 21
Site 7.8
Trichocoleus
16 6
52° 36' 5.38 99% KX7591
desertorum 51et
57.1104" E 22

34° 39'

55.3674" N
Site 7.1
- - -
17 2
52° 38' 127.8

1.3848" E

Site 7.2 Trichocoleus KX7591

34° 40' 99%
18 2 desertorum 52et 23

34
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31.9614" N 67.7

52° 41'

19.1358" E

34° 40'

27.8976" N
Site 6.5
195.7 - - -
19 2
52° 44'

22.059" E

34° 37'

45.2202" N
Site 6.5
192.6 - - -
20 9
52° 42'

38.2356" E

34° 36'

3.5028" N
Site 8.1 Leptolyngbya KX7591
3.27 99%
21 2 subtilissima 53et 24
52° 42'

48.1248" E

34° 34'

9.5376" N
Site 7.1
165.3 - - -
22 3
52° 45'

55.9902" E

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34° 32'

27.747" N
Site 7.8
10.96 - - -
23 2
52° 44'

56.6664" E

34° 30'

45.9216" N
Site 8.0 Phormidium KX7591
3.47 99%
24 5 autumnale 54et 25
52° 42'

13.518" E

34° 28'

47.7588" N
Site 7.9 Leptolyngbya scottii KX7591
2.50 99%
25 8 57et 27
52° 39'

45.2016" E

34° 26'

16.9332" N
Site 7.7
6.79 - - -
26 0
52° 36'

27.4494" E

34° 25' Trichocoleus KX7591

99%
Site 7.3
15.765" N desertorum 58et 28
55.7
27 8
99%
52° 31' Phormidium KX7591

36
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45.6492" E autumnale 79et 29

34° 25'

3.5322" N
Site 8.1 Phormidium KX7591
87.8 99%
28 8 autumnale 61et 32
52° 28'

13.065" E

34° 24' Trichocoleus KX7591

22.7442" N desertorum 64et 99% 33

Site 7.5
33.1
29 3
52° 24' Phormidium 100% KX7591

55.3098" E autumnale 65et 34

34° 25'

7.6116" N
Site 7.9 Trichocoleus KX7591
3.98 99%
30 7 desertorum 59et 30
52° 22'

12.1614" E

34° 24'

10.5078" N
Site 7.0
203 - - -
31 2
52° 19'

19.128" E

34° 25'
Site 7.9 Trichocoleus KX7591
2.87 99%
48.3924" N
32 7 desertorum 69et 35

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52° 16'

35.9832" E

34° 28'

27.3822" N
Site 7.6
98.2 - - -
33 5
52° 13'

28.1136" E

34° 31'

10.362" N
Site 8.2
2.91 - - -
34 1
52° 11'

4.7436" E

34° 34'

9.5376" N
Site 8.2 Phormidium KX7591
0.65 99%
35 9 autumnale 2et 13
52° 7'

32.1594" E

34° 38'

13.6968" N
Site 8.2 Phormidium KX7591
1.09 99%
36 1 autumnale 73et 36
52° 6'

32.8314" E

Site 8.0 Phormidium KX7591

34° 41' 0.23 99%
37 4 autumnale 74et 37

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37.0098" N

52° 4'

34.179" E

34° 45'

36.7374" N
Site 7.7 Phormidium KX7591
1.09 99%
38 9 autumnale 76et 38
52° 3'

29.9082" E

34° 49'

40.3356" N
Site 7.6 Leptolyngbya scottii KX7591
2.45 99%
39 6 56et 26
52° 3'

0.2484" E

34° 53'

19.3992" N
Site 7.4
55.9 - - -
40 8
52° 0'

7.2108" E

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Fig. 1. Map of sampling locations

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Figs 2-28. Diversity of cyanobacterial morphotypes from desert soil: (2-12) Ph. autumnale,

strains 2et, 44et, 54et, 79et, 60et, 61et, 65et, 73et, 76et, 40et, 74et; (13) Ph. animale, strain 48et;

(14) Microcoleus sp. strain 50et; (15-16) L. scottii strains 56et, 57et; (17) L. subtilissima; (18-27)

Tdesertorum strains 35et, 41et, 43et, 46et, 51et, 52et, 58et, 59et. 64et, 69et; (28) Tychonema sp.

strain 39et. All the figures have the same magnification; scale bar, 10 μm.

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Fig. 29. Phylogenetic tree of the partial 16S rRNA gene sequencing of cyanobacteria of this

research (■) and selected strains from GenBank. The E. coli sequence was designated as an

outgroup.

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