DOI: http://dx.doi.org/10.22092/ijb.2013.

4152

MORPHOLOGICAL AND PHYLOGENETIC DIVERSITY OF

CYANOBACTERIA IN FOUR HOT SPRINGS OF IRAN
F. Heidari, H. Riahi, M. Yousefzadi & Z. Shariatmadari
Received 26.03.2013. Accepted for publication 01.05.2013.

Heidari, F., Riahi, H., Yousefzadi, M. & Shariatmadari, Z. 2013 12 31: Morphological and phylogenetic diversity of
cyanobacteria in four hot springs of Iran. -Iran. J. Bot. 19 (2): 162-172. Tehran.

Hot springs have been a subject of intense discussion for biologists in the last decades. Thermophilic cyanobacteria
are scientifically valuable for their analogy to the ancient life forms on earth and also as a source of thermostable
biocompounds. Exploration of their biodiversity is an important step towards these goals. In a revision of the
cyanobacteria in hot springs of Iran, four hot springs in the Hormozgan (Bandar Abbas) and Mazandaran provinces
of Iran were sampled from October 2011 to July 2012. In total 43 species belonging to 20 genera, 11 families and 5
orders of the planktonic cyanobacteria were identified. Among these taxa Chroococcus with 7, Oscillatoria and
Phormidium with 5 species found to be more predominant and noticeable among other genera. All of these taxa are
new records for studied hot springs. We developed a combined molecular and morphological approach to
identification of cyanobacteria. In this study seventeen populations and 13 morphological characters were analyzed.
Molecular study based on 16S rRNA gene sequence does not disrupt morphological information and it confirms the
separation of studied taxa according to morphological characters.

Fateme Heidari <fateme.hidary@yahoo.com>, Hossein Riahi (correspondence <h-riahi@cc.sbu.ac.ir>) and

Zeinab Shariatmadari <zeshariatmadari@yahoo.com>, Faculty of Biosciences, Shahid Beheshti University,
Tehran, Iran. –Morteza Yosefzadi <morteza110110@gmail.com>, Faculty of Basic Science, Hormozgan
University, Bandar Abbas, Iran. P. O. Box: 3995.

Key words. Cyanobacteria, hot spring, morphological diversity, 16S rRNA, phylogeny.

‫ ﭼﺸﻤﻪ آب ﮔﺮم در اﻳﺮان‬4 ‫ﻣﻄﺎﻟﻌﻪ ﺗﻨﻮع ﻣﻮرﻓﻮﻟﻮژﻳﻜﻲ و ﻓﻴﻠﻮژﻧﻲ ﺳﻴﺎﻧﻮﺑﺎﻛﺘﺮي ﻫﺎي‬

.‫ ﻓﺎرغ اﻟﺘﺤﺼﻴﻞ ﻛﺎرﺷﻨﺎﺳﻲ ارﺷﺪ داﻧﺸﻜﺪه ﻋﻠﻮم زﻳﺴﺘﻲ داﻧﺸﮕﺎه ﺷﻬﻴﺪ ﺑﻬﺸﺘﻲ‬،‫ﻓﺎﻃﻤﻪ ﺣﻴﺪري‬
.‫ اﺳﺘﺎد داﻧﺸﻜﺪه ﻋﻠﻮم زﻳﺴﺘﻲ داﻧﺸﮕﺎه ﺷﻬﻴﺪ ﺑﻬﺸﺘﻲ‬،‫ﺣﺴﻴﻦ رﻳﺎﺣﻲ‬
.‫ اﺳﺘﺎدﻳﺎر داﻧﺸﻜﺪه ﻋﻠﻮم ﭘﺎﻳﻪ داﻧﺸﮕﺎه ﻫﺮﻣﺰﮔﺎن‬،‫ﻣﺮﺗﻀﻲ ﻳﻮﺳﻒ زادي‬
.‫ اﺳﺘﺎدﻳﺎر داﻧﺸﻜﺪه ﻋﻠﻮم زﻳﺴﺘﻲ داﻧﺸﮕﺎه ﺷﻬﻴﺪ ﺑﻬﺸﺘﻲ‬،‫زﻳﻨﺐ ﺷﺮﻳﻌﺘﻤﺪاري‬
‫ ﺳﻴﺎﻧﻮﺑﺎﻛﺘﺮيﻫﺎي ﺗﺮﻣﻮﻓﻴﻞ ﺑﺪﻟﻴﻞ ﺷﺒﺎﻫﺖ ﺑﺎ‬.‫ﭼﺸﻤﻪﻫﺎي آب ﮔﺮم در ﺳﺎلﻫﺎي اﺧﻴﺮ از ﺳﻮي زﻳﺴﺖﺷﻨﺎﺳﺎن ﺑﺴﻴﺎر ﻣﻮرد ﺗﻮﺟﻪ واﻗﻊ ﺷﺪهاﻧﺪ‬
،‫ از ارزش ﻋﻠﻤﻲ ﺑﺎﻻﻳﻲ ﺑﺮﺧﻮردار ﻫﺴﺘﻨﺪ‬،‫ﻓﺮمﻫﺎي زﻧﺪﮔﻲ اﺟﺪادي در زﻣﻴﻦ و ﻫﻤﭽﻨﻴﻦ ﺑﻪ ﻋﻨﻮان ﻣﻨﺒﻌﻲ از ﺗﺮﻛﻴﺒﺎت زﻳﺴﺘﻲ ﭘﺎﻳﺪار در ﺑﺮاﺑﺮ ﮔﺮﻣﺎ‬
‫ ﭼﺸﻤﻪي‬4 ‫ در اﻳﻦ ﺗﺤﻘﻴﻖ ﺳﻴﺎﻧﻮﺑﺎﻛﺘﺮيﻫﺎي ﻣﻮﺟﻮد در‬.‫و ﺑﺮرﺳﻲ ﺗﻨﻮع زﻳﺴﺘﻲ آﻧﻬﺎ ﮔﺎم ﻣﻔﻴﺪي در ﺟﻬﺖ رﺳﻴﺪن ﺑﻪ اﻫﺪاف ذﻛﺮ ﺷﺪه ﻣﻲﺑﺎﺷﺪ‬
‫ ﻋﻼوه ﺑﺮ ﻣﻄﺎﻟﻌﻪ ﺑﺮ اﺳﺎس‬.‫ ﺗﻴﺮه ﺷﻨﺎﺳﺎﻳﻲ ﺷﺪ‬11 ‫ ﺟﻨﺲ و‬20 ‫ ﮔﻮﻧﻪ ﻣﺘﻌﻠﻖ ﺑﻪ‬43 ‫ در اﻳﻦ ﺗﺤﻘﻴﻖ‬.‫آب ﮔﺮم در اﻳﺮان ﻣﻮرد ﻣﻄﺎﻟﻌﻪ ﻗﺮار ﮔﺮﻓﺖ‬
16S rRNA ‫ ﻣﻄﺎﻟﻌﺎت ﻣﻮﻟﻜﻮﻟﻲ ﺑﺮ اﺳﺎس ﺗﻮاﻟﻲ ژﻧﻮﻣﻲ‬،‫ ﺟﻬﺖ ﺷﻨﺎﺳﺎﻳﻲ دﻗﻴﻖﺗﺮ ﺳﻴﺎﻧﻮﺑﺎﻛﺘﺮيﻫﺎي ﺷﻨﺎﺳﺎﻳﻲ ﺷﺪه‬،‫ﺧﺼﻮﺻﻴﺎت ﻣﻮرﻓﻮﻟﻮژﻳﻚ‬
.‫ ﻧﺘﺎﻳﺞ ﺑﺪﺳﺖ آﻣﺪه در ﻫﺮ دو روش ﺑﻜﺎر ﺑﺮده ﺷﺪه ﻫﻢ راﺳﺘﺎ و ﺗﺎﻳﻴﺪ ﻛﻨﻨﺪه ﺑﻜﺪﻳﮕﺮ ﺑﻮدﻧﺪ‬.‫اﻧﺠﺎم ﺷﺪ‬

INTRODUCTION cyanobacteria strains in laboratory conditions as well as

Cyanobacteria, the only oxygenic photosynthetic the limited range of morphological features in this
bacteria, are interested due to their ability to grow in group of microalgae, complicate classification of these
high temperatures and in other extreme environments microorganisms (Ward & Castenholz 2000). During
(Sompong & al. 2008). Inability to culture some most of the 19th and 20th centuries, cyanobacterial
163 Cyanobacteria in hot springs of Iran IRAN. J. BOT. 19 (2), 2013

Fig. 1. Map of sampling locations and hot springs.

Table 1. Some physico-chemical parameters of four hot springs of Iran.

Studied Sites
Parameters Geno Chahahmad Khamir Ramsar

Location Hormozgan province, Hormozgan province, Hormozgan province, Mazandaran province,

Bandar Abbas city Khamir city Khamir city Ramsar city

Latiude/Longitude 26º 56´ 55˝ N 26º 56´ 31˝ N 26º 56´ 46˝ N 36º 55´ 20˝ N
55º 32´ 27˝ E 55º 26´ 17˝ E 55º 33´ 23˝ E 50º 39´ 30˝ E
Temp (°C ) 40 39 37 50
pH 7/2 6/9 7 6/8
EC ( ms cm-1 ) 14/02 20 20 13.3
Na+(mg L-1) 1600 2400 2200 410
K+(mg L-1 ) 580 400 200 240

Ca++(mg L-1 ) 800 3000 1440 1100

Mg+(mg L-1 ) 475 950 710 1100

Cl- (mg L-1 ) 4560 18520 9210 3504

PO 4-3 (mg L-1 ) 14/02 300 400 400
SO4 -2 (mg L -1) 1600 152 1374 1800
NO3 - (mg L -1 ) 0.7-1.0 1.1- 1.3 1.3-1.4 0.8-13.6
Total alkalinity 135 150 160 244
(mg L-1)

taxonomy was based on morphology (Geitler 1932; Komárek 1985, 1988; Komárek & Anagnostidis 1986,
Elenkin 1938; Desikachary 1959; Starmach 1966; 1989; Komárek 1994, 2003, 2011), which includes a
Kondrateva 1968). The taxonomic position of many combination of morphological, ecological and
morphologically defined species is unclear and some molecular characteristics. Resent molecular data
genera urgently need revision (Komárek & support the validity of many genera, e.g. Planktothrix,
Anagnostidis 1998, 2005). Moreover, the situation of Pseudananabaena, Microcystis and Spirulina (Willame
them is complicated by a conflict between & al., 2006; Komárek 2003, 2010) as defined by
bacteriological and botanical nomenclature and Komárek & Anagnostidis (1998, 2005), but at the
taxonomic practices (Stanier & al. 1978; Rippka & al. species level we often have insufficient morphological,
1979; Castenholz 2001). The most progressive system ecological and molecular data for reliable recognition
utilize a polyphasic approach (Anagnostidis & of species diversity. Thermophilic cyanobacterial mat
 IRAN. J. BOT. 19 (1), 2013 F. Heidari & al. 164

Table 2. Morphological characters and their character states in studied taxa.

Number Character Character Code

1 Life form 0- unicellular, 1- filamentous, 2- pseudo filamentous
2 Vegetative cell shape 0- spherical, 1- rod shape, 2- disco shape, 3- other forms
3 Sheathe 0- without sheathe, 1- with thick sheathe, 2- with thin sheathe
4 Filament form 0- without filament, 1- flat, 2- spiral
5 Apical cell form 0- without apical cell, 1- flat, 2- headed
6 Duplicate form 0- dichotomy, 1- homogony
7 Cell form 0- longer than width, 1- shorter than the width, 2- equal length with a width
8 Cross wall 0- without cross wall, 1- thick cross wall, 2- thin cross wall
9 Granule wall 0- without granule, 1- with granule
10 Spiral form 0- without spiral, 1- spiral loose, 2- spiral massive
11 Filament width 0- without filament, 1- 10 to 45 µm, 2- 5 to10 µm, 3- 2 to 5 µm, 4- 0.6 to 2 µm
12 Cell width/length 0- 0.1, 1- 1, 2- 1 to 2
13 Cell width 0- 1 µm, 1- 1 to 3 µm, 2- 3 to 5 µm, 3- 5 to 10 µm

communities occur in geothermal springs of Physico- chemical characteristics of the spring

neutral/alkaline pH and at temperatures of up to ~ 74 water
°C. Sometimes shared simple morphological characters Water samples were collected in a 1 liter polyethylene
as well as limitations of cultivation have made bottle from depth 10-30 cm and 30-50 cm from the
microscopic studies usefulness. One way to better water edge (Masoudi & al. 2011). Spring water
characterize these morphologically similar species is to temperature was measured at the site by laboratory
use molecular diversity information (Sompong & al. thermometer (mercury thermometer).The pH of the
2008; Ferris & al. 1997; Nubel & al. 1997; Norris & al. water samples was determined by digital pH meter
2002). It should also be noted that in studies where (DENVER pH meter) and electrical conductivity
near-complete 16S rRNA gene sequences have been determined by a conductivity bridge. Mineral content
used, conflicts between morphological and molecular samples such as potassium (K+), calcium
of water 600pb
identification of some cyanobacterial sequences have (Ca ) and sodium (Na+) were determined by flame
++

been found (Hongmei & al. 2005). Although a few hot photometer. Concentration of nitrate (NO3-) and
springs of the world have been studied, distinct sulphate (So4-2), dissolved oxygen (DO), chloride (Cl-)
phylogeographic groups have been extensively
S.aqu studied and inorganic phosphate (PO4-3) also were determined
O.sub
only in the continental USA, Iceland, New Zealand, for water samples following the methodology outlined
and some countries in Asia (Papke & al. 2003; Yim & by Clescerl & al. (1995) (Table 1).
al. 2006). Studies of the cyanobacterial mats in hot
springs in Iran have been minimally investigated. In Morphological study
present study, an attempt is to explain the Samples were streaking on agar nitrate free BG-11
morphological and phylogenetical diversity of medium (Stanier & al. 1971), and incubated in a culture
cyanobacteria in four hot springs of Iran using chamber at 25° and a 12/12 h light-dark cycle at
numerical taxonomy and molecular methods based on artificial illumination (37- 46 µmol m-2 s-1) for two
16S rRNA gene sequences. weeks. Isolation involved from removing colonies that
developed in medium and observed under light
MATERIALS AND METHODS microscope. Taxonomic determination was carried out
Sampling by light microscopy (Olympus, Model BM-2) and
Water samples were collected from four hot springs in based on Desikachary (1952) and Prescott (1962).
Geno, Khamir, Chahahmad and Ramsar (Table 1, Fig. Identification was carried out by morphometric
1). The physico-chemical properties of the water from method. Seventeen morphological characters and
each sampling site were measured. At each hot spring, numerical taxonomic studies were used for classifying
the sites were surveyed to characterize the temperature the various species of cyanobacteria. Taxonomic
profile range, and water temperature clines were analysis was performed with cluster analysis and
established in the range of 37-50°C. principal component analysis. A data matrix based on
165 Cyanobacteria in hot springs of Iran IRAN. J. BOT. 19 (2), 2013

the coded multiple states of characters were used in this

study (Table 2). Cluster analysis using the UPGMA
method (unweighted pair-group method with
arithmetical averages) was carried out. Phenetic
relationships among the species were constructed. All
analyses were carried out using SPSS (Ver.16).
DNA extraction
The bulk of each cyanobacterial culture isolation was
extracted by genomic DNA extraction kit AccuPrep
(Bioneer). 25 mg of each sample in a 1.5 ml tube with
200 µl lysis buffer and 20 µl of Proteinase K were
homogenized. The tubes were incubated for 1 h at 60
°C. 200 µl Binding Buffer was added. After mixing
gently for 10 minutes, the samples were incubated at 60
°C. 100 µl Isopropanol was added. The supernatant was
then transferred to a 2 ml tube. After a final gentle
mixing centrifugation for 5 min at 8,000 rpm, 150 µl
elution buffer was added. Extracted DNA was Fig. 2. GE banding patterns of O.sub (Oscillatoria
harvested at -20 °C temperature. subbrevis), J.met (Jaaginema metaphyticum) and S.aqu
(Synechocystis aquatilis).
PCR amplification of cyanobacterial 16S
rRNA The lowest species diversity was observed in
For DNA amplification, the 16S rRNA gene regions, Nostocales and the highest species diversity belonged
approximately 600 bp in length, were amplified by to Oscillatoriales. Among the genera were identified,
PCR using the A2 (5- AGAGTTTGATCCTGGCTCAG- Oscillatoria, Phormidium, Chroococcus and Spirulina
3) and S8 (5- TCTACGCATTTCACCGCTAC-3) showed the highest number of species. Oscillatoria
primers (Giovannoni & al. 1988). Each reaction subbrevis, Jaaginema metaphyticum (Syn.: Oscillatoria
contained 2.8 µl MgCl2, 150 mM dNTPs, 0.5 µM of angusta) and Spirulina subsalsa was observed in 3
each primer, approximately 50 ng template DNA, 3 µl stations of each hot spring. In present study, 32 taxa
Taq polymerase in a total volume of 100 µl. For PCR from Geno hot spring, 16 taxa from Chahahmad, 14
amplification cycle using the cyanobacterial primers taxa from Khamir and 9 taxa from Ramsar were
was 4 min at 95 °C, then 30 cycles of 1 min identified. The highest diversity of cyanobacteria was
denaturation at 95 °C, 1 min annealing at 59 °C, 2 min found in the Geno hot spring. The result also revealed
extension at 72 °C, and a final extension of 8 min at 72 that seasonal variation can affect the species diversity.
°C (Fig.2). Highest species diversity was recorded in autumn.
Several taxa of these microorganisms could not be
Phylogenetic analysis grown in the enrichment cultures and in the laboratory
Multiple alignments were created with reference to the condition. The present study has been done by focusing
selected GenBank sequences using BioEdit which on the importance of morphological characters to
implements the Clustal W multiple alignment recognize cyanobacterita, by numerical taxonomic
algorithm. Sequences were aligned with the software study. In total 17 morphospecies of cyanobacteria from
MEGA, version 5. The Neighbor-joining method was 4 hot springs by using 13 most stable quantitative and
used to compute evolutionary distances in present qualitative characters used for clustering analysis.
study. In this program, bootstrap analysis was used to Results of this study showed that the selective
evaluate the tree topologies by performing 1000 morophological characters separate genera but they did
resamplings. The tree was rooted using the Bacillus not separate them in species rank. Although, single cell
subtilis 16S rRNA sequence as an out-group. strains were separated completely from filamentous
RESULTS forms. Some of filamentous samples due to their
Morphological analysis morphological similarities were not completely
In this study, 43 distinct morphospecies of separated from each other. In numerical taxonomic
cyanobacteria were characterized using light study according to morophological characters, it was
microscopy (Fig. 5). All morphospecies and their consedered that the species Jaaginema metaphyticum
distribution are listed in Table 3. (Syn: Oscillatoria angusta) and Geitlerinema
IRAN. J. BOT. 19 (1), 2013 F. Heidari & al. 166

Fig. 3. Hierarchical cluster analysis dendrogram of cyanoprokaryota taxa based on morphological characters using
UPGMA method.

amphibium (Syn: Oscillatoria amphibian) are grouped region, the genome sequences of 22 samples were used
to their pervious genus Oscillatoria (Fig. 3). (Table. 6). Among the studied samples, 11 samples
To discover the most variable characters belonged to cyanobacteria of hot springs from four
among the several morphological features, PCA carried regions of Geno, Chahahmad, Khamir and Ramsar. In
out. The analysis revealed that the first five factors the phylogenetic tree by algorithm neigbhor-joining
comprise about 88.2% of total variance. In the first based on gene sequence 16S rRNA the distinctive
factor with about 25% of total variance, characters of primary clustering of the sample of out-group from taxa
trichome, vegetative cell, form of spiral and kind of of photosynthetic prokaryotes from cyanobacteria were
proliferation possessed the highest positive correlation. analyzed. In clusters, the cluster containing all of the
In the second factor with about 24% of total variance, analyzed cyanobacteria, two minor groups are
characters of apical cell shape and filament width recognizable. In one of minor clusters, all the
possessed the highest positive correlation (Table 4, 5). unicellular samples belonged to Synechococcus,
Synechocystis, Chroococcus with the bootstrap value of
Phylogenetic analysis 99% placed in a unique group, and in other cluster
In this section, for drawing cladogeram by using the Spirulina and Oscillatoria were presented (Fig. 4).
analysis of data of sequence of genome in 16S rRNA
167 Cyanobacteria in hot springs of Iran IRAN. J. BOT. 19 (2), 2013

 
Fig. 4. Phylogenetic relationship for the thermophilic cyanobacteria was constructed using partial 16S rRNA gene
sequences.

DISCUSSION present study, Synechococcous have been found in the

Temperature is one of the most important parameter for mats that grow between 30-40 ˚C. Some workers also
cyanobacterial species diversity in microbial mat of hot focus on the role of pH and combined nitrogen
springs. The studies revealed that cyanobacterial (especially ammonium), on the species distribution in
diversity and complexity decreased with increasing cyanobacterial mat community below 60 ˚C (Ward &
temperature (Ferris 1996; Ferris & Ward 1997; Ward Castenholz 2002; Sompong & al. 2005). Results of this
1998). Skimisdottir (2000) showed that in thermal study also support the previous studies. Diazotrophic
gradients from 50 ˚C to 75 ˚C, the layered mats are cyanobacteria are able to colonize in the springs where
characterized by the presence of unicellular forms like nitrogen levels are lower than proper condition for the
Synechococcus. The cyanobacterial mats occurring at other taxa. Conversely, they may be out-competed by
the lower end of thermophily (40-50˚C) are often non diazotrophic cyanobacteria in spring with sufficient
dominated by morphologically defined filamentous combined nitrogen (Ward & Castenholz 2002). In this
cyanobacteria like Phormidium, Oscillatoria, study in hot springs with high levels of combined
Pseudanabaena, Calothrix and Fischerella (Ward & nitrogen, diazotrophic cyanobacteria are absent. Results
Castenholz 2002; Sompong & al. 2005). However, of this study showed that cyanobacterial community
Norris & al. (2002) reported that cyanobacteria such as reduced in high temperature. In Ramsar hot spring with
Synechococcous also co-occurs with other unicellular 50 ˚C temperature, the lowest species diversity was
and filamentous forms at lower temperature. In the observed. In addition cyanobacterial species diversity
IRAN. J. BOT. 19 (1), 2013 F. Heidari & al. 168

10 µm F 

Fig. 5. A. Spirulina subsalsa, B. Synechococcus elongatus, C. Chroococcus sp., D. Chroococcus minimus, E.

Planktotrix rubescens, F. Oscillatoria subbrevies, G. Phormidium sp., H. Phormidium chalybeum, I. Phormidium
articulatum.

can be affected by physico-chemical characteristics of cladogram according to 16S rRNA gene sequencing
the spring waters, temperature and the regional well separated filamentous taxa from single cell taxa.
distribution of springs. In this study, because of the In filamentous taxa the polyphyly of oscillatorials were
temperature was below 60 °C, high species composition determined. But even molecular study cannot obviously
similarity was observed. Ramsar hot spring showed the separate Jaaginema metaphyticum (Syn: Oscillatoria
least of diversity due to high temperature and different angusta) from its previous group.
geographical location from the other springs. In conclusion, we report here that the
According to this fact that the grouping of taxa molecular study does not disrupt results of
according to morphological characters is not sufficient, morphological classification. Genomic sequences also
especially in complex taxa such as Jaaginema does not provide the necessary information for
metaphyticum, 16S rRNA gene sequencing was used separation of taxa from pervious groups (Oscillatoria
for better sample recognition. The phylogenetic tree angusta, Oscillatoria amphibian, Oscillatoria limentica
according to neighbor-joining algorithm, in appropriate and Phormidium tenue) and the separated taxa remain
way separates the studied genera and which is a close to their pervious groups. More exact recognition
confirmation on the morphological studies. The of them need more detail study on polyphasic.
169 Cyanobacteria in hot springs of Iran IRAN. J. BOT. 19 (2), 2013

Table 3. List of Cyanoprokaryota species recorded from Iran and their distributions in studied sites (1.Geno hot spring, 2.
Chahahmad hot spring, 3. Khamir hot spring, 4. Ramsar hot spring.*New Record from Iran).

Taxon 1 2 3 4
1 Chroococcus dispersus (Keissler) Lemmermann + +
2 *Chroococcus limenticus Lemmermann + + +
3 *Chroococcus macrococus (Kützing) Rabenhorst +
4 Chroococcus minimus (Keissler) Lemmermann + +
5 Chroococcus minor (Kützing) Nägeli +
6 Chroococcus turgidus (Kützing) Nägeli +
7 *Chroococcus varius A. Braun +
8 *Cyanobacterium cedrorum (Sauvageau) Komárek, Kopecky & Cepak +
9 *Entophysalis granulosa Kützing + +
10 *Geitlerinema acutissimum (Kufferath) Anagnostidis +
11 *Geitlerinema amphibium (C. Agardh ex Gomont) Anagnostidis +
12 *Jaaginema metaphyticum Komárek + + + +
13 *Jaaginema minimum (Gicklhorn) Anagnostidis & Komárek +
14 Johannesbaptistia pellucida (Dickie) W. R Taylor & Drouet + +
15 *Komvophoron schmidlei (Jaag) Anagnostidis & Komárek +
16 *Leptolyngbya tenuis (Gomont) Anagnostidis & Komárek + +
17 *Lyngbya versicolor Wartmann ex Gomont +
18 Microchaete sp. +
19 Nodularia spumigena Mertens ex Bornet & Flahault +
20 Oscillatoria anguina Bory ex Comont +
21 Oscillatoria limosa C. Agardh ex Gomont +
22 Oscillatoria princeps Vaucher ex Gomont + +
23 Oscillatoria subbrevis Schmidle + + + +
24 Oscillatoria tenuis C. Agardh ex Gomont + +
25 Phormidium articulatum (Gardner) Anagnostidis & Komárek + + +
26 Phormidium chalybeum (Mertens ex Gomont) Anagnostidis & Komárek +
27 *Phormidium formosum (Bory de Saint – Vincent ex Gomont) Anagnostidis & Komárek +
28 *Phormidium nigrum (Vaucher ex Gomont) Anagnostidis & Komárek + +
29 *Phormidium ornatum (Kützing) Anagnostidis & Komárek +
30 *Planktothrix agardhii (Gomont) Anagnostidis & Komárek + + + +
31 *Planktothrix prolific (Gomont) Anagnostidis & Komárek +
32 *Planktothrix rubescens (De Candolle ex Gomont) Anagnostidis & Komárek +
33 *Pseudanabaena catenata Lauterborn +
34 Pseudanabaena limnetica (Lemmermann) Komárek + +
35 *Pseudanabaena mucicola (Naumann & Huber-Pestalozzi) Schwabe +
36 Spirulina labyrinthiformis Gomont +
37 Spirulina major Kützing ex Gomont +
38 *Spirulina nordstedtii Nordstedt ex Gomont +
39 Spirulina subsalsa Oerstedt ex Gomont + + + +
40 *Synechococcus elongatus (Nägeli) Nägeli + +
41 Synechocystis aquatilis Sauvageau + +
42 *Trichodesmium lacustre Klebahn +
43 *Tychonema bornetii (Zukal) Anagnostidis & Komárek +

ACKNOWLEDGMENTS Anagnostidis, K. & Komarek, J. 1990: Modern

The authors gratefully acknowledge the financial approach to the classification system of
support of University of Shahid Beheshti. cyanophytes, 5-Stigonematales. Algological Studies
59: 1-73.
REFERENCES Anagnostidis, K. 1989: Geitlerinema, a new genus of
Anagnostidis, K. & Komarek, J. 1985: Modern Oscillatorialean cyanophytes. -Pl. Syst. Evol. 164:
approach to the classification system of 33-46.
cyanophytes, 1-Introduction. – Arch. Hydrobiol Brock, T. D. 1978: Thermophilic microorganisms and
Suppl. 71/Algological Studies 38/39: 291-302. life at high temperatures. -Berlin, Springer Verlag.
Anagnostidis, K. & Komarek, J. 1988: Modern Castenholz, R. W. 1996: Endemism and biodiversity
approach to the classification system of of thermophilic cyanobacteria. -Nova Hedwigia,
cyanophytes, 3-Oscillatoriales. – Arch. Hydrobiol. Beiheft 112: 33–47.
80/Algological Studies 50-53: 327-472. Castenholz, R. W. 2001: Phylum BX. Bacteria- In:
IRAN. J. BOT. 19 (1), 2013 F. Heidari & al. 170

Table 4. Total variance of factors according to principal component analysis of morphological characters.
Total Variance Explained
Initial Eigenvalues Extraction Sums of Squared Loadings Rotation Sums of Squared Loadings
Compone % of % of Cumulative Cumulative
nt Total Variance Cumulative % Total Variance % Total % of Variance %
1 5.144 39.566 39.566 5.144 39.566 39.566 3.256 25.045 25.045
2 2.131 16.392 55.958 2.131 16.392 55.958 3.172 24.401 49.446
3 1.696 13.046 69.004 1.696 13.046 69.004 1.730 13.305 62.751
4 1.372 10.557 79.561 1.372 10.557 79.561 1.690 12.997 75.748
5 1.135 8.734 88.295 1.135 8.734 88.295 1.631 12.547 88.295
6 .875 6.730 95.025
7 .282 2.167 97.192
8 .230 1.770 98.962
9 .086 .659 99.622
10 .041 .316 99.938
11 .007 .055 99.993
12 .001 .007 100.000
13 -
7.371 -5.670E-15 100.000
E-16
Extraction Method: Principal Component
Analysis.

Table 5. Rotated component matrix according to PCA analysis of morphological characters.

Rotated Component Matrixa

Component
1 2 3 4 5
VAR00001 .267 .309 .844 -.027 .045
VAR00002 .872 .367 .110 -.009 .210
VAR00003 -.072 .030 -.110 .938 .004
VAR00004 .943 .203 .178 .035 .148
VAR00005 .092 .973 .051 -.030 .076
VAR00006 .685 .599 .181 .097 .345
VAR00007 -.041 -.345 .660 .335 .370
VAR00008 .097 .898 .105 .275 .156
VAR00009 -.045 .075 .169 -.179 .694
VAR00010 .836 -.463 .064 -.061 -.175
VAR00011 .459 .625 .598 .001 -.081
VAR00012 .101 .181 .291 .755 -.174
VAR00013 .347 .157 -.064 .061 .854
Extraction Method: Principal Component Analysis.
Rotation Method: Varimax with Kaiser Normalization.
a. Rotation converged in 9 iterations.
171 Cyanobacteria in hot springs of Iran IRAN. J. BOT. 19 (2), 2013

Table 6. GenBank code of samples.

Sample GenBank code
1 Synechocystis sp. HQ900669.1
2 Synechocystis sp. AB039001.1
3 Synechocystis sp. HQ900668.1
4 Synechococcus elongatus str. Ramsar JQ771323.1
5 Synechococcus sp. AF448077.1
6 Oscillatoria sp. EF150796.1
7 Phormidium irriguum f. minor FN813342.1
8 Lyngbya sp. AY049752.1
9 Lyngbya sp. AY049751.1
10 Spirulina sp. DQ058861.1
11 Phormidium sp. HM217057.1

Boone, D.R. & Castenholz, R.W. (eds): Bergey s Suppl. 82/Algological Studies 56: 247-345.
Manual of Systematic Bacteriology, 2 nd Edition. – Komarek, J. & Anagnostidis, K. 2005:
pp. 473-599. -Springer Verlag. Cyanoprokaryota.2 Teil: Oscillatoriales,- In: Budel
Clescerl, L.S., Greenberg, A. E. & & Eaton, A. D. B., Gardner G., Krienitz l. & Schagerl M. (eds):
1995: Standard Methods for the Examination of Susswasserflora von Mitteleuropa, vol. 19/2.- 759
water and wastewater. American Public Health pp. -Elsevier Munchen.
Association. -Washington D.C., 1325 PP. Komarek, J. 1994: Current trends and species
Desikachary, T. V. 1959: Cyanophyta. – 686 pp. - delimitation in the cyanoprokaryote taxonomy. –
Indian Council of agricultural Research, New Delhi. Algological Studies 75: 11-29.
Elenkin, A. A. 1938: Monographia algarum Komarek, J. 2003: Problem of the taxonomic category
cyanophycearum aquidulcium et terretrium in “species” in cyanobacteria. -Algological Studies
finibus URSS inventarum. – 1908 pp. -Sumptibus 109: 281-297.
Academiae Scientiarum URSS, Leningrad. Komarek, J. 2011: Introduction to the 18 th IAC
Ferris, M., Ruff- Roberts, A., Kopczynski, E., Bateson, Symposium in Ceske Budejovice 2010 czech
M. & Ward, D. 1996: Enrichment culture and Republic. Some current problems of modern
microscopy ceneceal diverse thermophilic cyanobacterial taxonomy. –Fottea 11: 1-7.
Synechococcus populations in a single hot spring Komarek, J. & Anagnostidis, K. 1998:
microbial mat habitat. –Appl. Environ. Microbiol. Cyanoprokaryota. 1. Teil: Chroococcales. – In: Ettl,
62: 1045-1050. H.; Gardner, G.; Heying, H. & Mollenhauer, D
Garcia- Pichel, F., Prufert- Bebout, L. & Muyzer, G. (eds.): Susswasserflora von Mitteleuropa, vol. 19/1.
1996: Phenotypic and phylogenetic analyses show – 548 pp. -Gustav Fischer, Jena- Stuttgart- Lubeck-
Microcoleus chtonoplastes to be a cosmopolitan Ulm.
cyanobacterium. -Appl. Environ. Microb. 62: 3284- Kondrateva, N. V. 1968: Sin o zeleni vodorostli –
3291. Cyanophyta [blue-green algae- Cyanophyta]. – 522
Geitler, L. 1932: Cyanophyceae. – In: Rabenhorst, L. pp. -Vid, naukova dumka,, Kiev, UkrRSR.
(ed): Kryptogamen – Flora. – 1196 pp. - Marteinsson, V. T., Hauksdottir, S., Hoble, C. F. V.,
Akademische Verlags- gesellschaft, Leipzing. Kristamannsdottri, H., Hreggvidsson, G. O. &
Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Kristiansson, J. K. 2001: Phylogenetic diversity
Lane, D. J. & Pace, N. R. 1988: Evolutionary analysisof subterranean hot springs in iceland. -
relationships among cyanobacteria and green Appl. Environ. Microbiol. 67: 4242–8.
chloroplasts. -Bacteriol. 170 ( 8): 3584-3592. Masoudi, M., Ramezannejad Ghadi, R. & Riahi, H.
Hongmei, J., Aitchison, J. C., Lacap, D. C., 2011: Phytoplankton Flora of Miankaleh Wetland. -
Peerapornpisal, Y., Sompong, U. & Pointing, S. B. Iran. J. Bot. 18 (1): 141-148.
2005: Community phylogenetic analysis of Norris, T. B., Mcdermott, T. R. & Castenholz, R. W.
moderately thermophilic cyanobacterial mats from 2002: The long-term effects of UV exclusion on the
China, the Philippines and Thailand. - microbial composition and photosysthetic
Extremophiles 9: 325–32. competence of cyanobacteria in hot spring
Komarek, J. & Anagnostidis, K. 1989: Modern microbial mats. -FEMS Microbiol. Ecol. 1323: 1–
approach to the classification system of 17.
cyanophytes, 4-Nostocales. –Arch. Hydrobiol. Nubel, U., Garcia-Pichel, F., Muyzer, G. 1997: PCR
IRAN. J. BOT. 19 (1), 2013 F. Heidari & al. 172

primers to amplify 16S rRNA genes from -Bact. Rev., 35: 171-205.
cyanobacteria. -Appl. Environ. Microbiol. 63: Stanier, , R. Y., Sistrom, W. R., Hansen, T. A.,
3327-32. Whitton, B. A., Castenholz, R. W., Pfennig, N.,
Papke, R. T., Ramsing, N. B., Bateson, M. M. & Ward, Gorlenko, V. N., Kondrateva, E. N., Eimhjellen, K.
D. M. 2003: Geographical isolation in hot spring E., Whittenbury, R., Gehran, R. L. & Truper, H. G.
cyanobacteria. -Environ. Microbiol. 5: 650–9. 1978: Proposal to place the nomenclature of the
Prescott, G. W. 1970: Algae of the western great lake cyanobacteria (blue-green algae) under the rules of
area. -WM. C. Brown company publishers. 977pp. the International Code of Nomenclature of Bacteria.
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, –Int. j. Syse. Bacteriol. 28: 335-336.
& Stanier, R. Y. 1979: Generic assignments, strain Starmach, K. 1966: Cyanophyta- sinice.- 753pp. -PAN,
histories and properties of pure cultures of Panstw. Wyd. Nauk., Warszawa.
Cyanobacteria. – J. Gen. Microbiol. 111: 1-61. Ward, D. M., Castenholz, R. W. 2000: Cyano-3.
Skimisdottir, S., Hreggvidsson, G. O., Hiorleifsdottir, bacteria in geothermal habitats in Ecology of
S., Marteinsson, V. T., Petursdottir, S. K., Holst, O. cyanobacteria. Their diversity in time and space
& Kristjansson, J. K. 2000: Influence of sulfide and (Whitton B.A. & Potts M., eds.), pp 37–59. -
temperature on species composition and community Dordrecht, Kluwer Academic Publishers.
structure of hot spring microbial mats. -Appl. Williame, R., Boutte, C. H., Grubisic, S., Wilmotte, A.,
Environ. Microbiol. 66: 2835-2841. Komarek, J. & Hoffmann, L. 2006: Morphological
Sompong, U., Hawkins, P., Besley, C., Peerapompisal, and molecular characterization of planktonic
Y. 2005: The distribution of cyanobacteria across cyanobacteria from Belgium and Luxembourg. –
Physical and chemical gradients in hot spring in Appl. Phycol. 42 : 1312-1332.
northern Thailand. -FEMS Microbiology Ecology Willmotte, A. & Golubic, S. 1991: Morphological and
52: 365-376. genetic criteria in the taxonomy of cyanophyta /
Sompong, U., Anuntalabhochai, S., Cutler, R. W., cyanobacteria. -Arch. Hydrobiol. Algol. Stud. 92:
Castenholz, R. W. & Peerapornpisal, Y. 2008: 1–24.
Morphological and Phylogenic diversity of Yim, A. C., Hongmei, J., Aitchison, J. C. & Pointing,
cyanobacterial populations in six hot springs of S. B. 2006: Highly diverse community structure in a
Thailand. -Science Asia 34: 153-162. remote central Tibetan geothermal spring does not
Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen- display monotonic variation to thermal stress. -
Bazire, G. 1971: Purification and properties of FEMS. Microbiol. Ecol. 57: 80–91.
unicellular blue- green algae (order Chroococcales).