Systematic and Applied Microbiology 37 (2014) 100112

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Systematic and Applied Microbiology

journal homepage: www.elsevier.de/syapm

Phylogeny of culturable cyanobacteria from Brazilian mangroves

Caroline Souza Pamplona Silva, Diego Bonaldo Genurio,
Marcelo Gomes Marcal Vieira Vaz, Marli Ftima Fiore
University of So Paulo, Center for Nuclear Energy in Agriculture, Laboratory of Molecular Ecology of Cyanobacteria, 13400-970 Piracicaba, So Paulo, Brazil

a r t i c l e

i n f o

Article history:
Received 26 April 2013
Received in revised form 23 October 2013
Accepted 16 December 2013
Keywords:
Atlantic Rain Forest
16S rRNA gene sequencing
Taxonomy
Brackish water
Marine cyanobacteria and bioactivity

a b s t r a c t
The cyanobacterial community from Brazilian mangrove ecosystems was examined using a culturedependent method. Fifty cyanobacterial strains were isolated from soil, water and periphytic samples
collected from Cardoso Island and Bertioga mangroves using specic cyanobacterial culture media.
Unicellular, homocytous and heterocytous morphotypes were recovered, representing ve orders,
seven families and eight genera (Synechococcus, Cyanobium, Cyanobacterium, Chlorogloea, Leptolyngbya,
Phormidium, Nostoc and Microchaete). All of these novel mangrove strains had their 16S rRNA gene
sequenced and BLAST analysis revealed sequence identities ranging from 92.5 to 99.7% when they were
compared with other strains available in GenBank. The results showed a high variability of the 16S rRNA
gene sequences among the genotypes that was not associated with the morphologies observed. Phylogenetic analyses showed several branches formed exclusively by some of these novel 16S rRNA gene
sequences. BLAST and phylogeny analyses allowed for the identication of Nodosilinea and Oxynema
strains, genera already known to exhibit poor morphological diacritic traits. In addition, several Nostoc
and Leptolyngbya morphotypes of the mangrove strains may represent new generic entities, as they were
distantly afliated with true genera clades. The presence of non-ribosomal peptide synthetase, polyketide
synthase, microcystin and saxitoxin genes were detected in 20.5%, 100%, 37.5% and 33.3%, respectively,
of the 44 tested isolates. A total of 134 organic extracts obtained from 44 strains were tested against
microorganisms, and 26% of the extracts showed some antimicrobial activity. This is the rst polyphasic
study of cultured cyanobacteria from Brazilian mangrove ecosystems using morphological, genetic and
biological approaches.
2014 Elsevier GmbH. All rights reserved.

Introduction
Mangroves are transitional ecosystems between terrestrial and
marine environments that are found in tropical and subtropical
regions [14], covering approximately 6070% of shoreline [29]. In
Brazil, mangroves are included in the Atlantic Rain Forest Domain
and cover approximately 25,000 km2 , of which 240 km2 belong to
the coastline of the So Paulo state [64]. These ecosystems have
economic and ecological roles by providing nutrient inputs to maintain the estuarine and marine food webs [17,30]. However, there is a
broad range of variation in salinity and oxygen content in mangrove
environments, and these are the main factors constraining the
establishment and development of biota. Nevertheless, mangroves
have high primary production that can be assigned to cyanobacteria
[19].

Nucleotide sequence data reported are available in the GenBank

database under the accession numbers HQ730083, HQ730084, HQ730085 and
KC695831KC695877.
Corresponding author. Tel.: +55 19 34294657; fax: +55 19 34294610.
E-mail address: ore@cena.usp.br (M.F. Fiore).
0723-2020/$ see front matter 2014 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.syapm.2013.12.003

The phylum Cyanobacteria consists of oxygenic photoautotrophic organisms of the Bacteria domain, and some taxa
x atmospheric nitrogen. Such nutritional strategies and great
metabolic diversity allow these microorganisms to colonize many
terrestrial and aquatic ecosystems, including extreme environments as mangroves. To date, research on cyanobacterial diversity
in mangroves is scarce. Studies of its morphology have been conducted in Mexico and India using microscopic observation of
natural samples [68,75]. In addition, investigations have been performed in Singapore (tropical coastline) and Portugal (temperate
estuaries) that have applied isolation techniques and molecular
approaches to characterize the cyanobacterial strains [7,47,53].
In Brazil, few taxonomic studies were conducted using optical
light microscopic visualization of cyanobacteria present in mangrove and benthic marine environmental samples [1,36,13,51].
Recently, cyanobacterial communities in soil and phyllospheres
of Brazilian mangroves were assessed by molecular cultivationindependent approaches [59]. To our knowledge, there are no
studies on the isolation and cultivation of cyanobacteria from these
Brazilian environments.
During the last decades, marine cyanobacteria have proven
to be important sources of bioactive natural products, including

C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

substances with cytotoxic, antifungal, antibacterial and antiviral activities [8]. Among these natural products, there are toxins
that are harmful to humans and animals, such as microcystins
and saxitoxins [44,60], and compounds with antitumoral and
anti-inammatory activities [25,77,78]. Most of these substances
are synthesized by non-ribosomal peptide synthetase (NRPS) and
polyketide synthase (PKS) enzymes [49]. These multifunctional
enzymes are encoded by highly conserved genes and thus can be
used as templates for primer design to search for potential natural
substances of biotechnological interest.
This study aimed to isolate, cultivate and characterize novel
cyanobacterial strains that colonize mangrove environments. The
taxonomic position of the isolates was evaluated by morphological and molecular phylogenetic analyses. In addition, these strains
were screened for target genes involved in non-ribosomal synthesis and the inhibitory effect of their intra and extracellular extracts
were evaluated against microorganisms using bioactivity assays.
Microcystins and saxitoxins were examined using specic primer
set and ELISA.

101

resolution. Statistical condence of the inferred evolutionary relationships was assessed by bootstrapping (1000 replicates). The
novel nucleotide sequences were deposited in the NCBI GenBank
database under the accession numbers, HQ730083, HQ730084,
HQ730085 and KC695831KC695877.
Molecular screening of NRPS, PKS, microcystin and saxitoxin genes
The aminoacyl-adenylation domain of NRPS and ketoacyl
domain of PKS were examined by PCR using the degenerate primer
sets, MTF/MTR [50] and KSF/KSR [2], respectively. The amplication
and thermal cycling conditions were performed as described previously [70]. The saxitoxin (sxtI) and microcystin (mcyA) genes were
screened by PCR according to Kellmann et al. [35] and Tillett et al.
[74], respectively. Five milliliters of the PCR products was separated
by electrophoresis using a 1.2% agarose gel stained with ethidium
bromide. The gel prole was analyzed by a Fluor-S MultiImager
(Bio-Rad, Hercules, CA, USA) and recorded.
Enzyme-linked immunosorbent assay (ELISA)

Materials and methods

Sampling sites and cyanobacterial isolation
Water, periphyton and soil samples were collected in 2006 from
two mangrove ecosystems in the state of So Paulo, Brazil. The Cardoso Island mangrove is a pristine environment located in a natural
reserve of the Canania municipality (25 05 02 S, 47 57 42 W),
whereas the Bertioga mangrove is located near the city of Bertioga
(23 53 49 S, 46 12 28 W) and is a disturbed environment due to
an oil spill (Fig. 1).
Three culture media were used for cyanobacterial isolation,
ASN-III [61], SWBG-11 and MN [11], and each included the addition of cyclohexamide (70 mg L1 ). One hundred microliters of
homogenized soil in saline solution (NaCl 0.9%) or marine water
was spread onto solid media using Drigalskis handle. Subsamples of periphyton were streaked onto the media with a platinum
loop. Repeated streaking onto fresh solid media and microscopic
observation were performed until a mono-cyanobacterial culture
was established. In all isolation steps, cultured cells were grown
under a 14:10 h light:dark cycle with white uorescent irradiation
(40 mol photon m2 s1 ) at 24 1 C.
Morphological evaluation
Isolated cyanobacterial strains were identied using the diacritical morphological features for genera devised by Komrek and
Anagnostidis [3840] and the classication system of Hoffmann
[28]. Microscopic inspections were conducted on a Zeiss Axioskop
40 optical light microscope equipped with an AxioVisionLE 4.6 digital imaging system (Carl Zeiss, Jena, Germany).
DNA extraction and phylogenetic analysis using 16S rRNA gene
sequences
Total genomic DNA was extracted from cultured cyanobacterial strains using a modied CTAB-based extraction method
adapted for cyanobacteria [21]. PCR amplication, cloning and
sequencing of the 16S rRNA gene were also conducted as previously described [22]. The sequences obtained in this study and in
related studies were retrieved from GenBank, aligned and used
to infer their phylogenetic position on the basis of the Maximum Likelihood (ML) method included in the MEGA version 5.0
software package [73]. Four phylogenetic trees were constructed
on the basis of unicellular, homocytous, isopolar-heterocytous
and heteropolar-heterocytous features for a better phylogenetic

Lyophilized cells were resuspended in 2 mL of sterilized ultrapure water, microwaved for 1 min and then centrifuged for 5 min
at 10,000 g [69]. The supernatant was collected and analyzed by
ELISA using microplate kits for microcystins (Beacon Analytical Systems Inc., Portland, ME, USA) and saxitoxins (Branson, Smithkline
Co., Shelton, CT, USA) following the manufacturers recommendations with at least three replicates. The detection limit of these
methods was 0.1 g L1 for microcystins and 0.02 g L1 for saxitoxins.
Antimicrobial activity
The cyanobacterial strains were incubated in 500-mL asks
containing 300 mL of culture media in a shaker at 100 rpm and
maintained for 21 days in the same culture conditions described
above. After growth, the cyanobacterial culture was harvested by
centrifugation at 9000 g for 5 min at 4 C. Equal volumes of supernatant culture medium were extracted with ethyl acetate and
chloroform. The organic solvent fraction was collected and evaporated. The collected cells were submitted to methanol extraction
using glass beads ( 3 mm) followed by centrifugation at 9000 g
for 5 min at 4 C. The methanolic supernatant was collected and
evaporated [70].
Solid assays were performed to determine the inhibitory
effects of cyanobacterial organic extracts on bacteria (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Staphylococcus pasteuri,
Micrococcus luteus, Escherichia coli and Salmonella typhimurium) and
yeast (Candida cruzei). Bacterial and yeast cultures were spread
onto Petri plates containing LuriaBertani (LB) and YEPD media,
respectively. The cyanobacterial organic extracts (50 L) were
applied to Whatman no. 1 lter paper disks with a 6-mm diameter and were then transferred as the inocula to the Petri plates.
The bacterial and yeast plates were incubated at 37 C and 28 C,
respectively. Negative controls consisted of 50 L of the sterilized
culture medium extracted with the same solvents. After 14 h of
incubation, the presence of an inhibition halo was examined. All of
these experiments were conducted in triplicate.
Results and discussion
Morphological evaluation
Fifty cyanobacterial strains were isolated from environmental
samples taken from soil, water and periphytic material collected
from the mangrove ecosystems of Cardoso Island and Bertioga

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C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

Fig. 1. Mangrove sampling sites used in this study. A Cardoso Island mangrove (25 05 02 S, 47 57 42 W); B Bertioga mangrove (23 53 49 S, 46 12 28 W).

(Fig. 1, Table S1). According to morphological examination it

was isolated cyanobacterial morphotypes belonging to ve orders
(Synechococcales 31 strains, Chroococcales 3 strains, Oscillatoriales 1 strain, Pseudanabaenales 8 strains and Nostocales
7 strains), seven families (Synechococcaceae, Cyanobacteriaceae,
Entophysalidaceae, Pseudanabaenaceae, Phormidiaceae, Nostocaceae and Microchaetaceae) and eight genera (Synechococcus,
Cyanobium, Cyanobacterium, Chlorogloea, Leptolyngbya, Phormidium, Nostoc and Microchaete) (Table S1 and Fig. 2).
Supplementary data related to this article found, in the online
version, at http://dx.doi.org/10.1016/j.syapm.2013.12.003.
Several members of Synechococcus, Cyanobium, Leptolyngbya,
Phormidium and Nostoc recovered in this study were also isolated
from intertidal and estuarine zones of the Portuguese Atlantic coast
[7,47]. The few studies based on microscopic observation of natural samples from the Brazilian Atlantic coastline have also reported
the presence of the Synechococcus, Leptolyngbya, Phormidium and
Microchaete genera [3,5,6,13,51]. However, these authors did not
observe the genera Cyanobium, Chlorogloea, Cyanobacterium and
Nostoc. The isolation technique applied in this study seems to
favor the growth of pico-unicellular (orders Synechococcales and
Chroococcales) forms over the other cyanobacterial forms. In addition, the inuence of river ows and soil leaching on the mangrove
microbial communities may favor the presence of cyanobacteria from different habitats, such as the terrestrial and freshwater
strains of the genus Nostoc, isolated in this study.
Phylogenetic analyses
Near complete 16S rRNA gene sequences (13981432 bp) were
obtained for the fty strains isolated from the Brazilian mangroves.
BLAST analysis showed identities ranging from 92.5 to 99.7% among
the novel mangrove sequences when compared with those available in GenBank (Table S2). Phylogenetic trees based on 16S rRNA
gene sequences were constructed for unicellular, homocytous,
isopolar-heterocytous and heteropolar-heterocytous cyanobacterial forms (Figs. 36, respectively).
Supplementary data related to this article found, in the online
version, at http://dx.doi.org/10.1016/j.syapm.2013.12.003.
The unicellular phylogenetic tree grouped the sequences from
the mangrove cyanobacteria into four distinct clades (IIV)
(Fig. 3). Clade I was formed mainly by sequences obtained
from strains isolated from marine environments, whereas Clades
IIIV grouped sequences from freshwater habitats. Clade I clustered twenty-three mangrove cyanobacterial sequences that were
related to Cyanobium sp. PCC 6307 (reference strain of Cyanobium
group I) and Cyanobium sp. PCC 7001 (reference strain of Cyanobium
group II) [27,37]. Within this clade, three subclades were observed
(Ia, Ib and Ic). The Ia subclade assembled only the Brazilian

mangrove cyanobacterial sequences with a high bootstrap value

(89%). Subclade Ib grouped four Brazilian mangrove cyanobacterial sequences with a 90% bootstrap value into reference strain
Cyanobium group II (Cyanobium sp. PCC 7001) [23,37], Cyanobium
sp. NS01 [23], Cyanobium sp. LEGE 06137 and Cyanobium sp. LEGE
07318 [7,47], all of which were recovered from different parts of
the Atlantic Ocean. The reference strain, Cyanobium sp. PCC 7001
(group II), was originally identied as Synechococcus sp. PCC 7001
and was further renamed as Cyanobium sp. PCC 7001 [27]. Similarly,
the Brazilian mangrove cyanobacterial strains included in cluster Ib
were previously morphologically identied as Synechococcus spp.
and were then reclassied as the Cyanobium spp. Subclade Ic cluster with four Brazilian mangrove cyanobacterial sequences with
the Cyanobium sp. PCC 6307 (group I) [23,37] and other Cyanobium
sequences recovered from marine waters and saline lakes [18,54].
Typical marine groups consisting of Synechoccocus (Synechococcus sp. WH 8103, Synechococcus sp. WH 7805, Synechococcus sp.
WH 7803 and Synechococcus sp. WH 8101) and Prochlorococcus
[23] were distantly related to the Brazilian Cyanobium (clade I) and
Synechococcus isolates (clade II). The majority of the novel Brazilian
mangrove Synechococcus sequences (seven strains) were grouped
together with the Synechococcus sp. MLCB sequence (clade II), a
strain isolated from the alkaline and hypersaline Monolake in the
United States [9]. The authors of the latter study emphasized the
close relationship of Synechococcus sp. MLCB to Prochlorococcus and
typical marine Synechococcus strains. However, our results indicated that Synechococcus sp. MLCB was more closely related with
sequences from mangrove environments than those from the open
sea. The Synechococcus sp. CENA 140 sequence was loosely afliated
with clades I and II. Although BLAST analysis showed the best match
with Cyanobium sp. JJ2-3 (98.5%) and Synechococcus sp. CENA 108
(98.0%), the sequence from the Brazilian mangrove was not grouped
with these 16S rRNA gene sequences. This result indicates a novel
cyanobacterial entity.
The molecular evidence of polyphyly of the morphotype genus
Synechococcus was previously reported [18,23,31,63,65,76] and it
was also observed in this study. The tree from Fig. 3 was constructed based only on 16S rRNA gene sequences of unicellular
cyanobacteria and Synechococcus clades showed approximate congruence with the previously studies that used sequences from
unicellular and multicellular strains. For instance, the major group
of Synechococcus sequences of our study (CII) was inserted in the
group A4 described by Schirrmeister et al. [65]. However, unlike
obtained by these authors the thermophilic Synechococcus sp. P1
(isolated from Octopus Spring in Yellowstone, national park, USA)
was positioned together with the termophilic Synechococcus sp.
C9 (isolated from the same environment that P1) in accordance
with the tree performed by Turner et al. [76]. The type species for
this genus is the freshwater Synechococcus elongatus (type strain

C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

103

Fig. 2. Photomicrographs of representatives isolates of cyanobacteria from Brazilian mangroves. A Cyanobium sp. CENA 136; B Cyanobium sp. CENA 162; C Cyanobium
sp. CENA 177; D Synechococcus sp. CENA 179; E Synechococcus sp. CENA 140; F Cyanobacterium sp. CENA 169; G Cyanobium sp. CENA 145; H Chlorogloea sp. CENA
152; I Oxynema sp. CENA 135; J Nodosilinea sp. CENA 137; K Leptolyngbya sp. CENA 134; L Leptolyngbya sp. CENA 155; M Leptolyngbya sp. CENA 156; N Nostoc sp.
CENA 175; O Nostoc sp. CENA 184; P Microchaete sp. CENA 176. Scale bar = 20 m.

PCC 6301) which in our phylogenetic tree is the sister taxa of

Clades I and II. The Synechococcus genus has lineages related to
several environments (extremophiles, non-extremophiles, marine
and freshwater), which may reect in genetic diversity. This genus
needs re-evaluation and may be separated according to habitat
source.
The general topology of the Cyanobium (clade I) and Synechococcus (clade II) sequences obtained in this study was in agreement to

the phylogenetic analysis based on 58 concatenated core ortholog

genes [46] and on 16S rRNA gene sequences from 54 whole-genome
sequenced cyanobacterial strains [66].
The morphological features of the unicellular strain CENA169
observed by light microscopy were correlated with the genus
Cyanothece. However, according to our phylogenetic analysis the
CENA169 sequence clustered with three sequences of the genus
Cyanobacterium (including Cyanobacterium stanieri PCC7202, the

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C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

Fig. 3. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of unicellular strains. The studied strains are shown with a black circle. A bootstrap test
involving 1000 resamplings was performed and bootstrap values greater than 50% are given in front of the relevant nodes.

type species of the Cyanobacterium genus) [62]) with bootstrap

value of 63%. This cluster was positioned distantly from others Cyanothece sequences, as well from Cyanothece sp. ATCC
51142 sequence, which is the reference strain for this genus [37].

According to Komrek [37], many Cyanothece morphotypes have

been incorrectly identied based on Rippka and Cohen-Bazire
[62]. Moreover, these authors [37] have been recommended
that Cyanobacterium must be used to correct this taxonomic

C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

105

Leptolyngbya sp. LEGE 07314 (HM217061)

Leptolyngbya sp. LEGE 07298 (HM217044)
Leptolyngbya sp. LEGE 07084 (HM217072)
Leptolyngbya/Nodosilinea sp.PCC7104 (AB039012)
Leptolyngbya sp./Nodosilinea sp. 0BB24S04 (AJ639893)
52
Nodosolinea nodulosa UTEX 2910 (EF122600)
Nodosilinea sp. CENA 147 (1413 bp)
98
80
82
Nodosilinea sp. CENA 137 (1412 bp)
Nodosilinea sp. CENA 183 (1412 bp)
CI
74
100 Leptolyngbya antarctica ANT.LAC.1 (AY493588)
Leptolyngbya antarctica ANT.LACV6.1 (AY493589)
53
82
Nodosilinea sp. CENA 167 (1413 bp)
Nodosilinea sp. CENA 144 (1413 bp)
70
Leptolyngbya sp. LEGE 07309 (HM217053)
Leptolyngbya sp./N. bijugata Kovacik 1986/5a (EU528669)
74
97 Nodosilinea epilithica Kovacik 1998/7 (HM018677)
Nodosilinea sp. FI2-2HA2 (HM018678)
Leptolyngbya sp.CENA 156 (1414 bp)
Leptolyngbya sp. CENA 155 (1415 bp)
56
Leptolyngbya sp. PCC7375 (AB039011)
Leptolyngbya sp.CENA 134 (1417 bp)
Leptolyngbya sp. LEGE 07089 (HM217063)
Halomicronema sp. TFEP1 (AF320093)
99
Geitlerinema sp. BBD_HS217 (EF110974)
Geitlerinema sp. PCC7105 (AB039010)
Spirulina subsalsa AB2002/06 (AY575934)
Spirulina subsalsa PD2002/gca (AY575935)
100
Halospirulina sp. CCC Baja-95 Cl.3 (Y18790)
Halospirulina tapeticola CCC Baja-95 Cl.2 (NR_026510)
100 Leptolyngbya frigida ANT.LH70.1 (AY493574)
88
Leptolyngbya frigida ANT. MANNING.1 (AY493573)
100
Leptolyngbya sp. CENA 112 (EF088337)
Leptolyngbya sp. CENA 103 (EF088339)
78
Leptolyngbya sp. WJT25-NPbg1 (HM018682)
94
Leptolyngbya sp. FI5-2HA4 (HM018683)
Leptolyngbya sp. FI5-2HA3-2 (HM018684)
99
99 Plectolyngbya hodgsonii ANT.LPR2.2 (AY493583)
Plectolyngbya hodgsonii ANT.LG2.1 (AY493615)
100
99
Leptolyngbya crispata SEV4-3-C6 (AY239598)
Leptolyngbya crispata SEV1-1-C1 (AY239599)
86
Leptolyngbya tenerrima UTCC 77 (AF218368)
100
Leptolyngbya angustata UTCC 473 (AF218372)
Leptolyngbya boryanum (X84810)
Leptolyngbya sp. CENA 104 (EF088333)
C II
Leptolyngbya boryana UTEX B 485 (AF132793)
Leptolyngbya foveolarum VP1-08 (FR798945)
Leptolyngbya boryana IAM M-101 (AB245143)
100
Pseudanabaena sp. PCC7408 (AB039020)
Pseudanabaena sp. PCC6903 (AB039017)
99
Pseudanabaena sp. PCC7403 (AB039019)
Pseudanabaena sp. PCC6802 (AB039016)
Gloeobacter violaceus PCC 7421 (NC005125)
84

80

85

84
59
74

83

94

95

0.01

Fig. 4. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of homocytous strains. The studied strains are shown with a black circle. A bootstrap test
involving 1000 resamplings was performed and bootstrap values greater than 50% are given in front of the relevant nodes.

misidentication. Hence, in accordance with our phylogenetic

analysis the strain CENA 169 was named as Cyanobacterium sp.
CENA169.
Two strains of Chlorogloea, CENA 150 and CENA 152, were
recovered from two different Brazilian mangroves and formed
a unique clade (clade IV) with a bootstrap value of 99%. These
two 16S rRNA gene sequences were the only representatives
of the Chlorogloea genus and the Entophysalidaceae family, and
BLAST analysis showed low identities (92.9%) among these
sequences and those available in the NCBI database. Therefore, a more robust phylogenetic inference was not possible.
Nevertheless, this Chlorogloea cluster was more closely related
to the Cyanobacterium clade (clade III) obtained in this study
and to the clade of marine Synechococcus group III (Synechococcus sp. PCC 73109 and Synechococcus sp. PCC 7003)
[27,37].

In general, the unicellular Brazilian mangrove cyanobacterial

sequences were more related to marine Cyanobium than to typical marine Synechococcus strains and the freshwater Synechococcus
group (group I.1) [27]. In addition, our results demonstrated that
some clades (Ia and IV) were formed exclusively by the Brazilian
mangrove cyanobacterial sequences, reinforcing that they are not
afliated with any known cyanobacterial group. The existence of an
endemic cyanobacterial group based on 16S rRNA gene sequences
was also observed for Synechococcus isolated from the Red Sea [23].
The 16S rRNA gene sequences of ve Pseudanabaenacean
strains fell into the cluster formed by Leptolyngbya-like sequences
obtained from the Portuguese coastline and Antarctic and by the
sequence of the type-species of the novel genus Nodosilinea (N.
nodulosa UTEX 2910) (Fig. 4). This genus was recently described
from a separate phylogenetic lineage of Leptolyngbya strains that
share the formation of nodule, a common morphologic diacritic

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C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

Cyanospira capsulata 9NAT (FR774776)

Cyanospira rippkae PCC9501 (AY038036)
79
| Cyanospira rippkae CR86F7 (FR774774)
Anabaenopsis sp. 1A (AF516747)
88
76
Anabaenopsis sp. PCC 9215 (AY038033)
Nodularia harveyana BECID29 (AJ781146)
68
Nodularia harveyana Bo53 (AJ781143)
87
Nodularia spumigena BY1 (AF268004)
92
99 Nodularia sphaerocarpa Fae19 (AJ781144)
Nodularia sp. Lukesova 1/91 (AM711553)
Anabaena sp. 90 (AJ133156)
58
99
Anabaena circinalis NIES41 (AF247588)
Anabaena oscillarioides BECID32 (AJ630427)
Nostoc sp. 2LT05S03 (FM177500)
51
74
Nostoc sp. CENA 88 (GQ259207)
Nostoc sp. CENA 175 (1413 bp)
61
91
Nostoc sp. PCC 8112 (AM711537)
95
Nostoc elgonense TH3S05 (AM711548)
97 Nostoc sp. KU001 (AB087403)
Nostoc sp. PCC 8976 (AM711525)
Nostoc sp. CENA 184 (1414 bp)
Nostoc sp. Mollenhauer 1:1-067 (DQ185207)
73
Nostoc sp. CENA 186 (1412 bp)
C II
Nostoc sp. CENA 158 (1415 bp)
88
Nostoc sp. CENA 159 (1414 bp)
Nostoc sp. CENA 160 (1413 bp)
Anabaena oscillarioides BO HINDAK 1984/43 (AJ630428)
Nostoc carneum IAM M-35 (AB325906)
98 Nostoc sp. PCC7120 (NC003272)
Anabaena variabilis NIES23 (AF247593)
68
Nostoc sp. KK-01 (AB187508)
99
Nostoc sp. PCC 7423 (DQ185242)
91
Nostoc muscorum CENA 61 (AY218828)
99 Nostoc sp. 6S-05a (FR798937)
Nostoc sp. 5N-02c (FR798936)
Nostoc sp. 9E-03 (FR798938)
Nostoc calcicola TH2S22 (AM711529)
Nostoc sp. Mollenhauer 1:1-088 (DQ185208)
Mojavia pulchra JT2-VF2 (AY577534)
Nostoc verrucosum KU005 (AB494996)
Nostoc muscorum I (AJ630451)
83
Nostoc sp. 8964:3 (AM711541)
Nostoc sp. Al1 (AM711522)
Nostoc punctiforme SAG 60.79 (GQ287652)
Nostoc sp. ANT.L52B.7 (AY493623)
85
Nostoc calcicola III (AJ630447)
61
Nostoc edaphicum X (AJ630449)
Nostoc punctiforme PCC 73102 (NC_010628)
Nostoc sp. IO-102-I (AY566855)
Nostoc ellipsosporum V (AJ630450)
Gloeobacter violaceus PCC 7421 (NC005125)
84
97

71
53

CI

C III

C IV

0.01

Fig. 5. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of nostocacean strains. The studied strains are shown with a black circle. A bootstrap test
involving 1000 resamplings was performed and bootstrap values greater than 50% are given in front of the relevant nodes.

character observed under low light conditions [56]. Additionally,

these Leptolyngbya strains were recovered from diverse habitats
worldwide, such as marine phytoplankton, marine benthos, freshwater lakes, stone walls, and desert soils from Asia, Europe, and
North America [56]. The other three sequences of Pseudanabaenacean, Leptolyngbya sp. CENA134, Leptolyngbya sp. CENA156 and
Leptolyngbya sp. CENA155, were loosely afliated with Leptolyngbya boryana, the type-species of the true Leptolyngbya cluster
(Fig. 4). These three 16S rRNA gene sequences had identities below
95.8% when they were compared with those available in the GenBank database and formed three independent branches in the
phylogenetic tree. These results highlight the polyphyletic condition of the Leptolyngbya genus.
The only Oscillatoriacean strain isolated from the pristine
mangrove of Cardoso Island was morphologically identied as
Phormidium sp. CENA 135. The 16S rRNA gene sequence of this
strain showed a 98.8% identity with Phormidium lloydianum CCALA
960 isolated from saltworks in Petchaburi Province, Thailand, and
they grouped with a 100% bootstrap value in the phylogenic
analysis. The CENA 135 and CCALA 960 strains with their distinctly phylogenetic position regarding the true Phormiduim group,

their specic phenotypic characteristics (exhibiting elongated, narrowed and bent cells with pointed ends, without calyptra) and
their ecological features (halophytic and mineral localities) were
separated into the novel generic entity, Oxynema [12].
The 16S rRNA gene sequences obtained for six Nostocaceae
strains fell into two distinct Nostoc clusters (clades I and II) in the
phylogenetic analysis (Fig. 5). These two well-supported clades
were more closely related to the Nodularia, Anabaenopsis and
Cyanospira genera than to the typical Nostoc cluster (clade IV).
Nostoc sp. CENA 175 isolated from soil collected in the Bertioga
mangrove was placed in cluster I and formed a well-supported
group with six other Nostoc strains. This cluster corresponds to
clade IVb as reported by Papaefthimiou et al. [55] and is composed
of sequences of free-living members isolated from different habitats and countries. Additionally, this cluster grouped the sequence
of Brazilian microcystin-producing Nostoc sp. CENA 88, which was
isolated from a freshwater reservoir [24]. Cluster II was almost
exclusively formed by sequences generated in this study and were
obtained from strains isolated from water (Nostoc sp. CENA 184)
and soil (Nostoc sp. CENA 158, Nostoc sp. CENA 159, Nostoc sp.
CENA 160 and Nostoc sp. CENA 186) samples collected in the

C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

Rexia erecta CAT 1M (AY452533)

Hassallia sp. CCALA 954 (FR822751)
Coleodesmium sp. ANT.L52B.5 (AY493596)
59
Coleodesmium wrangelii (AF334703)
99 Tolypothrix distorta SAG 93.79 (GQ287651)
65
Hassallia byssoidea CCALA 823 (AM905327)
Hassallia sp. CCALA 957 (FR822754)
93
Hassallia sp. CCALA 957 (FR822754)
Hassallia sp. CCALA 955 (FR822752)
55
Calothrix sp. HA4356-MV2 (JN385289)
Gloeotrichia echinulata URA3 (AM230705)
61
99
Gloeotrichia echinulata PYH6 (AM230703)
Gloeotrichia echinulata PYH14 (AM230704)
Tolypothrix sp. UAM 335 (HM751850)
Tolypothrix sp. UAM 337 (HM751851)
84 Tolypothrix sp. UAM 334 (HM751849)
80 Tolypothrix sp. UAM 332 (HM751847)
Tolypothrix sp. PCC 7415 (AM230668)
Tolypothrix sp. UAM 340 (HM751853)
99 Camptylonemopsis sp. HA4241-MV5 (JN385292)
73
Camptylonemopsis sp. HA4241-MV5 (HQ847564)
Calothrix brevissima (AB074504)
52
Tolypothrix sp. IAM M-259 (AB093486)
Calothrix sp. PCC 7101 (AB325535)
Tolypothrix sp. TOL328 (AM230706)
Tolypothrix sp. PCC 7504 (AM230669)
Hassallia sp. CNP3-B3-C04 (HQ847556)
68
Spirirestis rafaelensis WJT-71-NPBG6 (JQ083655)
79 Hassallia sp. EM2-HA1 (HQ847555)
Hassallia sp. CM1-HA08 (HQ847554)
96 Calothrix sp. TJ12 UAM-372 (EU009154)
57
Calothrix sp. MU27 UAM-315 (EU009152)
55
Calothrix sp. PCC 7103 (AM230700)
50
Calothrix desertica PCC 7102 (AF132779)
Calothrix sp. 2T08 (FR798918)
76
Rivularia sp. VP4-08 (FR798919)
93
Calothrix sp. D253 (X99213)
99
Calothrix sp. PCC (AM230701)
Calothrix sp. PCC 7714 (AJ133164)
Calothrix sp. CCALA 953 (FR822750)
64 Calothrix sp. BECID1 (AM230680)
Calothrix sp. XSP10A (AM230679)
94
Calothrix sp. UKK3412 (AM230681)
99
Calothrix sp. BECID16 (AM230682)
Calothrix sp. BECID33 (AM230683)
Calothrix sp. CAL3363 (AM230684)
Microchaete sp.CENA 176 (1413 bp)
Calothrix sp. BECID 30 (AM230685)
Calothrix sp. PCC 7507 (AM230678)
94
Rivularia sp. PCC 7116 (AM230677)
54
Calothrix sp. CCMEE 5093 (AY147029)
96
Calothrix sp. ANT.LPR2.4 (AY493597)
68
Rivularia sp. XP16B (AM230676)
88 Calothrix sp. BECID14 (AM230671)
Calothrix sp. XP9A (AM230670)
Scytonematopsis contorta HA4292-MV4 (HQ847561)
Petalonema sp. HA4277-MV1 (HQ847568)
Petalonema sp. ANT.LG2.8 (AY493624)
Scytonema hofmanni PCC 7110 (AF132781)
Brasilonema octagenarum UFV-OR1 (EF150855)
Gloeobacter violaceus PCC 7421 (NC_005125)

107

97

68

52

88

70

51
64

CI

C II

0.01

Fig. 6. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of microchaetacean and rivulariacean strains. The studied strain is shown with a black
circle. A bootstrap test involving 1000 resamplings was performed and bootstrap values greater than 50% are given in front of the relevant nodes.

Cardoso Island mangrove. Only the sequence of Nostoc sp. Mollenhauer 1:1-067 isolated from the lichen, Peltigera didactyla, grouped
together in this cluster. The phylogeny of the Nostoc genus determined based on 16S rRNA, nifD, nifH and rbcLX gene sequences
has been shown to be polyphyletic [24,26,32,48,55,57,58,72]. These
results supported the division of Nostoc morphotypes into new
generic entities as noted for the genus Mojavia [58]. However, it

has been recommended that the separation of new taxonomic entities not be based only on genetic afliation but also on the use
of a polyphasic approach, including morphological diacritic features, thylakoid position and ecology [37]. Therefore, based on the
genetic separation of clusters I and II from the typical Nostoc (cluster IV) strains observed in this study, both clusters of Nostoc-like
may be reclassied into two new generic entities. However, an

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C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

Table 1
PCR tests of NRPS, PKS, microcystin (mcyA) and saxitoxin (sxtI) syntethase genes and ELISA results for microcystin and saxitoxin cyanotoxin.
Strain

NRPS

PKS

mcyA

ELISA microcystin

sxtI

ELISA saxitoxin

Cyanobium sp. CENA136

Cyanobium sp. CENA138
Cyanobium sp. CENA139
Synechococcus sp. CENA140
Cyanobium sp. CENA141
Cyanobium sp. CENA142
Synechococcus sp. CENA143
Cyanobium sp. CENA145
Cyanobium sp. CENA146
Cyanobium sp. CENA148
Cyanobium sp. CENA149
Cyanobium sp. CENA151
Cyanobium sp. CENA153
Cyanobium sp. CENA154
Cyanobium sp. CENA157
Cyanobium sp. CENA162
Cyanobium sp. CENA163
Cyanobium sp. CENA164
Cyanobium sp. CENA165
Cyanobium sp. CENA166
Cyanobium sp. CENA168
Synechococcus sp. CENA170
Synechococcus sp. CENA171
Synechococcus sp. CENA172
Synechococcus sp. CENA174
Cyanobium sp. CENA177
Cyanobium sp. CENA178
Synechococcus sp. CENA179
Synechococcus sp. CENA180
Cyanobium sp. CENA181
Cyanobium sp. CENA185

nd
nd

nd
+
nd

+
+
+
+
+
+
+
+
+
+
+
+
+
nd
nd
+
+
+
+
+
nd
+
nd
+
+
+
+
+
+
+
+

+
nd
nd
nd
nd
+

nd
+
nd
nd
nd

nd
nd
+
nd
nd
nd
nd
nd
nd
nd
nd
nd

nd
nd
nd
nd

+
nd
nd
nd
nd
+
+

+
nd
+
nd
nd
nd
nd
nd
+
+
nd
+
nd
+
+
+
nd
nd
nd
nd
+

+
nd
nd

nd
nd
nd

nd
nd

nd
nd

nd
nd
nd
nd
nd
nd

nd
nd

nd

nd
nd

nd

nd

Chlorogloea sp. CENA150

Chlorogloea sp. CENA152
Cyanobacterium sp. CENA169

+
+

+
+
+

nd

+
nd
+

+
nd

nd

Oxynema sp. CENA135

Leptolyngbya sp. CENA134

Nodosilinea sp. CENA137
Nodosilinea sp. CENA144
Nodosilinea sp. CENA147
Leptolyngbya sp. CENA155
Leptolyngbya sp. CENA156
Nodosilinea sp. CENA167
Nodosilinea sp. CENA183
Nostoc sp. CENA158
Nostoc sp. CENA159
Nostoc sp. CENA160
Nostoc sp. CENA175
Microchaete sp. CENA176
Nostoc sp. CENA184
Nostoc sp. CENA186

+
+
nd
nd

+
+
+
+
+
+
+
+
+
+
+
+
+
nd
nd

nd
nd
nd

nd
nd

nd
+
nd
nd
nd
nd

+
nd
nd
nd
nd
nd
nd
+

nd

nd
nd
nd

+
nd
nd

nd
nd
+
+
+
+
nd
nd
nd

nd
nd

nd
nd

nd
nd
nd

: negative results; +: positive results; nd: not determined.

autapomorphic diacritic feature must be identied in these

cyanobacteria strains to certify their separation from typical Nostoc
members.
The only heteropolar form isolated was the CENA 176 strain
that had a morphology resembling that of the Microchaete genus.
The only differences between this strain and the type-species,
Microchaete grisea, were the distinct constrictions at cross walls
and the bright bluegreen color of the cells [36]. Furthermore,
Microchaete sp. CENA 176 was isolated from soil samples collected
in the Bertioga mangrove (marine coastline), an ecologic environment that is similar to that from which M. grisea was recovered
[36]. The best mach upon BLAST analysis of the 16S rRNA gene
sequence of CENA176 was with Calothrix sp. BECID 30 (94.5% identity) isolated from rock surfaces in the littoral zone of the Baltic
Sea [67]. The phylogeny of the heteropolar forms arranged the
sequences into two different clusters (I and II) and placed CENA176
within cluster II, formed predominantly by sequences of strains

morphologically described as rivulariacean (Fig. 6). However, the

Microchaete genus belongs to the Microchaetaceae family. The evaluation of several Microchaetaceae members based on nucleotide
sequences has indicated that the whole family is phylogenetically heterogeneous and still requires taxonomic revision [45,52].
Because Microchaete sp. CENA176 was the only cultured strain with
identical morphological traits to the M. grisea type-species and had
a 16S rRNA gene sequence, it is suggested that the name Microchaete
be used only for sequences that fall into the Microchaete sp. CENA
176 clade.
The fty mangrove strains isolated were afliated to eight
cyanobacterial genera according to morphological traits, but their
phylogeny based on 16S rRNA gene sequences indicated that
15 genetic lineages were recovered. Therefore, the phylotypes
diversity was higher than morphotypes diversity. Similar studies
conducted in intertidal zones and in temperate estuaries of the
Portuguese coast recovered 12 and 21 phylotypes, respectively

C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

109

Table 2
Bioactivity assays conducted using a subset of cyanobacterial extracts against pathogenic bacteria and yeast.
Strains

Extract

BSa

BCb

SAc

SPd

MLe

ECf

STg

CCh

Cyanobium sp. CENA 136

Cyanobium sp. CENA 142

Extracellular EA
Intracellular
Extracellular CH
Extracellular EA
Extracellular CH
Extracellular EA
Extracellular CH
Extracellular EA
Intracellular
Extracellular CH
Intracellular
Extracellular CH
Extracellular EA
Intracellular
Extracellular CH
Extracellular EA
Intracellular
Extracellular CH
Extracellular EA
Intracellular
Intracellular
Extracellular CH
Extracellular EA
Intracellular
Extracellular CH
Intracellular
Intracellular
Extracellular EA
Intracellular
Extracellular CH
Extracellular EA
Intracellular
Extracellular CH
Extracellular EA

+
+

+
+

+
+

+
+

+
+

+
+
+
+
+
+
+
+
+

+
+

+
+
+
+
+
+
+
+

+
+

+
+
+

+
+
+
+
+
+

+
+

+
+

+
+
+

+
+
+

+
+
+

+
+

+
+

+
+

+
+
+

Cyanobium sp. CENA 149

Chlorogloea sp. CENA 152
Cyanobium sp. CENA 153
Cyanobium sp. CENA 165

Cyanobacterium sp. CENA 169

Synechococcus sp. CENA 170

Synechococcus sp. CENA180

Cyanobium sp. CENA 185

Leptolyngbya sp. CENA 134

Oxynema sp. CENA 135
Nodosilinea sp. CENA 137
Nodosilinea sp. CENA 147
Nostoc sp. CENA 158
Nostoc sp. CENA 159

Nostoc sp. CENA 160

+
+

+
+
+
+
+

: negative results; + positive results; EA: ethyl acetate extract; CH: chloroform extract.
a
Bacillus subtilis.
b
B. cereus.
c
Staphylococcus aureus.
d
S. pasteuri.
e
Micrococcus luteus.
f
Escherichia coli.
g
Salmonella typhimurium.
h
Candida cruzei.

[7,47]. Also, investigation on unicellular diazotrophic cyanobacteria performed in tropical coastline of Singapore showed only one
cluster [53]. Recent work investigating the uncultivated cyanobacterial community from the same Brazilian mangrove environments
[59] recovered 16S rRNA gene sequences that showed high identities with cultured Cyanobium, Cyanobacterium, Synechococcus,
Leptolyngbya, Nostoc and Phormidium/Oxynema strains isolated
in this study. A phylogenetic analysis (data not shown) including all 16S rRNA gene sequences retrieved from the studied
Brazilian mangroves presented 15 clades with seven containing
a mix of sequences obtained in this study and from Rigonato
et al. [59]. Therefore, isolation and uncultured techniques are
complementary and allow for a better estimation, as several
16S rRNA gene sequences obtained using uncultured molecular
approaches were not recovered using isolation techniques and vice
versa.
More systematic studies of cyanobacteria employing a polyphasic approach are urged in the attempt to construct a more
robust cyanobacterial classication system. Taxonomic studies of
cyanobacteria are increasing in tropical and subtropical areas,
but its diversity is still underestimated due to poor sampling or
misidentication of the species based on those from temperate
zones. In this study, agreement between morphological and phylogenetic analyses was not always observed, and some clades

contained only sequences of strains from Brazilian mangroves,

forming monophyletic groups. These data indicate that Brazilian
mangroves harbor a unique cyanobacterial phylotypes with some
of them being endemic to this extreme environment.
Molecular screening of the NRPS, PKS, mcyA and sxtI genes and
ELISA
Partial sequences of NRPS and PKS domains were PCR-amplied
from 20.5% and 100%, respectively, of the 44 mangrove strains
(Table 1). Fragments of mcyA were detected in 37.5% of a total of
16 strains, and sxtI fragments were amplied in 33.3% of a total
of 27 strains. ELISA analyses showed a positive immunoreaction
for microcystin and saxitoxin production in 78.2% (from 23 strains
tested) and 8.8% (from 34 strains tested), respectively, of the cultured cell extracts (Table 1).
Cyanobium sp. CENA 142 and Oxynema sp. CENA 135 strains
exhibited amplication of NRPS, PKS, mcyA and sxtI as well as
positive ELISA results for microcystin and saxitoxin production,
indicating the potential of both of these strains to produce toxins. Additionally, Cyanobium sp. CENA 136 and Cyanobium sp. CENA
146 gave positive results for the presence of the mcyA gene and
also for microcystin production (Table 1). However, some strains
showed positive amplication of mcyA and sxtI, but did not react

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C.S.P. Silva et al. / Systematic and Applied Microbiology 37 (2014) 100112

using the ELISA. In these cases, biological and/or chemical tests are
necessary to conrm the gene expression as well as the toxin production. On the other hand, other strains showed an inverse pattern
in which ELISA tests were positive, but mcyA and sxtI genes were
not amplied (Table 1).
Previous
studies
have
demonstrated
that
some
strains
do
not
contain
the
microcystin-producing
N-methyl-transferase domain of the mcyA gene, leading to
the production of demethyl microcystin variants, e.g., Nostoc sp.
CENA 88 [24], Planktothrix sp. [4143] and Anabaena sp. [20]. Thus,
to overcome this problem, other genes involved in microcystin
biosynthesis, such as mcyD, mcyE and mcyG, that are responsible
for the synthesis of the unique Adda moiety of microcystins
must be investigated [16]. In addition, despite the fact that
the sxtI gene (encoding the enzyme, O-carbamoyltransferase)
has been designated as a good candidate to investigate the
potential to produce saxitoxins [35], other sxt genes must be
evaluated.
Picoplanktonic cyanobacteria have been reported to be involved
in microcystin production [10], and the present study conrmed
this potential for at least three Cyanobium strains. It is known
that the Synechococcus and Cyanobium morphotypes are the most
abundant cyanobacteria in marine environments [15,34]. Therefore, the ability to produce toxins by some of the strains in these
genera could represent a health risk to animals and humans worldwide. Further investigation using mass spectrometry should be
conducted to conrm this production.

Bioactivity assay
A total of 134 organic extracts obtained from 44 of the mangrove
cyanobacterial strains were tested against pathogenic bacteria
and yeast, and 34 extracts showed some antimicrobial activity.
The active extracts belonged to 17 cyanobacterial strains comprising unicellular, homocytous and isopolar-heterocytous forms
(Table 2). Among the 34 organic extracts, 50% were active against
gram-positive bacteria (B. subtilis, B. cereus, S. aureus, S. pasteuri
and M. luteus), and 27% were active against gram-negative bacteria (E. coli and S. typhimurium). Seventeen percent were active
against yeast (C. cruzei) (Table 2). These results are in agreement with those reported in other studies on the antimicrobial
activity of extracts from cyanobacterial isolates [33,70]. A recent
study with the Brazilian mangrove cyanobacterial strains, Leptolyngbya sp. CENA134, Oxynema sp. CENA135 (identied earlier
as Phormidium sp.) and Cyanobium sp. CENA136 (Synechococcus sp.) showed that they were able to degrade different textile
dyes [71].

Conclusion
The Brazilian mangroves harbor a large number of morphotypes of culturable unicellular, non-heterocytous and heterocytous
lamentous cyanobacteria. The phylotypes recovered in this
study showed great genetic diversity and were distributed
in several branches, with some of them formed exclusively
by these unique Brazilian mangrove cyanobacterial 16S rRNA
gene sequences. These data contribute to a more robust
system of cyanobacterial classication by adding information
regarding their diversity in tropical areas. In addition, these
isolated strains allowed for the investigation of toxin production and bioactivity against pathogenic bacteria and yeast,
thereby representing a useful biological source for discovering new natural products with biotechnical and pharmaceutical
importance.

Acknowledgments
This study was supported by grants from the State of So
Paulo Research Foundation (FAPESP 2004/13910-6) and the Brazilian National Research Council (CNPq 559720/2009-2 and
478097/2010-7). C.S.P. Silva, D.B. Genurio and M.G.M.V. Vaz
were supported by FAPESP graduate scholarship 2006/01671-2,
2010/00321-3 and 2010/18732-0, respectively. M.F. Fiore would
also like to thank CNPq for a research fellowship (306607/2012-3).
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