Cladistics
Cladistics 28 (2012) 357–374
10.1111/j.1096-0031.2011.00386.x
Polyphasic approach and typification of selected Phormidium
strains (Cyanobacteria)
Katia Sciuto, Carlo Andreoli, Nicoletta Rascio, Nicoletta La Rocca and Isabella Moro*
Department of Biology, University of Padova, via U. Bassi 58, B-35131 Padova, Italy
Accepted 18 October 2011
Abstract
Cyanobacteria (phylum Cyanophyta ⁄ Cyanobacteria, class Cyanophyceae) are among the most widespread organisms and are
able to adapt themselves to different extreme environments. These micro-organisms have an important ecological role, given their
ability to perform oxygenic photosynthesis, and are employed in different fields based on their ability to produce several bioactive
compounds. Their prokaryotic nature, the presence of many cryptic species, and the coexistence of different nomenclature systems
make the taxonomic identification of cyanobacteria particularly difficult. Moreover, for several species, the original reference strains
(holotypes) are lacking. Increasingly, authors are using a polyphasic approach to characterize cyanobacteria, while typification is a
recent trend that is being used to solve the problem of missing holotypes in other micro-organisms. Here we focus on a filamentous
cyanobacterium, isolated from the Euganean Thermal District (Padova, Italy) and temporarily named strain ETS-02, using a
polyphasic approach that includes morphological, ultrastructural, biochemical (pigment and fatty acid content), physiological
(nitrogen fixation), and genetic (16S rRNA, 16S–23S ITS, cpcB-IGS-cpcA, rpoC1, gyrB, rbcL, nifD loci) analyses. The description of
Phormidium cf. irriguum CCALA 759 as the epitype of Phormidium irriguum was also used to complete the characterization of strain
ETS-02.
The Willi Hennig Society 2011.
Cyanobacteria (phylum Cyanophyta ⁄ Cyanobacteria,
class Cyanophyceae) are among the most widespread,
morphologically distinct, and abundant prokaryotes
(Whitton, 1992). They inhabit almost all environments,
playing an important role as primary producers. In
particular, they are able to adapt themselves to different
extreme habitats, such as desert soils, alkaline lakes,
hypersaline ponds, glaciers, and hot springs (Ward
et al., 1998; Paerl et al., 2000; López-Cortés et al., 2001;
Jungblut et al., 2005; Sánchez-Baracaldo et al., 2005;
Taton et al., 2006).
Cyanobacteria are also known to produce several
bioactive compounds, most of which have various
therapeutic applications (Li et al., 2005; Singh et al.,
2005; Khan et al., 2006; Eriksen, 2008; Gerwick et al.,
2008; Sivonen and Börner, 2008). Nearly half of the
active cyanobacterial compounds known thus far are
*Corresponding author:
E-mail address: [email protected]
The Willi Hennig Society 2011
produced by members of the order Oscillatoriales
(Gerwick et al., 2008). This order, representative genera
including Lyngbya, Oscillatoria, and Phormidium,
includes filamentous species which are not able to form
heterocysts (Komárek and Anagnostidis, 2005).
The identification of new cyanobacterial strains is a
complex issue, due to small cell sizes and morphologies
that can look very similar among phylogenetically
distinct entities.
Cyanobacteria show the phenomenon of ‘‘cryptic
species,’’ i.e. organisms that seem to belong to a
particular species from a morphological point of view
but are genetically distinct (Ward et al., 1998; Casamatta et al., 2005; Taton et al., 2006; Marquardt and
Palinska, 2007). Moreover, different ecotypes of the
same species can exploit separate ecological niches of the
same environment and ⁄ or can be found in geographically distant areas worldwide (Garcı́a-Pichel et al., 1996;
Palińska et al., 1996; Ward et al., 1998; Palińska and
Marquardt, 2008).
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K. Sciuto et al. / Cladistics 28 (2012) 357–374
The arbitrary use of two different nomenclature
codes, Botanical and Bacteriological, makes the situation even more complex. For nearly 30 years, taxonomists have been trying to find a solution that will lead to
the creation of a unique nomenclature system (Oren,
2004; Komárek, 2005, 2010; Marquardt and Palinska,
2007; Oren et al., 2009).
Moreover, the description of several strains deposited
in international culture collections relies only on morphological traits, without molecular characterization.
Conversely, many nucleotide sequences found in public
databases are attributed to strains that have not been
morphologically described (Wilmotte and Herdman,
2001; Rajaniemi et al., 2005). A further problem is that,
for many genera, the described type species are no
longer available for comparative purposes.
This picture is particularly true for the order Oscillatoriales, whose heterogeneous and polyphyletic nature
has been emphasized by many authors (Casamatta
et al., 2005; Teneva et al., 2005; Premanandh et al.,
2006; Palińska and Marquardt, 2008).
At present, many studies suggest the use of a
polyphasic approach to properly identify new cyanobacterial strains (Garcı́a-Pichel et al., 2001; Komárek,
2005, 2010; Palińska and Marquardt, 2008; Moro et al.,
2010; Sciuto et al., 2011). Moreover, a recently proposed
tendency to overcome the problem of unavailable
holotypes in other groups of micro-organisms, such as
fungi and microalgae, is to describe new epitypes (Hyde
and Zhang, 2008; Jahn et al., 2008; Tuji and Williams,
2008; Maistro et al., 2009; Rybalka et al., 2009), i.e.
specimens or illustrations selected to serve as interpretative types when the holotypes, or all original material
associated with a validly published name, are demonstrably ambiguous and cannot be critically identified for
purposes of the precise application of the name of a
taxon (McNeill et al., 2006).
The Euganean Thermal District (Padova, Italy) has
been known since Roman times for its therapeutic
properties, and many people come from all over the
world to benefit from thermal water and mud treatments. Some researchers have highlighted the important
contribution provided to the therapeutic properties of
the mud by cyanobacteria (Bellometti et al., 1997, 2000;
Galzigna et al., 1998), which during the mud maturation
process develop as conspicuous mats on its surface. In
particular, a filamentous strain isolated from this
environment and characterized as Phormidium sp.
ETS-05 (Ceschi-Berrini et al., 2004) showed large
amounts of polyunsaturated fatty acids in its membrane
glycolipids, conferring anti-inflammatory properties to
the mud (Lalli et al., 2004; Bruno et al., 2005; Marcolongo et al., 2006).
In spite of this, the biodiversity of the Euganean
Thermal District has been poorly studied. In fact,
knowledge of the Euganean cyanobacterial diversity is
based largely on reports dealing with morphological
identification (Andreoli and Rascio, 1975; Tolomio
et al., 2004), while, only more recently, analyses have
been carried out using more extensive approaches,
including ultrastructural, pigment, and genetic analyses
(Ceschi-Berrini et al., 2004; Moro et al., 2007a,b, 2010).
We were therefore interested in studying new cyanobacterial strains growing on the thermal mud surface
through more complete surveys, as well as gaining a
better understanding of their contribution to the
properties of the mud.
We carried out analyses using morphological, ultrastructural, biochemical, physiological, and molecular
approaches to characterize strain ETS-02, another
filamentous cyanobacterium isolated from the Euganean
Thermal District. For the molecular analyses, a multilocus approach, as in Lineau et al. (2010), was
adopted, considering seven different genomic regions:
16S rRNA, 16S–23S ITS, cpcB-IGS-cpcA, rpoC1, gyrB,
rbcL and nifD. The 16S rRNA gene and 16S–23S
intergenic transcribed spacer (16S–23S ITS) were
employed as molecular markers for cyanobacteria and,
more generally, for prokaryotes (Iteman et al., 2000;
Casamatta et al., 2005; Premanandh et al., 2006; Palińska and Marquardt, 2008). The cpcB-IGS-cpcA locus
(coding for a portion of the phycocyanin operon), the
rpoC1 gene (coding for the c subunit of the cyanobacterial RNA polymerase core), the gyrB gene (coding for
the b subunit of the DNA gyrase, a type II topoisomerase), and the rbcL gene (coding for the large subunit of
ribulose 1,5-bisphosphate carboxylase ⁄ oxygenase, the
enzyme controlling CO2 fixation during the Calvin
cycle) are single copy regions that have more recently
been used in cyanobacterial phylogeny (Toledo and
Palenik, 1997; Fergusson and Saint, 2000; Wilson et al.,
2000; Watanabe et al., 2001; Seo and Yokota, 2003;
Teneva et al., 2005; Premanandh et al., 2006; Tomitani
et al., 2006; Ballot et al., 2008). In addition, the nifD
gene, coding for two subunits of the enzyme complex
nitrogenase (an enzyme involved in N2 fixation) and
initially amplified as further proof of nitrogen fixation
ability, was employed for phylogenetic reconstruction,
as previously reported (Henson et al., 2004).
For a more precise identification of this cyanobacterium, we propose Phormidium cf. irriguum CCALA 759
as the epitype of the species P. irriguum (Kützing ex
Gomont) Anagnostidis and Komárek.
We show that the combination of a polyphasic
approach and the typification of cyanobacterial strains,
deposited in international culture collections and corresponding to the original descriptions, provides a good
‘‘combined method’’ to solve the taxonomic problems
associated with the phylum Cyanophyta ⁄ Cyanobacteria
and to identify new cyanobacterial strains when the
holotypes (i.e. the original specimens or illustrations
used by the author or designated by the author as the
K. Sciuto et al. / Cladistics 28 (2012) 357–374
nomenclatural types and conserved in one herbarium or
other collection or institution; McNeill et al., 2006) are
lacking or no longer suitable for a complete polyphasic
comparison.
Materials and methods
Cyanobacterial strains and growth conditions
Samples of microbial mats were collected from the
thermal mud surface in a tank of the Garden Hotel
(Montegrotto Terme, Italy) in May 2005. At the moment
of sampling, the water temperature was about 45 C and
the pH 6.8. From these samples, a filamentous strain was
isolated, grown in axenic cultures, and named strain ETS02 (‘‘ETS’’ = ‘‘Euganean Thermal Springs’’).
The following cyanobacterial strains were obtained
for comparison from international culture collections:
Lyngbya aestuarii PCC 7419, Lyngbya majuscula CCAP
1146 ⁄ 4, Oscillatoria sancta PCC 7515, Oscillatoria tenuis
PCC 9107, Phormidium cf. irriguum CCALA 759,
Phormidium ambiguum IAM M-71, and Phormidium
autumnale CCALA 143.
All of the cyanobacterial strains were cultured in
BG11 medium (Rippka et al., 1979) at 20 ± 2 C, with
a photon flux density of 16 lmol photons ⁄ m2 ⁄ s and a
12 ⁄ 12-h light–dark cycle. For the Lyngbya strains, the
culture medium comprised BG11 and ASN-III (1 : 1,
v ⁄ v) (Rippka et al., 1979).
A living culture of strain ETS-02 was deposited at the
Culture Collection of Autotrophic Organisms, Czech
Republic (CCALA 946), where it is available to the
public.
Light and fluorescent light microscopy
Light and fluorescent light microscopy observations
were performed using a DMR 5000 Leica (Wetzlar,
Germany) microscope, equipped with a digital image
acquisition system. The viability of cyanobacterial cells
was investigated through observation of chlorophyll red
autofluorescence when excited with UV light.
Morphological identification of strain ETS-02 was
based on the recent classification of Hoffmann et al.
(2005) and on the traits proposed by Komárek and
Anagnostidis (2005).
Electron microscopy
For electron microscopy [scanning (SEM) and transmission electron microscopy (TEM)], the samples were
prepared as previously described (Moro et al., 2007a,b,
2010). TEM analyses were carried out with an Hitachi
HS9 (Tokyo, Japan) transmission electron microscope
operating at 75 kV. SEM observations were conducted
359
with a Stereoscan 260 (Cambridge Instruments, Cambridge, UK) instrument operating at 25 kV.
Water-soluble pigment analyses
Phycobiliproteins (PBPs) were extracted from pellets
of cultured cells, finely ground in a mortar with liquid
nitrogen, using a phosphate buffer (0.01 m NaH2PO4,
0.15 m NaCl, pH 7). The extract absorption spectra
were analysed on a DU530 Beckman Coulter spectrophotometer (Fullerton, CA, USA).
Lipid-soluble pigment analyses
Chlorophyll a and carotenoid extracts were obtained
from cell pellets, ground as described above in 90%
acetone and then analysed by reversed-phase highperformance liquid chromatography (HPLC).
The analyses were carried out according to Färber
and Jahns (1998) and Komárek et al. (1999) with an
Agilent HPLC system (Waldbronn, Germany), composed of a Rheodyne valve (Rohnert Park, CA, USA), a
reversed-phase column (5-lm particle size; 25 · 0.4 cm;
250 ⁄ 4 RP 18 Lichrocart), a binary pump, and an Agilent
1100 series diode array detector.
Fatty acid content analyses
Dried cell pellets were disrupted with an Ultra-Turrax
instrument (IKA-Werke GmbH & Co., Staufen, Germany) in a mixture of dichloromethane ⁄ methanol (2 : 1,
v ⁄ v) to extract lipids. The total fatty acids were obtained
as methyl esters from these extracts according to
Christie (1982) and then analysed by gas-chromatography at the L.A.Z. Centre (Department of Animal
Sciences, Legnaro, Padova, Italy).
Nitrogen fixation and gliding motility tests
To test for nitrogen fixation ability, some cultures of
strain ETS-02 were grown under the described environmental conditions but in BG110, a medium in which
NaNO3 is replaced by an equimolar concentration of
NaCl (Rippka et al., 1979).
To assess the gliding ability of strain ETS-02 on a
solid surface and the nature of its photomovement,
some filaments were placed on a solid medium in the
centre of plates, whose surface was three-quarters
covered with a black sheet. The plates were exposed
to a continuous photon flux density of 16 lmol
photons ⁄ m2 ⁄ s and observed after 1–2 weeks.
Genetic analyses
Genomic DNA was extracted from cell pellets ground
in a mortar with liquid nitrogen, using the Genomic
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K. Sciuto et al. / Cladistics 28 (2012) 357–374
Table 1
List of taxa and GenBank accession numbers of the nifD gene
sequences used to design the NifD1 and NifD2 primers
Strain
nifD accession number
Anabaena cylindrica PCC 7122
Anabaena sp. PCC 7120
Calothrix sp. PCC 7101
Cylindrospermum majus PCC 7604
Leptolyngbya boryana PCC 6306
Leptolyngbya sp. PCC 7004
Leptolyngbya sp. PCC 7104
Leptolyngbya sp. PCC 73110
Leptolyngbya sp. PCC 7375
Lyngbya aestuarii PCC 7419
Nodularia spumigena PCC 73104
Nostoc sp. PCC 7120
Nostoc sp. PCC 7423
Oscillatoria sancta PCC 7515
Pseudanabaena sp. PCC 6802
Pseudanabaena sp. PCC 7403
Pseudanabaena sp. PCC 7409
Scytonema sp. PCC 7814
Symploca atlantica PCC 8002
AF442506
V01482
AY196953
AY196952
EF576854
EF576858
EF576859
EF576865
EF576867
EF576869
AF442509
AF442504
AF442503
EF576870
EF576857
EF576864
EF576868
AY196954
EF576872
DNA Purification Kit from Fermentas (Burlington,
ON, Canada).
The 16S rRNA gene, the 16S–23S ITS region, the
rpoC1 gene, the gyrB gene, the rbcL gene, and the cpcBIGS-cpcA locus were amplified from the DNA extracts
by PCR, as previously described (Seo and Yokota, 2003;
Ballot et al., 2004; Tomitani et al., 2006; Moro et al.,
2010).
For amplification of the nifD gene, the forward
primer NifD1F (5¢-TYGGWGGHGACAARAARCT-3¢)
and the reverse primer NifD2R (5¢-TARTCCCARG
AGTGCATYTG-3¢) were designed, based on multiple
alignment of several cyanobacterial nifD gene sequences, which included those of both heterocystous
and non-heterocystous filamentous forms (Table 1).
Standard PCR protocols were carried out in 25-lL
aliquots with Taq DNA polymerase (Fisher, Molecular
Biology, Trevose, PA, USA), according to the manufacturerÕs recommendations, on a Peltier Thermal
Cycler PTC-200 (MJ Research, St Bruno, Quebec,
Canada). Thermocycling conditions were as follows:
one cycle at 94 C for 5 min; 35 cycles of 94 C for 45 s,
50 C for 30 s, and 72 C for 45 s; and a final step at
72 C for 8 min. Approximately 80 ng of template DNA
per reaction was used.
The PCR product analyses, sequencing, and final
consensus sequence assembly were performed as
described by Moro et al. (2010).
The following different data sets were constructed,
using the obtained sequences (from the investigated
organism and the strains acquired from culture collections) plus suitable sequences found in the DDBJ ⁄ GenBank ⁄ EBI Data Bank: 16S rRNA, 16S–23S ITS, rpoC1,
gyrB, rbcL, cpcB-IGS-cpcA, nifD, and 16S rRNA +
rpoC1 + gyrB. The last-named was produced by concatenating the sequences shared by all of the corresponding single gene data sets, as described in Maistro
et al. (2007, 2009). In the construction of the data sets,
we took care to insert sequences obtained both from
members of the subclass Oscillatoriophycidae, family
Oscillatoriaceae, and from members of the subclass
Oscillatoriophycidae, family Phormidiaceae, as the
investigated organism could belong to both of these
groups, based on morphological and ultrastructural
data. Moreover, being filamentous non-heterocystous
cyanobacteria as well, members of the subclass Synechococcophycideae, family Pseudanabaenaceae, were
considered in the phylogenetic analyses. To root the
trees, each data set included members of the subclass
Nostocophycideae, a group of filamentous heterocystous cyanobacteria, long known to be monophyletic and
phylogenetically closely related to the filamentous nonheterocystous cyanobacteria (Hoffmann et al., 2005;
Tomitani et al., 2006; Gupta and Mathews, 2010). A
summary of all the organisms considered in the phylogenetic analyses is given in Fig. S1.
Unfortunately, we were able to concatenate only three
of the five genes used in our phylogenetic surveys, as
many of the sequences available for the considered
markers in public databases were obtained from different strains of the same species. As we are working with
prokaryotes, we consider that concatenating gene
sequences of the same species but obtained from
different strains is unacceptable. The effect of using
different strains of the same species in prokaryotic
phylogeny is shown also by Naum et al. (2008). Thus,
we have concatenated only the three genes for which
there were enough available sequences (in addition to
those we obtained here) for the same strains of a given
cyanobacterial species.
The multiple alignments for the phylogenetic analyses
were generated with the ClustalW computer program
(Thompson et al., 1994). Multiple alignment of the 16S
rRNA gene was analysed by the Gblocks program,
using default settings, and ambiguously aligned nucleotides were removed from the final data set (Castresana,
2000).
An a priori estimation of the phylogenetic signal
present in the multiple alignments was performed by
maximum-likelihood (ML) mapping (Strimmer and von
Haeseler, 1997). The phylogenetic signal was evaluated
using the Tree-Puzzle 5.2 program (Schmidt et al.,
2002).
Finally, only the 16S rRNA, the rbcL, the nifD, and
16S rRNA + rpoC1 + gyrB gene data sets were used
for the phylogenetic analyses.
The analyses were performed according to the maximum-parsimony (MP) method, using MEGA ver. 4.0
software (Tamura et al., 2007), and the ML method,
with the PHYML 2.4.4 program (Guindon and Gascuel,
K. Sciuto et al. / Cladistics 28 (2012) 357–374
2003), by applying the GTR + I + C evolutionary
model (Lanave et al., 1984). Non-parametric bootstrap
re-sampling (Felsenstein, 1985) was performed to test
the robustness of the tree topologies (1000 replicates).
Bayesian inference analyses were also carried out,
using MrBayes version 3.1 (Ronquist and Huelsenbeck,
2003). The substitution model in this case was again
GTR + I + C, and the Bayesian analyses were performed with four search chains for 1 000 000 generations, sampling trees every 100 generations. The first
2500 trees were discarded as burn-in. For the analysis of
the 16S rRNA gene data set, the chains were run for
10 000 000 generations, and the first 25 000 trees were
discarded as burn-in. Parameter stability was estimated
361
by plotting log-likelihood values against generation
time, and a consensus tree with posterior probabilities
was then produced. The nexus files for the Bayesian
analyses were generated with the Mesquite 2.71 software
package (Maddison and Maddison, 2009). The resulting
trees were visualized through NJplot (Perriére and
Gouy, 1996).
Results
The cultured strain ETS-02 appeared to be composed
of long, variously curved, bright blue–green trichomes
(Fig. 1), strictly packed to form mats in older cultures,
1
20 µm
2
10 µm
3
4
10 µm
5 µm
5
1 µm
6
1 µm
Fig. 1. Light micrograph of strain ETS-02. Flexuous, densely packed trichomes.
Fig. 2. Light micrograph of strain ETS-02. Reproduction by trichome fragmentation in short hormogonia through the formation of necridia
(arrow).
Fig. 3. Light micrograph of strain ETS-02. Trichomes surrounded by a colourless sheath, protruding from the filament itself (arrow).
Fig. 4. Scanning electron micrograph of strain ETS-02 showing trichome fragmentation in short hormogonia (arrow).
Fig. 5. Transmission electron micrograph of strain ETS-02. Necridic cell (arrow) between two neighbouring cells.
Fig. 6. Transmission electron micrograph of strain ETS-02. Trichome showing thylakoids (t) parallel to the longitudinal cell walls.
K. Sciuto et al. / Cladistics 28 (2012) 357–374
or more dispersed in liquid medium in younger cultures.
The aggregated filaments were able to float at the
surface of the liquid medium or grow attached to sides
and bottom of the flask. Trichomes were isopolar,
composed of cells that were wider (3–5 lm) than long
(1–2 lm) (Fig. 2), ending with rounded apical cells, and
surrounded by a thin colourless sheath, which could
protrude from the filament itself (Fig. 3).
Reproduction was by trichome fragmentation in short
hormogonia through the formation of necridia (Figs 4
and 5). Subsequently, each hormogonium reconstituted
the average length of a filament by binary fission of the
component cells. The daughter cells began a new
division before being completely separated.
TEM observations revealed that thylakoids did not
assume a particular arrangement in the cell, but could lie
parallel to the longitudinal cell walls (Fig. 6), parietal
(Fig. 5), radial, or variously distributed. Some cells
showed widened thylakoids, with a net-like appearance
(Fig. 6). No gas vesicles were observed in the cytoplasm.
Most of the features reported for strain ETS-02 were
observed also in Phormidium cf. irriguum CCALA 759,
although cells of the latter isolate were larger (9–12 lm
wide, 2–3 lm long) (Figs 7 and 8).
Strain ETS-02 was able to survive in medium without
organic nitrogen, as ascertained through the observation
of the chlorophyll red autofluorescence of cells maintained in this condition for 3 weeks. No heterocysts or
other specialized cells were observed by light microscopy
in this culture condition (not shown).
The organism was also able to move towards the light
on a solid surface (data not shown).
Spectrophotometric measurements of buffered saline
PBP extract of strain ETS-02 showed the presence of two
peaks at 576 and 608 nm (Fig. 9), corresponding to
phycoerythrocyanin (PEC) (Bryant, 1982; Garcı́a-Pichel
et al., 1996) and C-phycocyanin (C-PC) (Edwards et al.,
1996), respectively. The same spectrum was also found for
Phormidium cf. irriguum CCALA 759. Lipid-soluble
pigment analyses, carried out with HPLC, showed corresponding peaks in strain ETS-02 and Phormidium cf.
irriguum CCALA 759 (Table 2). Strain ETS-02 presented
an additional peak that was not possible to identify.
Preliminary fatty acid content analysis of strain ETS02 highlighted the presence of the following molecules:
C14:0 (myristic acid), C14:1 (myristoleic acid), C16:0
(palmitic acid), C16:1 (palmitoleic acid), C18:0 (stearic
acid), C18:1, D9 (oleic acid), C18:1, D11 (cis-vaccenic
acid), C18:2 (linoleic acid), C18:3a (a-linolenic acid),
and C20:0 (arachidic acid).
The gene sequences obtained in this study for the
different molecular markers were deposited in the
DDBJ ⁄ GenBank ⁄ EBI Data Bank with the accession
numbers reported in Table 3, which also includes the
accession numbers of the reference cyanobacterial
sequences used for comparison.
7
10 µm
8
10 µm
Fig. 7. Light micrograph showing trichomes of Phormidium irriguum
CCALA 759.
Fig. 8. Scanning electron micrograph showing trichomes of Phormidium irriguum CCALA 759.
0.5
C-PC
PEC
Absorbance
362
0
400
Wavelength (nm)
750
Fig. 9. Absorption spectrum of phycobiliprotein extract of strain
ETS-02 showing the presence of two peaks corresponding to
phycoerythrocyanin (PEC) and C-phycocyanin (C-PC).
The phylogenetic signal of the different multiple
alignments (both the single and the combined data sets)
was calculated with the ML mapping method, and the
corresponding results, given in quartet percentages, are
reported in Table 4.
K. Sciuto et al. / Cladistics 28 (2012) 357–374
363
Table 2
Comparison of the HPLC lipid-soluble extract peaks of strains ETS-02 and Phormidium cf. irriguum CCALA 759
Peak no.
Pigment
kmax (nm)
ETS-02
CCALA 759
1
2
3
4
5
6
7
8
Myxol 2¢-dymethyl-glycoside
Nostoxanthin
Caloxanthin
Zeaxanthin
Unknown pigment
Echinenone
trans-b-carotene
cis-b-carotene
296,
428,
428,
428,
304,
460
428,
426,
+
+
+
+
+
+
+
+
+
+
+
+
)
+
+
+
368,
452,
452,
452,
446,
448, 476, 506
480
480
480
470, 502
450, 476
444, 470
Peaks are listed according to their elution order and for each the maximum wavelengths are given (‘‘+,’’ presence; ‘‘),’’ absence).
Table 3
List of the sequences used in this study for strain ETS-02 and for each of the comparison strains obtained from the Culture Collection of Autotrophic
Organisms (CCALA), Institute of Molecular and Cellular Biosciences (IAM), and Pasteur Culture Collection of Cyanobacteria (PCC)—the
corresponding DDBJ ⁄ GenBank ⁄ EBI accession numbers are given
Taxon
Strain designation
16S rRNA
16S–23S ITS
cpcAB
rpoC1
gyrB
rbcL
nifD
Strain ETS-02
Phormidium cf. irriguum
Phormidium ambiguum
Phormidium autumnale
Oscillatoria sancta
Oscillatoria tenuis
Lyngbya aestuarii
Lyngbya majuscula
ETS-02
CCALA 759
IAM M-71
CCALA 143
PCC 7515
CCAP 1459 ⁄ 4
PCC 7419
CCAP 1446 ⁄ 4
FN813342
FN813343
AB003167
FN813344
AF132933
FN813345
AJ000714
AY768394
FN813339
FN813340
–
AM778716
EF178272
FN813341
AY768375
AY768384
FN813309
FN813310
–
FN813311
AJ401185
FN813312
AJ401187
–
FN813320
FN813321
FN813322
FN813323
FN813324
FN813325
FN813326
–
FN813313
FN813314
FN813315
FN813316
FN813317
FN813318
FN813319
–
FN813327
FN813328
FN813329
FN813330
FN813331
FN813332
AB075915
FN813333
FN813334
FN813335
FN813336
FN813337
EF576870
FN813338
EF576869
DQ078751
A dash indicates that the sequence was not obtained. Bold type indicates sequences obtained in this survey.
Table 4
Likelihood mapping results for the different multiple alignments
Data set
QFR (%) QPU (%) QFU (%)
16S rRNA
16S–23S ITS
cpcB-IGS-cpcA
rpoC1
gyrB
rbcL
nifD
16S rRNA + rpoC1 + gyrB
98.5
69.6
91.7
97.9
91.7
98.4
95.6
99.3
1.6
16.4
3.3
1.9
5.1
1.5
1.3
0.7
0.0
14.1
5.1
0.2
3.1
0.1
3.2
0.0
QFR, quartets fully resolved; QPU, quartets partially unresolved;
QFU, quartets fully unresolved.
In total, 10 000 quartets were evaluated in each calculation.
The 16S rRNA, rpoC1, rbcL, and 16S rRNA + rpoC1 + gyrB gene multiple alignments showed the
highest phylogenetic signals, with percentages of fully
resolved quartets ranging from 97.9% to 99.3%. The
nifD multiple alignment signal was lower, with 95.6% of
fully resolved quartets. The 16S–23S ITS locus showed
the lowest phylogenetic signal, with only 69.9% of
quartets fully resolved and the highest percentage of
fully unresolved quartets (14.1%). For the cpcB-IGScpcA and gyrB multiple alignments, the percentage of
fully resolved quartets was much lower (91.7%) than for
the other multiple alignments. However, for the phyco-
cyanin operon multiple alignment, the totally unresolved quartets were 5.1%, while the gyrB multiple
alignment showed this percentage for the partially
unresolved quartets.
The 16S–23S ITS locus and the cpcB-IGS-cpcA
operon were only used for nucleotide identity comparisons, aligning the sequences of strain ETS-02 and
Phormidium cf. irriguum CCALA 759 (Figs S2 and S3).
Both ETS-02 and CCALA 759 16S–23S ITS loci
presented a tRNAIle and a tRNAAla sequence inside,
each with 100% nucleotide identity between the two
strains. Moreover, the structurally important domains
D1, D1¢, D2, D3, and D4, and the anti-terminator
region boxA, reported in Iteman et al. (2000), were
conserved in the two organisms (100% identity). Some
differences were observed in the remaining parts of the
16S–23S ITS locus. In particular, strain ETS-02 presented a 7-bp gap in the region between the tRNAIle and
tRNAAla sequences, while strain CCALA 759 showed
two subsequent gaps of 5 and 14 bp after the tRNAAla
sequence.
In the phycocyanin operon, the IGS region was 94 bp
for strain ETS-02 and 78 bp for strain CCALA 759. The
alignment between the two strain operons revealed
15.2% identity for the cpcB region (25 substitutions on
164 aligned positions), 41.5% identity for the IGS
region (16 insertions ⁄ deletions and 23 substitutions on
364
K. Sciuto et al. / Cladistics 28 (2012) 357–374
94 aligned positions), and 6.5% identity for the cpcA
region (eight substitutions on 122 aligned positions).
16S rRNA, rbcL, and nifD gene data sets were used
for phylogenetic reconstructions, as well as the data set
joining the 16S rRNA, rpoC1 and gyrB gene sequences
(16S rRNA + rpoC1 + gyrB).
In the 16S rRNA gene trees (Figs 10 and 11), several
clades were clearly distinguishable. Clade A, including
strains belonging to the genera Leptolyngbya and
Pseudanabaena, was a sister taxon to clade B, comprising two Pseudanabaena strains. Within clade A, Leptolyngbya sp. IAM M-99 and Leptolyngbya sp. PCC
73110 were joined by a node with bootstrap values of
100% in both the MP and the ML trees (100% BTMP
and BTML, respectively) and a posterior probability (PP)
of 100%. The same was observed for Leptolyngbya sp.
PCC 7375 and Pseudanabaena persicina CCMP 638. The
remaining named clades (C–G), plus Planktothrix agardhii IAM M-244, represented other Oscillatoriales,
spanning the families Oscillatoriaceae and Phormidiaceae. Clade C, with 69 ⁄ 59 ⁄ 59% BTMP ⁄ BTML ⁄ PP,
included strain ETS-02, Phormidium cf. irriguum CCALA 759, and Phormidium ambiguum IAM M-71. Strain
ETS-02 and Phormidium cf. irriguum CCALA 759 were
sister taxa with 95 ⁄ 79 ⁄ 78% BTMP ⁄ BTML ⁄ PP. Clade D
(57 ⁄ 58 ⁄ 79% BTMP ⁄ BTML ⁄ PP) comprised two more
Phormidium strains: Phormidium uncinatum SAG 81.79
and Phormidium tergestinum CCALA 155. Clade E,
supported by 99 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP, included
three members of the species Phormidium autumnale. In
particular, Phormidium autumnale Arct-Ph5 and Phormidium autumnale SAG 78.79 were joined by a node
with 92 ⁄ 94 ⁄ 100% BTMP ⁄ BTML ⁄ PP, while Phormidium
autumnale CCALA 143 was associated with Oscillatoria
100 Leptolyngbya sp. IAM M-99 (AB003169)
Leptolyngbya sp. PCC 73110 (AB075988)
67
A
100 Leptolyngbya sp. PCC 7375 (AB039011)
Pseudanabaena persicina CCMP 638 (AB075994)
Pseudanabaena sp. PCC 7403 (AB039019)
67
Pseudanabaena sp. PCC 7367 (AB039018)
B
Phormidium cf. irriguum CCALA 759
95
C
ETS-02
69
Phormidium ambiguum IAM M-71 (AB003167)
Phormidium uncinatum SAG 81.79 (EF654086)
57
Phormidium tergestinum CCALA 155 (EF654083)
D
Planktothrix agardhii IAM M-244 (AB074507)
78 Oscillatoria nigro-viridis PCC 7112 (AB074509)
Phormidium autumnale CCALA 143
99
53
Phormidium autumnale Arct-Ph5 (DQ493873)
92
E
Phormidium autumnale SAG 78.79 (EF654084)
97
Arthrospira platensis IAM M-135 (AB074508)
Lyngbya aestuarii PCC 7419
100
F
Oscillatoria sancta PCC 7515 (AF132933)
Trichodesmium erythraeum IMS-101 (AB075999)
G
Anabaena cylindrica IAM M-253 (AF247592)
Nodularia spumigena PCC 73104 (AB039002)
Nostoc linckia IAM M-251 (AB074503)
96
85
90
Trichormus variabilis IAM M-204 (AB074502)
Trichormus variabilis IAM M-3 (AB016520)
Anabaenopsis circularis IAM M-4 (AF247595)
Calothrix brevissima IAM M-249 (AB074504)
99
Chlorogloeopsis fritschii PCC 6718 (AF132777)
Fischerella muscicola PCC 73103 (AB074505)
20
Fig. 10. Phylogenetic tree based on 16S rRNA gene sequences and reconstructed using the maximum-parsimony (MP) analysis. Bootstrap values
(‡ 50%) are provided for each node. Sequences determined in this work are indicated in bold. GenBank accession numbers are indicated in
parentheses. Bar represents 20 nucleotide substitutions per site.
K. Sciuto et al. / Cladistics 28 (2012) 357–374
100/100
365
Leptolyngbya sp. IAM M-99 (AB003169)
Leptolyngbya sp. PCC 73110 (AB075988)
60/83
A
100/100 Leptolyngbya sp. PCC 7375 (AB039011)
Pseudanabaena persicina CCMP 638 (AB075994)
Pseudanabaena sp. PCC 7403 (AB039019)
59/59
B
Pseudanabaena sp. PCC 7367 (AB039018)
Phormidium cf. irriguum CCALA 759
79/78
98
C
ETS-02
59/59
Phormidium ambiguum IAM M-71 (AB003167)
Phormidium uncinatum SAG 81.79 (EF654086)
58/79
Phormidium tergestinum CCALA 155 (EF654083)
D
80 Oscillatoria nigro-viridis PCC 7112 (AB074509)
Phormidium autumnale CCALA 143
100/100
54
E
Phormidium autumnale Arct-Ph5 (DQ493873)
59
94/100
Phormidium autumnale SAG 78.79 (EF654084)
Arthrospira platensis IAM M-135 (AB074508)
99/100
Lyngbya aestuarii PCC 7419
64
100/100
F
Oscillatoria sancta PCC 7515 (AF132933)
Trichodesmium erythraeum IMS-101 (AB075999)
G
Planktothrix agardhii IAM M-244 (AB074507)
Anabaena cylindrica IAM M-253 (AF247592)
Nodularia spumigena PCC 73104 (AB039002)
Nostoc linckia IAM M-251 (AB074503)
98/100
99/100
95/100
68
Trichormus variabilis IAM M-204 (AB074502)
Trichormus variabilis IAM M-3 (AB016520)
Anabaenopsis circularis IAM M-4 (AF247595)
Calothrix brevissima IAM M-249 (AB074504)
100/100
Chlorogloeopsis fritschii PCC 6718 (AF132777)
Fischerella muscicola PCC 73103 (AB074505)
0.02
Fig. 11. Phylogenetic tree based on 16S rRNA gene sequences and reconstructed using the maximum-likelihood (ML) analysis of evolutionary
distances determined by the GTR + I + C model. Bootstrap values (‡ 50%) and Bayesian posterior probabilities are provided for each node (ML
in bold and BI in normal font). Sequences determined in this work are indicated in bold. GenBank accession numbers are indicated in parentheses.
Bar represents 0.02 nucleotide substitutions per site.
nigro-viridis PCC 7112 with a node showing 78 ⁄ 80%
BTMP ⁄ BTML support. Finally, Arthrospira platensis
IAM M-135 and Lyngbya aestuarii PCC 7419 constituted clade F (97 ⁄ 99 ⁄ 100% BTMP ⁄ BTML ⁄ PP) and
Oscillatoria sancta PCC 7515 and Trichodesmium erythraeum IMS-101 formed clade G (100 ⁄ 100 ⁄ 100%
BTMP ⁄ BTML ⁄ PP).
For members of clades C–E, levels of 16S rRNA gene
sequence similarity were also calculated (Table 5).
Heterocystous cyanobacteria were found to be monophyletic (90 ⁄ 98 ⁄ 100 BTMP ⁄ BTML ⁄ PP) and constituted
the outgroup of the whole tree.
In January 2010, sequences for the 16S rRNA gene
(EU196638) and the 16S–23S ITS locus (EU196671) of
Phormidium cf. irriguum CCALA 759 were deposited in
the DDBJ ⁄ GenBank ⁄ EBI Data Bank. The alignment of
each of these new online sequences with the correspond-
ing sequences obtained during this study revealed that
they were completely different. In particular, the 16S–
23S ITS sequence of strain CCALA 759 (EU196671) did
not contain any tRNA sequence, in contrast to ours.
Moreover, accession EU196638 and the 16S rRNA gene
sequence obtained in this survey for strain CCALA 759
showed only 90.50% identity (1084 aligned positions). A
tree based on the 16S rRNA gene was also made adding
the EU196638 sequence to the data set. This clearly
showed that the online sequence was phylogenetically
distant from that obtained here (data not shown).
In the phylogenetic reconstruction based on the 16S
rRNA + rpoC1 + gyrB multiple alignment (Fig. 12),
four clades were observed. Clade A (97 ⁄ 97 ⁄ 100%
BTMP ⁄ BTML ⁄ PP), including three different members of
the family Pseudanabaenaceae, could be recognized.
Within this group, Leptolyngbya sp. IAM M-99 was a
366
K. Sciuto et al. / Cladistics 28 (2012) 357–374
Table 5
Levels of similarity (below the major diagonal) and corresponding levels of divergence (above the major diagonal) of 16S rRNA gene sequences,
calculated among members of clades C, D and E
Taxon
Clade C
1. Strain ETS-02
2. Phormidium cf. irriguum CCALA 759
3. Phormidium ambiguum IAM M-71
Clade D
8. Phormidium uncinatum SAG 81.79
9. Phormidium tergestinum CCALA 155
Clade E
4. Oscillatoria nigro-viridis PCC 7112
5. Phormidium autumnale CCALA 143
6. Phormidium autumnale Arct-Ph5
7. Phormidium autumnale SAG 78.79
1
2
3
8
9
4
5
–
97.6
93.3
2.4
–
93.4
6.7
6.4
–
90.8
90.2
91.2
90.4
90.6
90.4
91.1
91.6
90.7
90.5
91.3
91.5
6
7
9.2
8.8
9.9
9.8
9.6
10.1
9.4
9.3
10.3
9.6
9.5
10.5
8.9
8.8
9.9
8.4
8.5
9.6
90.2
89.9
–
91.2
8.8
–
8.7
10.3
9.2
10.7
8.3
10.7
8.3
10.5
89.7
89.5
90.1
90.4
91.3
90.9
91.8
91.7
89.7
89.3
89.3
89.5
–
99.1
97.3
97.0
0.9
–
97.1
96.8
2.7
2.9
–
98.8
3.0
3.2
1.2
–
Each strain considered is associated with a number in the first column, reported in the first line of the table. Values refer to a 1383-bp
alignment.
Leptolyngbya sp. IAM M-99 (AB074798, AB074775)
80/92/100
Leptolyngbya sp. PCC 7375 (AB074808, AB074785)
97/97/100
A
Pseudanabaena sp. PCC 7367 (AB074799, AB074776)
95/100/100
Pseudanabaena sp. PCC 7403 (AB074810, AB074787)
79/100
Oscillatoria nigro-viridis PCC 7112 (AB074802, AB074779)
100/100/100
Phormidium autumnale CCALA 143
97/100
98/100
Arthospira platensis IAM M-135 (AB074788, AB074765)
72/100/100
100/100
B
Oscillatoria sancta PCC 7515
Lyngbya aestuarii PCC 7419
99/100/100
C
Planktothrix agardhii IAM M-244 (AB074790, AB074767)
88/95/100
Phormidium cf. irriguum CCALA 759
Phormidium ambiguum IAM M-71
100/100/100
D
ETS-02
Anabaena cylindrica IAM M-253 (AB074793, AB074770)
Calothrix brevissima IAM M-249 (AB074791, AB074768)
100/100/100
Nostoc linckia IAM M-251 (AB074792, AB074769)
73/76/100
Nodularia spumigena PCC 73104 (AB074805, AB074782)
63/100
Anabaenopsis circularis IAM M-4 (AB074796, AB074773)
73
Trichormus variabilis IAM M-3 (AB074795, AB074772)
62/66/100
Trichormus variabilis IAM M-204 (AB074789, AB074766)
60/100
Fischerella muscicola PCC 73103 (AB074804, AB074781)
80/68/100
78/73/100
Chlorogloeopsis fritschii PCC 6718 (AB074801, AB074778)
0.05
Fig. 12. Phylogenetic tree based on the 16S rRNA + rpoC1 + gyrB multiple alignment, reconstructed using the maximum-likelihood analysis of
evolutionary distances determined by the GTR + I + C model. MP and ML bootstrap values (‡ 50%) and Bayesian posterior probabilities are
provided for each node (MP in grey, ML in bold, BI in normal font). Sequences determined in this work are indicated in bold. GenBank accession
numbers are indicated in parentheses. Bar represents 0.05 nucleotide substitutions per site.
sister taxon to Leptolyngbya sp. PCC 7375 with 80 ⁄ 92 ⁄
100% BTMP ⁄ BTML ⁄ PP, and the two Pseudanabaena
strains (PCC 7376 and PCC 7403) were joined by a node
with 95 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP.
Clade B (97 ⁄ 100% BTML ⁄ PP), consisted of Oscillatoria
nigro-viridis PCC 7112, Phormidium autumnale
CCALA 143, and Oscillatoria sancta PCC 7515, and
clade C (99 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP), including
Arthrospira platensis IAM M-135, Lyngbya aestuarii
PCC 7419, and Planktothrix agardhii IAM M-244,
represented a sister taxon to clade A (98 ⁄ 100%
BTML ⁄ PP).
K. Sciuto et al. / Cladistics 28 (2012) 357–374
Strain ETS-02, Phormidium cf. irriguum CCALA 759,
and Phormidium ambiguum IAM M-71 grouped in clade
D with 100 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP. Within this
clade, Phormidium cf. irriguum CCALA 759 was a sister
taxon to Phormidium ambiguum IAM M-71
(88MP ⁄ 95ML ⁄ 100% BT ⁄ BT ⁄ PP).
Also in this case, the outgroup, represented by
heterocystous cyanobacteria, was well supported
(100 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP).
The phylogenetic relatedness between strain ETS-02
and Phormidium cf. irriguum CCALA 759 was confirmed in the phylogenetic tree obtained with the nifD
gene multiple alignment (Fig. 13), where they clustered
with 100 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP. Leptolyngbya sp.
PCC 7004 and Symploca atlantica PCC 8002 grouped
with them in clade A (80 ⁄ 84 ⁄ 100% BTMP ⁄ BTML ⁄ PP).
Clade B (100 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP), comprising members of the genera Pseudanabaena, Oscillatoria,
Lyngbya, and Leptolyngbya, was joined to clade A by a
node with 67 ⁄ 85 ⁄ 100% BTMP ⁄ BTML ⁄ PP.
367
In this case, Phormidium ambiguum IAM M-71 was
placed distantly to strains ETS-02 and CCALA 759,
being included in clade C (57 ⁄ 96 ⁄ 100% BTMP ⁄
BTML ⁄ PP) with other oscillatoriacean cyanobacteria.
In particular, within this last group, Leptolyngbya
boryana PCC 6306 and Leptolyngbya sp. PCC 73110
clustered together with 100 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP.
The same was observed for Phormidium autumnale
CCALA 143 and Oscillatoria tenuis PCC 9107.
Finally, in the tree based on the rbcL gene (Fig. 14),
strain ETS-02 grouped with Phormidium ambiguum
IAM M-71 and Phormidium autumnale CCALA 143 in
clade A (99 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP). Phormidium
cf. irriguum CCALA 759 and Oscillatoria tenuis PCC
9107 clustered in clade B (94 ⁄ 74 ⁄ 59% BTMP ⁄ BTML ⁄ PP).
Lyngbya aestuarii PCC 7419 and Lyngbya majuscula
CCAP 1146 ⁄ 4 grouped in clade C (60 ⁄ 82 ⁄ 100%
BTMP ⁄ BTML ⁄ PP). Symploca atlantica PCC 8002 and
Pseudanabaena sp. PCC 7403 were joined by a node with
52 ⁄ 84% BTML ⁄ PP (clade D), while Oscillatoria sancta
100/100/100
ETS-02
Phormidium cf. irriguum CCALA 759
50/93/100
80/84/100
A
Leptolyngbya sp. PCC 7004 (EF576858)
Symploca atlantica PCC 8002 (EF576872)
Pseudanabaena sp. PCC 7403 (EF576864)
Pseudanabaena sp. PCC 6802 (EF576857)
67/85/100
100/100/100
Oscillatoria sancta PCC 7515
B
Lyngbya aestuarii PCC 7419 (EF576869)
Leptolyngbya sp. PCC 7375 (EF576867)
100/100/100
98
Leptolyngbya sp. PCC 7104 (EF576859)
Lyngbya majuscula CCAP 1446/4 (DQ078751)
Phormidium ambiguum IAM M-71
100/100/100
57/96/100
Leptolyngbya sp. PCC 73110 (EF576865)
Leptolyngbya boryana PCC 6306 (EF576854)
85/100
C
Oscillatoria tenuis CCAP 1459/4
100/100/100
Phormidium autumnale CCALA 143
Pseudanabaena sp. PCC 7409 (EF576868)
100/100/100
94/96/100
96/94/98
100/100/100
Anabaena sp. PCC 7120 (V01482)
Nostoc sp. PCC 7120 (AF442504)
Nostoc sp. PCC 7423 (AF442503)
Cylindrospermum majus PCC 7604 (AY196952)
Scytonema sp. PCC 7814 (AY196954)
89/98/100
62/72/95
Nodularia spumigena PCC 73104 (AF442509)
Anabaena cylindrica PCC 7122 (AF442506)
83/97/100
Calothrix sp. PCC 7101 (AY196953)
0.05
Fig. 13. Phylogenetic tree based on nifD gene sequences, reconstructed using the maximum-likelihood analysis of evolutionary distances determined
by the GTR + I + C model. MP and ML bootstrap values (‡ 50%) and Bayesian posterior probabilities are provided for each node (MP in grey,
ML in bold, and BI in normal font). Sequences determined in this work are indicated in bold. GenBank accession numbers are indicated in
parentheses. Bar represents 0.05 nucleotide substitutions per site.
368
K. Sciuto et al. / Cladistics 28 (2012) 357–374
Phormidium autumnale CCALA 143
92/61/59
A
Phormidium ambiguum IAM M-71
99/100/100
ETS-02
98/100/100
Phormidium cf. irriguum CCALA 759
94/74/59
B
Oscillatoria tenuis CCAP 1459/4
73/82/100
Lyngbya aestuarii PCC 7419 (AB075915)
60/82/100
C
Lyngbya majuscula CCAP 1446/4
67/97
Symploca atlantica PCC 8002 (AB075922)
52/84
85
Pseudanabaena sp. PCC 7403 (AB075920)
51/92
100/100/100
D
Oscillatoria sancta PCC 7515
Trichodesmium erythraeum IMS 101 (AB075920)
E
Pseudanabaena persicina CCMP 638 (AB075919)
85/69/85
Leptolyngbya sp. PCC 73110 (AB075914)
55/81/97
Geitlerinema sp. PCC 8501 (EU119380)
88/97/100
97/99/100
68/90/100
72/99
85/100
F
Cylindrospermum licheniforme UTEX 2014 (AB075909)
Nodularia sp. KAC 17 (AB075917)
Nostoc sp. PCC 7906 (AB075918)
Chlorogloeopsis fritschii PCC 6912 (AB075907)
Calothrix desertica PCC 7102 (AB075906)
Fischerella thermalis PCC 7521 (AB075913)
0.05
Fig. 14. Phylogenetic tree based on rbcL gene sequences, reconstructed using the maximum-likelihood analysis of evolutionary distances determined
by the GTR + I + C model. MP and ML bootstrap values (‡ 50%) and Bayesian posterior probabilities are provided for each node (MP in grey,
ML in bold, and BI in normal font). Sequences determined in this work are indicated in bold. GenBank accession numbers are indicated in
parentheses. Bar represents 0.05 nucleotide substitutions per site.
PCC 7515 and Trichodesmium erythraeum IMS-101 were
joined by a node with 100 ⁄ 100 ⁄ 100% BTMP ⁄ BTML ⁄ PP
(clade E). Clade F (85 ⁄ 69 ⁄ 85% BTMP ⁄ BTML ⁄ PP)
included Leptolyngbya sp. PCC 73110, Pseudanabaena
persicina CCMP 638, and Geitlerinema sp. PCC 8501.
The outgroup was again represented by heterocystous
cyanobacteria, grouped with 85 ⁄ 100% BTML ⁄ PP
support.
Discussion
The correct identification of cyanobacteria is an
important issue for microbiologists and ecologists, but
the nature of these micro-organisms makes this complicated. The situation is made even more difficult by the
arbitrary use of different systems of nomenclature, the
unavailability of many described type species for comparison, and disagreements relating to the use of
classical and molecular techniques. All this has often
led to confusion and erroneous identification of cyanobacteria.
The Euganean Thermal District has been exploited
for therapeutic purposes since ancient times. Recent
studies have underlined how the curative properties of
the muds are linked to the colonization of its surface by
cyanobacteria. In particular, the filamentous strain
Phormidium sp. ETS-05 has been shown to be rich in
polyunsaturated fatty acids with anti-inflammatory
activity (Lalli et al., 2004; Bruno et al., 2005). During
samplings to assay the biodiversity of the Euganean
Thermal District, another filamentous cyanobacterium
similar to strain ETS-05 was found. Comparison of their
16S rRNA gene sequences and more extensive morphological analyses revealed that the new organism was
distinct from strain ETS-05. The finding of distinct but
morphologically very similar cyanobacteria sharing
the same habitat is a common one, and cryptic species
are a widespread phenomenon in the phylum Cyanophyta ⁄ Cyanobacteria (Ward et al., 1998; Casamatta
et al., 2005; Taton et al., 2006; Marquardt and Palinska,
2007). Therefore, strain ETS-02 was subjected to a
polyphasic study in order to better identify it.
As a filamentous non-heterocystous cyanobacterium,
and given its thylakoid arrangement, strain ETS-02 was
assigned to the subclass Oscillatoriophycidae and to the
order Oscillatoriales. Features such as the presence of a
thin sheath surrounding each filament, cell width, and
the reproduction through the formation of necridic cells
led us to hypothesize that the organism could be
K. Sciuto et al. / Cladistics 28 (2012) 357–374
attributed to the families Oscillatoriaceae or Phormidiaceae as well, in agreement with Komárek and Anagnostidis (2005) and Hoffmann et al. (2005). As, except
for rRNA operons, very few nucleotide sequences were
available in public databases for cyanobacteria belonging to these families, we used strains of Lyngbya,
Phormidium, and Oscillatoria for molecular comparisons, choosing those morphologically more similar to
ETS-02 and ⁄ or more representative of their genera.
Of the molecular markers considered, the 16S–23S
ITS and cpcB-IGS-cpcA data sets gave the poorest
phylogenetic signal, indicating that the sequences
included in the analyses were too divergent and could
not be used for a reasonable phylogenetic reconstruction. This is not surprising, as the 16S–23S ITS and the
IGS of the phycocyanin operon non-coding genomic
regions are potentially more likely to accumulate mutations during evolution. This also confirms previous
suggestions by many authors of using these markers to
discriminate cyanobacteria at lower taxonomic ranks,
such as at the strain level (Robertson et al., 2001; Papke
et al., 2003; Teneva et al., 2005; Premanandh et al.,
2006).
Almost all of the phylogenetic reconstructions clearly
revealed relatedness between strain ETS-02 and Phormidium cf. irriguum CCALA 759. Phormidium irriguum
(Kützing ex Gomont) Anagnostidis and Komárek
belongs to the group VIII of the genus Phormidium. In
fact, given the taxonomic complexity of this taxon, the
genus Phormidium has been divided into eight groups
based mainly on apical cell morphology (Komárek and
Anagnostidis, 2005). Only in the rbcL phylogenetic tree
strain ETS-02 is not associated with strain CCALA 759,
but is in a group including two Phormidium strains:
P. autumnale CCALA 143 and P. ambiguum IAM
M-71. The latter, belonging to group VIII as well, is
phylogenetically related to both strains ETS-02 and
CCALA 759 in all the trees obtained, apart from the
reconstruction based on the nifD gene. Thus, based on
our phylogenetic analyses, strain ETS-02 belongs to
group VIII of the genus Phormidium.
The contrasting results obtained using different
molecular markers are probably due to their cellular
function. In fact, the molecular markers employed in the
phylogenetic analyses can be divided into two categories: the first, represented by the 16S rRNA, rpoC1 and
gyrB genes, is involved in replication and translation
reactions of the genetic material; and the second one,
consisting of the rbcL and nifD genes, is involved in
carbon and nitrogen fixation processes, respectively. The
two categories of genes, in all probability, followed
distinct evolutionary pathways, which are reflected by
the different phylogenies obtained. Perhaps, because the
replication and translation reactions are more basic for
the functionality of the cell, the genes involved in these
functions better reflect cyanobacterial history at the
369
phylum level, while the others are more suitable to
understand the relationships within certain taxa. In
support of this interpretation, besides the known
evidence that not all cyanobacteria are able to fix
nitrogen and so lack the enzyme complex nitrogenase,
some unicellular cyanobacteria unable to perform oxygenic photosynthesis, and lacking the genes coding for
the Calvin–Benson cycle and for photosystem II, have
recently been found (Bothe et al., 2010).
Nucleotide identity analyses carried out on the 16S
rRNA gene, 16S–23S ITS locus, and cpcB-IGS-cpcA
operon strengthen the relatedness between strain ETS02 and Phormidium cf. irriguum CCALA 759. In
particular, the conserved and structurally important
domains of the 16S–23S ITS and the tRNA sequences
found within this locus are identical in the two organisms. Moreover, the level of divergence (6.5%) of the
phycocyanin operon cpcA region between the two
strains is similar to those previously observed by Teneva
et al. (2005) among different strains of P. autumnale. In
the same study, the authors found a larger level of
divergence (44%) among different Phormidium species.
Following the proposed threshold levels of 16S rRNA
gene sequence similarity used to assign two cyanobacteria to the same genus (95%) or to the same species
(97.5%) (Stackebrandt and Goebel, 1994), the calculated similarity values confirm some previous findings,
i.e. that Oscillatoria nigro-viridis PCC 7112 belongs to
group VII of the genus Phormidium (Komárek and
Anagnostidis, 2005). However, focusing on strain ETS02 and those grouping with it in the 16S rRNA and 16S
rRNA + rpoC1 + gyrB phylogenetic trees and according to the above threshold values, strain ETS-02 is
considered to belong both to the same genus and to the
same species as strain CCALA 759, while P. ambiguum
IAM M-71, besides representing a distinct species,
belongs to a different genus from both ETS-02 and
CCALA 759. Given all of the phylogenetic reconstructions and the morphologies of the three strains, this
seems highly improbable. Evidently, 16S rRNA gene
sequence similarity threshold values can be taken into
account to identify cyanobacteria, but cannot constitute
sufficient indication alone.
The nucleotide differences found between the 16S
rRNA gene and 16S–23S ITS sequences published for
strain CCALA 759 and the corresponding ones obtained
here confirm the existence of multiple copies of rrn
operons in cyanobacteria. The fact that both in our survey
and in LokmerÕs (2007), only one of these operons was
amplified may be due to the different primers used in the
amplification reactions and to the previously documented
differential efficiency and specificity of amplification by
diverse primer pairs (Reysenbach et al., 1992; Suzuki and
Giovannoni, 1996; Nübel et al., 1997). Thus there is the
need to complement a more classical 16S rRNA gene
phylogenetic analysis with phylogenetic reconstructions
370
K. Sciuto et al. / Cladistics 28 (2012) 357–374
based on other single-copy molecular markers, such as the
rpoC1 and gyrB genes.
These last two loci, together, proved meaningful to
attribute strain ETS-02, Phormidium cf. irriguum
CCALA 759, and P. ambiguum IAM M-71 (90.2–
90.8% sequence identities among the rpoC1 + gyrB
gene fragments of these taxa) to the same genus in our
analyses, and thus they may prove important for the
identification of Phormidium strains at the genus level.
The level of phylogenetic relatedness found between
strain ETS-02 and strain CCALA 759 is also supported
by the biochemical, morphological, and ultrastructural
analyses. Indeed, the lipid-soluble pigment patterns are
highly similar, while the water-soluble pigment patterns
are identical, with both the organisms containing
phycoerythrocyanin, a rare PBP found only in a limited
number of cyanobacteria (Bryant, 1982; Garcı́a-Pichel
et al., 1996). Moreover, the 608-nm peak, corresponding
to C-PC and present in both organisms, is of note,
having been reported, to our knowledge, only in
Synechococcus lividus, a cyanobacterium found in Yellowstone National Park (Edwards et al., 1996).
The widened thylakoid aspect, called keritomy, is
present both in strain ETS-02 and in CCALA 759.
Keritomy is observed throughout the whole spectrum of
cyanobacterial taxonomic groups, but it appears to be
specific, genetically determined, although differentially
occurring during the life cycle and ⁄ or according to
environmental conditions (Komárek and Anagnostidis,
2005).
Table 6 summarizes the morphological, ultrastructural, physiological, biochemical, and molecular features
between strain ETS-02 and strain CCALA 759.
In conclusion, we can attribute strain ETS-02 to
group VIII of the genus Phormidium (subclass Oscillatoriophycidae, order Oscillatoriales, family Phormidiaceae). It seems also to belong to the same species as
Table 6
Basic characteristics of strain ETS-02 and Phormidium cf. irriguum
CCALA 759
Characteristic
Phormidium cf.
Strain ETS-02 irriguum CCALA 759
Cell morphology
Wider than
long
Cell width (lm)
3–5
Thylakoid arrangement Irregular
Sheath
+
Keritomy
+
Necridic cells
+
Heterocyst formation
)
PEC production
+
Isolation source
Thermal
mud surface
+
t-RNAIle
+
t-RNAAla
PEC, phycoerythrocyanin.
Wider than long
9–12
Irregular
+
+
+
)
+
Littoral zone of
sand pit lake
+
+
strain CCALA 759, i.e. P. irriguum (Kützing ex
Gomont) Anagnostidis and Komárek. This species is a
new combination by Anagnostidis and Komárek (1988,
p. 405) based on the description and illustration of
O. irrigua by Gomont (1892). As the holotype strain is
not a living culture, it cannot be used as a reference
strain according to our polyphasic method, which
focuses not only on external morphology, but also on
aspects such as ultrastructure, pigment composition,
and genetic features. When the original material associated with a validly published name is demonstrably
ambiguous and ⁄ or cannot be used for the precise
application of the name of a taxon, as in this case, the
International Code of Botanic Nomenclature allows the
institution of an epitype (McNeill et al., 2006). We
designate strain CCALA 759 as this epitype, as it
resembles the original description of P. irriguum according both to LokmerÕs (2007) observations and ours.
Nevertheless, cell size clearly distinguishes strain ETS02 from P. irriguum CCALA 759. Considering the
original description of P. irriguum and both LokmerÕs
analyses and ours, this species shows a range of cell
width that varies between 6 and 12 lm, while cells of
strain ETS-02 are smaller, with a width ranging between
3 and 5 lm, particularly if culture parameters, such as
organic nitrogen availability, temperature, and light
intensity, are changed (data not shown). However, in
our opinion the cell sizes are not sufficient alone to
consider strain ETS-02 as representing a novel species,
especially evaluating the whole spectrum of our observations. Some authors suggest, in certain well-documented cases, the creation of the subspecific taxonomic
categories of variety (‘‘the category variety could be
used in the cases of differentiation in one remarkable or
more features, usually combined with ecological or
phytogeographical deviation’’) or form (‘‘the category
form could be applied for a stable, recognizable deviation without ecological specificity’’) (Komárek and
Golubić, 2005). For this reason we propose the name
Phormidium irriguum f. minor for the Euganean strain
(where ‘‘f.’’ stands for forma, form).
Phormidium irriguum f. minor Sciuto et Moro
Thallus blue–green. Trichomes bright blue–green, long,
variously curved, strictly packed to form mats. Isopolar
trichomes, composed by cells wider (3–5 lm) than long
(1–2 lm), ending with rounded apical cells, and surrounded by a thin colourless sheath, sometimes protruding from the filament itself. Reproduction by trichome
fragmentation in short hormogonia through the formation of necridia. During cell division, daughter cells can
begin a new division before growing to the size of the
mother cell. Thylakoids with irregular arrangement,
sometimes keritomized. Able to produce phycoeritrocyanin.
K. Sciuto et al. / Cladistics 28 (2012) 357–374
Type material: A living culture is deposited at the
Culture Collection of Autotrophic Organisms (CCALA)
(Czech Republic) as strain CCALA 946.
Type locality: Phormidium irriguum f. minor was
isolated from cyanobacterial mats covering the thermal
muds of the Garden Hotel of Montegrotto Terme
(Padova, Italy).
Etymology: The specific form name refers to the cell
size, which is smaller than those of the species Phormidium irriguum (Kützing ex Gomont) Anagnostidis et
Komarek.
Another finding that results from the present survey is
the heterogeneity of the genus Phormidium Kützing ex
Gomont, as attested mainly by the phylogenetic reconstructions based on 16S rRNA gene sequences (Figs 10
and 11) in which more clades were detected within this
taxon. In particular, clade D, comprising P. uncinatum
(group VIII) and P. tergestinum (group V), and clade E,
including four Phormidium strains of group VII, placed
far from the Phormidium entities grouped in clade C
(group VIII). These clades should probably be ascribed
to different genera, but further investigation is required.
The genus Phormidium thus needs revision; several
studies by other authors have highlighted the polyphyletic and heterogeneous nature of this group, as it is
presently intended (Teneva et al., 2005; Palińska and
Marquardt, 2008; Komárek, 2010).
We thus make the following proposal. As the type
species of the genus Phormidium is P. lucidum Kützing
ex Gomont, a member of group VIII, the name
Phormidium should be maintained only for members
of group VIII. Moreover, as P. lucidum is based on an
illustration only, which is not suitable for a modern
approach including ultrastructural and molecular analyses, and we were not able to find a culture of P. lucidum
that could be subjected to a polyphasic analysis and then
typified, we suggest another type species for the future
new genus Phormidium. The new type species could be
P. irriguum, belonging to group VIII of the current
genus Phormidium (and thus probably being also phylogenetically related to P. lucidum), and the designed
type strain could be P. irriguum CCALA 759, on which
a polyphasic characterization has been carried out in
this study. If our proposal is accepted it could help
other researchers regarding the complex systematics of
Phormidium.
The proposed ‘‘combined approach’’ consists of the
following steps:
1. Preliminary morphological and ultrastructural
observations of the cyanobacterial strain of interest,
focusing on those characteristics (presence ⁄ absence of
thylakoids, thylakoid arrangement, presence of differentiated cells) that allow its immediate assignation to one of
the four subclasses of the phylum (Hoffmann et al., 2005):
Gloeobacterophycidae, Synechococcophycidae, Oscillatoriophycidae and Nostochophycidae.
371
2. More detailed morphological, ultrastructural, biochemical, and physiological analyses to allocate the
cyanobacterial strain of interest to more precise sub-taxa
(orders, families, genera, and ⁄ or species) within the
individual subclass or, at least, to reduce the number
of possible related taxa.
3. Phylogenetic multilocus analyses, based on the
classical 16S rRNA gene and other single-copy markers
(e.g. the rpoC1 gene). The data set reconstructions
should be determined based on morphological, biochemical, and physiological analyses made to this point,
amplifying the chosen molecular markers from selected
cyanobacterial strains, deposited in international culture
collections, if the corresponding sequences are not
already available in public databases.
4. Focus on the cyanobacterial strain (or strains) that
are phylogenetically related to the organism of interest,
extending the morphological, biochemical and physiological analyses.
5. If necessary, typify the considered cyanobacterial
strain (or strains) in order to provide a reference for
future studies. The form of typification should be based
on the specific case.
6. The conclusions and identification of the studied
cyanobacterium should take account of all available
information, detecting possible autapomorphic characters, and including a nomenclatural analysis.
At present, a polyphasic approach to characterize
cyanobacteria seems essential. This approach should not
only include different types of analyses, ranging from
the more classical (morphological, biochemical, and
physiological) to the molecular, but also comparisons
with deposited type species. Where the holotypes of the
described reference species are lacking or not suitable
for a detailed comparison, a typification with cultured
strains can be useful.
Therefore, far from proposing completely new analytical methods, the described ‘‘combined approach’’ joins
the more classical analyses to the more recent molecular
ones, making them complementary and equally important to the correct identification of new strains belonging
to the phylum Cyanophyta ⁄ Cyanobacteria, avoiding the
incongruities between only classical-based and only
molecular-based identifications. The amount of information produced by such an approach should facilitate
subsequent studies on cyanobacteria, contributing to
solve the taxonomic and classification problems of this
complex and interesting phylum.
Acknowledgements
This work was supported by grants provided by
Centro Studi Termali ‘‘Pietro dÕAbano,’’ Abano Terme
(Padova, Italy). We thank Dr Nancy Jenkins for help
with the English text.
372
K. Sciuto et al. / Cladistics 28 (2012) 357–374
References
Anagnostidis, K., Komárek, J., 1988. Modern approach to the
classification system of cyanophytes. 3. Oscillatoriales. Arch.
Hydrobiol. Suppl. 80, 327–472.
Andreoli, C., Rascio, N., 1975. The algal flora in the thermal baths of
Montegrotto Terme (Padua). Its distribution over one-year period.
Int. Revue ges. Hydrobiol. 60, 857–871.
Ballot, A., Dadheech, P., Krienitz, L., 2004. Phylogenetic relationship
of Arthrospira, Phormidium, and Spirulina strains from Kenyan and
Indian water bodies. Arch. Hydrobiol. Suppl. Algol. Stud. 113, 37–
56.
Ballot, A., Dadheech, P.K., Haande, S., Krienitz, L., 2008. Morphological and phylogenetic analysis of Anabaenopsis abijatae and
Anabaenopsis elenkinii (Nostocales, Cyanobacteria) from tropical
inland water bodies. Microb. Ecol. 55, 608–618.
Bellometti, S., Cecchettin, M., Galzigna, L., 1997. Mud pack therapy
in osteoarthrosis. Changes in serum levels of chondrocyte markers.
Clin. Chim. Acta 268, 101–106.
Bellometti, S., Poletto, M., Gregotti, C., Richelmi, P., Bertè, F., 2000.
Mud bath therapy influences nitric oxide, myeloperoxidase and
glutathione peroxidase serum levels in arthritic patients. Int. J.
Clin. Pharmacol. Res. XX, 69–80.
Bothe, H., Tripp, H.J., Zehr, J.P., 2010. Unicellular cyanobacteria
with a new mode of life: the lack of photosynthetic oxygen
evolution allows nitrogen fixation to proceed. Arch. Microbiol.
192, 783–790.
Bruno, A., Rossi, C., Marcolongo, G., Di Lena, A., Venzo, A., Berrie,
C.P., Corda, D., 2005. Selective in vivo anti-inflammatory action of
the galactolipid monogalactosyldiacylglycerol. Eur. J. Pharmacol.
524, 159–168.
Bryant, D.A., 1982. Phycoerythrocyanin and phycoerytrin: properties
and occurrence in cyanobacteria. J. Gen. Microbiol. 128, 835–
844.
Casamatta, D.A., Johansen, J.R., Vis, M.L., Broadwater, S.T., 2005.
Molecular and morphological characterization of ten polar and
near-polar strains within the Oscillatoriales (Cyanobacteria). J.
Phycol. 41, 421–438.
Castresana, J., 2000. Selection of conserved blocks from multiple
alignments for their use in phylogenetic analysis. Mol. Biol. Evol.
17, 540–552.
Ceschi-Berrini, C., De Appolonia, F., Dalla Valle, L., Komárek, J.,
Andreoli, C., 2004. Morphological and molecular characterization
of a thermophilic cyanobacterium (Oscillatoriales) from Euganean
Thermal Springs (Padua, Italy). Arch. Hydrobiol. Suppl. Algol.
Stud. 113, 73–85.
Christie, W.W., 1982. A simple procedure for rapid transmethylation
of glycerolipids and cholesteryl esters. J. Lipid Res. 23, 1072–1075.
Edwards, M.R., MacColl, R., Eisele, L.E., 1996. Some physical
properties of an unusual C-phycocyanin isolated from a photosynthetic thermophile. Biochim. Biophys. Acta 1276, 64–70.
Eriksen, N.T., 2008. Production of phycocyanin—a pigment with
applications in biology, biotechnology, foods and medicine. Appl.
Microbiol. Biotechnol. 1, 1–14.
Färber, A., Jahns, P., 1998. The xanthophyll cycle of higher plants:
influence of antenna size and membrane organization. Biochim.
Biophys. Acta 1363, 47–58.
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach
using bootstrap. Evolution 39, 783–791.
Fergusson, K.M., Saint, C.P., 2000. Molecular phylogeny of Anabaena
circinalis and its identification in environmental samples by PCR.
Appl. Environ. Microbiol. 66, 4145–4148.
Galzigna, L., Ceschi-Berrini, C., Moschin, E., Tolomio, C., 1998.
Thermal mud-pack as an anti-inflammatory treatment. Biomed.
Pharmacother. 52, 408–409.
Garcı́a-Pichel, F., Prufert-Bebout, L., Muyzer, G., 1996. Phenotypic
and phylogenetic analyses show Microcoleus chthonoplastes to be a
cosmopolitan cyanobacterium. Appl. Environ. Microbiol. 62,
3284–3291.
Garcı́a-Pichel, F., López-Cortés, A., Nübel, U., 2001. Phylogenetic
and morphological diversity of cyanobacteria in soil desert crusts
from the Colorado Plateau. Appl. Environ. Microbiol. 67, 1902–
1910.
Gerwick, W.H., Coates, R.C., Engene, N., Gerwick, L., Grindberg,
R.V., Jones, A.C., Sorrels, C.M., 2008. Giant marine cyanobacteria
produce exciting potential pharmaceuticals. Microbe 3, 277–284.
Gomont, M., 1892 Ô1893Õ. Monographie des Oscillariées (Nostocacées
homocystées). Ann. Sci. Nat. Bot. sér. 7 16, 91–264, plates 1–7.
Guindon, S., Gascuel, O., 2003. A simple, fast and accurate algorithm
to estimate large phylogenies by maximum likelihood. Syst. Biol.
52, 696–704.
Gupta, R.S., Mathews, D.W., 2010. Signature proteins for the major
clades of Cyanobacteria. BMC Evol. Biol. 10, 24.
Henson, B.J., Watson, L.E., Barnum, S.R., 2004. The evolutionary
history of nitrogen fixation, as assessed by NifD. J. Mol. Evol. 58,
390–399.
Hoffmann, L., Komárek, J., Kaštovský, J., 2005. System of cyanoprokaryotes (cyanobacteria)—state 2004. Algol. Stud. 117, 95–115.
Hyde, K.D., Zhang, Y., 2008. Epitypification: should we epitypify? J.
Zhejiang Univ. Sci. B 9, 842–846.
Iteman, I., Rippka, R., Tandeau de Marsac, N., Herdman, M., 2000.
Comparison of conserved structural and regulatory domains within
divergent 16S–23S rRNA spacer sequences of cyanobacteria.
Microbiology 146, 1275–1286.
Jahn, R., Mann, D.G., Evans, K.M., Poulı́čková, A., 2008. The
identity of Sellaphora bacillum (Ehrenberg) D.G. Mann. Fottea 8,
121–124.
Jungblut, A., Hawes, I., Mountfort, D., Hitzfeld, B., Dietrich, D.R.,
Burns, B.P., Neilan, B.A., 2005. Diversity within cyanobacterial
mat communities in variable salinity meltwater ponds of McMurdo
Ice Shelf, Antarctica. Environ. Microbiol. 7, 519–529.
Khan, M., Varadharaj, S., Ganesan, L.P., Shobha, J.C., Naidu, M.U.,
Parinandi, N.L., Tridandapani, S., Kutala, V.K., Kuppusamy, P.,
2006. C-phycocyanin protects against ischemia-reperfusion injury
of heart through involvement of p38 MAPK and ERK signaling.
Am. J. Physiol. Heart Circ. Physiol. 290, H2136–H2145.
Komárek, J., 2005. The modern classification of Cyanoprokaryotes
(Cyanobacteria). Oceanol. Hydrobiol. Stud. 34, 5–17.
Komárek, J., 2010. Recent changes (2008) in cyanobacteria taxonomy
based on a combination of molecular background with phenotype
and ecological consequences (genus and species concept). Hydrobiologia 639, 245–259.
Komárek, J., Anagnostidis, K., 2005. Cyanoprokaryota. Part 2.
Oscillatoriales. Elsevier, München, Germany.
Komárek, J., Golubić, S., 2005. Guide to the nomenclature and formal
taxonomic treatment of oxyphototroph prokaryotes (Cyanoprokaryotes). Proposal, Available at: http://www.cyanodb.cz/.
Komárek, J., Kopecký, J., Cepák, V., 1999. Generic characters of the
simplest cyanoprokaryotes Cyanobium, Cyanobacterium and Synechococcus. Cryptogam. Algol. 20, 209–222.
Lalli, A., Andreoli, C., Ceschi-Berrini, C., De Appolonia, F., Marcolongo, G., 2004. Anti-inflammatory active principles in Euganean
thermal MUD. European patent 05100038.8.
Lanave, C., Preparata, G., Saccone, C., Serio, G., 1984. A new method
for calculating evolutionary substitution rates. J. Mol. Evol. 20,
86–93.
Li, B., Zhang, X., Gao, M., Chu, X., 2005. Effects of CD59 on
antitumoral activities of phycocyanin from Spirulina platensis.
Biomed. Pharmacother. 59, 551–560.
Lineau, E.K., De Salle, R., Allard, M., Brown, E.W., Swofford, D.,
Rosenfeld, J.A., Sarkar, I.N., Planet, P.J., 2010. The mega-matrix
tree of life: using genome-scale horizontal gene transfer and
sequence evolution data as information about the vertical history
of life. Cladistics 26, 1–11.
K. Sciuto et al. / Cladistics 28 (2012) 357–374
Lokmer, A., 2007. Polyphasic approach to the taxonomy of the
selected oscillatorian strains (Cyanobacteria). Masters Thesis,
University of South Bohemia, České Budějovice, Czech Republic.
López-Cortés, A., Garcı́a-Pichel, F., Nübel, U., Vàzquez-Juàrez, R.,
2001. Cyanobacterial diversity in extreme environments in Baja
California, Mexico: a polyphasic study. Int. Microbiol. 4, 227–236.
Maddison, W.P., Maddison, D.R., 2009. Mesquite: a modular system
for evolutionary analysis, Version 2.71. Available at: http://
mesquiteproject.org/mesquite/mesquite.html.
Maistro, S., Broady, P.A., Andreoli, C., Negrisolo, E., 2007. Molecular phylogeny and evolution of the order Tribonematales (Heterokonta, Xanthophyceae) based on analysis of plastidial genes
rbcL and psaA. Mol. Phylogenet. Evol. 43, 407–417.
Maistro, S., Broady, P.A., Andreoli, C., Negrisolo, E., 2009. Phylogeny and taxonomy of Xanthophyceae (Stramenopiles, Chromalveolata). Protist 160, 412–426.
Marcolongo, G., De Appolonia, F., Venzo, A., Berrie, C.P., Carofiglio, T., Ceschi-Berrini, C., 2006. Diacylglycerolipids isolated from
a thermophile cyanobacterium from the Euganean hot springs.
Nat. Prod. Res. 20, 766–774.
Marquardt, J., Palinska, K.A., 2007. Genotypic and phenotypic
diversity of cyanobacteria assigned to the genus Phormidium
(Oscillatoriales) from different habitats and geographical sites.
Arch. Microbiol. 187, 397–413.
McNeill, J., Barrie, F.R., Burdet, H.M., Demoulin, V., Hawksworth,
D.L., Marhold, K., Nicolson, D.H., Prado, J., Silva, P.C., Skog,
J.E., Wiersema, J.H., 2006. International Code of Botanical
Nomenclature (Vienna Code) 2006. Available at: http://ibot.sav.sk/icbn/main.htm.
Moro, I., Di Bella, M., Rascio, N., La Rocca, N., Andreoli, C., 2007a.
Conferva duplisecta Pollini: rediscovery in Euganean Thermal
Springs (Italy) and new assignment to the Oscillatoria genus.
Caryologia 60, 133–136.
Moro, I., Rascio, N., La Rocca, N., Di Bella, M., Andreoli, C., 2007b.
Cyanobacterium aponinum, a new Cyanoprokaryote from the
microbial mat of Euganean thermal springs (Padua, Italy). Algol.
Stud. 123, 1–15.
Moro, I., Rascio, N., La Rocca, N., Sciuto, K., Albertano, P.,
Bruno, L., Andreoli, C., 2010. Polyphasic characterization of a
thermo-tolerant filamentous cyanobacterium isolated from the
Euganean thermal muds (Padova, Italy). Eur. J. Phycol. 45, 143–
154.
Naum, M., Brown, E.W., Mason-Gamer, R.J., 2008. Is 16S rDNA a
reliable phylogenetic marker to characterize relationships below
the family level in the Enterobacteriaceae? J. Mol. Evol. 66, 630–
642.
Nübel, U., Garcı́a-Pichel, F., Muyzer, G., 1997. PCR primers to
amplify 16S rRNA genes from Cyanobacteria. Appl. Environ.
Microbiol. 63, 3327–3332.
Oren, A., 2004. A proposal for further integration of the cyanobacteria
under the Bacteriological Code. Int. J. Syst. Evol. Microbiol. 54,
1895–1902.
Oren, A., Komárek, J., Hoffmann, L., 2009. Nomenclature of the
Cyanophyta ⁄ Cyanobacteria ⁄ Cyanoprokaryotes—what has happened since IAC Luxembourg? Algol. Stud. 130, 17–26.
Paerl, H.W., Pinckney, J.L., Steppe, T.F., 2000. Cyanobacterialbacterial mat consortia: examining the functional unit of microbial
survival and growth in extreme environments. Environ. Microbiol.
2, 11–26.
Palińska, K.A., Marquardt, J., 2008. Genotypic and phenotypic
analysis of strains assigned to the widespread cyanobacterial
morphospecies Phormidium autumnale (Oscillatoriales). Arch.
Microbiol. 189, 325–335.
Palińska, K.A., Liesack, W., Rhiel, E., Krumbein, W.E., 1996.
Phenotype variability of identical genotypes: the need for a
combined approach in cyanobacterial taxonomy demonstrated on
Merismopedia-like isolates. Arch. Microbiol. 166, 224–233.
373
Papke, R.T., Ramsing, N.B., Bateson, M.M., Ward, D.M., 2003.
Geographical isolation in hot spring cyanobacteria. Environ.
Microbiol. 5, 650–659.
Perriére, G., Gouy, M., 1996. WWW-query: an on-line retrieval system
for biological sequence banks. Biochimie 78, 364–369.
Premanandh, J., Priya, B., Teneva, I., Dzhambazov, B., Prabaharan,
D., Uma, L., 2006. Molecular characterization of marine cyanobacteria from the Indian subcontinent deduced from sequence
analysis of the phycocyanin operon (cpcB-IGS-cpcA) and 16S–23S
ITS region. J. Microbiol. 44, 607–616.
Rajaniemi, P., Hrouzek, P., Kaštovská, K., Willame, R., Rantala, A.,
Hoffmann, L., Komárek, J., Sivonen, K., 2005. Phylogenetic and
morphological evaluation of the genera Anabaena, Aphanizomenon,
Trichormus and Nostoc (Nostocales, Cyanobacteria). Int. J. Syst.
Evol. Microbiol. 55, 11–26.
Reysenbach, A.-L., Giver, L.J., Wickham, G.S., Pace, N.R., 1992.
Differential amplification of rRNA genes by polymerase chain
reaction. Appl. Environ. Microbiol. 58, 3417–3418.
Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier,
R.Y., 1979. Generic assignments, strain histories and properties of
pure cultures of Cyanobacteria. J. Gen. Microbiol. 111, 1–61.
Robertson, B.R., Tezuka, N., Watanabe, M.M., 2001. Phylogenetic
analyses of Synechococcus strains (cyanobacteria) using sequences
of 16S rDNA and part of the phycocyanin operon reveal multiple
evolutionary lines and reflect phycobilin content. Int. J. Syst. Evol.
Microbiol. 51, 861–871.
Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.
Rybalka, N., Andersen, R.A., Kostikov, I., Mohr, K.I., Massalski, A.,
Olech, M., Friedl, T., 2009. Testing for endemism, genotypic
diversity and species concepts in Antarctic terrestrial microalgae of
the Tribonemataceae (Stramenopiles, Xanthophyceae). Environ.
Microbiol. 11, 554–565.
Sánchez-Baracaldo, P., Hayes, P.K., Blank, C.E., 2005. Morphological
and habitat evolution in the Cyanobacteria using a compartmentalization approach. Geobiology 3, 145–165.
Schmidt, H.A., Strimmer, K., Vingron, M., von Haeseler, A., 2002.
TREE-PUZZLE: maximum likelihood phylogenetic analysis using
quartets and parallel computing. Bioinformatics 18, 502–504.
Sciuto, K., Rascio, N., Andreoli, C., Moro, I., 2011. Polyphasic
characterization of ITD-01, a cyanobacterium isolated from the
Ischia Thermal District (Naples, Italy). Fottea 11, 31–39.
Seo, P., Yokota, A., 2003. The phylogenetic relationships of cyanobacteria inferred from 16S rRNA, gyrB, rpoC1 and rpoD1 gene
sequences. J. Gen. Appl. Microbiol. 49, 191–203.
Singh, S., Kate, B.N., Banerjee, U.C., 2005. Bioactive compounds
from Cyanobacteria and microalgae: an overview. Crit. Rev.
Biotechnol. 25, 73–95.
Sivonen, K., Börner, T., 2008. Bioactive compounds produced by
cyanobacteria. In: Herraro, A., Flores, E. (Eds.), The Cyanobacteria: Molecular Biology, Genomics and Evolution. Caister Academic Press, Norfolk, pp. 159–197.
Stackebrandt, E., Goebel, B.M., 1994. Taxonomic note: a place for
DNA-DNA reassociation and 16S rRNA sequence analysis in the
present species definition in bacteriology. Int. J. Syst. Bacteriol. 44,
846–849.
Strimmer, K., von Haeseler, A., 1997. Likelihood-mapping: a simple
method to visualize phylogenetic content of a sequence alignment.
Proc. Natl Acad. Sci. USA 94, 6815–6819.
Suzuki, M.T., Giovannoni, S.J., 1996. Bias caused by template
annealing in the amplification of mixtures of 16S rRNA genes by
PCR. Appl. Environ. Microbiol. 62, 625–630.
Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4:
molecular evolutionary genetics analysis (MEGA) software version
4.0. Mol. Biol. Evol. 24, 1596–1599.
Taton, A., Grubisic, S., Balthasart, P., Hodgson, D.A., LaybournParry, J., Wilmotte, A., 2006. Biogeographical distribution and
374
K. Sciuto et al. / Cladistics 28 (2012) 357–374
ecological ranges of benthic cyanobacteria in East Antarctic lakes.
FEMS Microbiol. Ecol. 20, 1–18.
Teneva, I., Dzhambazov, B., Mladenov, R., Schirmer, K., 2005.
Molecular and phylogenetic characterization of Phormidium species (Cyanoprokaryota) using the cpcB-IGS-cpcA locus. J. Phycol.
41, 188–194.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Improving the
sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res. 22, 4673–4680.
Toledo, G., Palenik, B., 1997. Synechococcus diversity in the California
current as seen by RNA polymerase (rpoC1) gene sequences of
isolated strains. Appl. Environ. Microbiol. 63, 4298–4303.
Tolomio, C., De Appolonia, F., Moro, I., Ceschi-Berrini, C., 2004.
Thermophilic microalgae growth on different substrates and at
different temperatures in experimental tanks in Abano Terme
(Italy). Arch. Hydrobiol. Algol. Stud. 111, 145–157.
Tomitani, A., Knoll, A.H., Cavanaugh, C.M., Ohno, T., 2006. The
evolutionary diversification of cyanobacteria: molecular–phylogenetic and paleontological perspectives. Proc. Natl Acad. Sci.
USA 103, 5442–5447.
Tuji, A., Williams, D.M., 2008. Typification and type examination of
Synedra familiaris Kütz. and related taxa. Diatom 24, 25–29.
Ward, D.M., Ferris, M., Nold, S., Bateson, M., 1998. A natural view
of microbial biodiversity within hot spring cyanobacterial mat
communities. Microbiol. Mol. Biol. Rev. 62, 1353–1370.
Watanabe, K., Nelson, J.S., Harayama, S., Kasai, H., 2001. ICB
database: the gyrB database for identification and classification of
bacteria. Nucleic Acids Res. 29, 344–345.
Whitton, B.A., 1992. Diversity, ecology, and taxonomy of the
cyanobacteria. In: Mann, N.H., Carr, N.G. (Eds.), Photosynthetic
Prokaryotes. Plenum Press, New York, pp. 1–51.
Wilmotte, A., Herdman, M., 2001. Phylogenetic relationships among
the cyanobacteria based on 16S rRNA sequences. In: Boone, D.R.,
Castenholz, R.W. (Eds.), BergeyÕs Manual of Systematic Bacteriology, 2nd edn. Springer, New York, pp. 487–493.
Wilson, K.M., Schembri, M.A., Baker, P.D., Saint, C.P., 2000.
Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Appl.
Environ. Microbiol. 66, 332–338.
Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Fig. S1. Summary of the organisms considered in the
phylogenetic analyses.
Fig. S2. Comparison between the 16S-23S ITS
sequences of ETS-02 and CCALA 759 strains.
Fig. S3. Comparison between the cpcB-IGS-cpcA
sequences of ETS-02 and CCALA 759 strains.
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