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R E S E A R C H A R T I C L E
Life cycleas a stable trait in the evaluationofdiversityofNostocfrombio¢lms in riversPilar Mateo1, Elvira Perona1, Esther Berrendero1, Francisco Leganes1, Marta Martın1 & Stjepko Golubic2
1Departamento de Biologıa, Facultad de Ciencias, Universidad Autonoma de Madrid, Madrid, Spain; and 2Department of Biology, Boston University,
Boston, MA
Correspondence: Pilar Mateo,
Departamento de Biologıa, Facultad de
Ciencias, Universidad Autonoma de Madrid,
C Darwin no. 2., 28049 Madrid, Spain. Tel.:
134 914 978 184; fax: 134 914 978 344;
email: pilar.mateo@uam.es
Received 2 July 2010; revised 29 November
2010; accepted 20 December 2010.
Final version published online 24 January 2011.
DOI:10.1111/j.1574-6941.2010.01040.x
Editor: Riks Laanbroek
Keywords
morphological variability; phylogenetic
relationships; cyanobacterial diversity.
Abstract
The diversity within the genus Nostoc is still controversial and more studies are
needed to clarify its heterogeneity. Macroscopic species have been extensively
studied and discussed; however, the microscopic forms of the genus, especially
those from running waters, are poorly known and likely represented by many more
species than currently described. Nostoc isolates from biofilms of two Spanish
calcareous rivers were characterized comparing the morphology and life cycle in
two culture media with different levels of nutrients and also comparing the 16S
rRNA gene sequences. The results showed that trichome shape and cellular
dimensions varied considerably depending on the culture media used, whereas
the characteristics expressed in the course of the life cycle remained stable for each
strain independent of the culture conditions. Molecular phylogenetic analysis
confirmed the distinction between the studied strains established on morphologi-
cal grounds. A balanced approach to the evaluation of diversity of Nostoc in the
service of autecological studies requires both genotypic information and the
evaluation of stable traits. The results of this study show that 16S rRNA gene
sequence similarity serves as an important criterion for characterizing Nostoc
strains and is consistent with stable attributes, such as the life cycle.
Introduction
The genus Nostoc is a frequent member of many aquatic and
terrestrial ecosystems, showing considerable morphological
diversity (Mollenhauer et al., 1994; Dodds et al., 1995; Potts,
2000). Field populations of most Nostoc species are distin-
guished according to a characteristic set of macroscopic
properties, unknown in other genera. The life cycle of Nostoc
is complex and considered an important feature for distin-
guishing of species (Geitler, 1932; Lazaroff & Vishniac, 1961;
Komarek & Anagnostidis, 1989; Mollenhauer et al., 1994).
In addition, there are numerous microscopic Nostoc spp.,
probably as many as the macroscopic species, which play
different important roles in the biosphere (Mollenhauer
et al., 1994, 1999). However, the microscopic species in the
genus are understudied, poorly known and likely represent
many more species than currently described (Mollenhauer
et al., 1994, 1999; Rehakova et al., 2007). The information
stemming from laboratory studies is often problematic,
because many of the isolates were misidentified (Komarek
& Anagnostidis, 1989; Wilmotte, 1994) or the information
on the habitat and, especially, microenvironmental condi-
tions was inadequately recorded. Therefore, the isolation
and characterization of new cyanobacterial strains from
diverse biotopes remain important in studies of cyanobac-
terial diversity and ecology, even where culture-independent
techniques based on the rRNA operon have been used
successfully (e.g. McGregor & Rasmussen, 2008). Studies of
cultured isolates provide a link between genotypic and
phenotypic features to allow a better understanding of
cyanobacterial physiology and autecology. In addition,
characterizations based on polyphasic studies improve the
resolution of cyanobacterial taxonomy (Wilmotte, 1994;
Palinska et al., 2006) and currently constitute the best-
defined base-line for diversity and ecological studies (Taton
et al., 2006; Heath et al., 2010). In recent years, the studies
based on combined genetic and phenotypic properties of
new isolates have increased the reliability of identification,
but arrived at the recommendation that the genus Nostoc
needs to be revised (Rajaniemi et al., 2005; Rehakova et al.,
FEMS Microbiol Ecol 76 (2011) 185–198 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
MIC
ROBI
OLO
GY
EC
OLO
GY
2007; Papaefthimiou et al., 2008). The process of revision of
nostocacean genera has been introduced recently by
Komarek (2010a, b).
The ecophysiological characteristics of cyanobacterial
populations constitute important traits in polyphasic stu-
dies (Komarek, 2010a, b). Microbial ecology requires the
identification of organisms and their specific functions, but
depends on the recognition of consistent and reproducible
traits. The use of characteristics, such as life cycle, is
expected to provide more precise phenotypical and ecologi-
cal characterizations. Cyanobacterial life-cycle processes
have been studied in depth in aquatic ecosystem models
(Hense & Beckmann, 2010; Suikkanen et al., 2010).
The aim of this study was a thorough characterization of
two Nostoc strains isolated from epilithic biofilms of two
Spanish rivers, through the analysis of their genetic, mor-
phological and ecophysiological characteristics. As we strive
to increase our knowledge of the cyanobacterial diversity,
this polyphasic approach will allow us a better understand-
ing of the Nostoc diversity and the identification of the
occupants of particular ecological niches and their functions
in different environments.
We examined the morphological variability, sequence and
timing of life cycles in two different media: the BG110
medium (Rippka et al., 1979), most commonly used for
cyanobacteria, and a medium with a low nutrient concen-
tration: CHU 10 medium (Chu, 1942), which approached
the low nutrient conditions of the studied streams (Berren-
dero et al., 2008). The capability to produce the pigment C-
phycoerythrin was also determined. The life cycle was
followed by a daily microscopic observation of selected
hormogonia from the first day until the breaking of fila-
ments into new hormogonia. Genetic analyses were per-
formed by sequencing 16S rRNA genes.
Materials and methods
Environmental setting
Nostoc strains were isolated from the epilithic biofilm of two
different calcareous streams in east Spain: the Matarrana
stream (Teruel, northeast Spain) and the Amir stream (Alba-
cete, southeast Spain). Both streams are located in the
Mediterranean slope of Spain at a low altitude, in an area with
Mediterranean climate. These streams are characterized by the
dominance of cyanobacteria and the absence of any kind of
human perturbation or pollution. pH was about 8.5 in the
Matarrana stream and 7.9 in the Amir stream. The latter is
also characterized by brackish waters with high conductivity.
Strain isolation
The epilithic biofilm was removed by brushing the surface of
stones collected from the riverbed and resuspended in
culture media, which were later used for cyanobacterial
culturing on Petri dishes with different solid media (1.5%
agar concentration). This enrichment was allowed to grow,
and was examined under the microscope to distinguish
different morphotypes. Cultures were obtained by picking
material from the edge of discrete colonies that had been
growing for approximately 4 weeks on a solid medium. In
order to analyze variability in dependence of culture media,
two different growth media were tested: BG110 medium
(Rippka et al., 1979) and CHU 10 medium (Chu, 1942).
Stock cultures were maintained in liquid media in a culture
room (20 1C, 12 : 12 h light/dark, natural illumination of
10–15 mE m�2 s). Solid culture media were used for life-cycle
study, under the same light conditions. The strains were
named after the stream of their origin, and were included in
the culture collection of the University Autonoma de
Madrid (UAM): strain-MA4, Nostoc UAM 307, isolated
from the Matarrana stream, and strain-A8, Nostoc UAM
308, isolated from the Amir stream.
Observation of life cycles
Life cycle was studied by the daily microscopic observation
of selected hormogonia from the first day until the breaking
of filaments into new hormogonia. To study and evaluate
changes in the life cycle of the Nostoc isolates, several
filaments from stock cultures were placed on an agar surface
in Petri dishes 3 cm in diameter, and covered with a glass
cover slip. The Petri dishes were affixed onto microscope
slides and maintained for repeated observation. When
hormogonia developed a few days later, selected hormogo-
nia were located by x and y coordinates on the microscope
stage and followed throughout their development. Daily
examinations of developmental stages were carried out
using a photomicroscope Olympus BH-2 equipped using
camera lucida and a Leica digital camera and documented
by in-scale drawing and photomicrographs. Size measure-
ments of different cell types (vegetative trichome cells,
hormogonia cells, heterocysts, akinetes) were carried out.
Over the duration of the experiment, the changes in the cell
count of 50 different hormogonia of each strain were
recorded daily. Spacing between heterocyst as well as the
number of vegetative cells between two adjacent heterocysts
were monitored during the course of the development of
three separate hormogonia for each culture medium.
Pigment analysis
The capability to produce the pigment C-phycoerythrin was
determined because Nostoc strains may also be distinguished
by this property, which is not present in all strains of this
genus, producing phycoerythrocyanin instead (Rippka et al.,
1979; Lachance, 1981). For the analysis of C-phycoerythrin,
1-mL aliquots of culture were taken and permeated with
FEMS Microbiol Ecol 76 (2011) 185–198c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
186 P. Mateo et al.
glycerol. After 1 h in darkness at ambient temperature,
900 mL of distilled water was added, the samples were
centrifuged and the absorption spectrum of the supernatant
(400–700 nm) was recorded. The C-phycoerythrin content
of the supernatant was determined as described previously
(Gomez et al., 2009) using the extinction coefficients of
Bennett & Bogorad (1973).
Genotypic characterization
Genomic DNA was extracted following a modification of a
technique for extracting DNA from fresh plant tissue, using
cetyltrimethylammonium bromide (CTAB) (Doyle & Doyle,
1990). Cells were harvested by centrifugation after the
addition of about 150mL of sterile glass beads
(212–300mm; Sigma) and resuspended in 400 mL extraction
buffer [100 mM Tris/HCl, pH 8.0, 20 mM EDTA, 2.5%
CTAB (w/v), 1.4 M NaCl, 0.2% 2-mercaptoethanol (v/v)].
Samples were frozen in liquid nitrogen and then homoge-
nized in extraction buffer using a hand-operated homoge-
nizer (CSB-850-2RET, Bosch). Subsequently, they were
incubated at 60 1C for 30 min, followed by two extractions
with chloroform. DNA-containing phases were placed in
new tubes and an equal volume of 2-propanol was added.
The samples were centrifuged at 12 000 g for 5 min. The
pellet was resuspended in 400 mL sterile water and DNA
reprecipitated by the addition of 0.1 volume of 3 M sodium
acetate and two volumes of ethanol before being stored at
� 20 1C in 15 mL sterile water.
PCR amplification of cyanobacterial 16S rRNA gene was
carried out using the forward primer pA (Edwards et al.,
1989) for both strains, but the reverse 16S1494R (Wilmotte
et al., 2002) for strain A8 and the cyanobacterial-specific
B23S (Lepere et al., 2000) for strain MA4. The reaction
volume was 25mL and contained 6 mL DNA, 10 pmol of each
primer, 200 mM dNTP, 1mg bovine serum albumin,
Fig. 1. Photomicrographs of Nostoc strain-A8
UAM 308 showing phenotypic variability de-
pending on the culture medium. (a– c) BG110
medium; (d–f) CHU 10 medium. ak, akinetes
(after longer maintenance). Scale bar = 10mm.
FEMS Microbiol Ecol 76 (2011) 185–198 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
187Life cycle in the evaluation of Nostoc diversity
1.5 mM MgCl2, 2.5mL 10� polymerase buffer, 5mL 5�Eppendorf Taqmaster PCR enhancer and 0.75 U Ultratools
DNA polymerase (Biotools). The reaction conditions used
were those described by Gkelis et al. (2005). The 16S rRNA
gene was sequenced in several parts using the Big Dye
Terminator v3.1 Cycle Sequencing kit (Applied Byosistems)
using primers pA (Edwards et al., 1989), cyanobacterial-
specific CYA781R(a) (Nubel et al., 1997), 16S684F (50-
GTGTAGCGGTGAAATGCGTAGA-30) designed in this
study, 16S1494R (Wilmotte et al., 2002) and pGL2.1 (Ur-
bach et al., 1992) using an ABI Prism 3730 Genetic Analyzer
(Applied Biosystems) according to the manufacturer’s in-
structions. The sequences were obtained for both strands
independently. Nucleotide sequences obtained from DNA
sequencing were compared with sequence information
available in the National Center for Biotechnology Informa-
tion database using BLAST (http://www.ncbi.nlm.nih.gov/
BLAST). Multiple-sequence alignment was performed using
CLUSTAL_W (Thompson et al., 1994) of the current version of
the BIOEDIT program (Hall, 1999). The alignment was later
visually checked and corrected using BIOEDIT. The MALLARD
program (Ashelford et al., 2006) was used for identifying
anomalous 16S rRNA gene sequences within multiple se-
quence alignment.
Phylogenetic tree construction
Phylogenetic analysis of sequences was performed using
MEGA software (Tamura et al. 2007). Trees were constructed
using neighbor-joining (NJ), maximum likelihood (ML)
and maximum-parsimony (MP) algorithms (Saitou & Nei,
1987). Distances for the NJ tree were estimated by the
algorithm of Tajima & Nei (1984) using the complete
deletion option. Bootstrap analysis of 1000 replications was
performed for each consensus tree (Felsenstein, 1985).
Similar clustering was obtained with the NJ, ML and MP
algorithms; hence, we have chosen to represent the sequence
relationships with the NJ tree. The nucleotide sequences
obtained in this work have been deposited in the GenBank
database (accession numbers: HM623781 for strain A8 and
HM623782 for strain MA4).
Statistics
Student’s t-test was used to determine the significance of
differences between morphometric measurements of isolates
in different culture media.
Results
Variability of trichome morphology
The morphological variability of the two isolates under
study is shown in Figs 1 and 2. Morphological characteristics
relating to micromorphometric measurements of vegetative
cells, heterocysts, akinetes, size and numbers of cells in
hormogonia in the two different culture media are shown
in Table 1. Cell dimensions were the most variable feature,
and, in general, were higher in BG110 than in CHU 10
medium in both isolates (for significant differences, see
Table 1). Regarding trichome shape, sheaths and details of
cell shape, strain-A8 presented a high degree of morpholo-
gical variability in both media (Fig. 1). Trichomes with
different shapes could be observed: straight and slightly
Fig. 2. Photomicrographs of Nostoc strain-MA4 UAM 307 showing
phenotypic variability depending on the culture medium. (a) BG110
medium; (b, c) CHU 10 medium. ak, akinetes (after longer maintenance).
Scale bar = 10mm.
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188 P. Mateo et al.
curved trichomes (Fig. 1a–c) and flexuous or coiled tri-
chomes (Fig. 1d and e), sometimes densely entangled (Fig.
1b). The variable shapes could be observed in both media,
although a tendency for flexuosity was observed more
consistently in CHU 10 medium (Fig. 1d–f). Akinetes were
ellipsoidal (Fig. 1f). Individual sheaths were distinct only on
some trichomes in liquid culture (Fig. 1e).
Strain-MA4 showed less variability in trichome shapes,
which were mostly flexuous (Fig. 2). Similar characteristics
were found in both media, although trichomes tended to
show greater flexuosity in CHU 10 medium (Fig. 2b).
Akinetes were ellipsoidal (Fig. 2c). Individual sheaths were
more expressed than in strain A8 (Figs 3 and 4).
Life cycles
The development and life cycles of the two Nostoc strains
were followed daily. It was possible to continuously observe
the uninterrupted sequence of stages in the development of
trichomes from hormogonium stages until they were break-
ing up into new hormogonia. Similar behavior was found
for each strain in the two culture media studied, although in
the nutrient-richer BG110 medium, there was a clear ten-
dency for faster growth and shorter cycles than in the CHU
10 medium. Camera lucida drawings and photomicrographs
of these results are shown for strain-MA4 in BG110 medium
(Fig. 3) and CHU 10 medium (Fig. 4) and for strain-A8 in
Figs 5 (BG110 medium) and 6 (CHU 10 medium). The two
strains compared showed very different life cycles indepen-
dent of the culture medium used.
Strain-MA4 had a developmental cycle in which three
different stages could be defined: stage 1 represents the
development of new hormogonia with little conical terminal
cells with a sheath gel enclosing the cells (Figs 3 and 4). In
stage 2, the terminal cells begin to differentiate into terminal
heterocysts, while divisions of the intercalary cells form the
trichome; intercalary heterocysts begin to differentiate at
this stage. Stage 3 is characterized by the elongation of the
trichome due to the division of intercalary cells and by the
formation of strongly coiled filaments with the subsequent
differentiation of intercalary heterocysts; sheath gel con-
tinues enclosing the filament. This stage lasts for several
days, in which cell division continues producing long coiled
filaments until the mature filament breaks up at the posi-
tions of intercalary heterocysts, releasing motile hormogo-
nia in the process, while the heterocysts remain attached on
agar medium in their original positions. The cycle is
repeated for every new hormogonium.
A greater variability was found in strain-A8 (Figs 5 and
6), although a general cyclic sequence could be established
based on camera lucida drawings (Fig. 5). Stage 1: the
cells in hormogonia start to grow and divide; stage 2:
Table 1. Morphological characteristics of cells in Nostoc isolates cultured in BG110and CHU 10 media
Culture
MA4-Nostoc UAM 3O7 A8-Nostoc UAM 308
BG110 CHU 10 BG110 CHU 10
Breadth (mm) Length (mm) Breadth (mm) Length (mm) Breadth (mm) Length (mm) Breadth (mm) Length (mm)
VC
Mean� SD 5.9� 0.8�� 6.2� 1.3�� 4.7� 0.5�� 5.2�1.0�� 4.3�0.5� 5.4�0.9 3.4�1.0� 4.7� 1.1
Range 4–6.5 5–7 4–5.4 4.5–6 4–4.8 4–7 3–4 (5) 4–6
THe
Mean� SD 5.9� 0.9� 6.5� 1.3 4.6� 0.7� 6.3�0.6 4.3�0.4 5.4�1.1 3.7�0.8 4.7� 0.8
Range 4.5–6.5 5–7 4–5.8 5–7 4–4.8 4.8–7 3–4 (5) 4–6
IHe
Mean� SD 6.5� 0.7 7.5� 1.0� 6.0� 0.4 6.0�0.7� 4.6�0.7 5.8�0.8 4.1�0.4 5.3� 0.5
Range 5.5–7.5 5–8 5.5–6.5 5.5–6.5 4.5–5.5 5–7 3.5–4.5 4–6
Ak
Mean� SD 6.8� 0.9� 8.3� 1.4 5.6� 0.7� 8.7�1.0 5.1�0.7 6.5�0.8 4.9�0.2 6.8� 1.7
Range 5.5–8 7–10 5–7 8–11 4–6 6–8 4.5–5 5–9
HoC
Mean� SD 4.7� 0.3 5.4� 1.3 4.1� 0.8 5.5�0.6 3.0�0.3� 8.2�2.1� 2.2�0.3� 3.9� 0.5�
Range 4.5–5 4.5–6.5 4–5 4.5–6.2 2.7–3.6 3.2–12 2–2.5 3.3–4.6
NCHo
Mean� SD 53� 24�� 69� 16�� 24�10 28�16
Range 43–128 55–96 10–43 18–38
Mean� SD; range.��Significant differences (Po 0.001) in micromorphometric measurements in the culture media studied.�Significant differences (Po 0.05) in micromorphometric measurements in the culture media studied.
VC, vegetative cells; THe, terminal heterocysts; IHe, intercalary heterocysts; Ak, Akinetes; HoC, hormogonia Cells; NCHo, number of cells in
hormogonia.
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189Life cycle in the evaluation of Nostoc diversity
differentiation of terminal heterocysts begins at the same
time as the intercalary ones; stage 3: the intercalary cells
continue to increase in numbers by successive divisions,
producing clusters of cells within the parent sheath (Figs 5
and 6a, b, e, g), while the heterocysts continue to differenti-
ate. In this stage, morphological variations can be observed
(Fig. 6): elongation of curled filaments by successive division
of cells produces shorter (Figs 5 and 6a and h) or longer
contorted filaments (Fig. 6c and d), while the differentiation
of new heterocysts continues (Fig. 5). Some filaments show
crowding and apparent true branching (Fig. 6f, h, i).
Development continues until the filament breaks up next
to the intercalary heterocysts releasing hormogonia, while
the heterocysts remain in their original positions (Fig. 5).
New hormogonia may also form coils (Fig. 6j).
Differences in the developmental pattern of heterocyst differ-
entiation between both strains were also observed (Fig. 7). A
similar pattern was observed in both media for each studied
strain, although it varied from medium to medium by the
number of days needed for completion. The process in BG110
was selected as representative in Fig. 7: strain-MA4 showed a
gradual increase in the number of vegetative cells between
heterocyst (Fig. 7a) in contrast to strain-A8, which showed a
constant relationship between vegetative cells and heterocysts,
during the first days of development and subsequent exponen-
tial growth before the release of new hormogonia (Fig. 7b).
Pigment analysis
Both strains exhibited the presence of the pigment C-
phycoerythrin in both media under study (data not shown).
Phylogenetic position of Nostoc strains
The sequence of the 16S rRNA gene was determined for the
two isolates (1485 bp of strain-MA4 and 1455 bp of strain-
A8). When aligned with other cyanobacterial 16S rRNA gene
Fig. 3. Camera lucida drawings of the life-cycle
stages of Nostoc strain-MA4 UAM 307 in BG110
medium. Scale bar = 10 mm.
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190 P. Mateo et al.
sequences from the databases, our isolates clustered with
strains of the genus Nostoc in a cluster separated from
clusters with the genera Anabaena, Aphanizomenon and
Nodularia (Fig. 8). In the phylogenetic tree, strain-MA4
and strain-A8 were separated in different branches using
distance, MP and ML, with high bootstrap support on both
distance and likelihood trees, and a percentage of similarity
of 96% between both, indicating that our molecular data
were congruent with our morphological characterization.
Strain-A8 fell into a well-supported clade (clade A, Fig. 8)
containing strains of Nostoc commune, Nostoc punctiforme,
Nostoc calcicola, lichen phycobionts as well as other soil
representatives of Nostoc with sequence similarity ranging
from 97.1% to 100%, whereas MA4 was included in a clade
with strains of Nostoc muscorum and Nostoc sp. 8919 sharing
97.7–99.9% similarity (clade B, Fig. 8)
Discussion
Effect of culture conditions on morphologicalvariability
The results of our study showed that some morphological
characteristics, such as trichome shape and cellular dimen-
sions, vary depending on the culture medium used, whereas
other properties, such as life cycle, remain stable for each
strain independent of the culture conditions. Morphological
variability depending on the culture conditions has been
reported frequently (Whitton, 2002). Cultivated strains of
nominally the same morphospecies are often significantly
different from each other, either because of misidentifica-
tions or due to the loss of some properties during cultivation
under laboratory conditions (Komarek & Anagnostidis,
1989; Whitton & Potts, 2000; Wright et al., 2001).
Studies dealing systematically with the effect of growth
conditions on the morphology of cyanobacteria are few.
Within the Nostocales, these studies focused mainly on
species of Anabaena (Stulp & Stam, 1982; Zapomelova
et al., 2007, 2008a, b, 2010) and on the relationships between
genera Scytonema and Tolypothrix (Golubic & Kann, 1967;
Hoffmann & Demoulin, 1985; Zapomelova et al., 2008b). In
previous studies (Berrendero et al., 2008), we had also found
changes in morphology in Rivularia with regard to nutrients
in culture media. One of the possible approaches is to
establish the distinction between properties that vary under
different growth conditions from those that remain stable.
The other includes the attempt to construct media and other
Fig. 4. Photomicrographic documentations of the life cycle of Nostoc strain-MA4 UAM 307 in CHU 10 medium. Numbers indicate the day in the
sequence. Note that the heterocysts remain in the same position after the release of new hormogonia (day 21). Scale bar = 10 mm.
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191Life cycle in the evaluation of Nostoc diversity
culturing conditions as similar as possible to the conditions
in the environment of the origin of isolates. Some environ-
mental variables, such as temperature and light–dark re-
gimes, may be important to reproduce in culture; however,
it has been argued that the culture medium is of special
importance regarding the concentrations of nutrients
(Whitton & Potts, 2000; Whitton, 2002). It has been shown
that prolonged maintenance of heterocystous cyanobacteria
in a nitrate-containing medium (e.g. BG 11) may lead to the
selection of mutants, which have lost the ability to fix
nitrogen aerobically; they either formed abnormal hetero-
cysts or became aheterocystous (Rippka et al., 1979; Ques-
ada et al., 1992). Many studies and routine subculturing in
culture collections use media with too much phosphate to
ever become limiting during batch culture studies. This
means that morphological features influenced by phos-
phorus limitation are never seen (Whitton, 2002). Because
both of our culture media used had a lack of nitrate, on the
basis of the nitrogen-fixation ability of Nostoc, the vari-
ability found appears to be related to differences in the
P concentration. However, further studies based on gradient
P concentration assays are necessary to explain cause–effect
relationships.
Life cycle as a stable trait
Our results showed that the differences in the life cycle
between the studied strains remained consistent in both
media used, and confirm previous suggestions that life
cycles represent physiological and genetic capacities that are
unknown in other genera (Komarek & Anagnostidis, 1989;
Potts, 2000). Strain-A8 showed more variability with respect
to the developmental stages than strain-MA4. In the first
stages of the development, strain-A8 produced clusters of
four cells within the parental sheath: the tetrad stage (Figs 5
and 6). Lazaroff & Vishniac (1961) observed this special
stage and called it the ‘aseriate stage’; they suggested that
aseriate packets of cells differed from cells of hormogonia in
structure as well as in the plane of division, and functioned
as a spore-like reproductive stage, which could continue to
grow slowly in an unfavorable environment (Lazaroff,
1973). However, Mollenhauer et al. (1994) considered the
term inadequate and agreed with previous suggestions
(Geitler, 1932) that these cells divide in a single plane, but
are pushed together by the confinement and external
pressure of the envelopes, as it was evident in our results
(Fig. 6a). Herdman et al. (2001) also rejected the interpreta-
tion of three-dimensionally appearing packages by division
in a plane parallel to the longitudinal axis, citing the lack of
lateral heterocysts as evidence (lateral heterocysts are com-
mon in true branching of stigonematalean cyanobacteria).
Our study showed the appearance of true branching in
some trichomes of the strain A8 (Fig. 6h and i). The
occurrence of occasional true branching was reported by
Mollenhauer (1970), indicating an affinity to branching
cyanobacteria (Dodds et al., 1995). A mutant of N. muscor-
um exhibited true branching (Razdan & Dikshit, 1983).
Lazaroff (1973) suggested the existence of a general devel-
opmental cycle for Nostoc isolates that release hormogonia,
forming an aseriate stage in contrast to those Nostoc, which
do not form hormogonia and did not present this stage. The
suggestion was discussed not only on the basis of his
experiments but also on similar results found by Robinson
& Miller (1970) for N. commune and the study of Kantz &
Bold (1969), in which the authors attributed a punctifanue
(aseriate) stage to the 14 species of Nostoc examined. Other
differences in strain A8 regarding previous studies of
life cycles in Nostoc strains (Lazaroff & Vishniac, 1961;
Robinson & Miller, 1970) are that in those studies, the
gelatinous envelopes enclosing each filament cluster break
Fig. 5. Camera lucida drawings of the life-cycle sequence of Nostoc
strain-A8 UAM 308 in BG110 medium showing different stages. Scale
bar = 10 mm.
FEMS Microbiol Ecol 76 (2011) 185–198c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
192 P. Mateo et al.
and packets of aseriate cells are released, in contrast to
strain-A8, in which, after a short ‘aseriate’ stage, loops of
filaments between heterocysts are released by the disruption
of enclosing sheath, growing, and in a subsequent stage,
filaments break, leaving the heterocyst in the same place (see
Fig. 5).
In contrast, strain-MA4 developed linear hormogonia and
never presented the tetrad stage (Figs 2–4). Furthermore, clear
Fig. 6. Photomicrographs of life cycle of the more
variable Nostoc strain-A8 UAM 308: hormogonium
development from the first day of its release to the
12th day in CHU 10 medium (a); general view of
variability with different stages (b); long contorted
trichome similar to the (b) center (c); meandering
trichomes with intercalary and terminal heterocysts
(d, h); apparent ‘multiseriate’ stage (e, g); apparent
true branching (f, i, h) and coiling young hormogonia
(j). Scale bar in (b) = 50 mm, in all other
pictures = 10mm.
FEMS Microbiol Ecol 76 (2011) 185–198 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
193Life cycle in the evaluation of Nostoc diversity
differences were found compared with previous studies:
intercalary heterocysts differentiated in the firsts stages of the
development (Fig. 3) in contrast to N. muscorum-A and N.
commune 584, where the formation of intercalary heterocysts
occurred only in an advanced stage of packed cells (Lazaroff &
Vishniac, 1961; Robinson & Miller, 1970).
Distinctive characteristics of life cycles in Nostoc have been
proposed and discussed in the delimitation of species (Mollen-
hauer et al., 1994; Potts, 2000; Kondratyeva & Kislova, 2002).
Complicated life cycles were described in detail for some strains,
such as, for example, N. muscorum (Lazaroff, 1973), N.
commune (Robinson & Miller, 1970; Potts, 2000), and field
colonies of Nostoc sphaericum (Becerra-Absalon & Tavera,
2009) and Nostoc cordubensis (Prosperi, 1989). Hrouzek et al.
(2003) studied selected strains isolated from soils, not forming
distinct macroscopic colonies and growing mostly in slimy
mats, and they resolved three groups of strains, corresponding
to N. muscorum, Nostoc edaphicum and N. calcicola, respec-
tively, on the basis of their life cycle.
Phylogenetic analysis
In further studies, Hrouzek et al. (2005) also included in
morphological and phylogenetic analysis (16S rRNA gene)
new isolates of symbiotic origin as well as free-living strains,
showing many more clusters, thus indicating a wide genetic
diversity within the genus Nostoc (Hrouzek et al., 2005;
Rajaniemi et al., 2005; Papaefthimiou et al., 2008). In our
phylogenetic approach, Nostoc MA4 fell into the cluster
containing two N. muscorum strains from such previous
studies: cluster II of Papaefthimiou et al. (2008) and cluster
C of Hrouzek et al. (2005), with a similarity of 99.5% with
both strains. However, the life cycles described for those
strains in which granulated akinetes in long chains appeared
as typical of the N. muscorum group as defined by Hrouzek
et al. (2005) do not correspond to the life cycle of strain-
MA4 described in the present work.
Regarding the phylogenetic position, the strain-A8
plotted within a large cluster (Fig. 8 cluster A) together with
two strains of N. calcicola (Hrouzek et al., 2005; Rajaniemi
et al., 2005; Papaefthimiou et al., 2008), with N. punctiforme
PCC 73102, and N. commune from databases: cluster I of
Papaefthimiou et al. (2008) and cluster A of Hrouzek et al.
(2005). Similar clustering has been found in other Nostoc
studies (Rehakova et al., 2007) in which they found, in all
trees generated, the Nostoc clade containing N. commune
and Nostoc PCC 73102, lichen phycobionts and terrestrial
representatives of Nostoc. In our tree, cluster A was divided
Fig. 7. Change in the number and relations of vegetative cells (�) and heterocysts (�) during the course of development of Nostoc MA4 UAM 307 (a)
and Nostoc A8 UAM 308 (b), expressed as total numbers and as percentage of heterocysts of the number of cells in each filament. Bars show SD. Values
from BG110 medium are shown as representative. A similar developmental pattern was found for each strain in both the media studied.
FEMS Microbiol Ecol 76 (2011) 185–198c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
194 P. Mateo et al.
into two subclusters: A1 and A2. Nostoc strain-A8 was
included in subcluster A2, sharing high sequence similarity
(98.6–100%) to the components of this subcluster (Fig. 8).
However, only 96.4–98.3% of similarity was found between
the strain-A8 and the components of the subcluster A1,
indicating a higher genetic divergence regarding the compo-
nents of this subcluster. A threshold of 97.5% 16S rRNA
gene sequence similarity was suggested to separate prokar-
yotic species (Stackebrandt & Goebel, 1994) on the basis of
the fact that when two strains have genetic identity below
97.5%, they consistently have DNA–DNA hybridization
values below 70%, which has been used as a criterion for
recognizing bacterial species (Wayne et al., 1987). More
recently, Stackebrandt & Ebers (2006) suggested an increase
from 97.0 to 98.7–99.0% 16S rRNA gene sequence similarity
for the threshold to delineate separate species, but also
indicated that strains may belong to different genospecies
even if they share 100% 16S rRNA gene sequence similarity.
The evidence that different stable phenotypes of cyanobac-
teria are not reflected in 16S rRNA gene sequence compar-
isons is accumulating: this has been reported for
Merismopedia-like isolates (Palinska et al., 1996), Microcystis
strains (Otsuka et al., 1999), Nodularia spp.(Lehtimaki et al.,
2000; Lyra et al., 2005) and Leptolyngbya spp. (Casamatta
et al., 2005).
In our study, we found genetic similarity of our isolates
and other Nostoc from the database, but not phenotypic
coincidences. In addition, the habitat from where the strains
of this study were isolated was different from those found
for genetically similar strains. The importance of niche for
Fig. 8. NJ tree based on analysis of the 16S rRNA
gene showing the position of the sequences
obtained from the present study (in bold).
Numbers near nodes indicate bootstrap values
Z65% for NJ, ML and MP analyses.
FEMS Microbiol Ecol 76 (2011) 185–198 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
195Life cycle in the evaluation of Nostoc diversity
delimiting species has been discussed in the literature
(Mollenhauer et al., 1994; Komarek, 1985; Rehakova et al.,
2007). Accordingly, the habitat specificity could be a tax-
onomically informative characteristic, which needs to be
related to molecular, morphological and specific ecophysio-
logical properties.
It is clear that there are numerous limitations with the
current molecular characterization and identification of
Nostoc. On the one hand, on the basis of phylogenetic
analyses of the 16S rRNA gene, it has been found to be a
very wide and heterogeneous group, which may contain
more than one genus (Rajaniemi et al., 2005; Rehakova et al.,
2007; Komarek, 2010a). On the other, some Nostoc
genotypes seem to be phenotypically more diverse than can
be concluded from the 16S rRNA gene sequence data.
Clearly, 16S rRNA alone has a limited resolution
power to distinguish between cyanobacterial species as
traditionally based on phenotypic properties and ecology.
Cultures are important for taxonomic studies of cyanobac-
teria, especially when the isolates were not obtained from
macroscopic populations. However, because they vary de-
pending on the growth conditions, the studies of their
variability in culture need to identify the stable traits in
conjunction with the molecular characterizations also in-
cluding considerations of the habitat from which they were
isolated.
Studies of cyanobacterial populations complemented by
developmental studies in culture provide valuable informa-
tion on their cell differentiation and variability. This infor-
mation allows outlining and recognition of taxonomic
entities that are ecologically meaningful. The complexity
and variability of river and stream habitats have discouraged
systematic investigation. Consequently, much less is known
about the cyanobacterial diversity of such environments
than that of other ecosystems. The main difficulties in
studying cyanobacterial diversity are their morphological
variability and degree of polymorphism depending on
different geographical locations. As a result, the character-
ization and identification of cyanobacterial diversity is often
only possible using a combination of molecular, morpholo-
gical and ecophysiological approaches. The results of this
study show that a balanced approach to the evaluation of
diversity of Nostoc in the service of autecological studies
requires both genotypic information and the evaluation of
stable traits, such as the life cycle.
Acknowledgements
This work was supported by grants from Ministerio de
Educacion y Ciencia, Spain (CGL2004-03478/BOS,
CGL2008-02397/BOS) and from the Comunidad Autonoma
de Madrid (S-0505/AMB/0321 and S2009/AMB-1511). Spe-
cial thanks to Tomas Merino for technical assistance with
drawings.
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