Life cycle as a stable trait in the evaluation of diversity of Nostoc from biofilms in rivers

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: [email protected]

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

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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.


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.


188 P. Mateo et al.

http://www.ncbi.nlm.nih.gov/BLAST

http://www.ncbi.nlm.nih.gov/BLAST

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.



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.


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.



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.


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.



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.


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.



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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Life cycle as a stable trait in the evaluation of diversity of Nostoc from biofilms in rivers

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