Microb Ecol (2008) 55:608618

DOI 10.1007/s00248-007-9304-4

ORIGINAL ARTICLE

Morphological and Phylogenetic Analysis

of Anabaenopsis abijatae and Anabaenopsis
elenkinii (Nostocales, Cyanobacteria)
from Tropical Inland Water Bodies
Andreas Ballot & Pawan K. Dadheech & Sigrid Haande &
Lothar Krienitz

Received: 28 June 2007 / Accepted: 9 July 2007 / Published online: 18 August 2007
# Springer Science + Business Media, LLC 2007

Abstract Anabaenopsis spp. are heterocytous cyanobacteria commonly found in tropical, subtropical, and temperate
water bodies. So far, the knowledge about the phylogeny
of this genus is poor. Therefore, we have isolated 15
Anabaenopsis spp. strains from Kenyan and Mexican
alkaline and saline water bodies and from a Ugandan
freshwater body and studied the morphology and phylogeny in a polyphasic approach. Morphologically, the investigated strains could be discriminated in two groups. One
group was containing six Anabaenopsis abijatae and A. cf.
abijatae strains with up to more than 500 vegetative cells in
one filament, mostly single intercalary heterocyte formation, and the ability to branch out. The other group
comprised nine strains of Anabaenopsis elenkinii with short
filaments with up to 38 vegetative cells, intercalary heterocytes in pairs, and no ability to branch out. The morphological differences were reflected in the two distinct clusters,
which were found in the phylogenetic trees of 16S rDNA
A. Ballot (*) : L. Krienitz
Leibniz-Institute of Freshwater Ecology and Inland Fisheries,
Alte Fischerhtte 2,
16775 Stechlin, Germany
e-mail: ballot@igb-berlin.de
L. Krienitz
e-mail: krie@igb-berlin.de
P. K. Dadheech
Post-Graduate Department of Botany, Government College,
Ajmer 305001 Rajasthan, India
e-mail: pdadheech@rediffmail.com
S. Haande
Norwegian Institute for Water Research,
Gaustadallen 21,
N-0349 Oslo, Norway
e-mail: sigrid.haande@niva.no

and PC-IGS. While the high 16S rDNA similarity values

>97.5% found between all investigated A. abijatae and A.
elenkinii strains support the assignment of these two species
to one single genus, the morphological differences and the
low similarity values (<87.3) found in PC-IGS sequences
between the two clusters indicate two separate genera. A
close morphological and phylogenetic relationship was found
for A. abijatae and Anabaenopsis (Cyanospira) rippkae.

Introduction
The genus Anabaenopsis includes filamentous and heterocytous cyanobacteria. Originally, the genus was established
as a new section within the genus Anabaena [55], and later,
the new genus Anabaenopsis was created by Miller [34].
Heterocytous cyanobacteria are of monophyletic origin
based on investigations of 16S rDNA [50, 53, 54], and they
are included in section IV and section V in the classification
of the cyanobacteria defined by Rippka et al. [42, 43],
corresponding to the orders Nostocales and Stigonematales
of the Botanical code of Nomenclature [1, 26]. The section
IV of the cyanobacteria combines heterocytous filamentous
cyanobacteria that divide in one plane only [42], and section
V includes filamentous hetercytous cyanobacteria with cell
divisions in more than one plane [1, 42].
The phenotypic diversity of the genus Anabaenopsis has
been described by Jeeji-Bai et al. [22, 23] and revised by
Komark [25]. The type species of the genus is Anabaenopsis
elenkinii (Miller 1923). Anabaenopsis spp. are characterized
by mostly solitary free-floating filaments growing in irregular
or regular spirals or screw-like coils, and straight filaments
are rarely found [22]. Morphologically, Anabaenopsis spp.
resemble planktic Anabaena and Cylindrospermopsis species

Phylogeny of Anabaenopsis abijatae and Anabaenopsis elenkinii

but can be distinguished by a different and specific heterocyte

development as these are intercalary spherical, oval, or ovoid
and always in pairs. The filaments often disintegrate between
a pair of heterocytes, leading to trichomes with terminally,
secondary heterocytes [25, 26]. The genus comprises planktic
species with a main distribution in alkaline saline and
freshwater habitats in tropical and subtropical regions, e.g.,
Eastern Africa, India, Central Asia, and the Philippines [25],
whereas some findings of Anabaenopsis spp. are described
from temperate regions like Denmark, Germany, Greece,
Spain, and USA [9, 18, 28, 29, 35, 44]. Anabaenopsis
elenkinii is frequently found in the phytoplankton communities of East African alkaline saline lakes [2, 4, 33, 51, 52].
Ballot et al. [2, 4] have found Anabaenopsis abijatae Kebede
et Willn in three different alkaline Kenyan lakes, the Rift
Valley lakes Elmenteita and Nakuru and the volcanic craterlake Simbi. This species has been described for the first time
from Ethiopian alkaline saline Lake Abijatae by Kebede and
Willn [24]. In contrast to most Anabaenopsis spp., Anabaenopsis abijatae is characterized by irregularly coiled trichomes, which form botryoid-shaped macroscopic colonies
of up to 200 m in diameter [24, 25]. Cyanospira rippkae
Florenzano et al. [12], which is now included into the genus
Anabaenopsis as Anabaenopsis rippkae (Florenzano et al.)
comb. nova by Komark [25], was first detected in Lake
Magadi by Florenzano et al. [12].
Only a few studies have dealt with the phylogeny of
Anabaenopsis spp. Using sequences of the 16S rRNA gene
and RFLP of the 16S rRNA gene Iteman et al. [21] and
Rajaniemi et al. [41] have shown that members of the
genera Anabaenopsis, Nodularia, and Cyanospira are
closely related and are clearly separated from clusters formed
by species of Anabaena, Aphanizomenon, Nostoc, and
Cylindrospermopsis. Even the assignment of Anabaenopsis
and Cyanospira spp. to the same genus was suggested due to
the low sequence divergence between 16S rDNA of
Anabaenopsis and Cyanospira strains [21, 41]. However,
as the number of variable positions in the16S rDNA is low
and multiple copies of the gene often are present in one cell,
the suitability of 16S sequences for studying the relationship
between closely related species of cyanobacteria is widely
discussed [8, 13, 14, 37]. Therefore, the intergenic spacer of
the phycocyanin operon (PC-IGS) has been introduced for
the purpose of delineating inter- and intraspecific variations
within cyanobacterial genera [e.g., 6, 7, 36]). As the PC-IGS
region includes the noncoding highly variable intergenic
spacer region, it should be more suitable to show the
differences between closely related species than 16S rDNA
sequences, with more conserved regions [6, 36].
The present knowledge on the phylogeny of Anabaenopsis
spp. is very scarce. We therefore isolated 15 Anabaenopsis
spp. strains from Kenyan and Mexican alkaline saline water
bodies and a Ugandan freshwater habitat and studied the

609

strains morphologically and genetically (16S rDNA, PC-IGS)

in a polyphasic approach to increase the knowledge about
this genus.

Materials and Methods

Organisms and Culture Conditions
Fifteen strains of Anabaenopsis spp. were isolated from
water samples from Kenyan Lakes Elmenteita, Magadi,
Nakuru, Simbi and Sonachi, from Mexican Lake Texcoco
and from Kazinga Channel in Uganda (Table 1). The strains
were determined according to Kebede and Willn [24] and
Komark [25].
Using a microcapillary, single colonies of Anabaenopsis
spp. were isolated, washed, and placed in capped tubes
containing 10 ml culture medium. The cyanobacterial strains
from Kenya and Mexico were cultivated in Bourrelly
medium [19] modified with an addition of 0.3 g L1 Na2CO3
and 15 g L1 NaCl. The growth medium used for cultivation
of the Ugandan strains was the Z8 medium [27]. All strains
investigated in this study were maintained at 18C with a
photon flux density of 20 mol of photon m2 s1
(equivalent to 1500 lx). Morphological observations were
made using a Nikon Optiphot 2 light microscope (Nikon,
Tokyo, Japan).
Genomic DNA Extraction, PCR Amplification,
and Sequencing
Fresh culture material was boiled for 5 min and centrifuged.
After discarding the supernatant, the genomic DNA was
extracted and purified using Dynabeads DNA DIRECT
System I (Invitrogen/Dynal Biotech, Oslo, Norway) according to the manufacturers manual.
Polymerase chain reaction (PCR) amplification was done
using the Taq PCR Core Kit (Qiagen GmbH, Hilden,
Germany) in a Peltier Thermal Cycler PTC 200 (MJ Research,
Inc., San Francisco, USA). The reaction mixture contained
0.1 l Taq DNA polymerase (concentration 5 U per L),
0.6 L deoxyribonucleotide triphosphate (dNTP) mix
(10 mM), 2 L buffer (10 concentrated, containing Tris Cl,
KCl, (NH4)2SO4, and 15 mM MgCl2, 1 L forward and
reverse primer (10 M), and 1 L genomic DNA in a total
volume of 20 L. The primers pA and B23S were used for
the amplification of the 16S rRNA gene [11, 15], and the
primers PCf and PCr were used for the amplification of
the PC-IGS region [36].
The following program was used for amplification of the
16S rDNA fragment: 5 min at 94C for 1 cycle; 30 cycles of
30 s at 94C; 30 s at 50C; 1 min at 70C; and a final
elongation step of 72C for 3 min. For the PCR of the PC-

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A. Ballot et al.

IGS: 3 min at 94C for 1 cycle; 30 cycles of 20 s at 94C; 30 s

at 55C; 1 min at 72C with final elongation step of 72C for
5 min. PCR products were visualized by 1.5% agarose gel
electrophoresis with ethidium bromide staining and UV
illumination.
Amplified products of 16S rDNA and PC-IGS were
purified through Qiaquick PCR purification columns
(Qiagen, Hilden, Germany), and DNA was redissolved in
elution buffer according to the manufacturers protocol. The
purified product of 16S rDNA was sequenced using the
primers pA, pC, pE, pFr, pHr, [11] and a modified primer
pDr (GTC TTA CCG CGG CTG CTG). For sequencing of
PC-IGS the same primers as for the PCR were used. For
each PCR product, both strands were sequenced on ABI
3100 Avant Genetic Analyzer using BigDye Terminator
V.3.1 Cycle Sequencing Kit (Applied Biosystems, Applera
Deutschland GmbH, Darmstadt, Germany) according to the
manufacturers manual.

used as outgroup in the 16S tree and the PC-IGS tree,

respectively. For the analysis of 16S rDNA, additional 36
sequences from the GenBank were included.
Phylogenetic trees for 16S rDNA and PC-IGS were
constructed using maximum likelihood (ML) algorithm in
the program PAUP * v 10b [47]. For ML, the evolutionary
models of substitution were evaluated using the AIC
criterion in Modeltest v.3.06 [40]. The GTR+I+G evolutionary model was found to be the best fitting model for the
16S rRNA gene and the TrN+I evolutionary model to be
the best fitting model for PC-IGS. Due to limited computer
capacity for the ML analysis of the 16S tree in the program
PAUP * v 10b [47], 100 bootstrap replicates were
performed. For the ML analyses of the PC-IGS tree, 1000
bootstrap values were conducted. The sequence data were
submitted to the GenBank, and the accession numbers are
listed in Table 1.

Results
Phylogenetic Analysis
Sequences of partial 16S rRNA gene, and of parts of the
PC-IGS locus, were analyzed for all the strains. The
sequences were aligned using MS Windows based Manual
Sequence Alignment Editor Align Vers. 05/2006 [45], and
the sequence alignments were corrected manually. The
segments with highly variable and ambiguous regions and
gaps, where a proper alignment was impossible, were
excluded from the alignment. In the case of 16S rDNA, a
set containing 1301 positions was used, and the PC-IGS
alignment comprised 452 positions. Microcystis aeruginosa
(AB035549) and Nodularia spumigena (AF367146) were
Table 1 Anabaenopsis spp.
strains from different alkaline
and freshwater lakes used in
this study and genetic
properties

Alkaline saline
b
Freshwater

Strain

Species

AB2002/08
AB2002/09
AB2002/14
AB2002/15
AB2002/16
AB2002/17
AB2002/18
AB2002/25
AB2002/34
AB2002/35
AB2002/36
AB2002/37
AB2006/20

A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.

abijatae
abijatae
abijatae
abijatae
elenkinii
elenkinii
abijatae
cf. abijatae
elenkinii
elenkinii
elenkinii
elenkinii
elenkinii

NIVA-CYA 494
NIVA-CYA 501

A. elenkinii
A. elenkinii

Fifteen Anabaenopsis spp. strains isolated from Kenyan and

Mexican alkaline saline lakes and a Ugandan fresh water
body were cultivated and investigated for their morphological characteristics and their phylogenetic relationship
(Table 1). For the phycological identification of the
Anabaenopsis sp. strains, morphological features like cell
size of vegetative cells, size of heterocytes, size of akinetes,
and nature and shape of helix were taken into account
(Table 2). Two groups with different morphotypes could be
distinguished. On the basis of morphological features
observed in light microscope, the Kenyan strains AB2002/
08, AB2002/09, AB2002/14, AB2002/15, and AB2002/18

Habitat

Lake Simbia
Lake Simbia
Lake Nakurua
Lake Nakuru a
Lake Elmenteita a
Lake Nakuru a
Lake Elmenteitaa
Lake Magadia
Lake Sonachia
Lake Sonachia
Lake Sonachia
Lake Sonachia
Lake Texcoco
(Nabor Carillo)a
Kazinga Channelb
Kazinga Channelb

Country of origin

Accession Number
16S rDNA

PC-IGS

Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Kenya
Mexico

AM773295
AM773296
AM773297
AM773298
AM773299
AM773300
AM773301
AM773302
AM773303
AM773304
AM773305
AM773306
AM773307

AM773280
AM773281
AM773282
AM773283
AM773284
AM773285
AM773286
AM773287
AM773288
AM773289
AM773290
AM773291
AM773292

Uganda
Uganda

AM773308
AM773309

AM773293
AM773294

Straight-irregularly
coiled-helical
Straight-irregularly
coiled-helical
Straight-irregularly
coiled-helical
Straight-irregularly
coiled
Straight-irregularly
coiled-helical
Straight-irregularly
coiled
Coiled, straight
Coiled, straight
Regularly coiled
Regularly coiled,
straight
Regularly coiled,
straight
Regularly coiled,
straight
Coiled
Coiled
Coiled

AB2002/08

(4.0,
(4.0,
(4.0,
(4.0,

8.8)
8.8)
13.6)
11.2)

6.1 (3.2, 9.6)

8.7 (3.2, 20.0)
9.3 (6.4, 13.6)

8.1(4.8, 12.0)

6.4 (4.0, 8.8)

6.1
6.1
7.7
7.8

5.1 (4.0, 7.2)

5.6 (4.0, 8.8)

6.2 (4.8, 8.8)

5.6 (4.0, 9.6)

5.5 (3.2, 9,6)

5.5 (4.0,10.0)

(4.8,
(4.8,
(4.8,
(5.6,

7.2)
8.0)
8.0)
7.6)

4.1 (3.2, 4.8)

5.7 (4.0, 10.4)
5.3 (4.0, 8.8)

6.3 (5.6, 6.8)

6.4 (5.6, 7.2)

6.4
6.2
6.8
6.5

8.8 (7.2, 10.4)

6.6 (5.6, 8.0)

7.5 (6.4, 8,8)

8.0 (7.2, 9.6)

6.7 (5.6, 9.6)

6.8 (4.8, 9.6)

(5.6,
(4.8,
(5.2,
(5.0,

8.8)
7.2)
8.4)
11.2)

4.6 (3.6, 5.6)

5.3 (4.4, 6.4)
5.1 (4.0, 6.4)

7.1 (4.8, 9.2)

5.8 (4.8, 7.2)

6.6
5.5
6.6
8.3

8.6 (6.4, 10.4)

7.4 (5.6, 11.2)

8.2 (6.4, 11.0)

9.1 (7.2, 11.2)

8.2 (6.4, 11.2)

7.5 (4.8, 10.4)

Length (m)a

Length (m)a

Width (m)a

Heterocytes

Veg. cells

Numbers are means (minimum, maximum values);

+/ Filaments with and without capsule, n.o. not observed

AB2006/20
NIVA-CYA 494
NIVA-CYA 501

AB2002/37

AB2002/36

AB2002/16
AB2002/17
AB2002/34
AB2002/35

AB2002/25

AB2002/18

AB2002/15

AB2002/14

AB2002/09

Shape of
trichomes

Strain

(5.6,
(4.8,
(5.2,
(5.6,

8.0)
6.8)
8.4)
9.6)

4.3 (3.6, 5.2)

5.2 (4.2, 6.4)
5.0 (4.0, 5.6)

7.0 (4.8, 8.8)

5.8 (4.8, 7.2)

6.6
5.5
6.6
7.5

8.6 (6.4, 10.4)

7.4 (5.6, 11.2)

8.4 (7.2, 11.0)

9.1 (7.2, 11.2)

8.2 (6.4, 11.2)

7.5 (4.8, 10.4)

Width (m)a

8.7 (5.6, 12.0)

n.o.
n.o.

10.7 (9.6, 12.0)

10.5 (8.8, 11.2)

n.o.
n.o.
9.8 (8.8, 10.4)
13.1 (11.2, 15.2)

10.6 (8.8, 15.2)

n.o.

12.5 (8.8, 19.2)

n.o.

n.o.

n.o.

Length (m)a

Akinetes

6.5 (5.6, 10.0)

n.o.
n.o.

9.3 (8.8, 10.4)

9.9 (8.4, 11.2)

n.o.
n.o.
9.7 (8.8, 10.4)
11.5 (9.6, 13.6)

10.6 (8.8, 15.2)

n.o.

12.5 (8.8, 19.2)

n.o.

n.o.

n.o.

Width (m)a

Table 2 Morphological characteristics of Anabaenopsis spp. strains from Kenya, Mexico, and Uganda, grown under culture conditions

+
+
+
+
+
+
+/

+/
+/
+
+/
+
+
+

204
561
388
136
285
14
37
19
26
25
38
18
15
12

Capsule

233

Max. cell-nr
in trichomes

Branching

Phylogeny of Anabaenopsis abijatae and Anabaenopsis elenkinii

611

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A. Ballot et al.

were identified as Anabaenopsis abijatae during isolation.

These strains were growing as free-floating macroscopic
botryoidally shaped colonies with diameters up to 100 m
at the time of isolation (Fig. 1a). The trichomes were
irregularly coiled and tightly packed in mucilage. The
vegetative cells were spherical, ovoid, or tapered at one
end. The cell size was between 6836 m. Spherical or
deformed heterocytes were present, 710610 m in size.
Single filaments were not observed in the field samples,
only transitional stages between the botryoid and filamentous
form where observed in smaller colonies of A. abijatae.
Under natural conditions, the strain AB2002/25 was
growing with free-floating filaments, which were densely
aggregated but did not form botryoidally shaped colonies
(Fig. 1b). Conspicuous were long rows of spherical
akinetes, which partly possessed no cell content (Fig. 1b).
Under culture conditions, it developed similar features like
the A. abijatae strains, and accordingly, was determined as
A. cf. abijatae. In culture, all isolated strains of A. abijatae
(AB2002/08, AB2002/09, AB2002/14, AB2002/15,
AB2002/18 and AB2002/25) grew with helical, irregularly

coiled, or partly straight or straight filaments (Fig. 1c,d,

Table 2). Botryoidally shaped colonies were never observed. In all six A. abijatae strains, the filaments were
composed of gas vacuolated ovoid to spherical cells, 3.2
9.64.09.6 m in size. For all strains, the filaments
comprised more than 130 cells, and in the strain AB2002/
14, more than 500 vegetative cells were found in one
filament. All 6 A. abijatae strains were able to branch out
(Fig. 1d). Single terminal and mainly single intercalary
spherical heterocytes were observed, 4.811.2 m in
diameter. Rarely, two or three adjacent heterocytes were
observed (Fig. 1c,d,e). Terminal or intercalary spherical
akinetes were found only in the strains AB2002/15 and
AB2002/25. They were growing in rows from 1 up to 5
akinetes in the strain AB2002/15 and more than 19 akinetes
in the strain AB2002/25 (Fig. 1f). The filaments of all six
investigated strains of A. abijatae were encapsulated in thin
or thick mucilage (Table 2).
The Kenyan strains AB2002/16, AB2002/17, AB2002/
34, AB2002/35, AB2002/36, and AB2002/37, the strain
AB2006/20 from Mexico, and the strains NIVA-CYA 494

Figure 1 Micrographs of A. abijatae strains investigated in this study.

a A. abijatae: environmental botryoidally shaped colony, b A. cf.
abijatae: environmental colony from which strain AB2002/25 was
isolated, c A. abijatae (AB2002/08): coiled filament with terminal and

single intercalary heterocytes in culture, d A. abijatae (AB2002/15):

filament with true branching, e A. abijatae (AB2002/15): filament
with three adjacent heterocytes, f A. cf. abijatae (AB2002/25):
filament with 19 adjacent akinetes. (scale bars indicate 10 m)

Phylogeny of Anabaenopsis abijatae and Anabaenopsis elenkinii

Figure 2 Micrographs of A. elenkinii strains investigated in this

study. a A. elenkinii (AB2002/35): coiled filament with terminal
heterocytes and akinete, b A. elenkinii (AB2006/20): filament with
terminal heterocytes and two adjacent akinetes, c A. elenkinii (NIVACYA 501): filament with terminal heterocytes, d A. elenkinii
(AB2002/17): straight filament with intercalary heterocytes in pairs.
(scale bars indicate 10 m)

and NIVA-CYA 501 from Uganda were identified as

Anabaenopsis elenkinii (Table 1, Fig. 2a,b,c). These strains
were growing as single filaments, both in the natural
samples and under culture conditions. In culture, the
filaments of all the strains were mainly coiled, but in each
strain, a small percentage of straight filaments were
observed (Fig. 2d). Maximally 38 vegetative long ellipsoid
or long barrel-shaped cells were found in the filaments
together with spherical or ovoid terminally growing
heterocytes. Intercalary heterocytes were always growing
in pairs, but were rarely observed (Fig. 2d). Terminal or
intercalary growing spherical or oval akinetes were found
in the A. elenkinii strains AB2002/34, AB2002/35,
AB2002/36, and AB2002/37 (Fig. 2a). AB2006/20 possessed widely oval to slightly asymmetrical akinetes
(Fig. 2b), whereas no akinete production was observed for
the A. elenkinii strains AB2002/16, AB2002/17, NIVACYA 494, and NIVA-CYA 501 (Table 2).
The maximum likelihood tree of the 16S rDNA sequences
showed a tight cluster of Anabaenopsis spp. strains clearly

613

separated from all the other strains of the order Nostocales

included in this study (Fig. 3). The whole cluster was
supported by a bootstrap value of 65%. A close relationship to
members of the genus Nodularia could be observed. In the
Anabaenopsis cluster, two separate subclusters I and II could
be distinguished. These subclusters were divided in two
subclusters each: Ia, Ib, IIa, and IIb (Fig. 3). Cluster Ia
comprised the A. cf. abijatae strain AB2002/25 and cluster
Ib the A. abijatae strains AB2002/08, AB2002/09, AB2002/
14, AB2002/15 and AB2002/18 together with the strain of
Anabaenopsis rippkae (Cyanospira rippkae) (AY038036).
Cluster I was supported by a bootstrap value of 87%
(Fig. 3). All five strains of A. abijatae from lakes Elmenteita,
Nakuru, and Simbi, had identical 16S rDNA sequences
(100% similarity). The p-distances in cluster I ranged from
99.5 to 100%, with the lowest similarity found between A. cf.
abijatae AB2002/25 and A. rippkae (AY038036) (Table 3a).
Cluster II was clearly separated from cluster I. Subcluster IIa comprised the Ugandan freshwater strains NIVACYA 494 and NIVA-CYA 501 together with Anabaenopsis
sp. (AY038033) isolated from a coastal lagoon near
Valencia/Spain. Subcluster IIB included the Kenyan and
Mexican A. elenkinii strains AB2002/16, AB2002/17,
AB2002/34; AB2002/35, AB2002/36, AB2002/37, and
AB2006/20 together with Anabaenopsis sp. (AF516747).
Cluster II was supported by a bootstrap value of 90%
(Fig. 1). The p-distances in cluster II ranged from 98.2 to
100% (Table 3a).
In the PC-IGS tree, the investigated strains were
distributed in the same pattern as in the 16S tree (Fig. 4)
with two main clusters I and II. Cluster I included A. abijatae
strains AB2002/08, AB2002/09, AB2002/14, AB2002/15,
and AB2002/18 and A. cf. abijatae strain AB2002/25. The
tight cluster, which was supported by a bootstrap value of
100%, confirmed the close relationship of all six A. abijatae
strains. The p-distances (495 bp) were 100% for all six
strains. The p-distances between the strains of cluster I and
cluster II were between 85.4 and 87.3% (Table 3b). Cluster II
was divided into two subclusters. Subcluster IIa comprised
the A. elenkinii sp. strains NIVA-CYA 494 and NIVA-CYA
501 supported by a bootstrap value of 100% (Fig. 4).
Subcluster IIb included the A. elenkinii strains AB2002/16,
AB2002/17, AB2002/34; AB2002/35, AB2002/36, AB2002/
37, and AB2006/20, supported by a bootstrap value of 93%.
The similarity values in cluster II ranged from 91.9 to 100%
(Table 3b). PC-IGS sequences of other Anabaenopsis spp.
strains were not available from the GenBank.

Discussion
The Anabaenopsis strains isolated in this study were
identified as A. abijatae, A. cf. abijatae and A. elenkinii

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A. Ballot et al.

Calothrix desertica AF132779

Cylindrospermopsis raciborskii AF092504
Tolypothrix sp. AB093486
Anabaena variabilis AF247593
Nostoc muscorum AJ630451
Nostoc ellipsosporum AJ630450
Calothrix brevissima AB074504
Nostoc sp. AJ133161
Nostoc edaphicum AJ630449
86

Microcystis aeruginosa AB035549

Scytonema hofmanni AF132781

Nostoc commune AB113666
Cylindrospermum sp. AJ133163
99
Fischerella muscicola AB039003
Chlorogloeopsis fritschii AB093489
Anabaena sp. AJ133162
94 Aphanizomenon flos-aquae AY038035
Aphanizomenon gracile AJ630444
Aphanizomenon flos-aquae AJ630442
95
96
Anabaena spiroides AJ293118
77
Anabaena circinalis AF247576
Anabaena lemmermannii AJ133157
Anabaena flos-aquae AY038032
52
Aphanizomenon issatschenkoi AJ630446
Anabaena lemmermannii AJ293113
Anabaena circinalis AJ133156
Anabaena cylindrica AF247592
Anabaena bergii AF160256
Nodularia sp. AY038034
73
Nodularia baltica AJ133177
60
Nodularia spumigena AJ781134
54
Nodularia sphaerocarpa AJ781148
81
Nodularia harveyana AJ781146
a
Anabaenopsis cf. abijatae AB2002/25
87 Anabaenopsis abijatae AB2002/18
63
Anabaenopsis abijatae AB2002/15
I
b
Anabaenopsis abijatae AB2002/14
98 Anabaenopsis abijatae AB2002/09
65
Anabaenopsis abijatae AB2002/08
Cyanospira rippkae AY038036
97 Anabaenopsis sp. AY038033
a
Anabaenopsis elenkinii NIVA-CYA 494
90 60 Anabaenopsis elenkinii NIVA-CYA 501
Anabaenopsis elenkinii AB2006/20
Anabaenopsis elenkinii AB2002/16
II
87 Anabaenopsis elenkinii AB2002/17
b
Anabaenopsis sp. AF516747
54
Anabaenopsis elenkinii AB2002/37
Anabaenopsis elenkinii AB2002/36
Anabaenopsis elenkinii AB2002/35
Anabaenopsis elenkinii AB2002/34

0.1
Figure 3 Maximum likelihood tree based on partial 16S rDNA sequences of 52 cyanobacterial strains. Strains from this study are marked in bold.
Bootstrap values above 50 are included. Bar indicates 10% sequence divergence

[24, 25], and they showed different morphological characteristics and phylogenetic relationship.
Our study revealed that A. abijatae strains in culture are
characterized by morphological features which differ

conspicuously from those found under natural conditions.

The macroscopic spherical colonies described by Kebede
and Willn [24] grow in culture as single straight, irregularly
or regularly coiled filaments encapsulated in mucilage with a

Phylogeny of Anabaenopsis abijatae and Anabaenopsis elenkinii

615

Table 3 Calculated DNA similarities between cluster I and II (see

Fig. 3)
Clusters

Cluster I

a) 16S rDNA sequences (1347 bp)

Cluster I
99.5100
Cluster II
97.598.6
b) PC-IGS (495 bp)
Cluster I
100
Cluster II
85.487.3

Cluster II

98.2100

91.9100

high number of vegetative cells in one filament and mostly

solitary intercalary heterocytes. These morphological features are in contrast to those described for members of the
genus Anabaenopsis, comprising short filaments and paired
intercalary heterocytes [25, 43]. Interestingly, all five A.
abijatae strains and A. cf. abijatae (AB2002/25) have the
ability of to branch out. The cell division in more than one
plane has been described for cyanobacteria belonging to
subsection V of the proposed bacterial classification [20, 42]
or the order Stigonematales [1]. Gugger and Hoffmann [16]
have shown that the former classification most likely is not
valid and that true branching cyanobacteria are polyphyletic.
Their phylogenetic study on Stigonematales revealed that
cyanobacterial strains classified into subsections IV and V

Nodularia spumigena AF367146

Anabaenopsis abijatae AB2002/08

Anabaenopsis cf. abijatae AB2002/25
100 Anabaenopsis abijatae AB2002/15

Anabaenopsis abijatae AB2002/14

Anabaenopsis abijatae AB2002/18
Anabaenopsis abijatae AB2002/09
100 Anabaenopsis elenkinii NIVA-CYA501
Anabaenopsis elenkinii NIVA-CYA494
93

Anabaenopsis elenkinii AB2006/20

Anabaenopsis elenkinii AB2002/16

99
86

Anabaenopsis elenkinii AB2002/35

Anabaenopsis elenkinii AB2002/17

II
b

Anabaenopsis elenkinii AB2002/37

Anabaenopsis elenkinii AB2002/36
0.1

Anabaenopsis elenkinii AB2002/34

Figure 4 Maximum likelihood tree based on PC-IGS sequences of 16

cyanobacterial strains. Bootstrap values above 50 are included. Strains
from this study are marked in bold. Bar indicates 10% sequence
divergence

according to the presence or absence of true branching were

intermixed.
The morphological features of our cultured A. abijatae
strains resemble to features of Anabaenopsis (Cyanospira)
rippkae described by Florenzano et al. [12]. Members of
the genus Cyanospira have been found forming longer
trichomes with single intercalary heterocytes only [12]. The
ability to branch out is not mentioned for Cyanospira
strains [12]. Unfortunately, strains of Cyanospira sp. are not
available from the Pasteur Culture Collection (PCC) for
comparison (Rippka, personal communication).
The morphological features of the Anabaenopsis strains
AB2002/16, AB2002/17, AB2002/34, AB2002/35,
AB2002/36, AB2002/37, NIVA-CYA 494, NIVA-CYA
501, and AB2006/20 observed in our study correspond to
A. elenkinii as described earlier [22, 25, 26, 43]. The short
filaments with a maximum of 38 cells and the pairwise
intercalary heterocytes clearly distinguish these strains from
the A. abijatae strains and A. cf. abijatae strain AB2002/25
with filaments comprising more than 130 cells, mostly
single intercalary heterocytes, and with true branching.
The two distinct clusters I and II in the 16S rDNA tree
reflect the obvious morphological differences between the
Anabaenopsis strains observed in this study. However, the
range of the 16S p-distances (97.598.6%) between strains
of cluster I and strains of cluster II confirm a close
relationship of A. abijatae and A. (Cyanospira) rippkae to
other morphotypes of Anabaenopsis spp. [21, 41] and
suggests a single genus for all investigated strains.
Stackebrandt and Goebel [46] have defined a 16S rRNA
gene sequence similarity value of more than 97.5% as the
level at which two bacterial strains can be congeneric or
even could belong to the same species. On the other hand,
Oren et al. [38] described the existence of different bacterial
species sharing identical 16S rDNA. Therefore, 16S rDNA
sequences analysis is not necessarily a foolproof criterion to
guarantee species identity because closely related species
may not be recognizable with 16S rDNA as a genetic
marker [13, 38].
Even 16S rDNA sequences of Nodularia spp. and our
investigated Anabaenopis spp. strains share 16S rDNA
similarity values between 96.1 and 98.5% (data not shown).
The Nodularia strains were isolated from the Baltic See
with a brackish to marine environment [31]. The highest
similarity values ranging between 97.8 and 98.5% are found
between our strains and benthic Nodularia harveyana
(AJ781146). These findings suggest that either the defined
16S rRNA gene sequence similarity value of 97.5% [46] is
too low to define a genus, or members of the genera
Nodularia and Anabaenopsis are very closely related and
could be combined into one genus. The phylogenetic
investigation of morphologically distinguishable Anabaena
and Aphanizomenon strains resulted in high 16S rDNA

616

similarity values >97.5%, and a single genus was suggested

[31, 41].
Cluster I in our 16S rDNA tree reveals that Cyanospira
sp. is more related to A. abijatae than to other Anabaenopsis spp. with features resembling to A. elenkinii. It is
supported by a bootstrap value of 98% and a high DNA
similarity of 99.9%. A close relationship between members
of the genus Anabaenopsis and Cyanospira was found by
Iteman et al. [21] and Rajaniemi et al. [41], and a merging
of the two genera due to low sequence differences was
suggested. However, RFLP analysis of 16S rDNA showed
a closer relationship of Cyanospira spp. to Nodularia sp.
than to Anabaenopsis spp. with a true Anabaenopsis
morphotype [21].
The PC-IGS tree confirms the clustering found in the
16S rDNA tree, with one main cluster comprising A.
abijatae strains and the A. cf. abijatae strain AB2002/25,
and the other main cluster consisting of all other investigated Anabaenopsis strains. The p-distances with values
below 87.3% are much lower than those measured for 16S
rDNA sequences and do not support a close relationship
between Anabaenopsis strains of clusters I and II. Different
genotypes have also been found within several cyanobacterial genera like Microcystis, Cylindrospermospis, and Nodularia using PC-IGS sequencing [5, 10, 17, 49]. However,
PC-IGS similarity values measured in these genera were not
lower than 93.6% for Microcystis strains, 93.4% for
Nodularia strains, and 94.5% for Cylindrospermopsis strains
[5, 7, 10].
The DNA similarity of 100% of the PC-IGS sequences
between all five A. abijatae strains and A. cf. abijatae strain
AB2002/25 is higher than the similarity value of 99.6%
found for the 16S rDNA sequences of A. abijatae and A. cf.
abijatae strain AB2002/25. The high PC-IGS similarity
value supports the classification of the strain AB2002/25 as
A. abijatae. However, limited nucleotide variation in the
PC-IGS was found also for distinct morphospecies groups
of Nodularia and Phormidium [7, 48]. In the case of
Phormidium, identical PC-IGS sequences have been found
for closely related species, and a differentiation in species
or strains was not possible [48]. Accordingly, for the
discrimination of A. abijatae and Anabaenopsis sp. strain
AB2002/25 sequencing of PC-IGS seems to be a limited
tool only.
Our results suggest that beside morphology, the ecological background of the Anabaenopsis strains defines the
phylogenetic clustering in the 16S rDNA and PC-IGS tree.
The freshwater strains NIVA-CYA 494 and 501 are
forming subcluster IIa in both trees. The Anabaenopsis
strain PCC9215, which is included in subcluster IIa in the
16S tree was isolated from a coastal lagoon near Valencia/
Spain [39]. It is not clear if this habitat is saline or
freshwater, but most likely not alkaline. The Anabaenopsis

A. Ballot et al.

sp. strains from alkaline habitats are grouped in the distinct

cluster I or subcluster IIb. Genetic distinction is described
for several cyanobacterial genera (Nodularia, Spirulina,
Geitlerinema) living in habitats with different alkalinity
and salinity [e.g., 3, 30, 32]. However, Arthrospira spp.
from freshwater and alkaline saline habitats showed no
phylogenetic differences [3].
In summary our polyphasic approach, including morphological and phylogenetic data, gives contradictory
results for the classification of members of the genus
Anabaenopsis. The morphological data and the PC-IGS
sequencing data do not support a close relationship between
strains of A. abijatae and A. elenkinii. On the other hand,
the 16S rDNA similarity values above 97.5% between all
studied Anabaenopsis strains indicate one single genus for
all investigated species. According to our findings Anabaenopsis (Cyanospira) rippkae is more closely related to A.
abijatae than to other strains with a clear Anabaenopsis
morphotype.

Acknowledgements The authors thank the authorities of the

Republic of Kenya, especially the Ministry of Education Science
and Technology for providing research permission (No. MOEST 13/
001/31 C90). We thank the Uganda National Council for Science and
Technology for providing research permit (No. UNCST-EC584) and
Eberto Novelo and La Gerencia del Lago de Texcoco, CNA, Mxico,
for permission to collect phytoplankton samples. We thank Randi
Skulberg for providing the two NIVA-CYA strains from the NIVA
Culture collection of Algae. We are grateful to the German Federal
Ministry of Education and Research for financial support (grant No.
BIOLOG 01LC0001).

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