J. Phycol.

45, 928937 (2009)

 2009 Phycological Society of America
DOI: 10.1111/j.1529-8817.2009.00701.x

HETEROGENEITY OF THE CYANOBACTERIAL GENUS SYNECHOCYSTIS

AND DESCRIPTION OF A NEW GENUS, GEMINOCYSTIS 1
Jana Korelusova 2, Jan Kas tovsky
eske Budejovice, Czech Republic
University of South Bohemia, Faculty of Sciences, Institute of Botany, Branisovska 31, CZ-370 05 C

and Jir Komarek

eske Budejovice, Czech Republic
University of South Bohemia, Faculty of Sciences, Institute of Botany, Branisovska 31, CZ-370 05 C
Academy of Sciences of the Czech Republic, Institute of Botany, Dukelska 135, CZ 379 82 Trebon
, Czech Republic

The study and revision of the unicellular cyanobacterial genus Synechocystis was based on the type
species S. aquatilis Sauv. and strain PCC 6803, a reference strain for this species. Uniformity in rRNA
gene sequence, morphology, and ultrastructure was
observed in all available Synechocystis strains, with
the exception of the strain PCC 6308, which has
been considered by some to be a model strain for
Synechocystis. This strain differs substantially from
the typical Synechocystis cluster according to both
molecular (<90% of similarity, differences in
16S23S rRNA internal transcribed spacer [ITS] secondary structure) and phenotypic criteria (different
ultrastructure of cells). This strain is herein classified into the new genus Geminocystis gen. nov., as a
sister taxon to the genus Cyanobacterium. Geminocystis
differs from Cyanobacterium by genetic position
(<94.4% of similarity) and more importantly by its
different type of cell division. Because strain PCC
6308 was designated as a reference strain of the Synechocystis cluster 1 in Bergeys Manual, the members
of this genetic cluster have to be revised and reclassified into Geminocystis gen. nov. Only the members
of the Synechocystis cluster 2 allied with PCC
6803 correspond both genetically and phenotypically
to the type species of the genus Synechocystis
(S. aquatilis).

University of Texas at Austin, USA; WHOI, Woods

Hole Oceanographic Institute

Taxonomic classification is required for the recognition and evaluation of across-phylum biodiversity in cyanobacteria. Recently, 16S rRNA gene
sequence similarity has been an important component of recognition of cyanobacterial genera. When
combined molecular and phenotypic analyses of
cyanobacteria indicate that strains are not correctly
placed in the current systematic classification (i.e.,
their taxonomic designations cause taxa to be polyphyletic or paraphyletic), it is necessary to change
the names of the strains, even if it requires new taxa
to be named. However, nomenclature principles
(with application of scientific names) are still used
indispensably for correct designation of cyanobacterial taxa in both bacteriological and botanical studies, and consequently, revision must be done in
accordance with nomenclatoral rules (Oren 2004).
The simple cyanobacterial genus Synechocystis was
originally described as a botanical taxon by Sauvageau (1892) with the type species being S. aquatilis.
It is clearly defined morphologically: coccoid spherical cells with parietal thylakoids living solitary in various habitats and dividing into two perpendicular
planes in subsequent generations. Later authors
supplemented the descriptions of this genus by the
presence of a hexagonal S-layer in the cell wall and
division by pinching (Smarda et al. 1979, Vaara
1982, Komarek 1996). A total of 25 species were
described, which differ only by cell size and ecology
(Komarek and Anagnostidis 1998, Komarek and
Hauer 2009: http://www.cyanodb.cz). Numerous
strains were isolated recently, and the monophyly of
the Synechocystis cluster was supported by molecular
analyses (Castenholz 2001, Korelusova 2005, Rajaniemi-Wacklin et al. 2005). The simple morphology
and easy transfer in culture allowed for the use of
several Synechocystis strains as important models for
experimental work. Strain PCC 6803 was the first
cyanobacterial strain for which the total genome
was sequenced (Kaneko et al. 1995). Important

Key index words: 16S rDNA; Cyanobacterium;

Geminocystis; ITS; phylogeny; Synechocystis; taxonomy; thylakoids; ultrastructure
Abbreviations: ATCC, American Type Culture Collection (Manassas, VA, USA); BRNM HY, Czech
Central Herbarium of Algae, Moravian Museum,
Brno, Czech Republic; CALU, Collection of Algae
at Biological Institute, St. Petersburg, Russian
Federation; CCAP, Culture Collection of Algae
and Protozoa (Far Sawrey, Great Britain); GC,
guanin cytosin; ITS, internal transcribed spacer;
NCBI, National Center for Biotechnology Information; PCC, Pasteur Institute Algae Collections;
UTEX, The Culture Collection of Algae at the
1

Received 28 April 2008. Accepted 16 March 2009.

Author for correspondence: e-mail sebby@seznam.cz.

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ecophysiological and biochemical studies were also

realized with this strain (Hagemann et al. 1998,
1999, Huckauf et al. 2000, Nakamura and Hihara
2006, Nakasugi et al. 2006, Zabulon et al. 2007, and
many others). The selection of strain PCC 6803 as
an experimental model was successful in part
because it corresponds morphologically to the type
species S. aquatilis or to the closely related S. salina
Wisouch.
However, the present form-genus Synechocystis also
contains a second different morphotype, which is
represented by another model strain, PCC 6308,
and which was selected as a reference strain for cluster 1 of the genus Synechocystis in Bergeys Manual
(Castenholz 2001). The two clusters differ markedly.
Representatives of cluster 2 (Synechocystis sensu
stricto) have relatively smaller cells, 0.75(7) lm in
diameter, usually with recognizable chromatoplasma
and centroplasma (indicating peripheral thylakoid
arrangement), and they comprise the majority of
isolated strains assigned to Synechocystis, including
PCC 6803, and belong to the high GC Synechocystis
cluster (47.5 mol %; Rippka et al. 1979). Representatives of cluster 1 have relatively larger types with
cells (3)530 lm in diameter, with a homogeneous
or keritomic protoplast (indicating thylakoids distributed over the whole content of cells, usually parallel), and they comprise a more limited
group of strains (PCC 6308 and other strains PCC
6701, 6711, 6804, 6808), which belong to the low
GC cluster.
Phylogenetic analysis of the 16S rRNA gene
sequences is frequently utilized to identify clades in
ehakova et al. 2007, Kastovsky and
cyanobacteria (R
Johansen 2008). In cyanobacteria, as well as in most
eubacteria, the genes for rRNA are organized in an
operon with a single promoter region and occur in
the order 16S, 23S, and 5S. The three genes are separated by ITS regions (Gurtler and Stanisich 1996).
The 16S23S ITS contains many conserved domains
(D1-D5, Box B, Box A) and variable regions (V1-V3;
Iteman et al. 2000), which have proved to be useful
for phylogenetic studies (Rocap et al. 2002) as well
as identification of new taxa (Casamatta et al. 2006,
ehakova et al. 2007, Johansen et al. 2008). Primary
R
and secondary structures of these regions prevent
premature termination of transcription (essential
for maintaining transcription of the rRNA genes in
the correct stoichiometry; Berg et al. 1989, Condon
et al. 1995) and hold the secondary structure of
the nascent RNAs for processing to mature RNAs
(Apirion and Miczak 1993). The 16S23S rRNA
spacer region often contains one or two tRNA genes
(either tRNAGlu, tRNAAla, or both tRNAAla and
tRNAIle, see Boyer et al. 2001).
In the present study, morphology, ultrastructure,
16S rRNA gene phylogeny, and secondary structure
of the 16S23S rRNA spacer region were examined
for several coccoid taxa including Synechocystis
clusters 1 and 2 and Cyanobacterium (Rippka and

Table 1. Old and current designations of strains used in

text.
Strain

PCC 6308
PCC 6803
PCC 8806
PCC 7202
EF555569
PCC 6702
PCC 6805
PCC 6714
EF555570
EF555571

Old designation

Current designation

Synechocystis sp.
Synechocystis sp.
Synechococcus sp.
Cyanobacterium stanieri

Geminocystis herdmanii
Synechocystis sp.
Cyanobacterium sp.
Cyanobacterium stanieri
Geminocystis papuanica
Synechocystis sp.
Synechocystis sp.
Synechocystis sp.
Synechocystis sp.
Synechocystis sp.

Synechocystis sp.
Synechocystis sp.
Synechocystis sp.

Cohen-Bazire 1983). We present evidence below

that these belong to three separate genera, including Geminocystis gen. nov., Cyanobacterium, and
Synechocystis. To avoid confusion to the reader, we
will refer to all strains in the remainder of this
paper by our current, corrected genus designation,
not by past incorrect designations, even though
these strains may still persist in culture collections
under these incorrect taxonomic epithets. For easy
reference, equivalents are given in Table 1.
MATERIALS AND METHODS

Microscopy. Morphological variability was investigated using

LM (Olympus BX 51 with bright-field optics; Olympus Corporation, Tokyo, Japan). Strains were additionally studied using
TEM to determine the ultrastructure of cells. Cells were fixed
with 2.5% glutaraldehyde in a 0.1 M cacodylate buffer and later
postfixed with 2% osmium tetroxide. The fixed material was
dehydrated in an acetone series (30, 50, 70, 80, 90, 95, and
100%) and embedded in Spurrs resin (Spurr 1969). Ultrathin
cross-sections were contrasted with uranylacetate and Pb-citrate
and examined in a JEOL 1010 electron microscope (JEOL
Ltd., Tokyo, Japan).
DNA isolation and PCR amplification. Living cells were
broken up in a mini-beadbeater using a mixture of 0.1, 0.5,
and 1.0 mm diameter glass beads. Total genomic DNA was
isolated from cultured cyanobacterial cells using Invisorb Spin
Plant Mini Kit (Invitek, Berlin, Germany) according to
standard protocol and stored in )20C. The 16S rRNA gene
and the associated 16S23S ITS region of strains were amplified from the genomic DNA by PCR using the oligonucleotide
primers: primer 1 (CTC TGT GTG CCT AGG TAT CC)
(Wilmotte et al. 1993, Boyer et al. 2001) and primer 2 (GGG
GAA TTT TCC GCA ATG GG) (Nubel et al. 1997, Boyer et al.
2001). Amplification was performed in a Biometra T3
thermocycler (Biometra Gmbtti. L., Gottingen, Germany)
using 25 lL reactions containing 1020 ng of genomic DNA,
50 pmol of each oligonucleotide primer, 200 lM dNTP, 10
Taq reaction buffer, and 1 unit of Taq DNA polymerase.
Reactions were cycled with an initial denaturation step at 95C
for 5 min, followed by 35 cyc1es of DNA denaturation at 94C
for 1 min, primer annealing at 55C for 45 s, strand extension
at 72C for 2 min, and a final extension step at 72C for
10 min. PCR products were visualized on 1% agarose gels.
Sequencing of 16S rRNA. PCR products of expected size
(1,600 base pairs in length) were purified from the gel using
QIAquick gel extraction kits (Qiagen Gmbh., Hilden, Germany) and sequenced directly by cycle sequencing using
BigDyeTM Terminator Cycle Sequencing kit V3.1 (Applied
Biosystems Inc., Foster City, CA, USA). Primers used for the
cycle sequencing of 16S rRNA were primer 1 and primer 2

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930

(used for PCR amplification), and the internal primers: primer

5 (TGT ACA CAC CGG CCC GTC) (Wilmotte et al. 1993,
Flechtner et al. 2002), primer 6 (GAC GGG CCG GTG TGT
ACA) (reverse complement of primer 5), primer 7 (AAT GGG
ATT AGA TAC CCC AGT AGT C) (Nubel et al. 1997, Flechtner
et al. 2002), and primer 8 (AAG GAG GTG ATC CAG CCA CA)
(Wilmotte et al. 1993, Flechtner et al. 2002). The cycle
sequencing reaction started at 94C for 1 min followed by 30
cycles of the following: 30 s at 94C, 30 s at 50C, and 4 min at
60C. Both forward and reverse strands were sequenced, so that
each region was available as at least two independent reads.
The 16S rRNA sequenced fragments were assembled into
contigs using the software EditSeqTM and SeqManTM II
software (DNAStar, Madison, WI, USA). The sequences determined were deposited in GenBankTM under the accession
numbers EF555569EF555571.
Alignment and phylogenetic analysis. The nucleotide
sequences of 16S rRNA obtained in this study and related
ingroup and outgroup sequences obtained from GenBank
(http://www.ncbi.nlm.nih.gov) were aligned, and ambiguous
or hypervariable sites were removed using BioEdit 7.0.4.1 (Hall
1999). Alignment was analyzed by maximum-parsimony (MP),
neighbor-joining (NJ), and maximum-likelihood (ML) methods, using PAUP*4.0b10 (Swofford 2002) and PHYML v2.4.2
(Guindon and Gascuel 2003) software with 500 bootstrap
replications. The trees were viewed using TreeView version
1.6.6 (Page 1996) and rooted with a sequence for 16S rDNA of
Bacillus subtilis.
ML trees were constructed by PHYML v2.4.2, using the GTR
model for nucleotide substitutions with discrete gamma distribution in four categories; all parameters (gamma shape,
proportion of invariants, transition transversion ratio) were
estimated from the data set. ML bootstrap support was
computed in 500 replicates using the PHYML v2.4.2 program.
MP analysis was performed using PAUP*4.0b10. MP trees were
generated using a heuristic search constrained by random
sequence addition with TBR as a branch-swapping method and
500 bootstrap replicates; gaps were treated as missing data. An
NJ tree was constructed using PAUP*4.0b10 with 500 bootstrap
replications.
Secondary structure of ITS. ITS sequences were folded using
CLC RNA Workbench version 3.0.1 (CLC bio, Aarhus, Denmark), and variable hairpin structures for different genera
were compared.
RESULTS

The main group of Synechocystis strains, including

the experimental model strain PCC 6803 of Synechocystis (aquatilis salina) (Fig. 1a), is genetically uniform, forms a unique cluster in the phylogenetic
tree (Fig. 2, cluster C), forms unique secondary

Fig. 1. Scheme of thylakoid arrangement: (a) Synechocystis

type aquatilis PCC 6803 (comp. Fig. 2, cluster C); (b) Synechocystis
sp. PCC 6308 (=Geminocystis herdmanii; comp. Fig. 2, cluster A).

structure of 16S23S ITS (Fig. 5), and contains morphologically similar, simple types. The cells of all
strains from this cluster possess the same type of
ultrastructural patterns with parietally arranged thylakoids (Figs. 1a and 3, b, f, and g) and belong to
the high GC cluster mol % = 47.5 (Rippka et al.
1979, Rippka and Herdman 1992, http://www.
pasteur.fr/recherche/banques/PCC). Cells divide
into two perpendicular planes in successive generations (Fig. 4b). The similarity between all of these
strains is at least 98%, and their generic and specific
identity is supported by our results. Therefore, it
is possible to designate this cluster as a unique
taxon, which corresponds with the originally
described genus Synechocystis with the type species
S. aquatilis. Its morphological markers confirm the
botanical generic (diacritical) features. All strains
studied and included in cluster C (Fig. 2) could be
designated (according to traditional taxonomy) by
the specific name S. salina, which is closely related
to S. aquatilis and differs only by slightly smaller
dimensions.
In contrast, the strain Geminocystis herdmanii PCC
6308 (originally designated as Gloeocapsa alpicola
1051, lake water, Wisconsin, USA, 1949, http://
www.pasteur.fr/recherche/ banques PCC) is classified in quite different position in the phylogenetic
tree (Fig. 2, cluster A), belongs to the low GC
mol % cluster (34.7) sensu Rippka et al. (1979),
and forms different secondary structures of 16S
23S ITS from Synechocystis. The same phylogenetic
position of this strain was published by RajaniemiWacklin et al. (2005) and Moro et al. (2007); our
results fully confirm these data. The strain PCC
6308 also has more or less spherical cells like
Synechocystis, but slightly larger dimensions in comparison with cluster C (Fig. 2) ([3]3.54[4.5] lm
in diameter). The position of thylakoids is irregular
over the whole cell protoplast with slightly parallel
orientation (Figs. 1b and 3c). This strain (PCC
6308) is genetically close to the cluster with Cyanobacterium stanieri Rippka and Cohen-Bazire (1983),
which is the type species of the genus Cyanobacterium (Figs. 2, cluster B, and 3a; cf. strain PCC
7202). PCC 6308 differs distinctly from Synechocystis
by position in the phylogenetic tree, ultrastructure
(mainly by position of thylakoids in cells), and ITS
structure (Fig. 5), while it shares the thylakoid
arrangement typical for the type strain of C. stanieri
(Fig. 3a; Komarek et al. 1999) and other Cyanobacterium species [C. minervae, C. cedrorum; cf. Komarek
et al. 1999, 2004, Moro et al. 2007]. According to
secondary structure of 16S23S ITS, Geminocystis is
highly similar to Cyanobacterium (Fig. 5). Gemniocystis
is only 93.1%94.4% similar in 16S rDNA sequence
to C. stanieri strain PCC 7202 and differs in both
cell shape and number of planes of fission (see
below, Fig. 4). It evidently must be classified as a
separate
generic
unit
(genus
Geminocystis
gen. nov.).

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931

Fig. 2. Maximum-likelihood (ML) phylogenetic tree (50% majority rule consensus) as inferred from 16S rDNA sequences of selected
specimens of the cyanobacteria. GenBank accession numbers are written after names of strains. Numbers above branches indicate bootstrap support (neighbor joining [NJ] ML maximum parsimony [MP], 500 replicates). Only bootstrap values >50% are shown. Tree constructed from 1,455 characters (366 parsimony informative). loglk = )9257.46712. Gamma shape parameter alpha (0.503) and proportion
of invariants (0.420) estimated from the data set. Marked groups: A, Geminocystis cluster (with strain PCC 6308); B, Cyanobacterium cluster;
C, Synechocystis type aquatilis salina (including the type strain PCC 6803).

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Fig. 3. Arrangement of thylakoids (scale bar = 1 lm): (a) section through the cell of Cyanobacterium stanierii PCC 7202 (=type species
and type strain of the genus Cyanobacterium); (b) Synechocystis sp. PCC 6803; (c) Geminocystis herdmanii sp. nov. PCC 6308; (d) Synechococcus
sp. PCC 7502; (e) Geminocystis papuanica sp. nov. EF555569; (f) Synechocystis sp. EF 555570; (g) Synechocystis sp. EF 555571.

16S23S rRNA ITS secondary structure. For the

comparison of secondary structure of ITS, we used
the labeling of conserved and variable regions of
Iteman et al. (2000). Only the regions D1D1, box
B, and V3 are shown for comparison of different
genera in Figure 5. The variable stem V2 was not
observed in any of examined strains. Regions D2,
D3, D4, and Box A spacer were highly similar or
identical in all of the studied strains. However,
Geminocystis appears to have a unique primary
sequence in the D1D1 helix, which in all other
cyanobacteria sequenced to date has a beginning
sequence of 5-GACCU-3 or 5-GACCA-3. Both
Geminocystis and Cyanobacterium have a distinctively
shortened helix (1718 nucleotides, see Fig. 5), the
shortest such helices reported in any free-living
prokaryotic organism (Iteman et al. 2000 reported
the shortest sequence of 35 bases). This helix is
considerably distinct from that of Synechocystis (64
bases; Fig. 5). The same pattern could be observed
in the V3 helix. The V3 helix of Geminocystis is
much shorter than the V3 helix in Synechocystis
(Fig. 5). The box-B helices are fairly similar for all
three genera examined in this study. The 16S23S
rRNA spacer of Geminocystis and Cyanobacterium
contains tRNAAla and tRNAIle, whereas the ITS of
Synechocystis contains only tRNAIle. All these observations, based on both primary and secondary
structures of ITS, enable us to clearly distinguish
Geminocystis from Synechocystis. Both these genera

are also substantially different by 16S rDNA

sequencing and type of division.
Our study confirmed the diversity and heterogeneity of the present morphologically defined genus
Synechocystis, which must be taxonomically classified
into two clades, according to present molecular evidence. Separation on the level of genus is prescribed according to both modern bacteriological
(Castenholz 2001) and botanical (Komarek and
Anagnostidis 1998) criteria. Morphological uniformity and ultrastructure patterns are in agreement
with their genetic background. Both clusters should
be characterized as follows:
1.

2.

The typical cluster, which must be designated

as Synechocystis Sauv. 1892 with type species
S. aquatilis (and closely related S. salina), and
with the reference strain PCC 6803 (cluster C
in the phylogenetic tree in Fig. 2). It has solitary spherical cells 12.5(5) lm in diameter
and belongs to high GC cluster mol % = 47.5.
Three to 8(10) thylakoids are localized in cells
parietally. Cells always divide into two perpendicular planes in successive generations. The
typical strain (PCC 6803) was fully sequenced
by Kaneko et al. 1995 (CBS Genome Atlas
Database 2006; http://www.cbs.dtu.dk/services/
GenomeAtlas).
The strain PCC 6308 belongs to the newly
described genus Geminocystis gen. nov. (type

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TAXONOMIC DIAGNOSES

Fig. 4. Cell division pattern of strains belonging to studied

clusters: (a,b,c) division in two planes, (d) division in one plane
in subsequent generations. (a) Geminocystis papuanica EF555569,
(b) Synechocystis sp. PCC 6803, (c) Geminocystis herdmanii PCC
6308, (d) Cyanobacterium stanieri PCC 7202 (scale bar = 10 lm).

species G. herdmanii, position A of the phylogenetic tree in Fig. 2). The genus Geminocystis
has solitary, spherical, or slightly oval cells,
never forming colonies, and belongs to low
GC cluster mol % = 34.7. The organization of
thylakoids in cells is more or less parallel over
almost the whole cell volume. Geminocystis differs from the genus Cyanobacterium by bacteriological criteria (similarity of 16S rDNA
sequence is <95%) and also by division of cells
into two planes (Cyanobacterium with frequently
rod-shaped cells divides in one plane in subsequent generations, contrary to Geminocystis; cf.
also Allen 1968).

Geminocystis gen. nov.

Cellulae cyanoprocaryoticae, solitariae, sphaericae
vel paucim ovales, post divisionem hemisphaericae et
mox binae (quattuor) coniunctae, nunquam in coloniis, 310 lm in diametro, sine mucilagine, vel cum
involucris tenuis, diffluentibus, incoloris, circum cellulis solitariis. Cellulae aeruginosae, griseo-virides vel
subroseae. Contentu cellularum homogeneo, sine
separatio in centro- et chromatoplasma, interdum,
cum stiis longitudinalibus (positio thylakoidarum).
Systema thylokoidarum in cellulis longitudine plus
minusve paralleliter vel irregulariter ordinata. Divisio
cellularum in partes duas similares in planos duas in
generationes subsequentis; cellulae filiales, aequales,
hemisphaericae, crescentes in status originali
subgloboso ante proxima divisione. Cellulae semper
in cellulis duobus filialis dividuntur in planes perpendiculares in generaciones subseqventes. Typus generis: Geminocystis herdmanii.
Description: Cells cyanoprokaryotic, solitary, spherical or slightly oval, after division hemispherical and
for a short time two (four) together, never forming
colonies, 310 lm in diameter, without mucilage or
(mainly) with narrow, fine, colorless, and usually
diffluent and indistinct mucilaginous envelopes. Cell
content homogeneous, without separation of centroand chromatoplasma, sometimes with lengthwise
striation (thylakoids situated in cells lengthwise).
Color of cells is pale blue-green, bright-green, grey, or
pinkish. Cell division by binary fission (mainly by
pinching) into two morphologically equal, hemispherical daughter cells, which reach the original globular
shape before next division; cells always divide into two
perpendicular planes in successive generations.
Type species: Geminocystis herdmanii sp. nov.
Geminocystis herdmanii sp. nov. (Figs. 1b, 3c, and 4c).
Cellulae procaryoticae, sphaericae, post divisionem
hemisphaericae, aeruginosae, contentu homogeneo

Fig. 5. Comparison of secondary structures of 16S-23S rRNA internal transcribed spacer (ITS) of three genera (Cyanobacterium, Geminocystis, and Synechocystis); D1D1, Box B, and V3 represent variable regions presented in the work of Iteman et al. (2000).

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vel paucim keritomico, (3)3.54(4.5) lm in diametro.

Thylakoides plus minusve paralleliter vel irregulariter
ordinatae. Divisio in partes duas similares, cellulae
fimilares hemisphaericae. Holotypus hic designatus:
PCC 6308 deposited in preserved state in BRNM HY
1415. Habitatio: Planktice in lacubus oligotrophicis.
Locus classicus: Lacus in nominatus in Wisconsin,
USA, Species ad honorem Dr. Michael Herdman
(Paris) nominata.
Description: Solitary cyanoprocaryotic spherical
cells, after division hemispherical, blue-green with
homogeneous or slightly irregularly keritomic content, (3)3.54(4.5) lm in diameter. Thylakoids in
cells are arranged irregularly or parallely. Division
by binary fission in two morphologically similar,
hemispherical daughter cells.
Type: Strain PCC 6308 (deposited in preserved
state in BRNM HY 1415).
Habitat: Planktic in oligotrophic lakes, isolated
from lakes in Wisconsin, USA.
Reference and type strain: PCC 6308 (=ATCC 27150,
CCAP 1430 1, UTEX 1598, CALU 743).
Etymology: The species was named to the honor of
Dr. Michael Herdman, Pasteur Institute, Paris.
Geminocystis papuanica sp. nov. (Figs. 3e, 4a, and 6).
Cellulae solitariae vel in aggregationes parvis dispositae, sphaericae vel paucim ovales, hemisphaericae post divisionem, aeruginosae vel atro-griseae,
contentu interdum keritomico, praecipue irregulariter vittato, (3.5)45(6.2) lm in diametro. Divisio in
partes duas hemisphaericas similares. Holotypus hic
designatus: exicatum depositum in BRNM HY
1409); iconotypus: figura nostra 6. Habitatio: in
solis madidis in silvis tropicalibus prope oppidum
Wanang, Papua New Guinea.
Description: Cells solitary or in small clusters,
spherical or slightly oval, hemispherical after division, blue-green to dark grey, with slightly keritomic

Fig. 6. Geminocystis papuanica sp. nov., strain EF555569; iconotype. Scale bar = 10 lm.

content with parallely, slightly irregularly striated

protoplast, (3.5)45(6.2) lm in diameter. Division
by binary fission in two morphologically similar,
hemispherical daughter cells.
Type: In dried sample deposited in BRNM HY
1409); iconotype: our Figure 6.
Habitat: On surface of wet soil in tropical rainy forests in the vicinity of Wanang, Papua-New Guinea.
Etymology: The species was named according to
country of its original locality.
DISCUSSION

The reference strain of the genus Geminocystis,

PCC 6308, does not correspond to any other
sequenced cyanobacterial strain (with exception of
Geminocystis papuanica EF555569 presented in this
work), but it may be characterized morphologically
and ecologically as a close relative of Cyanobacterium
(Komarek et al. 2004). However, this strain differs
from Cyanobacterium substantially genetically (similarity of 16S rDNA is only 93.1%94.4%) and also by
type of cell division (cell division in Cyanobacterium
stanieri PCC 7202 proceeds perpendicularly to the
long axis of the cells and always occurs in the same
plane in successive generations, Fig. 4d; Komarek
et al. 1999, Herdman et al. 2001). Cells of Geminocystis divide in two planes (Fig. 4, a and c; cf. Allen
1968); this type of division is the autapomorphic
marker separating Geminocystis from Cyanobacterium.
The new description of such a newly recognized
cyanobacterial taxon is therefore adjured and in
agreement with the requests of validation of present
cyanobacterial taxa according to botanical (Johansen
and Casamatta 2005) as well as bacteriological
(Wayne et al. 1987, Stackebrand and Goebel 1994,
Oren 2004) criteria. The Geminocystis morphotype is
also evidently known from the older literature, but
not described at the generic level. Copeland (1936)
recorded and illustrated Synechococccus minervae var.
bigeminatus with lengthwise striation and division in
two perpendicular planes, and it very probably
belongs to the same genus.
Our strain EF555569 (Figs. 2, cluster A; 3e; 4a;
and 6) also belongs to the genus Geminocystis. It differs morphologically, molecularly, and ultrastructurally from the genera Synechocystis and Cyanobacterium.
It is evidently nonconspecific with PCC 6308, from
which it differs by larger dimensions and distinct
ecology (aerophyte from tropical rain forest of
Papua-New Guinea; PCC 6308 was isolated
from plankton of Lake Wisconsin). The strain
EF555569 was therefore described as a separate
species of genus Geminocystis (G. papuanica). Unfortunately, the strain was lost after sequencing and
characterization.
As mentioned earlier, the strain Geminocystis herdmanii PCC 6308 does not correspond to all other
typical Synechocystis strains and species and therefore
was classified according to molecular, cytological,

C Y A N O B A C T E R I A L G E N E R A S YN E C H O C Y S T I S A N D G E MI N O C Y S T I S

and phenotype markers as the type and reference

strain of the new genus Geminocystis. The problem is
that this strain was designated as a reference strain
of cluster 1 of the form-genus Synechocystis in
Bergeys Manual (Castenholz 2001, p. 513). Because
the type-species of the genus Synechocystis is S. aquatilis (which corresponds with strain PCC 6803, but
from which the strain PCC 6308 is clearly different
according to position in phylogenetic tree and ultrastructure), the whole cluster 1 in Bergeys Manual
(based on the strain PCC 6308) has to be reclassified into the genus Geminocystis. We agree with designation of PCC 6803 as the reference strain of the
genus Synechocystis.
The right taxonomic assignment of experimental
strains is a common problem in modern cyanobacterial science. The taxonomic names of strains are
used in world databases of cyanobacteria, but their
identification should be revised. If the name is
accepted, it must be a symbol of a special taxonomic
entity. However, numerous names do not agree with
characters of the respective strains in molecular
databases. Synechocystis is a good example of this
phenomenon; for example, in the NEWT database
(Phan et al. 2003) both strains, PCC 6308 and PCC
6803, are in one list. There is no good methodological or formal method for correcting the names.
However, the names should be corrected in all culture collections and databases.
Another problem is with the phenotypic characters
of various Synechocystis strains and species cited in the
literature. The cell diameter of various natural populations of typical Synechocystis morphospecies has been
reported in the past as from 0.7 to 5(7) lm. The
strains from cluster C (Fig. 2) have cells 2.32.5 lm
in diameter according to the PCC Web page (http://
www.pasteur.fr/recherche/banques/ PCC) and 1.5
3 lm according to our measurements, which indicates that they belong to one specific taxon
corresponding mostly to S. salina (1.84.5 lm in
diameter), closely related to the type species S. aquatilis (with cells 37 lm in diameter). At least the
strain PCC 6308, which belongs to the cluster Geminocystis (our cluster 2A) according to molecular analyses, has cells (3)3.54(4.5) lm in diameter according
to our repeated and precise measurements.
A similar inaccuracy is in Bergeys Manual for the
genus Cyanobacterium. The dimensions of spherical
to rod-shaped cells of the whole genus Cyanobacterium (Castenholz 2001, p. 497) should be 1.72.3
lm in diameter. We have received the type strain
PCC 7202 twice in 1998 and 2005 from the PCC collection, and this strain is still cultured in our laboratory. The dimensions of the elongated cells were
2.26.1 1.94.4 lm. Therefore, the dimensions of
PCC strains (http://www.pasteur.fr/recherche/banques/PCC) are unfortunately not reliable.
In the genus Cyanobacterium (cluster B in Fig. 2),
PCC 8806 (Table 1) is 98.7% similar in 16S rDNA
sequence to the type strain of Cyanobacterium stanieri

935

and should therefore be considered as the same

specific entity. The currently known Cyanobacterium
species are summarized in Komarek et al. (2004).
However, a majority of these species have not yet
been sequenced, and their classification in the genus
Cyanobacterium is therefore provisional on the basis of
morphological markers. In contrast, the recently
described Cyanobacterium aponinum Moro et al.
(2007) belongs to the genus Cyanobacterium. Perhaps
a few other traditionally large morphospecies of
the genus Synechocystis also correspond phenotypically
to the genus Cyanobacterium or Geminocystis, especially
Synechocystis septentrionalis Skuja and S. sallensis Skuja
(cf. Komarek and Anagnostidis 1998). Their final taxonomic status must be resolved by future combined
studies. Strain PCC 7502, which is classified in Bergeys
Manual (Castenholz 2001) as a special type of the
genus Cyanobacterium, is problematic because it is
without definition and validation of the species. The
specific name of this strain is lacking. According to
Bergeys Manual (Castenholz 2001, p. 497), the genus
Cyanobacterium is represented by two strains, PCC
7202 (type strain of C. stanieri) and PCC 7502, which
represent probably different species of the same
genus. These data are evidently incorrect. According to the CBS Genome Atlas Database (http://
www.cbs.dtu.dk/services/GenomeAtlas), and also
according to our results, PCC 7502 belongs to
another clade in the phylogenetic tree (Fig. 2). This
strain has more cylindrical cells, the position of the
thylakoids is parietal (Fig. 3d), and it morphologically corresponds to Cyanobacterium synechococcoides
(however, the type material of this morphospecies
has not been sequenced). Because this clade differs
from typical Synechococcus, Cyanobacterium, and Thermosynechococcus, it probably represents a special taxon at
the generic level (comp. Komarek et al. 2004, cluster 12), but surely does not belong to Cyanobacterium
or Geminocystis.
Another form-genus, which is phenotypically
related to the Geminocystis strain PCC 6308, is a marine Crocosphaera (Fig. 2; cf. solitary spherical cells and
ultrastructure: Waterbury et al. 1988, Zehr et al.
2001; Cyanobacteriology at WHOI 2006 [http://
www.whoi.edu/science/B/people/ewebb/Croco.html],
NCBI Sequence Viewer 2006 [http://www.ncbi.nlm.
nih.gov/entrez/viewer], not yet validly described).
The phylogenetic position of this taxon is clearly distant from Synechocystis as well as from Geminocystis and
Cyanobacterium and therefore cannot be designated
under any of these generic epithets (cf. Fig. 2).
The study was supported by grants MSM 600 766 5801, GACR
206 08 0318, and IAA60005704. We thank Dr. Keith Edwards
for the language correction.
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