1. No category

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2326–2333
DOI 10.1099/ijs.0.045468-0
Chalicogloea cavernicola gen. nov., sp. nov.
(Chroococcales, Cyanobacteria), from low-light
aerophytic environments: combined molecular,
phenotypic and ecological criteria
M. Rolda´n,1,2 M. Ramı´rez,2 J. del Campo,3 M. Herna´ndez-Marine´2
and J. Koma´rek4
Correspondence
M. Rolda´n
1
[email protected]
2
Servei de Microsco`pia, Universitat Auto`noma de Barcelona, Edifici C, Facultat de Cie`ncies,
08193 Bellaterra, Spain
Dep. Productes Naturals, Biologia Vegetal i Edafologia, Unitat de Bota`nica, Facultat de Farma`cia,
Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
3
Institut de Biologia Evolutiva, CSIC-UPF, Passeig Marı´tim de la Barceloneta, 37–49,
08003 Barcelona, Spain
4
Institute of Botany, Academy of Sciences of the Czech Republic, Dukelska´ 135, CZ-37982
Trˇebonˇ, Czech Republic
This work characterizes a unicellular cyanobacterium with nearly spherical cells and thin-outlined
sheaths that divide irregularly, forming small packets immersed in a diffluent mucilaginous layer. It
was isolated growing on calcite speleothems and walls in a show cave in Collbato´ (Barcelona,
Spain). Spectral confocal laser and transmission electron microscopy were used to describe the
morphology, fine structure and thylakoid arrangement. The pigments identified were
phycoerythrin, phycocyanin, allophycocyanin and chlorophyll a. Three-dimensional
reconstructions, generated from natural fluorescence z-stacks, revealed a large surface area of
nearly flat, arm-like thylakoidal membranes connected to each other and forming a unified
structure in a way that, to our knowledge, has never been described before. Phylogenetic analyses
using the 16S rRNA gene sequence showed 95 % similarity to strain Chroococcus sp. JJCM
(GenBank accession no. AM710384). The diacritical phenotypic features do not correspond to
any species currently described, and the genetic traits support the strain being classified as the
first member of an independent genus in the order Chroococcales and the family
Chroococcaceae. Hence, we propose the name Chalicogloea cavernicola gen. nov., sp. nov.
under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants. The
type strain of Chalicogloea cavernicola is COLL 3T (5CCALA 975T 5CCAP 1424/1T).
Cyanobacteria are a dominant component of diversely
structured photosynthetic biofilms living in low-light
environments. Examples of these communities can be
found in natural caves and artificially illuminated show
caves and catacombs, in which they are affected by the
physical, chemical and biological conditions (Herna´ndezMarine´ et al., 2001; Lamprinou et al., 2009; Rolda´n et al.,
2004a; Rolda´n & Herna´ndez-Marine´, 2009; Urzı` et al.,
2010). These parameters are interdependent, and deterAbbreviations: CLSM, confocal laser scanning microscopy; ML,
maximum-likelihood; TEM, transmission electron microscopy.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain COLL 3T is JQ967037.
A supplementary figure is available with the online version of this paper.
2326
mine which organisms can thrive (Decho et al., 2010).
Photosynthetic communities thrive mainly on the surfaces,
which appear blue, greenish or grey and cover substrates
irregularly. Since the famous study of green sickness
(Lefe`vre et al., 1964), which affected the prehistoric murals
in the caves of Lascaux, France, the presence of photosynthetic micro-organisms on the walls and paintings of caves
and monuments has been widely reported (Rolda´n &
Herna´ndez-Marine´, 2009). Uncontrolled growth of these
organisms can cause biodeterioration of and/or aesthetic
damage to surfaces. Determining the roles they play
requires knowledge of individual isolates, and the information gained can be used for protecting cultural heritage.
Molecular methods can be used to classify cyanobacteria
taxonomically, especially for groups that have very few
045468 G 2013 IUMS Printed in Great Britain
Chalicogloea gen. nov., from dim-light environments
morphologically differentiating characters (Wilmotte et al.,
1992; Garcia-Pichel et al., 1998; Koma´rek & Anagnostidis,
1998; Nu¨bel et al., 2000; Abed et al., 2002; Berrendero et al.,
2008; Oren, 2011). In addition, genetically identified items
need to correlate with phenotypic and ecological data
(Hoffmann et al., 2005; Johansen & Casamatta, 2005;
Koma´rek & Kasˇtovsky´, 2003; Koma´rek et al., 2004;
Koma´rek, 2010b; Koma´rkova´ et al., 2010). The specific
phenotypic traits used depend on the organism and the
tools employed to characterize it. These traits can be used
to differentiate among genera or morphospecies.
Features that are in agreement with phylogenetic relationships in unicellular cyanobacteria include the type of
cell division and the thylakoid arrangement (Koma´rek
& Anagnostidis, 1998; Koma´rek & Kasˇtovsky´, 2003).
Photosynthetic pigments may also be used as a taxonomic
marker in cyanobacteria (Bryant, 1982; Wilmotte, 1994;
Rolda´n et al., 2004b), although this depends on whether
information on the pigment type is available at the genus or
species level, and whether there are changes in pigments at
different life-cycle stages (Wolf & Schu¨ßler, 2005) or under
different environmental conditions (Ramı´rez et al., 2011).
During an intervention intended to clean speleothems in
an illuminated tourist cave, colonies of a unicellular
cyanobacterium, COLL 3T, were detected. Here, we report
their isolation and taxonomic characterization.
Samples were collected from a speleothem illuminated
during cave visits with white fluorescent bulbs. Samples
were collected at the deepest part (length 549 m and slope
20 m) of the Salpetre Cave in Collbato´ (Catalonia, northeast Spain; 41u 349 31.720 N 1u 509 10.140 W). Salpetre first
began to be exploited in the 16th century for potassium
nitrate and, in 1934, the cave was artificially illuminated
and made into a tourist attraction. The cave shows
considerable micro-environmental stability throughout
the annual cycle (data-logger Testo 177-H1). The mean
air temperature was 14.7 uC, with an annual variation of
4.1 uC; the mean stone temperature was 15.5 uC, with an
annual variation of 1.2 uC; the mean ambient humidity
(94.0 %; range 90.5–96.9 %) was high in the sampling zone;
and the mean environmental CO2 concentration was
588.2 p.p.m. (data-logger Testo 400). The photosynthetic
photon flux density was ,2.5 mmol photon m22 s21.
Samples were placed immediately in Petri dishes on a
2 mm layer of BG11 medium (Stanier et al., 1971)
solidified with agar (1 %; Merck). COLL 3T was isolated
from natural biofilms and grown on 1 % agarized BG11
medium at 17 uC under 10 mmol photon m22 s21 with a
light/dark cycle of 12 : 12 h.
Materials were examined using an Axioplan microscope
(Carl Zeiss) and photographed with an AxioCam MRc5
digital camera system. Cell measurements were taken from
photomicrographs.
Confocal images were taken with a Leica TCS-SP5 CLSM
(Leica Microsystems) equipped with a 636/1.4 (oil
http://ijs.sgmjournals.org
HC6PL APO lambda blue) objective. Autofluorescence
from photosynthetic pigments was viewed in the red
channel (590–800 nm emission) using the 561 nm excitation laser line. Concanavalin A–Alexa 488 (Molecular
Probes) (0.8 mM) targets cell-associated mucilage and was
recorded in the green channel (excitation, 488 nm;
emission, 495–530 nm). Stacks were subsequently processed with the Imaris version 6.1.0 software (Bitplane AG
Zu¨rich) to obtain maximum-intensity projections. The
isosurface module of Imaris was used to build 3D models.
Lambda stacks (xyl) were taken in order to determine the
emission spectra of the photosynthetic pigments (chlorophyll a, phycoerythrin, phycocyanin, allophycocyanin)
(Rolda´n et al., 2004b). The excitation wavelength used
was the 488 nm line of an Ar laser. Emission detection was
set from 520 to 780 nm. For each xy focal plane, confocal
laser scanning microscopy (CLSM) was used to measure
the emission variation every 10 nm (lambda step size
5.2 nm). The emission spectrum analysis was processed
using the LAS AF software. To analyse cells, 50 regions of
interest of 1 mm2 were delimited to calculate the mean
fluorescence intensity in relation to the wavelength.
For transmission electron microscopy (TEM), samples
were fixed in glutaraldehyde (2.5 %) and paraformaldehyde
(2 %) in 0.1 M cacodylate buffer for 2–4 h, washed in this
buffer, post-fixed in osmium tetroxide, dehydrated by a
graded acetone series and embedded in Spurr’s resin.
Ultrathin sections, cut from the block, were then stained
with 2 % uranyl acetate and lead citrate and examined
using a JEOL 1010 TEM at 100 kV accelerating voltage.
Genomic DNA for phylogenetic analysis was extracted from
15 ml cyanobacterial culture after centrifugation using 3 %
cetyltrimethylammonium bromide (Doyle & Doyle, 1987).
Samples were kept at 280 uC. 16S rRNA gene amplification
and sequencing analyses were performed as described
previously (Ferrera et al., 2004). 16S rRNA genes were
amplified using the primers 27f and 1492r. Amplified 16S
rRNA gene products were precipitated with ethanol and
resuspended in 20 ml sterile water. The PCR product from
the COLL 3T culture was sequenced completely using
primers 358f, 517r, 907f and 1492r by MACROGEN
Genomics Sequencing Services. The resulting sequence was
double checked using CHECK_CHIMERA (Maidak et al., 2001)
and by BLAST search with different sequence regions.
Environmental 16S rRNA gene sequences of cyanobacteria
were obtained from GenBank in a two-step screening. First,
sequences found by the NCBI Taxonomy application were
retrieved and checked by BLAST (Altschul et al., 1997) to
confirm their placement. Second, we used these and other
published sequences from cultures or environmental surveys
that belong to the target groups (but are not labelled as such
in GenBank) to retrieve additional sequences by BLAST.
Neighbour-joining phylogenetic trees were reconstructed
with wide taxon coverage to determine whether or not
ambiguous divergent sequences belonged to a given group.
Related sequences from cultured organisms were also
retrieved from GenBank and pruned to keep only a few
representatives for phylogeny.
2327
M. Rolda´n and others
16S rRNA gene sequences were aligned using MAFFT (Katoh
et al., 2002) with a close relative as outgroup. Alignments
were checked with Seaview 3.2 (Galtier et al., 1996) and
highly variable regions of the alignment were removed
using Gblocks (Castresana, 2000). Maximum-likelihood
(ML) phylogenetic trees with complete sequences were
reconstructed with RAxML (Stamatakis, 2006) using the
evolutionary model GTRMIXI. Phylogenetic analyses were
carried out in the freely available University of Oslo
Bioportal (http://www.bioportal.uio.no). Repeated runs on
different starting trees were carried out in order to select
the tree with the best topology (the one with the best
likelihood of 1000 alternative trees). A bootstrap ML
analysis was carried out with 1000 pseudoreplicates and the
consensus tree was computed with MrBayes (Huelsenbeck
& Ronquist, 2001). Trees were edited with FigTree version
1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).
Optical observations (Figs 1 and 2) showed that the irregular
colonies were immersed in a diffluent mucilaginous layer
that grew on calcite speleothems and walls in Collbato´, alone
or intermixed with coccoid and filamentous cyanobacteria
and moss protonemata. Cells were arranged in small, bright
bluish-green aggregates, mostly consisting of two to sixteen
cells. Envelopes followed the outline of individual cells or the
siblings after the division (Fig. 1d). Under CLSM, some cells
exhibited green concanavalin A fluorescence in the outer
sheath layers (Fig. 2a, c), while others did not (Fig. 2b). Their
thickness varied, apparently depending on the cell’s position
within the colony, from a diffluent, slightly lamellate slime,
up to 14 mm thick, to almost no apparent individual sheath.
Cells divided irregularly in different planes and did not
return to their original shape before the next division (Figs
1a, b and 2b). The cell shape was more or less spherical,
slightly elongated in pre-divisional cells or irregular in
outline, but never pear-shaped. The cells displayed different
cell dimensions, ranging from 2.6 to 4.3 mm (mean
3.3±0.4 mm; n554) in diameter. Neither nanocyte formation nor resting stages were observed.
The cellular content of fluorescent pigment was not
homogeneous (Fig. 2a–c). Three-dimensional models
showed that the thylakoids form a structure consisting of
interconnected layers, orientated at different angles, that
leave elongated or star-shaped non-fluorescent areas (Fig.
2d). The in vivo pigment spectral profile determined from
cultured material showed a small shoulder at 579 nm
corresponding to phycoerythrin and an emission peak at
662 nm corresponding to phycobiliproteins and chlorophyll a (Fig. S1, available in IJSEM Online).
TEM (Fig. 3) showed that the sheath near the cell consisted
of thin, poorly compacted fibres. The cell wall had a total
width of about 33 nm and, as is usual for cyanobacteria,
consisted of three layers, in which the electron-dense
peptidoglycan layer measured about 6.6 nm. The thylakoids were arranged in irregular arrays that intersected at
several angles (Fig. 3b). The thylakoid arrays crossed each
other, leaving an irregular central area, depending on the
position of the thylakoids and the orientation of the image,
where the nucleoid was located. Binary fission occurred via
a constrictive pinching mechanism (Fig. 3b). The division
Fig. 1. (a, b) Confocal 3D images of
Chalicogloea cavernicola gen. nov., sp. nov.
COLL 3T. General views of colonies, showing
the irregular division planes. The cells were
solitary or arranged in amorphous colonies
composed of a few cells. Neither nanocyte
formation nor resting stages were observed.
(c, d) Optical photomicrographs of strain
COLL 3T. (c) Cells or siblings enveloped by
concentric sheaths. (d) Sheaths stained with
methylene blue. Bars, 10 mm.
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International Journal of Systematic and Evolutionary Microbiology 63
Chalicogloea gen. nov., from dim-light environments
Fig. 2. Confocal 3D images of strain COLL 3T.
(a) 3D extended projection showing colonies
growing directly on the surface as flat biofilms,
alone and with or without sheaths. The
thickness of the biofilm is 18.5–27 mm. (b)
Maximum intensity projection showing the
shape of the thylakoidal region. (c) Simulated
fluorescence projection showing the sheaths
following the outline of individual cells or
siblings after cell division. Some cells exhibited
strong fluorescence in the outer sheath layers
when labelled with concanavalin A–Alexa 488.
(d) Isosurface reconstruction of thylakoid
fluorescence. The 3D representation was
composed of 15 slices acquired in steps of
160 nm. Bars, 10 mm.
process was initiated by a symmetrical invagination of both
the cytoplasmic membrane and the peptidoglycan layer,
while ingrowing of the outer membrane was delayed (Fig.
3c, d). Sections through cells also showed carboxysomes,
numerous glycogen granules between thylakoid membranes and polyphosphate bodies.
Fifty chroococcalean cyanobacterial sequences from the
public domain were added to the analysis for reference
purposes; the 16S rRNA gene sequence of an unidentified
strain of Pseudomonas aeruginosa (GenBank accession no.
AB364957) was used as an outgroup to root the phylogenetic
tree (Fig. 4). In the ML phylogenetic tree, COLL 3T showed
the highest similarity to Chroococcus sp. JJCM (95 %). The
16S rRNA gene sequence of COLL 3T was divergent enough
from that of any other cyanobacterium available in GenBank
to suggest that it represents a novel and differentiated taxon.
Here, we propose the name Chalicogloea cavernicola (M.
Rolda´n, M. Herna´ndez-Marine´ & J. Koma´rek) gen. nov., sp.
nov., to be published under the provisions of the
International Code of Nomenclature for Algae, Fungi and
Plants. The isolate Chalicogloea cavernicola COLL 3T is
deposited at the Culture Collection of Autotrophic
Organisms as CCALA 975T and at the Culture Collection
of Algae and Protozoa as CCAP 1424/1T.
Description of Chalicogloea, gen. nov.
Etymologia: Chalicogloea (Cha.li.co.gloe9a. Gr. n. chalix chalk;
Gr. n. gloios glutinous substance; N.L. fem. n. Chalicogloea
glutinous substance growing on chalk).
http://ijs.sgmjournals.org
Cellulae procaryoticae, in coloniis irregularibus, microscopicis,
irregulariter dense aggregatae, vagina circumdatae; cellulae
rotundatae, rotundae vel leviter oblongae, differente diametro,
contentu aeruginoso, in parte centrali sine coloure. Tegumentum
mucilaginosum, translucens, achromaticum, indistincte vel
dense lamellosum. Thylakoides paucim fasciculata, irregulariter
vel praecipue in partes parietalibus disposita. Reproductio:
Divisio cellulis rotundatis (primis) regulariter in planis
perpendicularibus, post, in coloniis adultis, irregulariter in
generationes successivis. Habitatio: Species aerophyticae. Typus
generis: Chalicogloea cavernicola species nova.
Description of Chalicogloea cavernicola, spec.
nova
Etymologia: Chalicogloea cavernicola [ca.ver.ni9co.la. L. n.
caverna grotto; L. suff. -cola (from L. n. incola) inhabitant,
dweller; N.L. n. cavernicola an inhabitant of a cavern].
Coloniae ad 100 mm in diametro, cellulae 2.6–4.3 mm in
diametro, contentu leve granuloso. Tegumenta achroa, hyalina,
vel parcim lamellosa. Habitatio: Hispania, Barcino, spelunca
Collbato´ dicta, aerophytica ad substrato calcareo adhaerens.
The phylotype strain is COLL 3T (5CCALA 975T 5CCAP
1424/1T), isolated from a Spanish cave.
Herbarium of Spain: BCN-Phyc 6164. Icona typica: figura
nostra 2a.
The genus Chalicogloea belongs to the group of typical
cyanobacteria that form irregular, microscopic packets in
2329
M. Rolda´n and others
Fig. 3. Transmission electron micrographs of
strain COLL 3T. In (a), it can be seen that
groups of more than four cells were not
covered by a common sheath. In (b–d), binary
fission can be seen to occur via a constrictive
pinching mechanism in which all cell wall
layers were involved. Lipid or cyanophycin
granules were scarce. The thylakoids were
put aside by the ingrowing layers. Thylakoids
were irregularly distributed, either parallel or
perpendicular to the cell wall. Bars, 1 mm (a),
0.5 mm (b), 0.2 mm (c, d).
aerophytic and subaerophytic communities on stony and
rocky substrates. It is defined by a separate position in the
phylogenetic tree, which is also supported by cytological
specificities. The recognized phylogenetic clusters of cyanobacteria (mostly on a generic level) are also commonly
separated by distinct autapomorphic features, and can also
be identified by optical microscopy. The genus Chalicogloea
differs from phenotypically similar generic taxa by the
following morphological markers.
(i) It is separated from all genera that reproduce facultatively
or obligatorily by baeocytes due to the total absence of
multiple fission. This important marker is also supported by
the large distance of these generic units from Chalicogloea in
the 16S rRNA gene phylogenetic tree. All baeocytic species
have a position among more complicated genera. This
covers, for example, the genera Stanieria (aquatic, mostly
marine), Chroococcidium (submersed, never forming compact packets) and especially Chroococcidiopsis, known from
various extreme habitats.
(ii) Of the non-baeocytic types, Chalicogloea can be confused
phenotypically with genera that have the following diacritical markers.
the next division (Golubicˇ, 1967). Gloeocapsa belongs to a
different family than Chalicogloea, and these genera are
distinctly separated phylogenetically.
Cyanosarcina. This genus is mostly similar to Chalicogloea
in its uncoloured, firm, thin sheaths, but, after the irregular
division, the cells remain in polyhedric-rounded form in
aggregated, dense packets. The cell morphology is different,
and the colonies usually have a more irregular shape. Almost
all Cyanosarcina species have a different ecology. They occur
in aquatic habitats, sometimes in mineral and thermal
waters. The only exception is Cyanosarcina parthenonensis,
described from aerophytic limestone substrates in Greece,
but this one species could belong to the genus Chalicogloea
(cf. Anagnostidis & Pantazidou, 1991).
Pseudocapsa. Members of this genus form more or less
irregular, packet-like colonies with colourless sheaths, but
the cells first divide radially and form characteristic colonies
(especially younger stages) with radially arranged cells (e.g.
Kova´cˇik, 1988). There are both aquatic and aerophytic types
in Pseudocapsa, although they are not yet sequenced.
However, the form of the cells and their organization is
distinctly different from other similar genera.
Gloeocapsa. The cells are in colonies that are more or less
distant from one another, each enveloped by their own
mucilaginous sheath. They are usually coloured by sheath
pigments. The cells divide regularly into three perpendicular planes in subsequent generations and the cells grow in
the original, more or less spherical shape and size before
Chlorogloeocystis. This is an aquatic genus that occurs in
2330
International Journal of Systematic and Evolutionary Microbiology 63
mineral waters and has colonies impregnated by ferric
precipitates (Brown et al., 2005). The spherical cells in
colonies are organized more or less in irregular rows,
without coloured envelopes. This genus has a known
Chalicogloea gen. nov., from dim-light environments
Cyanothece sp. 109 DQ243688
Cyanothece sp. 104 DQ243687
Cyanothece sp. 115 DQ243690
Dactylococcopsis sp. PCC8305 AJ000711
Cyanothece sp. PCC7418 AF296872
Euhalothece sp. ZM001 EU628548
88
Halothece sp. PCyano42 DQ058891
Rubidibacter lacunae EF126149
Geminocystis papuanica EF555569
Geminocystis herdmanii AB039001
Cyanobacterium aponinum AM238427
83
Cyanobacterium stanieri AF132782
Chroococcus turgidus HUW799 DQ460703
Gloeocapsa sp. PCC7310 AF132784
Limnococcus limneticus GQ375048
85
Chondrocystis sp. ANTLH59B1 AY493599
Chroococcus sp. JJCV AM710385
Chroococcus minutus GQ375047
64
Chroococcus membraninus GQ375049
Cyanothece sp. WH8902 AY620238
Gloeothece sp. KO68DGA AB067580
Gloeothece sp. KO11DG AB067577
Gloeothece sp. SK40 AB067576
Cyanothece sp. WH8904 AY620239
Cyanothece sp. PCC8801 AF296873
56 Aphanothece sacrum AB116658
Aphanothece sp. 2-0.5-3 EU015877
Microcystis panniformis EU541974
Microcystis wesenbergii U40334
94
Chroococcus sp. JJCM AM710384
Chalicogloea cavernicola CCALA 975T JQ967037
55
Gloeothece sp. PCC6909.1 EU499305
84 Gloeothece membranacea X78680
Cyanothece sp. PCC7424 AF132932
Snowella rosea AJ781042
Snowella litoralis AJ781041
Woronichinia naegeliana AJ781043
96
Merismopedia glauca AJ781044
Synechocystis trididemni AB011380
Aphanocapsa feldmani AF497568
60
Chamaesiphon subglobosus AY170472
Chroogloeocystis siderophila AY380791
Synechococcus lividus AF132772
‘Candidatus Synechococcus spongiarum’ EU307509
Aphanothece minutissima FM177488
57
Merismopedia tenuissima AJ639891
100
Aphanothece sp. 0BB21S01 AJ639901
52
Synechococcus nidulans EU078507
Synechococcus elongatus AF132930
Microcystis holsatica U40336
Microcystis elabens U40335
Pseudomonas aeruginosa AB364957
.
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.
.
.
.
.
.
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0.2
.
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.
Fig. 4. ML phylogenetic tree of cyanobacteria reconstructed from 52 complete 16S rRNA gene sequences (1427 informative
positions) showing the position of strain COLL 3T. ML bootstrap values below 50 % are shown for the deeper clades and the
corresponding nodes are indicated by filled squares. Bar, 0.2 substitutions per position.
phylogenetic position that is very distant from that of
Chalicogloea.
Gloeocapsopsis. This genus is similar to Chalicogloea in
its ecology, the form of its colonies and the special
envelopes around single cells; however, the cells are more
irregular and the laminated sheaths are usually intensely
coloured by sheath pigment (yellow–brown, reddish or
violet). The phylogenetic position of Gloeocapsopsis is not
yet well established, and certain differences are visible
within the genus between the marine types and aerophytic
populations usually described from wet stony walls.
Moreover, Gloeocapsopsis crepidinum (Thuret) Geitler ex
Koma´rek, the type species, is halophilic and lives on coastal
marine rocks (Silva e Silva et al., 2005, Ramos et al., 2010).
The thylakoid arrangement was rarely considered before
the availability of TEM (Lang & Whitton, 1973; Koma´rek,
2010a), although it is one of the phenotypic characteristics
http://ijs.sgmjournals.org
that is consistent with molecular criteria (Koma´rek &
Kasˇtovsky´, 2003). A large number of coccoid cyanobacteria
have a concentric-like thylakoid arrangement, although few
studies to date have employed advanced bioimaging
protocols to evaluate fine spatial arrangements in cellular
organization (Mullineaux, 1999; Nevo et al., 2007; Liberton
et al., 2011). It is difficult to observe the thylakoid
organization by optical microscopy or even with TEM;
nevertheless, optical images and TEM figures provide a
valid approach to determining the internal structure, which
could be studied better using CLSM serial sections to
reconstruct entire cells. The 3D reconstructions generated
from natural fluorescence z-stacks revealed a large surface
area of nearly flat, arm-like thylakoidal membranes
connected to each other and forming a unified structure
in a way that has, to our knowledge, never been described
before. This membrane arrangement in Chalicogloea
accounted for the changing directions of the thylakoids
seen in TEM and can be used as an autapomorphic
2331
M. Rolda´n and others
character for comparison with organisms with molecular
similarities (Koma´rek & Kasˇtovsky´, 2003) or for separation
from strains that show parietal thylakoidal arrangements.
Moreover, such a tight organization within a small cell
volume could be an advantage for living in dim caves.
Although not discussed here, phycoerythrin enables organisms to adapt to different qualities of light and to absorb
extra energy in a low-light environment (Albertano &
Herna´ndez-Marine´, 2001) and has been identified as part
of the present description. The pigments detected can be used
to monitor biodeteriogens in dim caves (Rolda´n et al., 2006).
The closest related sequence to COLL 3T is that of
Chroococcus sp. JJCM, a packet-forming organism isolated
from plankton from a reservoir, which was originally
determined to be Chroococcus cf. minor and later assigned
to Eucapsis sp. (Koma´rkova´ et al., 2010). There is 95 % 16S
rRNA gene sequence similarity between COLL 3T and
Chroococcus sp. JJCM, which is good evidence of an
independent evolutionary history (Johansen & Casamatta,
2005; Stackebrandt & Goebel, 1994). COLL 3T has the same
morphology and habitat as a cyanobacterium reported
under the name Gloeocapsa NS4 (Cox et al., 1981), which
was isolated at Juentica Cave (Asturias, Spain). Although
strain COLL 3T and Gloeocapsa NS4 have the same type of
cell division, thylakoid arrangement and ecology, there is
no molecular support to classify them under the same
genus.
Strain COLL 3T was identified here with an integrated
approach that brings together morphological characters and
molecular phylogeny and which is based on analysing the
complete 16S rRNA gene sequence. It should be highlighted
that diacritical characters, and the advanced bioimaging
protocols used to determine these characters, are very
important for identifying the species within the
Chroococcales. Overall, the polyphasic approach suggests
that strain COLL 3T belongs to a new genus and represents a
novel species of this genus. In addition, it should be classified
as an independent taxon in the order Chroococcales and
family Chroococcaceae.
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This study was supported by the Ministerio de Educacı´on y Ciencia,
Spain (grant CGL06-07424), and as a long-term research development
project of the Institute of Botany, Acadamy of Sciences of the Czech
Republic (no. RVO67985939). We are grateful to the Scientific and
Technical Services of the University of Barcelona and the Autonomous
University of Barcelona. Lastly, the authors thank Vanessa Balague´,
Antonia Navarro, M. Jose´ Garcı´a and the Ajuntament of Collbato´.
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