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Chromosome numbers and nuclear DNA contents in
the red microalgae Cyanidium caldarium and three
Galdieria species
Olga Muravenko
a
, Irina Selyakh
b
, Neonila Kononenko
c
& Igor Stadnichuk
d
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov st.,
117984 Moscow, Russia
b
Lomonosov Moscow State University, 119899 Moscow, Russia
c
Institute of Agricultural Biotechnology, 42 Timiryazevskaya st., 127550 Moscow, Russia
d
Bach Institute of Biochemistry, Russian Academy of Sciences, 33 Leninsky prosp., 117071
Moscow, Russia
Published online: 03 Jun 2010.
To cite this article: Olga Muravenko , Irina Selyakh , Neonila Kononenko & Igor Stadnichuk (2001) Chromosome numbers and
nuclear DNA contents in the red microalgae Cyanidium caldarium and three Galdieria species, European Journal of Phycology,
36:3, 227-232, DOI: 10.1080/09670260110001735378
To link to this article: http://dx.doi.org/10.1080/09670260110001735378
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Eur. J. Phycol. (2001), 36: 227232. Printed in the United Kingdom 227
Chromosome numbers and nuclear DNA contents in the red
microalgae Cyanidium caldarium and three Galdieria species
OLGA V. MURAVENKO
1
, IRINA O. SELYAKH
2
, NEONILA V. KONONENKO
3
AND IGOR N. STADNICHUK
4
" Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov st., 117984 Moscow, Russia
# Lomonosov Moscow State University, 119899 Moscow, Russia
$ Institute of Agricultural Biotechnology, 42 Timiryazevskaya st., 127550 Moscow, Russia
% Bach Institute of Biochemistry, Russian Academy of Sciences, 33 Leninsky prosp., 117071 Moscow, Russia
(Received 1 June 2000; accepted 1 January 2001)
An image analysis system was used to count and measure acetoorcein-stained mitotic chromosomes of the
acidothermophilic unicellular red algae Galdieria maxima, G. partita, G. sulphuraria and Cyanidium caldarium
(Cyanidiophyceae). Chromosome numbers fell into two groups: nl2 for all three species of Galdieria and nl5(7) for C.
caldarium. It seems that a separation of C. caldarium from Galdieria is karyologically justied. A chromosome number of 2
appears to be indicative of the genus Galdieria and could be used as a marker to distinguish this taxon from Cyanidium.
The two smaller chromosomes in the karyotype of C. caldarium were about 0n4 m long whereas the other three were
0n50n7 m long. In karyotypes of Galdieria species, the two chromosomes diered in length, the smaller chromosome
ranging from 0n8 to 1n8 m and the larger one from 1n2 to 2n3 m. The visualization of these extremely small chromosomes
was possible due to pretreatment of the cells with the DNA intercalator 9-aminoacridine. The mean absolute length of each
chromosome of the three members of Galdieria had statistically signicant interspecies dierences. Nuclear 1C DNA
contents were estimated in the algal cells by the Feulgen technique. All species investigated had genome sizes of
1n502n25i10
# pg. Thus it seems that the members of Cyanidiophyceae have the smallest known genomes of all
photosynthetic eukaryotes.
Key words: chromosome number, Cyanidium caldarium, DNA content, Galdieria maxima, Galdieria partita, Galdieria
sulphuraria, genome size
Introduction
Chromosome number is one of the characteristics
that is used to delimit species. Nevertheless, many
algal taxa have not been karyotyped and both their
genome sizes and chromosome numbers remain
unknown (Sedova, 1996). Determination of the
chromosome number may be helpful for classifying
algal species whose systematic position is un-
resolved. This requires both morphological and
biochemical criteria as well as karyological charac-
teristics, the problem being exemplied by the red
unicellular algal class Cyanidiophyceae.
The Cyanidiophyceae are unusual among algae,
showing characters of primitive unicellular rhodo-
phytes with an ability to inhabit hot acidic waters,
unique among photosynthetic eukaryotes
(Seckbach, 1999). These acidothermophilic algae
occur world-wide, but exclusively in isolated areas
of hot sulphur springs and volcanic calderas with
Correspondence to: I. N. Stadnichuk. Fax: j7 095 9542732.
e-mail : Stadnichuk!inbi.ras.ru
pH values from 0n5 to 3 and temperatures of
2555 mC (Gross et al., 1998). The taxonomic
position of Cyanidiophyceae has been discussed for
several decades (Ott & Seckbach, 1994). For a long
time, all members of this small class were confused
under the name Cyanidium caldarium (Tilden)
Geitler, owing to their coexistence in mixed popu-
lations, the resemblance of the life cycles, and their
identical pigment composition. Merola et al. (1981)
distinguished three dierent species in natural popu-
lations of Cyanidium caldarium, and classied
them as Galdieria sulphuraria (Galdieri) Merola,
Cyanidium caldarium and Cyanidioschyzon merolae
De Luca, Taddei & Varano. Cyanidioschyzon
merolae is a rare species and apparently only a
minor component of algal biomass in acidic sulphur
springs (De Luca et al., 1978). Galdieria and
Cyanidium are distributed more widely. They were
attributed to two dierent genera in the family
Cyanidiaceae (Merola et al., 1981), mainly on the
basis of their morphological characters including
spore formation. All Cyanidiophyceae propagate
asexually by mitotic spores that are formed within
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228 O. V. Muravenko et al.
the envelope of mature cells. The number of
autospores is species-specic and varies from 2 to
16, reaching 32 in G. sulphuraria (Merola et al.,
1981). Sentsova (1991, 1994) discovered in pre-
viously uninvestigated volcanic geographical areas
some unicellular thermoacidophilic algae with
characters of the Cyanidiophyceae. They were
identied as various new species of Galdieria: G.
daedala Sentsova, G. maxima Sentsova and G.
partita Sentsova. All the new species were dis-
tinguished according to the following criteria: cell
size, lobed chloroplast shape, dierent numbers of
autospores within maternal cells, and shape of the
empty cell envelopes after spore release (Sentsova,
1991, 1994).
This identication to species level for strains
isolated from dierent and restricted geographical
regions faces certain diculties. It was assumed
that G. daedala, G. maxima and G. partita are
not well dened taxa in terms of morphological
criteria, but represent morphologically variable
ecotypes, of one and the same Galdieria species
(Ciniglia et al., 1999). At the same time, one of these
new taxa, namely G. partita (Stadnichuk et al.,
1997; Sugawara et al., 1999), is now identied as
well as G. sulphuraria (Gross et al., 1999) in
dierent biochemical and genetic studies.
Galdieria can be distinguished from Cyanidium
only by one known signicant biochemical feature:
Galdieria grows auto- and heterotrophically,
whereas Cyanidium is an obligatory autotroph
(Gross, 1999). Ott & Seckbach (1994) concluded
that heterotrophy was not sucient to separate
Galdieria and Cyanidium and returned Galdieria to
Cyanidiumbecause both taxa are thermoacidophilic
and morphologically similar. An alternative classi-
cation of this algal group with six or seven species
of Cyanidiumwas proposed (Ott &Seckbach, 1994).
The inability of morphological criteria and bio-
chemical markers to resolve relationships within
this group of algae led us to quantify and charac-
terize nuclear genomes in the Cyanidiaceae. The
present study was initiated to provide information
on the karyotypes and DNA contents of four
asexual species of this family.
Materials and methods
Cell cultures
Cyanidium caldarium strain P-511, Galdieria maxima
strain P-507 and G. partita strain P-500 were obtained
from the IPPAS Culture Collection of Microalgae,
Institute of Plant Physiology, Russian Academy of
Sciences. G. sulphuraria strain 074W was a gift from
Dr W. Gross (Freie Universita$ t Berlin, Germany). The
geographical origins of the algal strains were: Kamchatka
Peninsula, Russia (C. caldariumandG. partita) ; Kunashir
Island, Russia (G. maxima) ; and Jawa Island, Indonesia
(G. sulphuraria). The four algal species were cultured
in 100 ml Allens medium (Allen, 1959) at pH 2n5 in
250 ml asks shaken under continuous white light
(80 mol m
# s
"), all at 35 mC except for G. sulphuraria
074W, which was grown at 25 mC (Gross, 1999).
Growth of G. maxima, G. partita and G. sulphuraria was
stimulated by the addition of 1%b-glucose to the mineral
medium.
Karyology
In mid-log phase, 0n1% (w\v) pepsin (Merck, USA)
was added for 3 h to the culture asks to digest the
cell walls. Then, to obtain a high resolution of
chromosomes 1 g ml
" of the DNA intercalator 9-
aminoacridine (Sigma, USA) was added and the asks
shaken for 12 h. After this pretreatment cells were
harvested by centrifugation at 3000 g for 5 min and
resuspended for 4 h in fresh culture medium with 0n05%
(w\w) of the mitotic inhibitor colchicine (Merck, USA).
The harvested cells were xed in 3: 1 ethanol\acetic
acid (v\v) and stained for 2448 h with 1% (w\v) aceto-
orcein at room temperature. Temporary chromosome
slides were prepared from these cell suspensions by
squashing in a drop of fresh acetoorcein solution. To
prevent rapid drying, cover slips were sealed with a
rubber glue (Muravenko et al., 1999). Between 30
and 60 late prometaphasemetaphase division gures
were observed for each species using an Amplivall micro-
scope (Carl Zeiss, Germany) connected to a special
chromosome image analysis system Video Test-Karyo
1n5 (Video Test, Russia). Fifteen best metaphase plates
with a good chromosome spread were selected and
photographed with a nal magnication of i1250. The
mean value and standard error of the mean for absolute
(m) and relative (%) lengths were calculated for each
chromosome and species using automated measurements
of chromosome images (Muravenko et al., 2000). The
MannWhitney non-parametric statistic test (Tyurin
& Makarov, 1999) was used to estimate signicant
dierences between the means (l0n05).
Microspectrophotometry
After harvesting, algal cells were xed in ethanol\acetic
acid (3: 1, v\v) for 4 h at 4 mC. Preliminary analysis
of the hydrolysis curves showed the treatment with 1 N
HCl at 50 mC for 30 min to be optimal for these algae.
The cells were stained with Schis reagent at 4 mC for
3 h. The Feulgen absorption of the nuclei was mea-
sured by an Opton MSP-20 microspectrophotometer
(Germany) with an analysis system for automatic
measurements. Telophase and metaphase plates were
selected manually to determine absorption values for
1C and 2C DNA contents, respectively. These data
were veried by intephase nuclei measurements, and
the software Statistika for Windows (5n0) was used to
obtain the absorption frequency distributions of nuclei
in G1, S, and G2 phases of the cell cycle (Khoudoleeva
et al., 2000). Nuclei in G1 phase correspond by
Feulgen absorption values to telophase plates, and nuclei
in G2 phase correspond to metaphase plates. Approxi-
mately 6070 nuclei were examined for each species.
Microspectrophotometric data obtained with the same
instrument for Saccharomyces cerevisiae with a known
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Chromosomes and nuclear DNA in Cyanidium and Galdieria 229
DNA content of 1n7i10
# pg (Sherman, 1991) were
used to convert Feulgen arbitrary units into picograms
of DNA.
Results
The mitotic chromosomes of G. maxima, G. partita,
G. sulphuraria and C. caldarium were visualized
during autospore formation (Figs 18). A new
technique was developed involving cell pre-
treatment with pepsin and 9-aminoacridine which
was crucial for obtaining high-quality squash slides
with elongated chromosome spreads and provided
adequate chromosome morphometric data (Tables
1, 2).
A chromosome number of nl2 was found in all
three Galdieria species. In each species, the chromo-
somes can be divided by their mean absolute lengths
into two groups (Figs 16). The chromosome mean
absolute lengths were 1n102n36 m for chromo-
some I and 0n771n10 m for chromosome II; G.
maxima had the largest chromosomes (Table 1).
The centromere positions in the chromosomes were
not visible. The mean absolute lengths of each
chromosome in the karyotypes of G. maxima, G.
partita and G. sulphuraria had signicant inter-
species dierences ( p 0n05), but the relative
lengths of both chromosomes (as % of total)
were approximately equal in all Galdieria species
(Table 1).
It was more dicult to calculate the chromosome
number in C. caldarium. Dot-like chromosomes
were observed in late prophase and early metaphase
that were smaller than in Galdieria. The image in
Figs 18. Acetoorcein-stained mitotic chromosomes and their computer drawings in Galdieria species and C. caldarium. Figs
1, 2. Galdieria maxima: prometaphase and metaphase chromosomes, nl2. Figs 3, 4. Galdieria partita: early metaphase
chromosomes, nl2. Figs 5, 6. Galdieria sulphuraria: metaphase chromosomes, nl2. Figs 7, 8. Cyanidium caldarium,
showing 5 clearly visualized metaphase chromosomes. Scale bars represent 3 m.
Fig. 7 indicates that each mitotic cell has at least ve
chromosomes. The mean absolute lengths for the
two smaller chromosomes were about 0n4 m, and
about 0n50n7 mfor the three larger ones (Table 2).
In some cells, C. caldarium seemed to have two
additional minute chromosomes that were barely
visible (data not shown). The chromosome number
nl5 should be considered as tentative due to the
problems associated with distinguishing these ex-
tremely small (less than 0n2 m) chromosomes that
could not be reliably counted. In addition, it was
very dicult to obtain metaphase plates with
satisfactory chromosome spreading in the squash
slides. The chromosomes of C. caldarium tend to
stick together, which hampered their counting. For
these reasons, the possibility that the C. caldarium
cells contain seven rather than ve chromosomes
cannot be excluded.
DNA quantication was carried out on mitotic
algal cells and nuclei in G
"
, S and G
#
phases of the
cell cycle. The distribution of nuclei in dierent
phases of the cell cycle was approximately the same
for all Galdieria species, but the number of C.
caldarium nuclei in the G1 phase was higher (Figs
912). Automatic calculation of the percentage of
nuclei in each phase allowed the estimation of the
1C and 2C values for the DNA content for each of
the four species under study. In a typical set of
observations, the absorption values for stained 1C
nuclei in G. maxima, G. partita, G. sulphuraria and
C. caldarium yielded a genome size estimate in the
range of 1n502n25i10
# pg (Tables 1, 2). G.
maxima cells had the highest 1C DNA content,
whereas the smallest genome was found in G.
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230 O. V. Muravenko et al.
Table 1. Chromosome sizes and DNA contents in Galdieria
Alga
Mean chromosome lengths
1C DNA content
(pgi10
#pSE)
mpSE % of total lengthpSE
I II I II
G. maxima 2n36p0n35 1n78p0n25 57p9 43p6 2n25p0n09
G. partita 1n56p0n23 1n26p0n21 55p11 45p13 1n56p0n06
G. sulphuraria 1n20p0n15 0n87p0n09 59p14 41p12 1n35p0n05
Table 2. Chromosome sizes and DNA content in C. caldarium
Mean absolute chromosome lengths ( mpSE)
Total length
( mpSE)
1C DNA content
(pgi10
#pSE) I II III IV V
0n69p0n05 0n63p0n02 0n50p0n03 0n43p0n04 0n36p0n05 2n61p0n06 1n49p0n03
9 10
11 12
Figs 912. Frequency distributions of relative DNA values for nuclei after Feulgen staining and the percentage of nuclei in
dierent phases of cell cycle. Fig. 9. Galdieria maxima. Fig. 10. Galdieria partita. Fig. 11. Galdieria sulphuraria. Fig. 12.
Cyanidium caldarium.
sulphuraria cells. Genome sizes estimated for G.
partita and C. caldarium were approximately equal.
1C DNA contents and total chromosome lengths in
the species studied were found to be positively
correlated.
Discussion
High-quality squash slides of plant cells can be
obtained only after digestion of the cell wall. The
cell pretreatment with pepsin used in this work
seems to be adequate for Cyanidiophyceae. The
envelopes of these algae are primarily proteinaceous
without signicant amounts of cellulose (Baily &
Staehelin, 1968), and pepsin is a non-specic pro-
tease active at pH 13 and 3640 mC, conditions
that completely coincide with those of algal growth.
Most karyological studies are conducted on
species with medium-sized or large chromosomes
(56 m). Chromosome lengths 4 m are con-
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Chromosomes and nuclear DNA in Cyanidium and Galdieria 231
sidered as small (Muravenko et al., 1999). Such dot-
like chromosomes without a primary constriction
are typical for unicellular algae (Sedova, 1996). In
our case visualization of chromosomes of the red
microalgae by the routine acetoorcein technique
was hindered by their short length. Therefore, we
applied pretreatment with the non-specic DNA
intercalator 9-aminoacridine, which is extremely
important for producing elongation and spreading
of small plant chromosomes (Muravenko et al.,
2000). The use of animage analysis systemimproved
chromosome visualization and measurement. This
approach also allowed us to reveal dierences in
chromosome lengths.
The chromosome numbers are known for no
more than 56% of all red algal species (Sedova,
1996). In the present investigation, chromosomes
were visualized and counted for four species of the
family Cyanidiaceae (class Cyanidiophyceae,
Rhodophyta) whose karyology has not been studied
before. The chromosome numbers fell into two
groups: nl2 for three species of Galdieria and nl
5 for C. caldarium. The estimated dierence in
chromosome numbers is a strong karyological
criterion of a well-dened species. It appears that a
separation of C. caldarium from Galdieria is
justied. The chromosome count testied that all
Galdieria species are closely related and that they
are distinct from C. caldarium. The primary physio-
logical dierence between the two genera is the
ability of Galdieria to grow heterotrophically
(Gross, 1999). Our karyological data, together with
this dierence, warrant recognition of Galdieria and
Cyanidium.
Karyological data for three of the four presently
known (Merola et al., 1981; Sentsova, 1994)
Galdieria species demonstrate that the chromosome
number in this genus is 2, which is the smallest
chromosome complement known in micro- and
macroalgae (Sedova, 1996). This group represents a
single genus of the Cyanidiophyceae. The statistic-
ally signicant size dierences of the two chromo-
somes among three Galdieria forms argues for
morphological recognition of separate Galdieria
species fromdierent world regions (Moreira, 1994;
Seckbach, 1994; Sentsova, 1994) rather than geo-
graphical races (Ciniglia et al., 1999).
Although most karyological investigations of
algae are limited to chromosome numbers (Sedova,
1996), genome size is a useful addition to this
information. The Feulgen reaction is one of the
most frequently used techniques for microspectro-
photometrical DNA quantication. The dierences
obtained in the Feulgen absorption values reect
absolute dierences in the nuclear DNA content
because Schis reagent, which binds to purine
residues, is not aected by the GC\AT ratio.
Cytophotometric DNA quantication has not
previously been applied to Galdieria, but the
genome size of one strain of G. sulphuraria has been
determined by pulsed-eld gel electrophoresis of
digested DNA (Moreira et al., 1994). DNA of C.
caldarium strain RK-1, practically identical to our
C. caldariumP-511 (Sentsova, 1991), was quantied
spectrouorometrically after staining with 4h,6-
diamidino-2-phenylindole (DAPI) (Suzuki et al.,
1992). According to these karyotypings, the nuclear
DNA of C. caldarium RK-1 and of G. sulphuraria
strain 17n91 averaged 13 Mbp (megabase pairs) and
9n8 Mbp, respectively. To compare our results with
these data we took into account that the
1n7i10
# pg DNA present in the nucleus of S.
cerevisiae (Sherman, 1991) equals the genome size
of 13n614n0 Mbp (Link & Olson, 1991; Gotta &
Gasser, 1996). The same pg\Mbp proportionality
applied to the Cyanidiophyceae indicates that the
nuclear DNA content in C. caldarium P-511
represents 12n0 Mbp and 10n8 Mbp in G. sulphuraria
074W. These values may be considered as a sat-
isfactory agreement with the genome size reported
for C. caldarium and G. sulphuraria (Suzuki et al.,
1992; Moreira et al., 1994). Only the genomes of
yeast and protozoa are also known to be so small
(Link & Olson, 1991). The genome size of several
lower unicellular eukaryotes, including Chlorella,
ranges from 35 to 47 Mbp (Takahashi et al., 1993).
Thus Cyanidiaceae, which have the smallest plant
genomes, can be considered as the most primitive
of photosynthetic eukaryotic organisms.
Acknowledgements
This work was supported in part by a grant fromthe
Russian Foundation for Basic Research (N 00-15-
97833). The authors are grateful to Prof. Alexander
V. Zelenin for helpful discussion and recommen-
dations.
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