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Hereditas 139: 3540 (2003)

Karyotypic analysis of two algae species Scenedesmus incrassatulus


Bohl. and Scenedesmus antennatus Breb. (Chlorophyta,
Chlorococcales)
BALIK DZHAMBAZOV
1,2
, DETELINA BELKINOVA
3
and RUMEN MLADENOV
3
1
Section for Medical Inammation Research, Department of Cell and Molecular Biology, Lund Uni6ersity,
Lund, Sweden
2
Cell Biology Lab, Department of De6elopmental Biology, Uni6ersity of Plo6di6, Plo6di6, Bulgaria
3
Department of Botany, Uni6ersity of Plo6di6, Plo6di6, Bulgaria
Dzhambazov, B., Belkinova, D. and Mladenov, R. 2003. Karyotypic analysis of two algae species Scenedesmus incrassatulus
Bohl. and Scenedesmus antennatus Breb. (Chlorophyta, Chlorococcales). Hereditas 139: 3540. Lund, Sweden. ISSN
0018-0661. Received May 5, 2003. Accepted August 25, 2003.
The karyotypes (number, morphology and size of the chromosomes) of two algae species of Scenedesmus genus, S.
incrassatulus and S. antennatus, were studied. The karyotype of S. incrassatulus (n=4) was asymmetric, characterized by
two large metacentric, one large submetacentric and one small metacentric chromosomes. The karyotype assembly of S.
antennatus (n=6) reveals two metacentrics and four submetacentrics. This karyotype was symmetric. The general
chromosomal formulae of both species, as well as the total average metaphase length of their haploid set are presented.
The results of chromosomal studies of other related species are compared and discussed. Data from the karyotypic
analysis showed that S. incrassatulus, S. antennatus and S. obliquus are separate biological species from taxonomical point
of view.
Balik M. Dzhambazo6, Section for Medical Inammation Research, Department of Cell and Molecular Biology, Lund
Uni6ersity, I 11, BMC, SE-221 84 Lund, Sweden. E-mail: balik.dzhambazov@inam.lu.se, balik@pu.acad.bg
The Scenedesmus Meyen (Chlorophyta, Chloro-
coccales) genus was described still at the beginning of
19th century. It comprises species which are widely
spread in the plankton of different freshwater basins.
The species of Scenedesmus genus are strongly poly-
morphic KOMA

REK and FOTT (1983). A number of


studies are dedicated to the variability of morpholog-
ical characteristics of its cultures under the inuence
of different factors: temperature (KRIENITZ 1987;
TRAINOR 1992, 1993), pH (TRAINOR and ROSKOSKY
1967), feeding of zooplankters (LU

RLING and VAN


DONK 1997; LU

RLING 1999, 2002), concentration of


the nutrient medium (MLADENOV and BELKINOVA
1997) and others. By means of these studies, the
stability of taxonomical characteristics is established,
and many taxa of this genus are revised. The kary-
ological investigations of Scenedesmus genus, whose
beginning was put by DZHAMBAZOV et al. (2001),
provide much more precise taxonomical characterisa-
tion of these algae species. The present work is
dedicated to two species, which are rarely met in
nature. Their variability in cultures is poorly
investigated.
The species Scenedesmus incrassatulus Bohl. 1897
and Scenedesmus antennatus Breb. 1848 belong to
Obliquodesmus Mlad. subgenus. Both species have a
long taxonomy history, the main problematic points
of which are presented below.
As a basic taxonomy characteristic of S. incrassat-
ulus, BOHLIN (1897) pointed out the spindle-like shape
of the coenobiums peripheral cells, as well as the
specic bending of their apices towards the coenobi-
ums centre (Fig. 1A). Later on, a number of varieties,
which differ from the holotype mainly by the oval
shape of their cells and smaller cell dimensions, were
described: S. incrassatulus var. mononae G.M. Smith
1916, S. incrassatulus var. o6alis Svir. 1924, S. incras-
satulus var. reniformis Khrist. 1926 and S. incrassat-
ulus (var. mononae) f. robustus Uherk. 1956. On the
basis of the alternative arrangement of cells in the
four-cell coenobia, S. incrassatulus var. alternans De-
dus. 1953 was differentiated. Obviously, the taxonomy
of S. incrassatulus is not fully cleared out, since in case
of natural materials study PHILIPOSE (1967) pre-
sented coenobia, which were identical with those of S.
incrassatulus, as S. arcuatus var. capitatus G.M. Smith
1918. This fact was taken in consideration by KO-
MA

REK and FOTT (1983), who noted S. arcuatus var.


capitatus sensu Philipose as a synonym of S. incrassat-
ulus. On the other hand, HORTOBA

GYI (1973) pre-


sented coenobia, typical for S. incrassatulus, under the
name of S. dactyllococoides R. Chod. Later on, HIN-
DA

K (1990) referred S. dactyllococoides sensu Horto-


bagyi to the group of synonyms of S. incrassatulus.
In addition, the same author also assigned S.
incras satulus var. alternans and S. antillarum Comas
1981 to the synonyms of S. incrassatulus.
B. Dzhambazo6 et al. 36 Hereditas 139 (2003)
Fig. 1. Iconotypes of Scenedesmus incrassatulus Bohl., 1897
(A) and Scenedesmus antennatus Breb. in Ralfs, 1848 (B).
tion, their relationship with other Scenedesmus spe-
cies from a taxonomy point of view is discussed.
Since the species from Scenedesmus genus are repre-
sented by haploid forms throughout their ontoge-
netic cycle, the present work analyses their haploid
set of chromosomes (n).
MATERIAL AND METHODS
Clonal synchronous cultures
Clonal cultures of S. incrassatulus and S. antennatus
were studied. Clone No. 8216, isolated as S. incras-
satulus from the strain Vodenicharov 8216 (loc.
Bulgaria, Plovdiv, shpond) and kept in PACC un-
der No. 8216 named S. incrassatulus Bohl. Clone
No. 8919, isolated as S. acutus var. globosus Hor-
tob. from strain Mladenov 8919 (loc. Bulgaria, lake
Srebarna near Danube) and kept in PACC under
No 8919 named S. antennatus Breb.
The clonal cultures were obtained according to
the method of MLADENOV and FURNADZIEVA
(1995). The algal cultures cultivation and synchro-
nisation were carried out with an installation, de-
scribed by DILOV et al. (1972). Cultures were
synchronised by altering light/dark periods of 15/9
hours. The temperature was 33C and 22C during
the light and dark period, respectively. The inten-
sity of light during the light period was 12000 lux
(cool white uorescence). A BBM-nutrient medium
was used for culture (ARCHIBALD and BOLD 1970).
The cultures density was controlled at the begin-
ning of the light period by diluting with nutrient
medium to a concentration of 2.510
5
cells ml
1
.
The suspensions were aerated with 100 litres of air
per hour per one litre of suspension, adding 1 %
CO
2
during the light cycle.
Karyotype analysis
The karyotype analysis was performed on
metaphase plates, prepared from synchronised
clonal cultures in vitro. At the 10th hour of the
light period the cultures were treated with 0.2 mg/ml
colcemid for 2 hours. The metaphase spreads were
prepared according to the methods of MOORHEAD
et al. (1960), PAUSHEVA (1988) and CONKIE et al.
(1989) adapted for plant cells of in vitro culture.
Briey, cells were centrifuged at 1500 rpm for 5
min, treated with 75 mM KCl solution for 15 min
and hydrolysed in 5N HCl for 5 min. After xation
and rinsing in methanol/acetic acid (3:1), the cell
suspension was pipetted onto microscope slides and
allowed to dry under controlled conditions for opti-
mized spreading using the air-dried technique.
Chromosomes were stained with 10 % Giemsa solu-
Based on the existence of mucous globules on
cells apices, S. antennatus Breb. in RALFS (1848)
was described (Fig. 1B). KOMA

REK and FOTT


(1983) also put forward the mucous globules and
the specic shape of peripheral cells (moonlike, with
tortiled outside of the coenobiums centre apices) as
diagnostic characteristics for S. antennatus. Al-
though they preserved its species independence, the
same authors made an assumption that the appear-
ance of mucous globules is an eco-physiological
morphosis, related to denite conditions. In their
opinion, such morphoses are observed in a number
of species from the same subgenus: S. acutus (var.
globosus Hortob. 1954), S. acuminatus (f. globosus
Hortob. et Nem. 1963), S. tetradesmiformis
(Wolosz.) Chod. 1926 and others.
Based on the above exposition, the unsettled tax-
onomy status of S. incrassatulus and S. antennatus
is evident. The difculties in solving this problem
come from the fact that one of the species S.
antennatus is quite rarely observed and is known
only from natural materials. The existence of cul-
tures of these two species in the PACC (Plovdiv
Algal Culture Collection) allowed the accomplish-
ment of the present karyological investigation.
Until now, a cytogenetic analysis of six species
from the Scenedesmus genus was carried out: for
the establishment of their taxonomy status, initially
their morphology was studied, and afterwards
their karyological characteristics (MLADENOV and
FURNADZIEVA 1995, 1997, 1999; MLADENOV and
BELKINOVA 1997; DZHAMBAZOV et al. 2001,
2002a,b). The main goal of this research is to study
the karyotypes organisation and morphometry of
S. incrassatulus and S. antennatus, in order to es-
tablish whether these are two separate species and
what are their karyotype characteristics. In addi-
Karyotypic analysis of Scenedesmus 37 Hereditas 139 (2003)
tion in phosphate buffer (pH=7.2) for 10 min. For
the establishment of the morphometrical character-
istic, 30 metaphase plates of each of S. incrassatulus
and S. antennatus were analysed. For the karyotype
analysis, metaphase plates of single cells or
metaphase plates of coenobial cells, in which the
chromosomes were clearly differentiated and within
the cells, were chosen. Chromosome lengths were
measured on photographic prints.
The morphometrical characteristic of the chromo-
somes was assessed according to LEVAN et al.
(1964) and contains data about the absolute length
of each chromosome (M
L
), its relative length (Lr=
length of the chromosome/length of all chromo-
somes), arm ratio (Mr=length of the longer
arm/length of the shorter arm), and centromeric in-
dex (Ic=length of the shorter arm/absolute length
of the chromosome).
RESULTS
Karyotype of Scenedesmus incrassatulus Bohl.
The karyotype is asymmetric, characterised by a
haploid set of n=4 (Fig. 2). The morphometric
data are shown in Table 1. Based on the chromo-
some morphology of LEVAN et al. (1964), we di-
vided the chromosomes into two groups (I and II)
and three subgroups (A, B and C). Chromosomes
1, 2 and 3 belong to the rst group (I). Subgroup
A includes the rst two chromosomes (1 and 2).
They are large and metacentric. The third chromo-
some, which belongs to subgroup B, is large and
submetacentric. The fourth chromosome belongs to
the second group (II) and subgroup C. It is small
and metacentric. The general chromosomal formula
of S. incrassatulus is 3(2M+1SM) +1(m), and the
total average metaphase length of the haploid set is
4.6890.04 mm.
Karyotype of Scenedesmus antennatus Breb.
This species is characterized by a haploid set of
n=6 and symmetric karyotype (Fig. 3). Its chro-
mosomes are of similar size and belong to one and
the same group (I). The average values, received
from the morphometric analysis of the single chro-
mosomes, are shown in Table 2. Based on the mor-
phology of the chromosomes, they are divided into
two subgroups (A and B). Subgroup A includes the
rst two chromosomes, (1 and 2), which are meta-
centric. The rest of the chromosomes (3, 4, 5 and
6), which are submetacentric, belong to subgroup
B. The morphometrical data showed that the total
average metaphase length of the haploid set is
Fig. 2. Karyogram (A) and idiogram (B) of S. incrassatulus
(n=4) [Scale 2 mm].
10.0990.05 mm. The general chromosomal formula
of S. antennatus is 6(2M+4SM).
DISCUSSION
Most karyologically investigated Scenedesmus spe-
cies of the Obliquodesmus Mlad. subgenus have n=
4, n=5 or n=6 chromosomes (Table 3).
The small number of comparative karyological
investigations of these species is due to the small
size of their chromosomes, as well as to the fact
that most of these species form coenobia, which
additionally impede their karyotype analysis.
HEGEWALD (1974), KOMA

REK and FOTT (1983)


and KRIENITZ (1990) accept the spindle-like shape
and the tortiled towards the centre of the coeno-
bium apices of their peripheral cells as diagnostic
characteristics of S. incrassatulus (Fig. 1). The study
of the stability of cultures morphological character-
istics established certain closeness and transition
state between S. incrassatulus and S. obliquus
B. Dzhambazo6 et al. 38 Hereditas 139 (2003)
Table 1. Mean 6alues of morphological indices for chromosomes of the karyotype of Scenedesmus incrassatulus
Bohl. (M- large metacentric, SM large submetacentric, m small metacentric).
Chromosome Absolute length Relative length Group Subgroup Centromeric index Arm ratio Type
(r) M
r
9m
r
(%) L
r
9l
r
(%) I
c
9i
c
(mm) M
l
9m
l
1 1.4390.06 30.5590.83 I 40.5690.68 A 1.4690.07 M
2 1.3090.04 27.7890.65 38.4690.80 1.6090.08 M
3 1.0890.04 23.0890.76 25.9390.62 B 2.8690.04 SM
4 0.8790.02 II 18.5990.58 41.3890.78 1.4290.11 m C

L abs.n
=4.6890.04 mm.
Fig. 3. Karyogram (A) and idiogram (B) of S. antennatus
(n=6) [Scale 2 mm].
us complete reasons to consider this species as an
independent taxon.
Although the number of chromosomes in both
species is one and the same (n=4), their morphology
is too different (Table 3). While the karyotype of S.
obliquus is characterised by two big submetacentric
and two small metacentric chromosomes (2(SM)+
2(m)), the karyotype of S. incrassatulus comprises
two big metacentric chromosomes, one big submeta-
centric and one small metacentric chromosomes
(3(2M+1SM) +1(m)).
The real existence of the S. antennatus species is
contested by HEGEWALD (1979), HEGEWALD and
SILVA (1988) and HINDA

K (1990), who consider S.


antennatus as a synonym of the strongly polymorphic
S. obliquus (Turp.) Ku tz. From the carried out kary-
otype analysis it is evident that both species differ in
the number of chromosomes, as well in their mor-
phology (Table 3).
The present study gives a proof about the indepen-
dence of both taxa S. incrassatulus and S. antennatus,
but the question about the clear morphological char-
acteristics, according to which these two species can
be easily determined by hydro-biologists and ecolo-
gists is left open.
Recent karyotype studies of the Scenedesmus genus
provide the morphological basis for future cytoge-
netic investigations that can be useful in a taxonomic
and phylogenetic context.
(BELKINOVA 1998, 2001). The present karyological
study of S. incrassatulus rejects any doubts about the
identity of S. incrassatulus with S. obliquus, and gives
Table 2. Mean 6alues of morphological indices for chromosomes of the karyotype of Scenedesmus antennatus
Breb. (M metacentric, SM-submetacentric).
Type Arm ratio Group Subgroup Relative length Chromosome Absolute length Centromeric index
(%) L
r
9l
r
(mm) M
l
9m
l
(%) I
c
9i
c
(r) M
r
9m
r
1.1290.10 M I 47.2290.76 A 1 1.8090.07 17.8490.72
45.4590.92 1.2090.06 M 2 1.6590.07 16.3590.54
1.9390.04 B 3 1.7990.05 17.7490.68 34.0890.74 SM
1.8690.08 SM 34.9190.32 4 1.6990.03 16.7590.36
34.3890.60 1.9190.06 SM 5 1.6090.06 15.8690.66
30.7790.88 2.2590.08 6 1.5690.04 SM 15.4690.28

L abs.n
=10.0990.05 mm.
Karyotypic analysis of Scenedesmus 39 Hereditas 139 (2003)
Table 3. Karyological data of Scenedesmus species: haploid number and general chromosomal formulae.
Karyotype Reference n Species
2(2SM)+2(1m+1sm) DZHAMBAZOV et al. 2001 Scenedesmus regularis 4
3(3M)+2(1m+1sm) DZHAMBAZOV et al. 2001 5 Scenedesmus pectinatus
2(1M+1SM)+4(1m+3sm) DZHAMBAZOV et al. 2002a Scenedesmus nygaardii 6
4(3M+1SM) DZHAMBAZOV et al. 2002a 4 Scenedesmus bernardii
4 Scenedesmus obliquus 2(SM)+2(m) DZHAMBAZOV et al. 2002b
5(3M+2SM) DZHAMBAZOV et al. 2002b 5 Scenedesmus obtusiusculus
4 Scenedesmus incrassatulus 3(2M+1SM)+1(m) this study
Scenedesmus antennatus 6 6(2M+4SM) this study
REFERENCES
Archibald, P. and Bold, H. 1970. Phycological studies, XI.
The genus Chlorococcum Meneghini. The Univ. of
Texas publication, No. 7015: 1115.
Belkinova, D. 1998. The inuence of temperature on the
variability of Scenedesmus incrassatulus Bohl. (Chloro-
phyta, Chlorococcales) in clonal cultures. Trav. Sci.
Univ. Plovdiv. Plant. 6: 3746.
Belkinova, D. 2001. A study on the morphological variabil-
ity and taxonomy of subgenus Acutodesmus Hegew.
(Scenedesmus, Chlorophyta). Ph.D. Thesis, Univ. of
Plovdiv, (in Bulgarian).
Bohlin, K. 1897. Die Algen der ersten Regnellschen Expe-
dition. I. Protococcoideen. Bihang til Kongliga Sven-
ska Vetenskaps-Akademiens Handlingar 23.
Conkie, D., Erba, E., Freshney, R. et al. 1989. Animal cell
culture. A practical approach. IRL Press, Oxford-
Washington DC.
Dilov, Chr., Bozkova, M. and Staev, St. 1972. Prakticeskij
blok dlja kultivirovanija mikrovodoroslej v labora-
tornych uslovijach. [A practical block for cultivation of
microalgae in laboratory conditions]. Dokl. Akad.
selskoch. Nauk Bolg. 4: 169175.
Dzhambazov, B., Mladenov, R., Belkinova, D. et al. 2001.
Karyotype study of Scenedesmus regularis Svir. and
Scenedesmus pectinatus Meyen (Chlorophyta, Chloro-
coccales) in clonal cultures in vitro. Arch. Hydrobiol./
Algolog. Studies 101: 121128.
Dzhambazov, B., Mladenov, R. and Belkinova, D. 2002a.
Scenedesmus nygaardii Hub.-Pest. und Scenedesmus
bernardii G.M. Smith (Chlorophyta, Chlorococcales)-
zwei verschiedene Arten. Arch. Hydrobiol./Algolog.
Studies 106: 131139.
Dzhambazov, B., Belkinova, D. and Mladenov, R. 2002b.
Sind Scenedesmus obtusiusculus Chod. und Scenedesmus
obliquus (Turp.) Ku tz. (Chlorophyta, Chlorococcales)
verschiedene Arten? Arch. Hydrobiol./Algolog. Stud-
ies 106: 141150.
Hegewald, E. 1974. Bericht u ber seltene Gro nalgen aus
Dortmund, mit einem Schlu ssel fu r die Gattung
Scenedesmus. Dortm. Beitr. Landesk., Naturw. Mit-
teil. 76: 6174.
Hegewald, E. 1979. Vergleichende Beobachtungen an Herb-
material und Freilandmaterial von Scenedesmus.
Arch. Hydrobiol./ Suppl. 56: 264286.
Hegewald, E. and Silva, P. 1988. Annotated catalogue of
Scenedesmus and nomenclaturally related genera includ-
ing original descriptions and gures. Bibl. Phycol. 80:
1587.
Hindak, F. 1990. Studies on the chlorococcal algae
(Chlorophyta) V. Biol. Prace, Bratislava, Publ. House
Veda, Bratislava 36: 1225.
Hortobagyi, T. 1973. Neue Chlorococcalen aus den Absetz-
und Grundwasser-anreicherungsbecken der Budapest
Wasserwerke. Acta Bot. Acad. Sci. Hungaricae 18:
119130.
Komarek, J. and Fott, B. 1983. Chlorophyceae (Gru nal-
gen), Ordnung Chlorococcales. Das Phytoplankton des
Su sswassers. In: Thinemann, A. (ed.), Die Binnenge-
wasser, Band XVI. Schweizerbartsche Verlagsbuchh.,
Stuttgart, (7 Teil, 1. Halfte): 11044.
Krienitz, L. 1987. Studien zur Morphologie und Tax-
onomie der Untergattung Acutodesmus (Chlorellales).
Arch. Hydrobiol./Algological Studies 46: 137.
Krienitz, L. 1990. Coccale Gru nalgen der mittleren Elbe.
Limnologica 21: 165231.
Levan, A., Fredga, K. and Sandberg, A. 1964. Nomencla-
ture for centromeric position on chromosomes.
Hereditas 52: 201220.
Lu rling, M. 1999. The smell of water. Grazer-induced
colony formation in Scenedesmus. Ph.D. thesis, Wa-
geningen Univ.
Lu rling, M. 2002. Excreta of the grazer Daphnia magna
increase the frequency of coenobia in the green alga
Scenedesmus obliquus of various physiological ages.
Arch. Hydrobiol./Algological Studies 104: 109122.
Lu rling, M. and Van Donk, E. 1997. Morphological
changes in Scenedesmus induced by infochemicals re-
leased in situ from zooplankton grazers. Limnol.
Oceanogr. 42: 783788.
Mladenov, R. and Furnadzieva, S. 1995. Morphologische
Veranderlichkeit klonaler Kulturen von Scenedesmus
falcatus Chod. und Scenedesmus acuminatus (Lagerh.)
Chod. Arch. Hydrobiol./Algolog. Studies 79: 93107.
Mladenov, R. and Belkinova, D. 1997. Variabilitat von
Scenedesmus acuminatus (Lagerh.) Chod. und Scenedes-
mus pectinatus Meyen in Nahrlo sung mit verschiedenem
NaCl-Gehalt. Arch. Protistenk. 147: 393399.
Mladenov, R. and Furnadzieva, S. 1997. Morphologische
Veranderlichkeit klonaler Kulturen von Scenedesmus
pectinatus Meyen. Arch. Hydrobiol./Algolog. Studies
84: 113121.
Mladenov, R. and Furnadzieva, S. 1999. Ontogenetische
Veranderungen in klonalen Kulturen von Scenedesmus
acuminatus (Lagerch.) Chod. und Scenedesmus pectina-
tus Meyen. Arch. Hydrobiol./Algolog. Studies 92:
3546.
Moorhead, P. S., Nowell, P. C., Mellman, W. J. et al. 1960.
Chromosome preparations of leucocytes cultured from
B. Dzhambazo6 et al. 40 Hereditas 139 (2003)
human peripheral blood. Exp. Cell Res. 20: 613
616.
Pausheva, Z. P. 1988. Praktikum po citologii rastenij. [A
practical approach in cytology of plants]. 4 ed. BO
Agropromizdat, Moskow.
Philipose, M. T. 1967. Chlorococcales. Indian Council of
Agric. Res., New Delhi.
Ralfs, J. 1848. The British Desmidieae. London. XXII.
Trainor, F. R. 1992. Cyclomorphosis in Scenedesmus com-
munis Hegew. Ecomorph expression at low temperature.
Br. Phycol. J. 27: 7581.
Trainor, F. R. 1993. Cyclomorphosis in Scenedesmus sub-
spicatus R. Chod. Cell behaviours during the unicell-
colony transformation of a phenotypicly plastic
organism. Arch. Protistenkd. 143: 5561.
Trainor, F. R. and Roskosky, F. G. 1967. Control of
unicell formation in a soil Scenedesmus. Can. J.
Botany 45: 16571664.