The karyotypes, C-banding patterns and positions of nucleolus organizer regions (NORs) of two European species of toads, Bufo bufo and Bufo viridis from seven localities in Yugoslavia were investigated. These karyotypes and chromosomal markers were compared with those in other species and subspecies of the Bufo bufo complex. The results suggest that C-banded patterns are characters specific for those species. A marker band on chromosome 5 in the karyotype of B. bufo bufo is shown.Ispitivani su kariotipovi, C-trake i položaj nukleolusnih organizatora (NOR) dve evropske vrste žaba, Bufo bufo i Bufo viridis, sa 7 jugoslovenskih lokaliteta. Kariotipski i hromozomalni markeri ovih vrsta poređeni su sa podacima koji se odnose na druge vrste i podvrste. Rezultati ukazuju da je raspored C-traka specifične za ispitivane vrste. Kod vrste B. bufo bufo nađena je marker traka na petom hromozomu.Projekat ministarstva br. 03E0

Bilić, Grozdana

Lazić, Tanja

Spasić-Bošković, Olivera

Vujošević, Mladen

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

UDC 575.853; 597.82
Original scientific paper
C-BANDING KARYOTYPES AND LOCATION OF NOR'S IN THE TOAD
SPECIES BUFO BUFO AND BUFO VIRIDIS FROM YUGOSLAVIA
Olivera SPASIC-BOSKOvic, Tanja LAZlC, Grozdana BILIC,
and Mladen VUJOSEVIC
Department of Genetics, Institute for Biological Research "Sinisa Stankovic",
I I060 Belgrade, Yugoslavia
Spasic-Boskovic 0., T. Lazic, G. ame, and M. Vujosevic
(2000): C-banding karyotypes and location of NOR's in the toad species
Bufo bufo and Bufo viridis from Yugoslavia. - Genetika., Vol. 32, No.2,
145-153.
The karyotypes, C-banding patterns and positions of nucleolus
organizer regions (NORs) of two European species of toads, Bufo bufo
and Bufo viridis from seven localities in Yugosiavia were investigated.
These karyotypes and chromosomai markers were compared with those in
other species and subspecies of the Bufo bufo complex. The results sug-
gest that C-banded patterns are characters specific for those species. A
marker band on chromosome 5 in the karyotype of B, bufo bufo is shown.
Key words: Bufo bufo, Bufo viridis, constitutive heterochromatin, NORs
INTRODUCTION
Karyotypic conservatism characterizes many systematic groups of Am-
phibia (MORESCALCHl, 1973; WILSON et al., 1974). The karyotypes of many am-
phibian species consist of meta- and submetacentric elements that can roughly be
divided into "large" and "small" groups. Eighty three percent of toad species have
a complement of 2n=22-26 chromosomes (KURAMOTO, 1990). The considerable
Corresponding author: Mladen Vujosevic, Department of Gentics, Institute tor Biological
Research "Sinisa Stankovic", 29. Novembra 142, 11060 Belgrade, Yugoslavia
e-mail: mladenvu@ibiss.bg.ac.yu
146 GENETIKA, Vol. 32, No.2. 145-153, 2000.
differences in distribution of constitutive heterochromatin (C-bands) between some
species (SCHMID, 1978; MATSUI et al., 1985; GREEN and SESSIONS, 1991) question
the notion of conservatism based on chromosome number and morphology. In spite
of accumulating data on differences in C-band patterns between species there is
little information about interpopulation variability of C-banding patterns in Am-
phibia (RAGGHIANTI et al., 1980; KING, 1980; GREEN, 1985; MIURA, 1995a).
The Bufo bufo complex includes many subspecies. Two subspecies in-
habit the Balkan Peninsula. The described differences in karyotypes and C-banding
patterns between B. bufo subspecies revealed intraspecific chromosomal variations
(OBARA et al., 1975; MATSUI, 1980; BIRSTEIN and MAZIN, 1982).
The taxonomy of green toads (Bufo viridis group) still lacks final clarifi-
cation. Following a suggestion of ROTH (1996) real distinguishing is possible only
between diploid and tetraploid toads (STOCK, 1998; CASTELLANO et al.. 1(98).
The subject of the present study was cytogenetical characterization of two
species that inhabit the Northern Balkans, namely the common toad B. hula and the
green toad B. viridis.
MATERIALS AND METHODS
A total of 17 specimens of toads from 7 different localities in Yugoslavia
were cytogenetically analyzed (Table 1 ).
Tahle I. - Collection localities and number ofspecimens examined
~cies Locality males females total
Bufo bufo Deliblatska pescara 1 I
Lesnica I I
Petnica 2 2 4
Pasina ravan I I
Tara (Mitrovac) 2 3
Zlotska klisura I
Prokletije (Milisevacki potok) I
Bufo viridis Petnica 2 3 5
The animals were treated with 0.3% colchicine solution 18-24 hours be-
fore chromosome preparation. C-bands were prepared according to the method of
SCHMID (1978), with the modification described previously by SPASIC et al.
(1996). The nucleolus organizer regions (NORs) were stained according to the pro-
cedure described by HOWELL and BLACK (1980). Constitutive heterochromatin
was analysed on 20 selected metaphase figures. Measurements and comparisons of
photometric profiles of chromosomes were done using a cooled SBIG ST7 CCI)
camera and the ANALYST computer program (MALKOV et al., 1(95).
O.SPASIC-BOSKOVIC et al.: C-BANDING KARYOTYPES AND LOCATION OF NOR'S 147
RESULTS
The examined B. hula and B. viridis specimens had symmetrical karyo-
types that consisted of 2n = 22 chromosomes (Fig. I). Chromosomal pairs 1-6 were
large and represented 80% of the genome, whereas small chromosomes 7-I I repre-
sented only 20 % of the genome.
Fig. 1. Karyotype of Bufo bufo, conventional staining
4
5
7
8
3~
a b
6 11
a b a b
Fig. 2. C-banded chromosomes of A: Bufo viridis, B:. Bufo bufo
The karyotypes of the examined species showed differences in the amount
and position of constitutive heterochromatin (Fig. 2). In both species, constitutive
148 GENETIKA, Vol. 32, No.2, 145-153,2000.
hetrochromatin was found only in the pericentrorneric region of the small chromo-
some pairs, while the large chromosomes possessed constitutive hetrochromatin in
the centromere, as well as in the arms, The chromosome complement of B. bufo
had more distinct C-positive heterochromatin in the centromeric regions than B.
viridis. Comparative analysis of the distribution of constitutive heterochromatin
provided information for a cytogenetic determination of the species (Fig. 3). The
karyotype of B. bufo was characterized by C-bands at 5q (intercalary), 6q (telorn-
eric), 2p (pericentromeric) and 7q (intercalary),
B. buio
7 8
.'
10
•
11
9 10 11
B. vindis ..
876
....... {
-
l ."'{
.." •i
t
I
I r"'--~"~'lt:..:....
1 2 3 4 5
Fig. 3. ldiograms ofC - banded karyotypes of the examined species
O.SPASIC-BOSKOVIC el al.: C-BANDING KARYOTYPES AND LOCATION OF NOR'S 149
B. viridis had very characteristic chromosome pairs Nos. I and 3 with
many C positive regions. Clear C-bands in the karyotype of B. viridis were located
in the telomere on the short arm of the 4th chromosomal pair and in the pericentro-
meric region on the long arm of the 6th chromosomal pair. The most important dif-
ference in the distribution of constitutive heterochromatin was a very strong band
in the intercalar position on the long arm of the 5th chromosome pair in B. bufo.
Comparisons of photometric profiles of the 5th chromosome confirmed that (Fig.
5). This band was absent in the chromosome complement of B. viridis. Chromo-
some pair No. 5 also displayed a significant difference in relative chromosomal
lengths, as well as in centrometic index.
Fig. 4. Ag-NOR stained metaphase from Bufo bufo: Arrows show Ag-stained NORs of
chromosome No.6
Interpopulation chromosome polymorphism, concerning general chromo-
some morphology and heterochromatin distribution, was not found in either spe-
cies. Both species had the same position of NORs and corresponding secondary
constrictions in the subtelomeric region on the long arms of the 6th chromosome
pair (Fig. 3). In contrast to many populations where tetraploids of B. viridis were
found, only diploid toads were found in the examined localities.
150 GENETIKA, Vol. 32,No.2, 145-153,2000.
Fig. 5. Chromosome No.5 selected for analysis and photometric profiles of chromosome
No 5 of Bufo viridis (up) and B. hufo (down). Arrow shows marker band
DISCUSSION
Compared to other families of Anura, species of the family Bufonidae
have been poorly karyologically and cytotaxonomically investigated. Much of the
available data refers to Asian species and subspecies (OBARA et al., 1975; MATSUI,
1980; MIURA, 1995a; 1995b: CASTELLANO et al., 1998). The karyotype character-
istics of European toads from the genus Bufo were described in considerable detail
by SCHMID (1978). BIRSTEIN and MAZIN (1982) studied chromosome polymor-
phism of 840 hufo. KING (1991) summarized that in the genus Bufo all species
have dark paracentromeric C-bands, gray telomeric bands, C-bands associated with
NORs, and that many chromosomes have interstitial bands in varying positions
throughout the genome
Comparative chromosome analysis of species and subspecies of the genus
Bufo revealed a conservative karyotype model. AIl of the chromosomes are either
meta- or submetacentric, and C-positive heterochromatin is mostly found in cen-
tromeric and pericentromeric regions of chromosomes. All forms of European
toads of the genus Bufo have NORs in the telomeric region in the 6th chromosome
pair.
In spite of the conservatism of overall karyotype morphology, the distri-
bution of constitutive heterochromatin varied considerably between the examined
O.SPASIC-BOSKOVIC et al.. C-BANDING KARYOTYPES AND LOCATION OF NOR'S lSI
species. B. hula had more distinct C-positive heterochromatin in the centromeric
regions than B. viridis. In the karyotype of B. viridis the chromosomes had many
weak C-positive regions along the chromosome arms. In addition, B. viridis is the
onfy European toad with a brightly fluorescing heterochromatin (SCHMID, 1978).
Examined specimens of B. bufo showed a very clear C-band on the long
arm ofthe Sth chromosome pair (Fig. 2). The same position of the C-positive region
was reported previously by SCHMID (1978) and BIRSTEIN and MAZIN (1982), who
examined specimens from Germany and some localities near Moscow. MATSUI et
al. (198S) described a Sp pc band in the karyotype of B. bufo verrucosissimus. The
same Sp pc band was also observed in the karyotype of B. japonicus (MIURA,
1995a). According to observations reported by other authors and data presented
here, we suggest that karyotype characteristics of the examined species based on
the constitutive heterochromatin distribution are more regional - specific, than sub-
species-specific.
While comparing the karyotypes of the Bufo species from different re-
gions in Europe and Asia, we observed that I p pc and 6q tel C - positive regions
are stable within the genus in spite of the observed differences in genome size
(OLMO, 1973; BALDARI and AMALDl, 1976; BIRSTEIN and MAZIN, 1982).
Since subspecies of the Bufo bufo complex, as well as the Bufo viridis
group, are morphornetrically closely related, further cytogenetic, biochemical and
bioacoustic analyses will be useful in identifying and characterizing the different
forms in these complex groups oftoads.
Acknowledgment - This work was supported by the Research Science Fund of
Serbia Contract No. 03E02.
Received April II'h, 2000
Accepted October 2"d, 2000
REFERENCES
BALDARI C.T. and F. AMALDI, (1976): DNA reassociation kinetics inrelation togenome size in tour
amphibian species. Chromosoma 59, 13-22.
BIRSTEIN V.J. and AL MAZIN. (1982): Chromosomal polymorphism of Bufo bufo and C - banding
pattern ofB. b. verrucosissima. Genetica 59, 93-98.
CASTELLANO S., C. GtACOMA, T. DUJSEBAYEVA, G. ODIERNA, and E. BALLETTO (1998):
Morphometrical and acoustical comparison between diploid and tetraploid green toads.
Biological J. ofthe Linnean Society 63, 257-281.
GREEN D.M. (1985): Differentiation in amount ofcentromeric heterochromatin betweensubspecies of
the redlegged frog, Rana aurora. Copeia 4, 1071-1074.
GREEN D.M. and S.K. SESSIONS (1991): Amphibian Cytogenetics and Evolution. San Diego, Cali-
fornia, Academic Press.
152 GENETIKA, Vol. 32, No.2, 145-153,2000.
HOWELL W.M. and D.A BLACK (1980): Controlled silver staining of nucleolus organizer regions
with a protectivecolloidal devlopcr: a l-step method. Experientia36, 1014-1015.
KING M. (1980): C-banding studies on Australian hylid frogs: secondary constriction structure and the
concept of euchromatin transformation.Chromosoma 80, 191-217.
KING M. (1991 ): The evolution of heterochromatin in the amphibian genome. In: Green D.M. and
S.K. Sessions, Eds. Amphibian Cytogenetics and Evolution. pp. 539-392. San Diego, Cali-
fornia, Academic Press.
KURAMOTO M. (1990): A list of chromosome numbers of anuran amphibians. Bull. Fukuoka Univ.
Educ. Part 1Il39, 83-127.
MALKOV S., M. VUJOSEVI(:, and A. JOVANOVIC. (1995): One method for automatic chromosome
analysis and comparison. Proceedingsof 12th Panhelenic Conference in Mathematics Educa-
tion (Mathematics and Other Sciences), Heraclion. Crete, Greece,371-378.
MATSUI M. (1980): Karyology of Euroasian toads of the Bufo bufo complex. Annotnes ZooI. .lap. 53,
65-68.
MATSUI M., T. SETO, Y. KOHSAKA, and LJ. BORKIN (1985): Bearing of chromosome C-banding
patterns on the classification of Euroasian toads of the Bufo bufo complex. Amphibia-Rep-
tilia 6, 23-33.
MIURA I. (I 995a): Two differentiated groups of the Japanese toad, Bufojaponicusjaponicus, demon-
strated by C-banding analysis of chromosomes. Caryologia 48 (2), 123-136.
MIURA I. (1995b): The late replication banding patterns of chromosomes are highly conserved in the
genera Rana, Hyla, and Bufo (Amphibia, Anura). Chromosome 103, 567-574.
MORESCALCHI A. (1973): Amphibia. In: Chiarcll, A.B. and E. Capanna, Eds. Cytotaxonomy and
Vertebrate Evolution, pp, 233-347. New York, Academic Press.
OBARA Y, K. KOGAME, and K. SAITOH (1975): Distribution pattern of the constitutive hetrochro-
marin in the chromosomes of Bufo bufo japonicus Schlegel. Chromosome lnf. Serv.N 18,
15-17.
OLMO E. (1973): Quantitative variations in the nuclear DNA and phylogenesis of the Amphibia
Caryologia 26, 43-68.
RAGGHIANTI M, S. BUCCI-INOCENTl,and G. MANCINO (1980): Chromosome polymorphism in
the Italian newt, Triturus italicus. Chromosorna 77, 333-345.
ROTH P. (1996): An overview of the systematics of the Bufo viridis - group in Middle and Central
Asia. In: Rocek Z., Ed. Studies in Herpetology. pp. 127-130. Prague, Charles university.
SCHMID M. (1978): Chromosome banding in amphibia. I. Constitutive heterochromatin and nucleolar
organizer region in Bufo and flyla. Chrornosoma66, 361-388.
SPASIC 0., N. TANIC, .I. BLAGO.ll:::VIC, and M. VUJOSEVIC (1996): Constitutive heterochromatin
and nucleolus organizer regions in Rana graeca (Anura, Amphibia) from the north Balkans
(Yugoslavia). Copeia (I), 223-226.
STOCK M. (1998) Mating call differences between diploid and tetraploid green toads (BIY;) viridis
complex) in Middle Asia. Amphibia-Reptilia 19 (1),29- 42.
WILSON x.c., M. VINCENT, K. SARICI-I, and L. R. MAXON (1974): Thc importance of gene rear-
rangement in evolution: Evidence from studies on the rates of chromosomal, protein and
anatomical evolution. Proc. Nat. Acad. Sci USA 71,3028-3030.
O.SPASIC-BOSKOVIC et al. C-BANDlNG KARYOTYPES AND LOCATION OF NOR'S 153
RASPORED C-TRAKA I LOKACIJA NUKLEOLUSNIH
ORGANIZATORA KOD DVE VRSTE ZABA, BUFO BUFO I BUFO
VIRIDIS IZ JUGOSLAVlJE
Olivera SPASIC-BOSKOVIC, Tanja LAZIC, Grozdana BILlC
i Mladen VUJOSEVIC
Odeljenje za genetiku, Institut za bioloska istrazivanja "SiniSa Stankovic", 11060
Beograd, Jugoslavija
lzvod
lspitivani su kariotipovi, C - trake i polozaj nukleolusnih organizatora
(NOR) dye evropske vrste zaba, Bufo bufo i Bufo viridis, sa 7 jugoslovenskih loka-
liteta. Kariotipski i hrornozomalni markeri ovih vrsta poredeni su sa podacima koji
se odnose na druge vrste i podvrste. Rezultati ukazuju da je raspored C-traka speci-
ficane za ispitivane vrste. Kod vrste B. bufo bufo nadena je marker traka na petom
hromozomu.
Primljeno 1I. IV 2000.
Odobreno 2. X 2000.


C-banding karyotypes and location of NOR's in the toad species Bufo bufo and Bufo viridis from Yugoslavia

http://ibiss-r.rcub.bg.ac.rs/bitstream/handle/123456789/434/Rad_konverzija_480.pdf?sequence=1&isAllowed=y

C-banding karyotypes and location of NOR's in the toad species Bufo bufo and Bufo viridis from Yugoslavia

Abstract

Similar works

Full text

Available Versions

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)