Comparative cytogenetic analysis of European brown frogs: Rana temporaria, R. dalmatina and R. graeca

CARYOLOGIA

Vol. 50, n. 2: 139-149, 1997

Comparative cytogenetic analysis of European brown frogs: Rana temporaria, Re dalmatina and R graeca OLIVERA SPASIC-BOSKOVIC, NIKOLA TANIC, ]ELENA BLAGOJEVIC and MLADEN VUJOSEVIC Department of Genetics, Institute for Biological Research «Sinisa Stankovic», 11060 Belgrade, Yugoslavia

SUMMARY - A cytogenetic study, based on the comparative analysis of chromosome morphology, amount and distribution of C-positive heterochromatin and positions of NORs, was undertaken on three species of brown frogs (Rana temporaria, R. dalmatina and R. graeca) from different Balkan localities. In contrast to their uniform karyotypes with respect to chromosome number, important interspecies differences have been observed in centromeric indices, the amount of constitutive heterochromatin, the distribution of C-bands and the positions ofNORs. Analysis of these karyotype characteristics suggests that mostly intrachromosomal rearrangements occurred during karyotype differentiation of the examined species.

INTRODUCTION

The genetic characterization of brown frogs that inhabit the Balkan peninsula has been insufficient. In recent years many authors have studied differentisozymes (CAPULA 1991; MENSI et at. 1992; GREEN and BORKIN 1993; ARANO et at. 1993) and chromosome differentiation (ODIERNA 1989; NISHIOKAet at. 1987; HERRERO et at. 1990; BELTCHEVA and SOFIANIDOU 1990; MIURAet at. 1995; SPASIC et at. 1996) in attempts at resolving the evolutionary relationships of Eurasian brown frogs. The majority of species of the genus Rana share a standard number of 2n = 26 chromosomes (MORESCALCHI) 1973, 1990) with a few exceptions that have 22 or 24 chromosomes (KURAMOTO 1972, 1990; SCHMID 1980). Although a great majority ofRana species have 26 large meta- and submetacentric chromosomes, comparative cytogenetic studies show considerable differences in the amount and distribution of constitutiveheterochromatin and the positions of secondary constrictions and nucleolusorganizers (NORs). Among karyotypycally conservative groups, such as the genusRana, comparative cytogenetic analysis based on the differential staining ofconstituve heterochromatin and NORs is particularly helpful.

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The present paper presents a cytogenetic analysis of R. dalmatina and R. temporaria from Yugoslav, and R. graeca from both Yugoslav and Greek localities. Ag-NOR staining and Cbanding were used to obtain more information on the phyletic relationships and heterochromatin evolution in European brown frogs.

MATERIALS AND METHODS A total of 87 frogs were used for chromosomal studies (Table 1). The animals received an intraperitoneal injection of 0.3% colchicine 24 h before chromosome preparation. Chromosome preparations were obtained from the intestine, bone marrow and testes according to the procedure of SCHMID (1978a). Ag-NOR staining was performed using the «one-step» method of HOWELL and BLACK (1980). C-bands were produced usingBa(OH)2 denaturation at 30° C for 15 s to 1 min, and incubation for 1 h in 2 X SSC at 65° C. Measurement of chromosome length, amount of constitutiveheterochromatin and estimation of the centromeric index were done from photographs of selected metaphase figures.

RESULTS

1. Karyotypes . The quantitative karyotype characteristics of Rana temporaria, R. dalmatina and R. graeca are summarized in Table 2. The general uniformity of thekaryotypes based on chromosome number (2n = 26) and morphology (five large and eight small pairs ofbiarmed chromosomes) was accompanied by important differences in thecentromeric indices of certain chromosomes. In the group of small chromosomes R. dalmatina had one metacentric chromosome pair (no. 13). R. graeca two (nos. 11 and 13), while R. temporaria was without metacentric elements. The observed differences in the centromeric indices for the lOth and 3rd chromosome pairs are due to the position of secondary constrictions and NORs. In comparison to the karyotypes of R. dalmatina and R. temporaria, the karyotype of R. graeca displayed significant differences in the centromeric index for most chromosomes. In contrast to the centromeric index, the relative chromosome length did not show significant differences among the karyotypes of the examined species. The stability of relative chromosome lengths and the variability ofcentromeric indices suggests thatintrachromosomal re arrangements occurred during differentiation of the chromosome complements in this group of frogs. CYTOGENETIC ANALYSIS OF EUROPEAN BROWN FROGS

TABLE 1 - Collection localities and number of specimens examined.

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2. C-positive heterochromatin. In contrast to the relative morphological uniformity of karyotypes of the examined species, important differences were observed in the amount of constitutiveheterochromatin, as well as in the position and size of C-bands. Inthe karyotypes of R. temporaria, R. dalmatina and R. graeca, constitutive heterochromatin was predominantly found in thecentromeric, paracentromeric and telomeric regions of chromosomes. R. graeca showed the highest amount of C-positiveheterochromatin (Table 3). In the karyotype of R. temporaria, the constitutive heterochromatin was

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TABLE 2 - Quantitative characteristics of chromosomes inkaryotypes of the examined species.centromeric index/lOO (length of short arm/length of whole chromosome), and percent length of chromosome pairs in relation to total genome length. Standard deviations are in parentheses.

mostly located in the centromeric and telomeric regions of the chromosomes and represented only 17.25% of the genome. The distribution of constitutiveheterochromatin is presented in Fig. 2. The karyotypes of the examined species had marker chromosomes or C-bands which characterized these complements. Comparative analysis ofheterochromatin distribution provided information for a cytogenetic determination of the examined species. R.temporaria had marker C-bands on 3p, 9q, and a characteristic distribution of constitutiveheterochromatin on the 5th chromosome pair. The karyotype of R. dalmatina was

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.

Fig. 1. - Map of collecting localities of Rana temporaria, R. dalmatina, and R. graeca. The numbers designate the localities listed in Table 1

characterized by pericentromeric bands on the short arm of the 7th andlOth chromosomes. R. graeca had very characteristic chromosomes 4, 7, 10 and 11. The metacentric chromosome 11 had a strong C-positive band which served as a chromosome marker for thekaryotype of R. graeca. A comparative analysis of karyotypes showed that the distribution ofCbands of chromosome 2 was very similar between species, but varied strongly on chromosomes 7 and 11. Chromosome 7 displayed a tendency to become moreheterochromatic in the centromere and short arm: in Rana temporaria it had the narrowest band on the short arm, in R.dalmatina it had a clearly expressed pericentromeric band, and in R. graeca it had distinct bands in the pericentromeric region. Cbanded metaphase chromosomes are presented in Fig.3.

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3. Nucteotus organizer regions (NORs). Secondary constrictions often correspond to NORs and are important characteristics of karyotypes. In the karyotypes of the examined species, the positions of theNORs served as markers for cytogenetically identifying the species. R.temporaria had NORs on the long, and R. graeca on the short arms of the loth, and R. dalmatina on the short arm of the 3rd chromosome pairs (Fig. 3).

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Fig. 3. - C-banded chromosomes of a) R.dalmatina, b) R. temporaria, c) R. graeca, and the localization of NORs in the karyotypes of d) R. dalmatina, e) R. temporaria f) R: graeca.

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DISCUSSION

Most amphibians possess a small number of large meta- and submetacentric chromosomes (MORESCALCHI 1973, 1993; SCHMID 1978a, b; GREEN and BORKIN 1993). Species belonging to the Rana genus mostly have 24 or 26 chromosomes and are considered a very conservative group with respect to chromosome number andkaryotype morphology. Analysis of the chromosome complements of R. temporaria, R. dalmatina and R. graeca revealed that the uniform morphology ofkaryotypes was accompanied by major differences in the centromeric indices of particular chromosomes. Differences in thecentromeric indices with unchanged relative chromosome lengths suggests that intrachromosomal rearrangements, such as pericentromeric inversions, occurred during the differentiation ofkaryotypes in this group of species. Chromosomes whose centromeric indices varied between karyotypes, also involved distinct differences in the amount and distribution of C-positiveheterochromatin. Comparative analysis of karyotypes suggests that the differences in the amount of constitutive heterochromatin are more distinct in centromeric regions than in the chromosome arms. These characteristics suggest that most of the chromosomal rearrangements took place in the centromeric regions of chromosome, i.e. in regions of constitutiveheterochromatin. Since no polymorphism in the distribution and amount of constitutive heterochromatin was found, it appeared that marker chromosomes or marker bands were a species specific trait of the examined species. The intraspecies stability of the amount and distribution of constitutiveheterochromatin suggests a very specific function of this genome fraction, as well as a role in theorganization and structure of the karyotypes in this group of species. ODIERNA (1989) presented thekaryotype of R. graeca (italica) that, in comparison to R. dalmatina and R. iberica, had the highest amount of genetic material and constitutiveheterochromatin. The karyotype of R. graeca presented here has twice the amount of centromeric heterochromatin than either R.dalmatina or R. temporaria. The amount and distribution of constitutive heterochromatin could be a specific characteristic of karyotypes of R. graeca italica and R. graeca graeca. In spite of their morphological (DUBOIS 1985) and isozyme (PICARIELO et at. 1990) differences, these two taxa inhabit the same biotopes (NOLLERT and NOLLERT 1992). GOINet at. (1968) and OELDOF et at. (1978) suggested that species with large genomes have long larval periods and are found inbiotopes with cold water. R. graeca prefers clear, cold mountain waters and in the Rana genus it represents a species with a very long larval period (BESCHKOV 1970, 1972). The existence of B chromosomes in the karyotype of R. temporaria was observed by some authors (ULLERICH 1967; SCHMID 1979b; BELCHEVAet at. 1982) . B chromosomes were not found in the chromosome complement of 20

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specimens from 4 different localities in Yugoslavia. R.temporaria from Yugoslav localities did not have supernumerary chromosomes or they were not present in the examined populations in high frequencies. 1ikewise, MIURAet at. (1995) did not observe the presence of B chromosomes in the karyotype of R. temporaria in Russian localities. Most of the species of the genus Rana share «standard» or «primitive» NOR location intercalary on the long arm of thelOth chromosome pair (SCHMID1978b; BIRSTEIN 1984). As in European water frogs, in R. temporaria the NOR presents its standard position. In contrast, R. arvatis has NORs on the 2nd (ULLERICH 1967; GREEN and BORKIN 1993), and R.dalmatina on the 3rd chromosome pair (GUILLEMIN 1967; ODIERNA 1989). SCHMID(1978b) assumed that changes in the position of NORs in the karyotype of R. dalmatina resulted from a reciprocal translocation of the short arm of the 3rd chromosome and of the NOR-bearing region on ht e long arm of the loth. An alternative hypothesis (MACGREGOR and SCHERWOOD 1979; ODIERNA 1989) was that the different localization of the NOR in thekaryotype of R. dalmatina was due to the amplification of a pre-existing NOR sitelocalized on the 3rd chromosome and accompanying deletion of the site on the loth pair. The characteristic changes in thecentromeric index and Cband distribution of chromosomes 3 and 10 in thekaryotypes of R. dalmatina and R. temporaria, that are presented in this paper, support the first hypothesis. In the karyotypes of R. graeca (itatica) NORs were found on the long arm of the lOth chromosome (VITELLI et at. 1982; ODIERNA 1989). Also, BELCHEVA and SOFIANIDOU (1990) reported the same position of NORs in the karyotype of R. graeca from localities in Bulgaria and Greece. However, SPASICet at. (1996) suggested that the NOR was localized on the short arm of the lOth chromosome pair. Other investigations, presented in this study of R. graeca karyotypes from other Balkan localities, strongly support the notion thatNORs are situated on the short arm of the l0th chromosome pair. The distribution of constitutive heterochromatin and the centromeric index of the l0th chromosome suggest that pericentric inversion occurred in the NOR bearing chromosome region. The different positions of NORs in R. graeca graeca compared to R. graeca itatica and other brown frogs, suggest a specific cytotaxonomic status for this subspecies. MENSIet at. (1992) estimated that the first split in the main stock of brown frogs occurred in 1ate Pliocene, when the ancestors of R. temporaria and R. iberica separated from other Mediterranean brown frogs. ARANO et at. (1993) suggested that this process occurred earlier. However, R.graeca was the first to split from the rest and remain isolated in the South of the Balkan Peninsula. On the basis of electrophoretic (PICARIELLO et at. 1990; CAPULA 1991), morphological (DUBOIS 1985; NOLLERT and NOLLERT 1992) and cytogenetic evidence presented here, it appears that R. graeca itatica derived later and became isolated in the Italian Peninsula. Differences in karyotypes, iso-

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zymes and morphology between R.graeca graeca and R. graeca itatica provide enough evidence to establish R. itatica as a separate species. In conclusion, karyotypes of frogs are not as conservative as was often suggested (WILSONet at. 1974, 1975; BUSH et at. 1977). The authors' assumption is based on the fact that re arrangements that change the number of arms or chromosomes occurred in frogs very rarely compared to other vertebrates. However, intrachromosomal rearrangements, as well as changes in regions of constitutive heterochromatin occur very often. KING (1980) has also suggested that there are no two different species of frogs with the same distribution of constitutiveheterochromatin in their karyotypes. Also, differences in NOR positions arecytotaxonomically informative. Furthermore, cytogenetic analysis in frogs and analysis of the significance of changes in regions of constitutive heterochromatin can provide more information for taxonomic studies. Acknowledgements. - We wish to express our thanks to Prof. Theodora S.Sofianidou (Aristotle University of Thessaloniki, Greece) and toImre Krizmanic (Nature Protection Institute of Serbia) for cooperation. Work was supported by Research Science Funds of Serbia, Contract No. 03E02. REFERENCES ARANO B., ESTEBAN M. and HERRERO P., 1993. -Evolutionary divel&ence in the Iberian brown frogs. Ann. Sci. Natu. Zool. (Paris), 14: 49-57. BELCHEVA R.G., ILIEVA H.L. and BESCHKOV V.A., 1982. -B-chromosomes in the karyotype of Rana temporaria L. from population in Bulgaria. Comptes Rendus Acad. Bulg. Sci., 35: 827-829. BELCHEVA R.G. and SOFIANIDOU T.S., 1990. - Karyological investigation of the brown frogs species (Anura-Ranidae) from Bulgaria and Greece. In: «Cytogenetics of Amphibian and Reptiles», ed. by E.Olmo, pp. 141-146. Birkhauser Verlag, Basel- Boston - Berlin. BESCHKOV A.V., 1970. - Biologie und Verbreitung des griechischen Froches (Rana graeca) 2. Untersuchungen iiber die Fortpflanzung und die Larven. Bull. Inst Zool. Musee Sofia, 32: 159-180. -, 1972. - Biologie und Verbreitung des griechischen Froches (Rana graeca) 3. Untersuchungen iiber ihre Okologie und ihre Verbreitung. Bull. Inst. Zool. Musee Sofia, 36: 125-136. BIRSTEIN V.J., 1984. - Localization of NORs in karyotypes of four Rana species. Genetica, 64: 149-154. BUCH G.L., CASE S.M., WILSON A.C. and PATTON J.L., 1977. -- Rapid speciation and chromosomal evolution in mammals. Proc. Natl. Acad. Sci USA, 74.3942-3946. CAPULA M., 1991. - Allozyme variation in Rana latastei populations (Amphibia: Ranidae) from Northern Italy and Istra (NW Yugoslavia): Biogeografic inferences from electrophoretic data. Zool. Anz., 227: S. 1-12. DUBOIS A., 1985. - Notes sur les Grenouilles brunes (grupe de Rana temporaria Linne, 1758) IV. Note preliminare sur Rana graeca Boulenger, 1891. Alytes, 4: 135-138. GREEN D.M. and BORKIN L.J., 1993. - Evolutionary relationships of Eastern Palearctic brown frogs, genus Rana: paragraphyly of the 24-chromosome species group and the significance of chromosome number change. Zool.J. Linn. Soc., 109: 1-25. GOIN O.B., GOINJ.C. and BACHMANN K., 1968. - DNA and amphibian life history. Copeia, 532-540. GUILLELMIN C., 1967. - Caryotypes de Rana temporaria (L.) et de Rana dalmatina (Bonaparte). Chromosoma, 21: 189-207. HERRERO P., ARANO B. and ESTEBANM.L., 1990. - Karyotypic characterization of brown frogs from the Iberian peninsula (Ranidae: Rana). In: «Cytogenetics of Amphibians and Reptiles», ed. by E.Olmo, pp. 135-140, Birkhauser Verlag, Basel- Boston – Berlin

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