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1 July 2010 The karyotype and NOR phenotype of Telestes ukliva (Cyprinidae)
Damir Valić, Damir Kapetanović, Davor Zanella, Milorad Mrakovčić, Emin Teskeredžić, Višnja Besendorfer, Marie Rábová, Petr Ráb
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The karyotype and major ribosomal sites as revealed by silver staining and CMA3-fluorescence of Croatian leuciscine cyprinid endemic to Cetina River, Telestes ukliva were studied. The diploid chromosome number was invariably 2n = 50. Karyotype consisted of eight pairs of metacentric, 13 pairs of submetacentric and four pairs subtelocentric chromosomes. The largest chromosome pair of the complement was subtelocentric, which is a characteristic cytotaxonomic marker for all representatives of the cyprinid lineage Leuciscinae. The nucleolar organizer regions (NORs) were detected in the telomeres of two pairs of mediumsized submetacentric chromosomes. Staining with CMA3 revealed four positive signals that corresponded to NOR sites. No heteromorphic sex chromosomes were found. The karyotype pattern of T. ukliva is nearly identical to that found in most other representatives of the Eurasian leuciscine cyprinids, while the multiple NOR phenotype appears to be more derived as opposed to a uniform one NOR-bearing chromosome pair, ubiquitous in this group.


Ten freshwater fish species locally endemic to the Adriatic basin in Croatia includes also the ukliva dace, Telestes ukliva (Heckel, 1843). This species is present throughout the Cetina River, with the exception of the river mouth. Most species of genus Telestes occur in the eastern Adriatic drainages and suffer from habitat alterations and loses. Many of them were earlier included in the genus Leuciscus, but present molecular and morphological data indicate that the genus Telestes is well defined monophyletic lineage being more closely related to Chondrostoma, Achondrostoma, Pseudochondrostoma, Parachondrostoma, Protochondrostoma, Iberochondrostoma and Phoxinellus (Freyhof et al. 2006) than to Leuciscus. Three species earlier referred to Phoxinellus have recently been transferred to Telestes (Freyhof et al. 2006). However, the systematics of Telestes and its actual diversity has not yet been well resolved (Kottelat & Freyhof 2007).

Extensive cytotaxonomic screening on the ´Leuciscus´-like species from Bosnia and Herzegovina, namely Squalius cephalus, S. albus, L. idus, L. leuciscus, T. souffia, T. turskyi and S. svallize from the area of Adriatic basin was conducted by Sofradžija (1977). This and some other cytogenetic studies showed that all investigated species have an invariable diploid chromosome number 2n = 50 and very similar karyotype (Lieder 1954, Wolf et al. 1969, Fontana et al. 1970, Berberović & Sofradžija 1972, Sofradžija 1977, Collares-Pereira et al. 1998).

Fig. 1.

Metaphase plates (A, B, D) and inter-phase nuclei (C) of Telestes ukliva from Cetina River. Metaphase chromosomes after Giemsa staining (A), or staining with silver nitrate (B). Arrows indicate nucleolar organizer regions (NORs) on short arms of two submetacentric chromosome pairs. C) Interphase nuclei with different number and size of nucleoli after staining with silver nitrate. D) Metaphase chromosomes and interphase nuclei after CMA3 staining. CMA3-positive signals are terminally located on short arms of two submetacentric chromosome pairs (arrows). Interphase nuclei with four CMA3-positive signals.


Fig. 2.

Karyotype of Telestes ukliva arranged from Giemsa-stained chromosomes.


Giemsa, C-banding, fluorochrome and silver staining are the most widely used techniques for differential chromosome banding in fishes (e.g. Boroń 1999, Boroń 2001, Ocalewicz et al. 2004, Kirtiklis et al. 2005). Nucleolar organizer regions (NORs) are widely used chromosomal markers to infer inter- and intra-specific relationships in various fish taxa (e.g. Collares-Pereira & Ráb 1999, Boroń 2001). NORs in neotelosteans are associated with GC-rich heterochromatin and therefore easily detected by fluorescent staining with GC-specific antibiotic CMA3 (Ráb et al. 1999). Silver staining of NORs is not only used as a chromosomal marker to characterize the karyotype, but also to detect transcriptionally active NORs (e.g. Birstein & Vasil'ev 1987, Gromicho & Collares-Pereira 2004). However, recent molecular studies show that not all sites of rDNA are detected with CMA3 or silver staining (Pendás et al. 1993, Reed & Philips 1995, Ráb et al. 1996).

The aim of this study was to describe karyotype and some other chromosomal characteristics of the ukliva dace, including number and location of NORs using Ag-staining and CMA3-fluorescence.

Material and Methods

Specimens sampled by electrofishing performed every two months in the Cetina River from 1997 to 2000 were analysed. A total of 10 unsexed juvenile specimens used for cytogenetic analysis were transported alive to the laboratory and kept in wellaerated aquarium until analysis. The specimens analyzed are deposited as vouchers in the Department of Zoology, Division of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia.

Mitotic chromosome preparations were obtained from fish kidneys and gills following modified method by Vitturi et al. (1986). Slides were examined under light microscope Opton III (Zeiss) and microphotographs were used for morphometric analysis. Chromosomes were classified according to Levan et al. (1964).

After destaining of Giemsa-stained slides with fixative and series of alcohol, those were stained with chromomycin A3 (CMA3) (Sigma) and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma) according to the modified method of Schweizer & Ambros (1994). Fluorescence was observed under the fluorescent microscope Opton Axioplan (Zeiss). Modified method described by Hizume et al. (1980) was used for the silver staining of chromosomes and nucleoli. For nucleoli, a total of 735 cells from 4 specimens were analyzed.


The chromosome complement in all investigated specimens of T. ukliva possessed diploid chromosomes number 2n = 50 (Fig. 1A and 2). The karyotype consisted eight pairs of metacentric (m), 13 pairs of submetacentric (sm) and four pairs of subtelocentric (st) chromosomes (Fig. 2). Total number of chromosome arms in the complement was NF = 92. The largest chromosome pair of the complement was subtelocentric, which is a characteristic cytotaxonomic marker for all representatives of the cyprinid lineage Leuciscinae.

Staining with CMA3 revealed CMA3-positive signals at the terminal position of the short arms of two sm chromosome pairs (Fig. 1D). Differences in size of CMA3-positive signals between homologous chromosomes were not detected. Four CMA3-positive signals were also recorded in interphase nuclei.

DAPI staining did not reveal any DAPI-positive signals on T. ukliva chromosomes (picture not shown). Silver staining revealed four positive NOR sites located terminally on the short arms of two sm pairs of chromosomes, which corresponded to four CMA3-positive signals. In most cells, all four NORs were active and of similar size (Fig. 1B). After silver staining, the interphase cells with one to five nucleoli were recorded (Fig. 1C). Cells with one (31.97%) and two (31.16%) nucleoli were the most frequent, indicating the association of NORs, while the number of cells with five nucleoli was very low (2.04%). Cells with three and four nucleoli were somewhat less frequent, 23.40% and 11.43% respectively.


The chromosome number 2n = 50 and the karyotype of species under study corresponds entirely to that found to be characteristic for representatives of the subfamily Leuciscinae examined as yet, including easily recognizable largest pair of chromosomes — cytotaxonomic leuciscine marker (Ráb et al. 2008 and references therein). The largest m and smallest m pairs of chromosomes are also easily recognizable in all species investigated. Results of extensive cytogenetic study by Sofradžija (1977) correspond with findings in our paper. Morphologically differentiated sex chromosomes were not observed, what could be explained by the fact that the assignment of particular chromosome into chromosome categories in cyprinids is sometimes very difficult due to their small size and hence identification of possible sex chromosomes is problematical without other genetic tools (Ráb & Collares-Pereira 1995).

Differential staining of chromosomes with CMA3 indicated four positive CMA3-signals located terminally on the short arm of two pairs of sm chromosomes. It should be mentioned that not all CMA3 positive sites represent rDNA cistrons, but indicate either transcriptionally inactived rRNA loci or just GC-rich heterochromatic blocks (Collares-Pereira & Ráb 1999), as well as not all rDNA sites are detected with CMA3 or silver staining, what some authors interpreted as a polymorphism (Boroń 2001). Silver staining revealed four AgNORs corresponding to CMA3-positive signals. In the most of the interphase cells, one to four nucleoli were observed, indicating various levels of rRNA activity. The association of NORs, which results from the formation of less than the maximum number of nucleoli, is also characteristic of NOR behaviour in the genomes of other fish species (e.g. Woznicki et al. 2000). Silver staining also showed that there was no size difference of positive Ag-NOR signals between homologous and nonhomologous NOR-bearing chromosomes. In a small number of interphase nuclei, five nucleoli were detected. This finding points to the possibility that an additional rDNA locus exists, which was not detected either by CMA3 or silver staining of metaphase chromosomes.

Presently, the number and location of NORs is frequently used in fish descriptive cytotaxonomy as a useful marker (e.g. Boroń 2001, Rábová et al. 2001, Rábová et al. 2003, Gromicho & Collares-Pereira 2004). In most analyzed European leuciscins, one NOR-bearing sm to st chromosome pair has been detected (e.g. Ráb & Collares-Pereira 1995, Rábová et al. 2003, Bianco et al. 2004). This character is considered as ancestral. Multiple NORs have been considered as a derived character and has been reported in several leuciscine and phoxinine European species (Rodriges & Collares-Pereira 1996, Boroń et al. 1997, Collares-Pereira & Ráb 1999, Boroń 2001, Bianco et al. 2004, Gromicho & Collares-Pereira 2004, Gaffaroglu et al. 2006).

To reliably establish the exact number of rDNA loci, fluorescence in situ hybridisation (FISH) with the appropriate probe for rDNA detection on chromosomes has to be applied. This technique was successfully applied in determination of number and position of rDNA loci in a various fish species, including those from Cyprinidae family (e.g. Collares-Pereira & Ráb 1999, Rábová et al. 2003, Ocalewicz et al. 2004).

Staining with DAPI did not show any positive signals (AT-rich heterochromatin regions) along T. ukliva chromosomes. The same results were found for chromosomes of other cyprinid species (Mayr et al. 1986, Ocalewicz et al. 2004) and together with other findings it could be concluded that this is another common feature for genomes of cyprinids. The minor differences in the karyotype between T. ukliva from the Cetina River and those of other species from Bosnia and Herzegovina indicate that speciation of these species, isolated from the last glaciation period was accompanied by certain, but at present level of detection nearly invisible chromosomal rearrangements. In addition, FISH experiments have to be included in future studies in order to determine the exact number and position of NORs, since this chromosome marker seems to be the most informative in the reconstruction of evolutionary events and relationships between closely related fish species at the chromosome level (Phillips & Reed 1996, Collares-Pereira & Ráb 1999, Rábová et al. 2001, Fontana et al. 2003, Rábová et al. 2003).



Berberović Lj. & Sofradžija A. 1972: Pregled podataka o hromosomskim garniturama slatkovodnih riba Jugoslavije [A review of the karyological data regarding freshwater fishes of Jugoslavia]. Acta Biol. Jug.-Ichthyologia 4 (3): 1–21. (in Serbo-Croatian with English summary) Google Scholar


Bianco P.G., Aprea G., Balletto E., Capriglione T., Fulgione D. & Odierna G. 2004: The karyology of the cyprinid genera Scardinius and Rutilus in southern Europe. Ichthyol. Res. 51: 274–278. Google Scholar


Birstein V.J. & Vasil'ev V.P. 1987: Nucleolar organizer regions, their function and polyploidy in fishes. J. Obshchey Biol. 6: 729–747. Google Scholar


Boroń A. 1999: Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland. Genetica 105: 293–300. Google Scholar


Boroń A. 2001: Comparative chromosomal studies on two minnow fish, Phoxinus phoxinus (Linnaeus, 1758) and Eupallasella perenurus (Pallas, 1814); an associated cytogenetic-taxonomic considerations. Genetica 111: 387–395. Google Scholar


Boroń A., Jankun M. & Kusznierz J. 1997: Chromosome study of swamp minnow Eupallasella perenurus (Dybowski, 1916) from Poland. Caryologia 50: 85–90. Google Scholar


Collares-Pereira M.J., Próspero M.I., Biléu R.I. & Rodrigues E.M. 1998: Leuciscus (Pisces, Cyprinidae) karyotypes: Transect of Portuguese populations. Genetics and Molecular Biology 21 (1): 63–69. Google Scholar


Collares-Pereira M.J. & Ráb P. 1999: NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae) — re-examination by FISH. Genetica 105: 301–303. Google Scholar


Fontana F., Chiarelli B. & Rossi A.C. 1970: Il cariotipo di alcune specie Cyprinidae, Centrarchidae, Characidae studiate mediante colture “in vitro”. Caryologia 23: 549–564. Google Scholar


Fontana F., Lanfredi M., Congiu L., Leis M., Chicca M. & Rossi R. 2003: Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescence in situ hybridization in six sturgeon species. Genome 46: 473–477. Google Scholar


Freyhof J., Lieckfeldt D., Bogutskaya N.G., Pitra C. & Ludwig A. 2006: Phylogenetic position of the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminichthys (Teleostei: Cyprinidae). Mol. Phylogenet. Evol. 38: 416–425. Google Scholar


Gaffaroglu M., Yuksel E. & Ráb P. 2006: Note on the karyotype and NOR phenotype of leuciscine fish Acanthobrama marmid (Osteichthyes, Cyprinidae). Biologia 61: 207–209. Google Scholar


Gold J.R. & Avise J.C. 1977: Cytogenetic studies of North American minnows (Cyprinidae). I. Karyology of nine California genera. Copeia 1977: 541–549. Google Scholar


Gromicho M. & Collares-Pereira M.J. 2004: Polymorphism of major ribosomal gene chromosomal sites (NOR-phenotypes) in the hybridogenetic fish Squalius alburnoides complex (Cyprinidae) assessed through crossing experiments. Genetica 122: 291–302. Google Scholar


Hizume M., Sato S. & Tanaka A. 1980: A highly reproducible method of nucleolus organizing regions staining in plants. Stain Technol. 55: 87–90. Google Scholar


Kirtiklis L., Boroń A. & Porycka K. 2005: Chromosome banding patterns of the gudgeon, Gobio gobio (Actinopterygii, Cyprinidae). Acta Ichthyologica et Piscatoria 35 (2): 119–123. Google Scholar


Kottelat M. & Freyhof J. 2007: Handbook of European freshwater fishes. Kottelat, Cornol, Switzerland and Freyhof, Berlin , GermanyGoogle Scholar


Levan A., Fredga K. & Sandberg A.A. 1964: Nomenclature for centromeric position on chromosomes. Hereditas 52: 201–220. Google Scholar


Lieder U. 1954: Chromosomenstudien an Knochenfischen. II. Über die Chromosomenzahl and Morphologie der Plötze (Leuciscus rutilus L.) und einiger ihrer Bastarde mit anderen Cypriniden. Z. Fischerei Hilfswiss. 3: 479–488. Google Scholar


Mayr B., Ráb P. & Kalat M. 1986: NORs and counterstain-enhanced fluorescence studies in Cyprinidae of different ploidy level. Genetica 69: 111–118. Google Scholar


Ocalewicz K., Jankun M. & Boroń A. 2004: Karyotypic characterization of bream, Abramis brama (Pisces, Cyprinidae). Folia Zool. 53: 329–334. Google Scholar


Pendás A.M., Morán P. & Garcia-Vázquez E. 1993: Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout. Chromosome Res. 1: 63–67. Google Scholar


Phillips R.B. & Reed K.M. 1996: Application of fluorescence in situ hybridisation (FISH) techniques to fish genetics: a review. Aquaculture 140: 197–216. Google Scholar


Ráb P. & Collares-Pereira M.J. 1995: Chromosomes of European cyprinid fishes (Cyprinidae: Cypriniformes): a rewiew. Folia Zool. 44: 193–214. Google Scholar


Ráb P., Karakousis Y., Rábová M. & Economidis P.S. 1996: Banded karyotype of cyprinid fish Leuciscus borysthenicus. Ichthyol. Res. 43: 463–468. Google Scholar


Ráb P., Rábová M., Pereira C.S., Collares-Pereira M.J. & Pelikánová Š. 2008: Chromosome studies of European cyprinid fishes: interspecific homology of leuciscine cytotaxonomic marker — the largest subtelocentric chromosome pair as revealed by cross-species painting. Chromosome Res. 16: 863–873. Google Scholar


Ráb P., Rábová M., Reed K.M. & Phillips R.B. 1999: Chromosomal characteristics of ribosomal DNA in the primitive semionotiform fish, longnose gar Lepisosteus osseus. Chromosome Res. 7: 475–480. Google Scholar


Rábová M., Ráb P. & Ozouf-Costaz C. 2001: Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi Cobitidae) detected by different banding techniques and fluorescent in situ hybridisation (FISH). Genetica 111: 413–422. Google Scholar


Rábová M., Ráb P., Ozouf-Costaz C., Ene C. & Wanzenböck J. 2003: Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae). Genetica 118: 83–91. Google Scholar


Reed K.M. & Phillips R.B. 1995: Molecular cytogenetic analysis of the double-CMA3 chromosome of lake trout, Salvelinus namaycush. Cytogenet. Cell. Genet. 70: 104–107. Google Scholar


Rodrigues E.M. & Collares-Pereira M.J. 1996: NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces, Cyprinidae). Genetica 98: 59–63. Google Scholar


Schweizer D. & Ambros P.F. 1994: Chromosome banding. In: Gosden J.R. (ed.), Methods in molecular biology. Vol 29: Chromosome analysis protocols. Humana Press Inc. , Totowa, New York : 97–112. Google Scholar


Sofradžija A. 1977: Kariologija i citotaksonomija vrsta roda Leuciscus iz voda Bosne i Hercegovine [Karyology and cytotaxonomy of the Leuciscus species from the waters in Bosnia and Herzegovina]. Godišnjak Biol. Inst. Univ. u Sarajevu 30: 113–211. (in Serbo-Croatian with English summary)  Google Scholar


Vitturi R., Catalano E., Macaluso M. & Zava B. 1986: Karyotypes of nine species of the family Labridae (Pisces, Perciformes). Biol. Zbl. 108: 223–226. Google Scholar


Wolf U., Ritter H., Atkin N.B. & Ohno S. 1969: Polyploidisation in the fish family Cyprinidae, order Cypriniformes. I. DNA content and chromosome sets in various Cyprinidae. Humangenetik 7: 240–244. Google Scholar


Woznicki P., Sanchez L., Martinez P., Pardo B.G. & Jankun M. 2000: A population analysis of the structure and variability of NOR in Salmo trutta by Ag, CMA3 and ISH. Genetica 108: 113–118. Google Scholar
Damir Valić, Damir Kapetanović, Davor Zanella, Milorad Mrakovčić, Emin Teskeredžić, Višnja Besendorfer, Marie Rábová, and Petr Ráb "The karyotype and NOR phenotype of Telestes ukliva (Cyprinidae)," Folia Zoologica 59(2), 169-173, (1 July 2010).
Received: 5 March 2009; Accepted: 1 June 2009; Published: 1 July 2010

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