First cytogenetic studies of the genus Heptapterus (Actinopterygii, Siluriformes): karyotype differentiation and review of cytogenetic data on the Heptapteridae family

The Neotropical fish Heptapterus mustelinus, collected in the Pindorama stream of the upper Paraná River basin (Brazil), was studied cytogenetically, verifying 54 chromosomes (26m + 18m + 4st + 6a). This diploid number has not been reported among the Heptapteridae that have been studied to date. Unlike most species of the family, there were multiple Ag-nucleolar organizer regions (NOR) and heterochromatin present in the centromeric region of most of the chromosomes of the complement, being Chromomycin A(3) (CMA(3) (+) )/4',6-diamidino-2-phenylindole (DAPI(-) ) in nine chromosomal pairs, besides the one that coincides with the Ag-NORs. The data presented in this work reveal a different path in the karyotypic evolution of H. mustelinus when compared to the others Heptapteridae genera.     

Evolutionary trends in Hoplerythrinus unitaeniatus (Agassiz 1829) (Characiformes, Erythrinidae)

Hoplerythrinus unitaeniatus is a species of economic importance, widely distributed in the Brazilian territory. Many studies have reported the karyotypic variation that occurs in the species, which forms two groups according to the diploid number, one with 2n = 52 and the other with 2n = 48. In this work, three populations of H. unitaeniatus from different hydrographic basins were cytogenetically analyzed. The diploid number found was 2n = 48 for all populations, however, differences in karyotypic formula and fundamental number and heterochromatin distribution were also observed. Ag-NORs were multiple, GC-rich and when applied FISH with 18S rDNA bright signals in six chromosomes were observed. Data from this study confirm the occurrence of interpopulation variability in H. unitaeniatus. The basal number and the possible divergence among the different cytotypes analyzed for this species to date, as well as the forward the proposal of biogeographic and tectonic history for the continental margin are discussed.     

A new karyotype for <Emphasis Type="Italic">Cavia magna</Emphasis> (Rodentia: Caviidae) from an estuarine island and <Emphasis Type="Italic">C. aperea</Emphasis> from adjacent mainland

We describe a new karyotype for Cavia magna Ximenez, 1980 from an estuarine island and the karyotype of Cavia aperea Erxleben, 1777 from an adjacent mainland. The species have differences in diploid number (2n), autosomal fundamental number, quantity, and distribution of heterochromatin as dissimilar distributions of the nucleolus-organizing regions (Ag-NORs). The C. aperea karyotype has a diploid number of 64 as previously reported for C. aperea and most other Cavia species. In contrast, this new C. magna karyotype exhibits a variant diploid number of 2n = 62, considering that previous work reported a karyotype of 2n = 64 for C. magna. The discovery of a distinct diploid number within C. magna represents the first record of intra-specific chromosomal variation in a species of Caviidae. The diploid number of 2n = 62, heterochromatin quantity, Ag-NOR distributions, and inversed X chromosome from this population of C. magna are as seen in the geographically proximate (Cavia intermedia Cherem Olimpio and Ximenez; intermediate Cavy). These data provide further evidence supporting C. magna as the sister species of C. intermedia.     

XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil

Potamotrygonidae is a representative family of South American freshwater elasmobranchs. Cytogenetic studies were performed in a Potamotrygon species from the middle Negro River, Amazonas, Brazil, here named as Potamotrygon sp. C. Mitotic and meiotic chromosomes were analyzed using conventional staining techniques, C-banding, and detection of the nucleolus organizing regions (NOR) with Silver nitrate (Ag-NOR). The diploid number was distinct between sexes, with males having 2n = 67 chromosomes, karyotype formula 19m + 8sm + 10st + 30a, and fundamental number (FN) = 104, and females having 2n = 68 chromosomes, karyotype formula 20m + 8sm + 10st + 30a, and FN = 106. A large chromosome, corresponding to pair number two in the female karyotype, was missing in the male complement. Male meiotic cells had 33 bivalents plus a large univalent chromosome in metaphase I, and n = 33 and n = 34 chromosomes in metaphase II. These characteristics are consistent with a sex chromosome system of the XX/XO type. Several Ag-NOR sites were identified in both male and female karyotypes. Positive C-banding was located only in the centromeric regions of the chromosomes. This sex chromosome system, which rarely occurs in fish, is now being described for the first time among the freshwater rays of the Amazon basin.     

XY<Subscript>1</Subscript>Y<Subscript>2</Subscript> chromosome system in <Emphasis Type="Italic">Salinomys delicatus</Emphasis> (Rodentia,Cricetidae)

Salinomys delicatus is considered a rare species due to its restricted and patchy distribution, poor records and low abundances. It is also the phyllotine with the lowest known diploid chromosome number (2n = 18), however its sex chromosome system has never been described. Here, we studied the chromosomes of six females and three males with bands G, C, DAPI/CMA₃ and meiosis. In males, the chromosome number was 2n = 19, with one large metacentric X-chromosome and two medium-sized acrocentrics absent in females. The karyotype of females was the same as previously described (2n = 18, FN = 32), with X-chromosomes being metacentric and the largest elements of the complement. In males, the two acrocentrics and the large metacentric form a trivalent in meiotic prophase. This indicates that S. delicatus has XY₁Y₂ sex chromosomes, which is confirmed by G and DAPI bands. Constitutive heterochromatin (CH) is restricted to small pericentromeric blocks in all chromosomes. The X-chromosome shows the largest block of centromeric CH, which could favor the establishment of this X-autosome translocation. This sex chromosome system is rare in mammals and, compared with other phyllotine rodents, S. delicatus seems to have undergone a major chromosome restructuring during its karyotypic evolution.     

Karyotype variation in 11 species of the Vernonieae Cass. tribe (Asteraceae Bercht. & J. Presl)

The Vernonieae tribe presents strong taxonomic delimitation problems as it is considered one of the most complex groups of the Asteraceae family, comprising approximately 1100 species distributed across 129 genera. In this study, a comparative analysis of the Vernonieae species was performed to understand the events involved in the chromosome evolution of these species and to further deduce their taxonomy. The representatives were cytogenetically characterized via analyses of morphology, karyotype asymmetry and differential staining with fluorochromes CMA and DAPI as well as FISH. According to morphometric data, all species showed symmetrical karyotypes with prevailing metacentric chromosomes, even in species belonging to different genera. Variability in diploid chromosome number was detected (2n = 18 to 2n = 60), and chromosome sizes were observed to be between 1.00 and 4.09 μm. Additionally, variation in the pattern of heterochromatin was observed mainly in relation to CMA⁺ bands, in which the number varied from 4 to 16 heterochromatic regions. Only one species, Vernonia scorpioides, presented positive DAPI bands, which were located in the terminal position in most of the chromosomes. The differences in the sizes and quantities of heterochromatic bands may be related to small structural rearrangements during karyotype evolution of the Vernonieae tribe.     

Chromosome polymorphism in <Emphasis Type="Italic">Rineloricaria pentamaculata</Emphasis> (Loricariidae,Siluriformes) of the Paraná River basin

Cytogenetic analysis in three Rineloricaria pentamaculata populations revealed diploid number 2n = 56 chromosomes, karyotype formula 8m/sm + 48st/a and FN = 64. Owing to the presence of the heteromorphic chromosome pair with a big submetacentric chromosome and a small acrocentric one in both males and females, 42.9% of specimens in the Tauá Stream population had the karyotype formula 9m/sm + 47st/a and FN = 65. Analysis of the nucleolus-organizing region by Ag-NOR and FISH techniques showed a single NOR system at pair 5 for R. pentamaculata populations of the Keller River and the Tauá Stream. However, specimens of populations of the Tatupeba Stream had multiple NOR systems at pairs 5 and 8. A constitutive heterochromatin pattern in R. pentamaculata is mainly distributed in the pericentromeric and telomeric regions with interstitial markers in certain chromosomes. Heterochromatin is located in the telomeric and centromeric positions of the acrocentric chromosome in the heteromorphic pair of the Tauá Stream population. In the submetacentric chromosome the markings are located in the telomeric (short arm), pericentomeric and interstitial (long arm) positions. The origins of polymorphisms are discussed.     

Cytogeography and chromosomal variation of the endemic East Asian herb Lycoris radiata

Information on the spatial distribution of cytotypes and karyotype variation in plants is critical for studies of the origin and evolution of polyploid complexes. Here, the spatial distribution of cytological races and intraspecific variation in the karyotype of Lycoris radiata, an endemic species to East Asia, is investigated. Conventional karyotype analysis methods were used to determine ploidy level and karyotypical characteristics in 2,420 individuals from 114 populations of L. radiata nearly covering the whole distribution areas in China. Of 114 populations studied, 52 (45.61%), 58 (50.88%), and 4 (3.51%) are diploid, triploid, and mixoploid populations, respectively, with 1,224, 1,195, and 1 individuals being diploid, triploid, and tetraploid, respectively. The triploid possesses a much wider distribution range than the diploid, with the former almost occupying the entire range of this complex species in East Asia and the latter distributing in the middle and east regions of China. Triploids tend to occur at high altitudes, and the relationship between the ploidy and altitude is significantly positive but low (r² = 0.103, p < 0.01). About 98.6% of examined bulbs have a common karyotype consisting of 22 or 33 acrocentric (A) chromosomes. Some aberrant chromosomes which should be generated from A‐type chromosome have been found including metacentrics (m), small metacentrics (m′), and B‐type chromosome. The results can provide a fundamental cytogeographic data for further studies on the evolutionary origins and adaptive divergences of polyploids, especially the triploid, within L. radiata using molecular and/or ecological methods in the future.     

Karyotypic differentiation via 2n reduction and a finding of a case of triploidy in anurans of the genus <Emphasis Type="Italic">Engystomops</Emphasis> (Anura,Leiuperidae)

The genus Engystomops is divided into two groups, namely the Duovox clade and the Edentulus clade. The species of Edentulus clade have karyotypes with 2n = 22, while E. pustulatus and E. puyango, which belong to Duovox clade, have 2n = 20. To investigate if 2n = 20 is a synapomorphy of Duovox clade, we cytogenetically analyzed all the species of this group, except for E. puyango, in the present study. All of them had 2n = 20, differing from the species of Edentulus clade. Since the species already karyotyped of the genus Physalaemus, which is considered to be the sister group of Engystomops, also have 2n = 22, we conclude that the 2n reduction is a synapomorphy of Duovox clade. Despite the karyotypes of all the species of Duovox clade were very similar, they varied in the NOR pattern. In E. coloradorum, an additional NOR was found in one homologue of the chromosome pair 10 exclusively in all females, indicating that this could possibly be a sexual pair of the ZZ/ZW system. Also in this species, it was found the first case of natural polyploidy of the genus Engystomops.     

Notes on chromosomes of ten species of the genus chrysolina mots. (Coleoptera:Chrysomelidae)

SixChrysolina species from Catalonia and the Canary Islands (Spain), viz.americana, gemina, femoralis, cerealis, menthastri andpolita, have similar diploid karyotypes of 24 (sub)metacentric chromosomes, and show Xy sex-determining system.C. banksi andobsoleta have 2n ()=23; their karyotype is presumably derived from that of the former group by loss of the y chromosomes. InC. haemoptera andC. carnifex 40 elements appear in the diploid set. It seems that 2n=24 is the most frequent number in theChrysolina. Higher chromosome numbers have possibly originated through centric fissions, as the acrocentric shape ofC. carnifex chromosomes seems to suggest. The 2n=23 and 24 species feed onLabiatae, while the two higher chromosome number species are associated with plants belonging to other families.     

Chromosomal characteristics and karyotype evolution of Oxyopidae spiders (Araneae, Entelegynae)

We made a cytogenetic analysis of four species of Oxyopidae and compared it with the karyotype data of all species of this family. In Hamataliwa sp, the mitotic cells showed 2n♂ = 26+X(1)X(2) and telocentric chromosomes. The 2n♂ = 28, which has been described for only one oxyopid spider, is the highest diploid number reported for this family. Peucetia species exhibited distinct karyotype characteristics, i.e., 2n♂ = 20+X(1)X(2) in P. flava and 2n♂ = 20+X in P. rubrolineata, revealing interspecific chromosome variability within this genus. However, both Peucetia species exhibited telocentric chromosomes. The most unexpected karyotype was encountered in Oxyopes salticus, which presented 2n♂ = 10+X in most individuals and a predominance of biarmed chromosomes. Additionally, one male of the sample of O. salticus was heterozygous for a centric fusion that originated the first chromosomal pair and exhibited one supernumerary chromosome in some cells. Testicular nuclei of Hamataliwa sp and O. salticus revealed NORs on autosomal pairs, after silver impregnation. The majority of Oxyopidae spiders have their karyotype differentiated by both reduction in diploid number chromosome number and change of the sex chromosome system to X type; however, certain species retain the ancestral chromosome constitution 2n = 26+X1X2. The most remarkable karyotype differentiation occurred in O. salticus studied here, which showed the lowest diploid number ever observed in Oxyopidae and the second lowest registered for Entelegynae spiders.     

Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes

Chromosome rearrangements may result in both decrease and increase of chromosome numbers. Here we have used comparative chromosome painting (CCP) to reconstruct the pathways of descending and ascending dysploidy in the genus Boechera (tribe Boechereae, Brassicaceae). We describe the origin and structure of three Boechera genomes and establish the origin of the previously described aberrant Het and Del chromosomes found in Boechera apomicts with euploid (2n = 14) and aneuploid (2n = 15) chromosome number. CCP analysis allowed us to reconstruct the origin of seven chromosomes in sexual B. stricta and apomictic B. divaricarpa from the ancestral karyotype (n = 8) of Brassicaceae lineage I. Whereas three chromosomes (BS4, BS6, and BS7) retained their ancestral structure, five chromosomes were reshuffled by reciprocal translocations to form chromosomes BS1‐BS3 and BS5. The reduction of the chromosome number (from x = 8 to x = 7) was accomplished through the inactivation of a paleocentromere on chromosome BS5. In apomictic 2n = 14 plants, CCP identifies the largely heterochromatic chromosome (Het) being one of the BS1 homologues with the expansion of pericentromeric heterochromatin. In apomictic B. polyantha (2n = 15), the Het has undergone a centric fission resulting in two smaller chromosomes – the submetacentric Het′ and telocentric Del. Here we show that new chromosomes can be formed by a centric fission and can be fixed in populations due to the apomictic mode of reproduction.     

Karyology and cytotaxonomy of the genus Passiflora L. (Passifloraceae)

 The chromosomes of 31 species of Passiflora, distributed throughout the subgenera Astrophea, Calopathanthus, Distephana, Dysosmia, Passiflora, Plectostemma and Tacsonia were analysed. Three different karyotypes were observed: 2n = 12, 24, 36; 2n = 18, 72 and 2n = 20. The karyotype of these species was almost always constituted of metacentric and submetacentric chromosomes with variable karyotype symmetry. In the group with x = 6, represented by the subgenus Plectostemma, six diploid species with 2n = 12, one tetraploid with 2n = 24 (P. suberosa) and an intraspecific polyploid with 2n = 12, 36 (P. misera) were analysed. P. pentagona (subgenus Astrophea) may also be included in this karyological group since it presents 2n = 24 and may be of polyploid origin, with x = 6. The interphase nuclei in this group were areticulate, except those of P. morifolia and P. pentagona with semi-reticulate characteristics. Two small terminal heterochromatic blocks, positive for chromomycin A₃, were identified in the largest chromosome pair of P. capsularis and P. rubra, species very closely related, while P. tricuspis displayed four chromosomes with proximal blocks. In the group with x = 9, represented mainly by subgenus Passiflora, 20 species with 2n = 18 and one with 2n = 72 were studied. They presented chromosomes larger than those species with x = 6 and interphase nuclei of semi-reticulate type, except for P. mixta with areticulate nuclei. Four terminal CMA⁺ blocks were observed in P. edulis, six blocks in P. caerulea and P. racemosa, while five blocks were observed in the single P. amethystina plant analysed. P. foetida (subgenus Dysosmia), the only species with 2n = 20, exhibited six chromosomes with CMA⁺ blocks and interphase nuclei of the areticulate type. The meiotic analysis of representatives of the three groups (P. foetida, P. suberosa, P. cincinnata and P. racemosa) always presented regular pairing and regular chromosome segregation, except in P. jilekii where a tetravalent was observed. The analysis of the chromosome variation within the genus and the family suggests that the base number of Passiflora may be x₁ = 6 or x₁ = 12, whereas x₂ = 9 is only an important secondary base number. Received April 11, 2000 Accepted October 5, 2000     

Extreme degree of chromosome number variability in species of the spider genus Scytodes (Araneae, Haplogynae, Scytodidae)

Genus Scytodes includes most species of the spider family Scytodidae. Until now, 187 species of the genus have been described. In spite of this great diversity, only three Scytodes species were karyotyped so far. The present paper provides for the first time karyotype analysis of two synanthropic species, Scytodes fusca and Scytodes itapevi. Furthermore, new data on karyotype of Scytodes globula are also provided using conventional and differential cytogenetical procedures. The diploid number in the genus Scytodes varied considerably, namely from 2n = 13 to 2n = 31. The diploid number found in S. globula (2n♂ = 13) is the lowest in haplogyne spiders with monocentric chromosomes. Except S. globula, this number has been found only in one haplogyne spider with monocentric chromosomes, namely Ochyrocera sp. (Ochyroceratidae). On the contrary, the diploid number of S. fusca (2n♂ = 31) is one of the highest diploid numbers recorded in haplogyne spiders. The degree of intrageneric variation found in the genus Scytodes is the highest recorded in araneomorph spiders with monocentric chromosomes so far. Some karyotype characteristics (diploid number, chromosome morphology, total chromosome length, and distribution of constitutive heterochromatin) allowed us to postulate a close relationship between S. globula and S. itapevi. According to the karyotype data, S. fusca is not closely related to these two species. This conclusion corroborates a recent taxonomic work that grouped S. globula, S. itapevi, and other four Scytodes species in the ‘globula group’.     

(T2AG3)n Telomeric Sequence Hybridization Suggestive of Centric Fusion in Karyotype Marsupials Evolution

It has been suggested that the karyotype of the marsupials derived from a low diploid number (2n = 14) which originated, through fissions of biarmed chromosomes, the karyotypes with a higher 2n. The telomeric sequence (T₂AG₃)_nwas in situhybridized to the chromosomes of Gracilinanus microtarsusand G. emiliae, Micoureus demeraraeand Marmosa murina, species with 2n = 14, in Monodelphissp., M. domestica, M. kunsiand M. brevicaudatawith 2n = 18, and in Lutreolina crassicaudata, Didelphis albiventris, Chironectes minimus, Philander opossumand P. frenata, all of them with 2n = 22. The probe hybridization occurred in the telomeric regions of both arms, short and long, of all chromosomes of the complement of all individuals of all species analysed. However, in some pairs of the karyotypes of Gracilinanus microtarsusand Micoureus demerarae(with 2n = 14), and in Monodelphissp., M. domestica, M. kunsiand M. brevicaudata(2n = 18) ectopic signs of hybridization were detected proximal to the centromeres, suggesting the retention of this telomeric sequence in the centromeric regions of some chromosomes of these species. Based on these results, it is proposed that the karyotype of marsupials evolved from a 2n = 22 to a 2n = 14, by means of chromosomal fusions. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Role of Fusion in Ant Chromosome Evolution: Insights from Cytogenetic Analysis Using a Molecular Phylogenetic Approach in the Genus Mycetophylax

Among insect taxa, ants exhibit one of the most variable chromosome numbers ranging from n = 1 to n = 60. This high karyotype diversity is suggested to be correlated to ants diversification. The karyotype evolution of ants is usually understood in terms of Robertsonian rearrangements towards an increase in chromosome numbers. The ant genus Mycetophylax is a small monogynous basal Attini ant (Formicidae: Myrmicinae), endemic to sand dunes along the Brazilian coastlines. A recent taxonomic revision validates three species, Mycetophylax morschi, M. conformis and M. simplex. In this paper, we cytogenetically characterized all species that belongs to the genus and analyzed the karyotypic evolution of Mycetophylax in the context of a molecular phylogeny and ancestral character state reconstruction. M. morschi showed a polymorphic number of chromosomes, with colonies showing 2n = 26 and 2n = 30 chromosomes. M. conformis presented a diploid chromosome number of 30 chromosomes, while M. simplex showed 36 chromosomes. The probabilistic models suggest that the ancestral haploid chromosome number of Mycetophylax was 17 (Likelihood framework) or 18 (Bayesian framework). The analysis also suggested that fusions were responsible for the evolutionary reduction in chromosome numbers of M. conformis and M. morschi karyotypes whereas fission may determines the M. simplex karyotype. These results obtained show the importance of fusions in chromosome changes towards a chromosome number reduction in Formicidae and how a phylogenetic background can be used to reconstruct hypotheses about chromosomes evolution.     

Heteromorphic sex chromosomes of lizardfish (Synodontidae): focus on the ZZ-ZW1W2 system in Trachinocephalus myops

The karyotype and DNA content of four lizardfish species (family Synodontidae), that is, Saurida elongata, Synodus ulae, Synodus hoshinonis and Trachinocephalus myops, were analyzed. The karyotype of T. myops significantly differed from that of the other three species having diploid chromosome number of 48 with mainly acrocentric chromosomes and the ZZ-ZW sex chromosome system. The chromosome number of male T. myops was 2n=26, while that of female T. myops was 2n=27. The karyotype consisted of 11 pairs of metacentrics, one pair of acrocentrics and, in addition, two large metacentrics in the male and a single large metacentric, a distinctly small subtelocentric and a microchromosome in the female. C-banding demonstrated that in the female the subtelocentric chromosome and the microchromosome were heterochromatic. The karyotype of T. myops was thought to be derived from a 48 chromosome type synodontid fish through the involvement of Robertsonian rearrangement; the rearrangement of the sex chromosomes proceeded during karyotype evolution. Among the chromosomes, the large metacentrics were determined to be neo-Z (a fusion of the original Z and an autosome), the microchromosomes the W₁ (originally W), and the subtelocentric chromosomes the W₂ (derived from an autosome pair). The miniaturization of W₁ and W₂ chromosomes and their heterochromatinization suggested that sex chromosomes in this species have been already highly differentiated. The findings on DNA content implied that the karyotype of T. myops evolved by centric fusion events without loss in DNA amount.     

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera,Belostoma)

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome–autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elegans (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elongatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG)_n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere–telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.     

Comparative molecular cytogenetic analysis of three <Emphasis Type="Italic">Leuciscus</Emphasis> species (Pisces,Cyprinidae) using chromosome banding and FISH with rDNA

A comparative molecular cytogenetic analysis was performed on three species of the genus Leuciscus viz. ide L. idus, chub L. cephalus and dace L. leuciscus distributed in Poland, using C-, Ag- and chromomycin A₃ (CMA₃)-stainings and fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Although the three species examined shared 2n = 50 chromosomes and the largest acrocentric chromosome pair in the complement, they were characterized with karyotypic differences in terms of the number of uni- and biarmed chromosomes and the localization of nucleolar organizer regions (NORs) revealed by Ag-staining and FISH. L. idus and L. cephalus showed the rDNA sites on the long arms of one submetacentric (SM) chromosome pair and on the short arms of one subtelocentric (ST) chromosome pair, respectively. These NORs were CMA₃-positive, GC-rich and C-positive heterochromatic sites in both species. Such chromosome banding features were also true for four NORs localizing on one of each SM and ST pair in L. leuciscus, but considerable numerical NOR polymorphism became apparent with Ag-staining and FISH due to a different combination of these NOR-bearing SMs and STs in this dace. The present results indicate that the molecular cytogenetic analysis applied herein may become useful to elucidate the karyotype evolution and phylogenetic relationships among the species in the genus Leuciscus and other related groups.