Chromosomal Location of Ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n Telomeric Repeats in the Neogastropod Fasciolaria Lignaria (Mollusca: Prosobranchia)

This paper reports on a successful application of fluorescent in situhybridization (FISH) with three repetitive DNA probes (ribosomal DNA (rDNA), (GATA)_nand (TTAGGG)_n) in the chromosomes of Fasciolaria lignaria(Mollusca: Prosobranchia: Neogastropoda). rDNA FISH consistently identified four chromosome pairs per spread in the three examined specimens. The telomeric sequence (TTAGGG)_nhybridized with termini of all chromosomes. GATA FISH revealed abundant, dispersed minisatellite regions which were not associated to the XY sex-determining mechanism as indicated by the absence of a Y specific pattern of labelling. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA) n , (TTAGGG) n and ribosomal genes by one-color and two-color FISH

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA) _n , and (TTAGGG) _n , and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG) _n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA) _n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.     

Fluorescence in situ hybridization of rDNA, telomeric (TTAGGG)n and (GATA)n repeats in the red abalone Haliotis rufescens (Archaeogastropoda: Haliotidae)

The physical location of 18S-5.8S-28S rDNA, telomeric sequences with (TTAGGG)n DNA probe and (GATA)n microsatellites were performed by fluorescence in situ hybridization in chromosomes of red abalone Haliotis rufescens. The karyotype of red abalone showed a diploid number of 36 (8M+9SM+1ST). FISH performed with rDNA probe, showed the location of major ribosomal clusters in the terminal region of the large arms of two submetacentric pairs (chromosome 4 and 5). Localization of heteromorphisms of FISH-rDNA was found between chromosome homologues and sister chromatids in all metaphases analyzed. This indicates that rDNA clusters are variable within the red abalone genome. The variability in the NOR-bearing reported using silver staining in other gastropods and our result are discussed. In addition, the presence of microsatellite (TTAGGG)n and (GATA)n was demonstrated after FISH treatment by DNA probes. The telomeric sequence occurred at the ends of all mitotic chromosomes, while the (GATA)n repetitive was found on chromosomal interstitial zones as well as at the telomeres in abalone chromosomes.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

FISH mapping of 18S-28S and 5S ribosomal DNA, (GATA)n and (TTAGGG)n telomeric repeats in the periwinkle Melarhaphe neritoides (Prosobranchia, Gastropoda, Caenogastropoda)

Spermatocyte chromosomes of Melarhaphe neritoides (Mollusca, Prosobranchia, Caenogastropoda) were studied using fluorescent in situ hybridization (FISH) with four repetitive DNA probes (18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n). Single-colour FISH consistently mapped one chromosome pair per spread using either 18S or 5S rDNA as probes. The telomeric sequence (TTAGGG)n hybridized with termini of all chromosomes whereas the (GATA)n probe did not label any areas. Simultaneous 18S-5S rDNA and 18S-(TTAGGG)n FISH demonstrated that repeated units of the three multicopy families are closely associated on the same chromosome pair.     

Chromosome analysis using different staining techniques and fluorescent in situ hybridization in Cerithium vulgatum (Gastropoda: Cerithiidae)

In the present paper one population of the large" subtidal mollusc Cerithium vulgatum Bruguière, 1792 (Gastropoda: Cerithiidae) from the Northwestern coast of Sicily was investigated from a karyological point of view. The chromosome complement was Giemsa stained, conventionally karyotyped in 18 homomorphic chromosome pairs (10 bi-armed and 8 mono-armed), and subsequently analysed using silver, CMA3 and DAPI staining, and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal sites (18S-28S rDNA) in the terminal region of the short arms of one small sized mono-armed chromosome pair. Ribosomal DNA was transciptionally active as indicated by its preferential impregnation with silver nitrate (Ag-NOR) and did not contain a high amount of GC base pairs as suggested by the lack of a bright CMA3 fluorescence. The (TTAGGG)n telomeric probe was hybridized to the termini of nearly all chromosomes, thus demonstrating that, in C. tulgatum, this sequence has been conserved during the genomic evolution. The finding of the telomeric hexanucleotide in six species belonging to the three high taxa of Gastropoda supports the notion that this sequence is widespread within this class. The (GATA)n probe did not label any chromosome regions except for a minute terminal area of a single bivalent at pachytene stage.     

Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)

The chromosome set of Patinopecten yessoensis (Jay, 1857) wascharacterized using Giemsa staining, DAPI staining and fluorescencein situ hybridization (FISH) with three repetitive DNA probes[18S–28S rDNA, 5S rDNA and telomeric (TTAGGG)_n]. DAPIstaining showed that AT-rich regions were located on the centromereof almost all chromosomes and interstitial banding was not observed.FISH showed that 18S–28S rDNA spread over the short armsof two subtelocentric chromosome pairs and 5S rDNA was locatedon the long arm of one subtelocentric chromosome pair. SequentialFISH demonstrated that 18S–28S and 5S rDNA were locatedon different chromosomes. FISH also showed that the vertebratetelomeric sequence (TTAGGG)_n was located on both ends of eachchromosome and no interstitial signals were detected. Sequential18S–28S rDNA and (TTAGGG)_n FISH indicated that repeatedunits of the two multicopy families were closely associatedon the same chromosome pair. (Received 4 January 2007; accepted 1 September 2007)     

Cytogenetic characterisation of the razor shells Ensis directus (Conrad, 1843) and E. minor (Chenu, 1843) (Mollusca: Bivalvia)

The European razor shell Ensis minor (Chenu 1843) and the American E. directus (Conrad 1843) have a diploid chromosome number of 38 and remarkable differences in their karyotypes: E. minor has four metacentric, one metacentric–submetacentric, five submetacentric, one subtelocentric and eight telocentric chromosome pairs, whereas E. directus has three metacentric, two metacentric–submetacentric, six submetacentric, six subtelocentric and two telocentric pairs. Fluorescent in situ hybridisation (FISH) using a major ribosomal DNA probe located the major ribosomal genes on one submetacentric chromosome pair in both species; FISH with a 5S ribosomal DNA (5S rDNA) probe rendered one chromosomal (weak) signal for E. minor and no signal for E. directus, supporting a more dispersed organisation of 5S rDNA compared to the major ribosomal genes. The vertebrate telomeric sequence (TTAGGG) _n was located on both ends of each chromosome, and no interstitial signals were detected. In this work, a comparative karyological analysis was also performed between the four Ensis species analysed revealing that the three European species studied so far, namely E. minor, E. siliqua (Linné 1758) and E. magnus Schumacher 1817 show more similarities among them than compared to the American species E. directus. In addition, clear karyotype differences were found between the morphologically similar species E. minor and E. siliqua.     

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae)

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA₃-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA₃-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.     

Comparative Molecular Cytogenetic Analysis of Two Congeneric Species, <Emphasis Type="Italic">Mugil Curema</Emphasis> and <Emphasis Type="Italic">M. Liza</Emphasis> (Pisces,Mugiliformes), Characterized by Significant Karyotype Diversity

Two congeneric mullet species, Mugil liza and M. curema, respectively with an all-uniarmed and an all-biarmed karyotype, were cytogenetically studied by base-specific fluorochrome staining and FISH-mapping of 45S and 5S ribosomal RNA genes (rDNA) and the (TTAGGG)_n telomeric repeats. Whereas 45S rDNA sites might be homeologus in the two species, 5S rDNA sites are not, as they are localized on chromosome arms of different size. In both species, the (TTAGGG)_n telomeric probe hybridized to natural telomeres and was found scattered along the NORs. In metacentric chromosomes of M. curema, no pericentromeric signals of the telomeric probe were detected. Data are discussed in relation to the karyotype evolution in Mugilidae and to the mechanisms and the evolutionary implications of Robertsonian rearrangements in M. curema.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)<Subscript>n</Subscript> and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family

In the present study dual-colour fluorescence in situ hybridization (FISH) was performed to study the chromosomal distribution of 18S and 5S rDNAs, (GATA)_n and 5S rDNA, and U2 snRNA and 18S rDNA in four species of Batrachoididae family: Amphichthys cryptocentrus, Batrachoides manglae, Porichthys plectrodon and Thalassophryne maculosa. The 18S rDNA signals were present in only one pair of chromosomes in all the four Batrachoididae species. The 5S rDNA was mapped on one pair of chromosomes, except in B. manglae, which showed a hybridization signal in two pairs. The two ribosomal genes are located on different chromosome pairs, except in A. cryptocentrus, in which they appear co-located. In all the cases, the (GATA)_n probe produced disperse hybridization signals in all four species. The U2 snRNA signals appear very widely scattered in A. cryptocentrus, P. plectrodon, but show a degree of clustering in a specific chromosome pair in B. manglae. In T. maculosa, they are thinly dispersed and strong hybridization signals are observed co-located to the 18S rDNA-bearing chromosomes. Finally, a double-colour FISH with U2 snRNA and 5S rDNA probes was performed in B. manglae, and this showed that these genes were not co-located. These results have been compared with those from another Batrachoididae species, and evolutive processes of these species are discussed.     

Ribosomal DNA distribution in somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) and molecular-cytotaxonomic relationships to some other cycad genera

Somatic chromosomes ofStangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescentin situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe.Stangeria eriopus showed a chromosome number of 2n=16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study ofS. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genusStangeria may be more closely related to the genusCeratozamia than the genusBowenia or the genusMicrocycas previously hypothesized.     

Constitutive heterochromatin, 5S and 18S rDNA genes in Apareiodon sp. (Characiformes, Parodontidae) with a ZZ/ZW sex chromosome system

Karyotype, sex chromosome system and cytogenetics characteristics of an unidentified species of the genus Apareiodon originating from Piquiri River (Paraná State, Brazil) were investigated using differential staining techniques (C-banding and Ag-staining) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The diploid chromosome number was 2n = 54 with 25 pairs of meta- (m) to submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes. The major ribosomal rDNA sites as revealed by Ag-staining and FISH with 18S rDNA probe were found in distal region of longer arm of st chromosome pair 26, while minor 5S sites were observed in the interstitial sites on chromosome pairs 2 (smaller cluster) and 7 (larger one). The C-positive heterochromatin had pericentromeric and telomeric distribution. The heteromorphic sex chromosome system consisted of male ZZ (pair 21) and female middle-sized m/st Z/W chromosomes. The pericentric inversion of heterochromatinized short arm of ancestral Z followed by multiplication of heterochromatin segments is hypothesized for origin of W chromosome. The observed karyotype and chromosomal markers corresponded to those found in other species of the genus.     

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm,Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.     

Molecular cytogenetics of <Emphasis Type="Italic">Oligosarcus hepsetus</Emphasis> (Teleostei,Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraíba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A₃ (CMA₃)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5 S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA₃- stainings and FISH with 18 S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5 S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA₃-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.     

NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae) – re-examination by FISH

Chromosomal polymorphism regarding the number of chromosomal NOR sites in the cyprinid fish Chondrostoma lusitanicum reported previously (Rodrigues & Collares-Pereira, 1996) was re-examined using fluorescence in situ hybridization (FISH) with a ribosomal DNA (rDNA) probe. All positive CMA3-bands contained ribosomal DNA documented by either two or four FISH positive signals in the respective karyotypes. This polymorphism suggests the occurrence of structural rearrangements of translocation type in rDNA region from one ancestral NOR-bearing chromosome pair ubiquitous among leuciscine cyprinid fishes to another pair. The absence of individuals heterozygous for this polymorphism is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

Chromosome Localization of 5S and 45S Ribosomal DNA in the Genomes of Linum L. Species of the Section Linum (Syn. Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18S–5.8S–26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA were colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum, L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.     

Chromosomal rearrangements and the first indication of an ♀X1X1X2X2/♂X1X2Y sex chromosome system in Rineloricaria fishes (Teleostei: Siluriformes)

Rineloricaria is the most diverse genus within the freshwater fish subfamily Loricariinae, and it is widely distributed in the Neotropical region. Despite limited cytogenetic data, records from southern and south-eastern Brazil suggest a high rate of chromosomal rearrangements in this genus, mirrored in remarkable inter- and intraspecific karyotype variability. In the present work, we investigated the karyotype features of Rineloricaria teffeana, an endemic representative from northern Brazil, using both conventional and molecular cytogenetic techniques. We revealed different diploid chromosome numbers (2n) between sexes (33♂/34♀), which suggests the presence of an ♀X₁X₁X₂X₂/♂X₁X₂Y multiple sex chromosome system. The male-limited Y chromosome was the largest and the only biarmed element in the karyotype, implying Y-autosome fusion as the most probable mechanism behind its origination. C-banding revealed low amounts of constitutive heterochromatin, mostly confined to the (peri)centromeric regions of most chromosomes (including the X₂ and the Y) but also occupying the distal regions of a few chromosomal pairs. The chromosomal localization of the 18S ribosomal DNA (rDNA) clusters revealed a single site on chromosome pair 4, which was adjacent to the 5S rDNA cluster. Additional 5S rDNA loci were present on the autosome pair 8, X₁ chromosome, and in the presumed fusion point on the Y chromosome. The probe for telomeric repeat motif (TTAGGG)_n revealed signals of variable intensities at the ends of all chromosomes except for the Y chromosome, where no detectable signals were evidenced. Male-to-female comparative genomic hybridization revealed no sex-specific or sex-biased repetitive DNA accumulations, suggesting a presumably low level of neo-Y chromosome differentiation. We provide evidence that rDNA sites might have played a role in the formation of this putative multiple sex chromosome system and that chromosome fusions originate through different mechanisms among different Rineloricaria species.