Cytogenetic characterization and description of an XX/XY1Y2 sex chromosome system in catfish Harttia carvalhoi (Siluriformes, Loricariidae)

Karyotypic and cytogenetic characteristics of catfish Harttia carvalhoi (Paraíba do Sul River basin, S?o Paulo State, Brazil) were investigated using differential staining techniques (C-banding, Ag-staining) and fluorescent in situ hybridization (FISH) with 18S and 5S rDNA probes. The diploid chromosome number of females was 2n = 52 and their karyotype was composed of nine pairs of metacentric, nine pairs of submetacentric, four pairs of subtelocentric and four pairs of acrocentric chromosomes. The diploid chromosome number of males was invariably 2n = 53 and their karyotype consisted of one large unpaired metacentric, eight pairs of metacentric, nine pairs of submetacentric, four pairs of subtelocentric, four pairs of acrocentric plus two middle-sized acrocentric chromosomes. The differences between female and male karyotypes indicated the presence of a sex chromosome system of XX/XY1Y2 type, where the X is the largest metacentric and Y1 and Y2 are the two additional middle-sized acrocentric chromosomes of the male karyotype. The major rDNA sites as revealed by FISH with an 18S rDNA probe were located in the pericentromeric region of the largest pair of acrocentric chromosomes. FISH with a 5S rDNA probe revealed two sites: an interstitial site located in the largest pair of acrocentric chromosomes, and a pericentromeric site in a smaller metacentric pair of chromosomes. Translocations or centric fusions in the ancestral 2n = 54 karyotype is hypothesized for the origin of such multiple sex chromosome systems where females are fixed translocation homozygotes whereas males are fixed translocation heterozygotes. The available cytogenetic data for representatives of the genus Harttia examined so far indicate large kayotype diversity.     

Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy

Basic and molecular cytogenetic analyses were performed in specimens of Characidium cf. zebra from five collection sites located throughout the Tietê, Paranapanema and Paraguay river basins. The diploid number in specimens from all samples was 2n = 50 with a karyotype composed of 32 metacentric and 18 submetacentric chromosomes in both males and females. Constitutive heterochromatin was present at the centromeric regions of all chromosomes and pair 23, had additional interstitial heterochromatic blocks on its long arms. The nucleolar organizer regions (NORs) were located on the long arms of pair 23, while the 5S rDNA sites were detected in different chromosomes among the studied samples. One specimen from the Alambari river was a natural triploid and had two extra chromosomes, resulting in 2n = 77. The remarkable karyotypic similarity among the specimens of C. cf. zebra suggests a close evolutionary relationship. On the other hand, the distinct patterns of 5S rDNA distribution may be the result of gene flow constraints during their evolutionary history.     

Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes,Characidae)

Hollandichthys is a fish genus of the family Characidae that was until recently considered to be monotypic, with cytogenetic, morphological, and molecular data being restricted to a few local populations. In the present study, the karyotype of a population of Hollandichthys multifasciatus was analyzed using classical and molecular cytogenetic approaches for the investigation of potential markers that could provide new perspectives on the cytotaxonomy. H. multifasciatus presented a diploid number of 2n=50 chromosomes and a karyotype formula of 8m+10sm+32st. A single pair of chromosomes presented Ag-NORs signals, which coincided with the 18S rDNA sites visualized by FISH, whilst the 5S rDNA sequences were mapped in two chromosome pairs. The distribution of the U snRNA genes was mapped on the Hollandichthys chromosomes for the first time, with the probes revealing the presence of the U1 snDNA on the chromosomes of pair 20, U2 on pairs 6 and 19, U4 on pair 16, and U6 on the chromosomes of pair 11. The results of the present study indicated karyotypic differences in comparison with the other populations of H. multifasciatus studied previously, reinforcing the need for further research to identify isolated populations or the potential existence of cryptic Hollandichthys species.     

Chromosomal diversification in populations of Characidium cf. gomesi (Teleostei, Crenuchidae)

Comparative cytogenetic studies carried out in two populations of Characidium cf. gomesi from Botucatu region, SP, Brazil, showed a similar karyotypic structure in a diploid number of 50 chromosomes, 32 metacentric and 18 submetacentric chromosomes for males and 31 metacentric and 19 submetacentric chromosomes for females as well as a ZZ-ZW sex chromosome system. Differences between both populations, however, were found in relation to the occurrence of B chromosomes and the distribution of 18S and 5S ribosomal DNA (rDNA) sites. Characidium cf. gomesi from the Alambari Stream, a component of the Tietê River basin, revealed 18S rDNA on Z and W chromosomes, while this gene was located on autosomes in the sample from the Paranapanema River basin. The 5S rDNA sites were observed in a single chromosomal pair (number 25) in the populations from Paranapanema and in two pairs in the specimens from Tietê (numbers 20 and 25). Besides that, in the sample from Paranapanema, both inter and intra-individual variations were found due to the occurrence of up to four heterochromatic supernumerary chromosomes in the cells. The life mode of this fish, restricted to headwaters and subjected to frequent breakdown into sub-populations, may have contributed to the fixation of such chromosomal differences. The karyotypic similarities found in the analysed populations, however, suggest that all are descended from the same ancestor group whereas their differences indicate that they are already existing in reproductively isolated populations.     

Karyotypic diversity and evolution of Loricariidae (Pisces, Siluriformes)

We present cytogenetic analyses of four fish species, belonging to four Loricariidae subfamilies: Neoplecostomus microps (Neoplecostominae) with 2n=54 chromosomes, Harttia loricariformis (Loricariinae) with 2n=56 chromosomes, Hypostomus affinis (Hypostominae) with 2n=66 chromosomes and Upsilodus sp. (Upsilodinae), with 2n=96 chromosomes. In addition to karyotypes, data on the location of 18s rDNA sites are presented, derived from indirect (silver nitrate impregnation) and direct (FISH) methods. There is only one pair of nucleolar organizing regions (NORs) per species, except in H. affinis. Diversity and NOR macrokaryotypic evolution in the species analyzed are discussed in relation to the evolution of the Loricariidae as a whole. In addition, a revision of the cytogenetic data available for this family is presented.     

Origin of the X1X1X2X2/X1X2Y sex chromosome system of Harttia punctata (Siluriformes,Loricariidae) inferred from chromosome painting and FISH with ribosomal DNA markers

Harttia is a genus in the subfamily Loricariinae that accommodates fishes popularly known as armored catfishes. They show extensive karyotypic diversity regarding interspecific numerical/structural variation of the karyotypes, with the presence of the XX/XY₁Y₂ multiple sex chromosome system, as found in H. carvalhoi. In this context, this study aimed to characterize Harttia punctata chromosomally, for the first time, and to infer the rearrangements that originated the X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system present in this species. The data obtained in this study, with classical (Giemsa, C-banding and AgNORs) and molecular methodologies (fluorescence in situ hybridization) and chromosome microdissection, indicated that a translocation between distinct acrocentric chromosomes bearing rRNA genes, accompanied by deletions in both chromosomes, might have originated the neo-Y chromosome in this species. The data also suggest that the multiple sex chromosome systems present in H. carvalhoi and H. punctata had an independent origin, evidencing the recurrence of chromosome alterations in species from this genus.     

The role of the Robertsonian rearrangements in the origin of the XX/XY1Y2 sex chromosome system and in the chromosomal differentiation in Harttia species (Siluriformes, Loricariidae)

Harttia is a genus of the subfamily Loricariinae that posses a broad chromosomal variation. In addition to interspecific karyotype diversity within this group, a multiple sex chromosome system, XX/XY₁Y₂, has been described for Harttia carvalhoi. Thus, this study aimed to determine the role of chromosomal rearrangements in karyotype differentiation in Harttia by classical and molecular cytogenetic procedures. The results show that Robertsonian rearrangements have a prominent role in the chromosomal diversification of the species analysed, which initially leads to hypothesize a diploid number reduction in Harttia torrenticola and H. carvalhoi. The metacentric chromosome 1, shared between H. torrenticola and H. carvalhoi, could have originated from centric fusions from the ancestral karyotype. A centric fission event associated with the first metacentric pair allowed for the origination of a multiple sex chromosome system XX/XY₁Y₂, specific to H. carvalhoi. This study highlights the relevance of Robertsonian rearrangements in karyotypic differentiation of the species studied and demonstrates that the occurrence of a centric fission, as opposed to a previously hypothesised chromosome fusion, is directly implicated in the origin of the sex chromosome system of H. carvalhoi.     

Karyotypic variability in Iheringichthys labrosus (Teleostei, Pimelodidae) from the Tibagi River basin (Paraná State, Brazil)

Cytogenetic analyses were carried out in a populational sample of Iheringichthys labrosus from the Guaraúna River (Upper Tibagi River; Paraná State, Brazil) in order to provide a karyotypic comparison with another previously studied population from the Lower Tibagi River, characterized by the presence of 32m + 8sm + 6st + 10a (2n = 56, FN = 102) and occurrence of supernumerary chromosomes (80% of individuals). The 17 specimens of I. labrosus (6 females, 10 males and 1 of unknown sex) from the Upper Tibagi River showed 2n = 56 chromosomes, a karyotype formula of 14m + 32sm + 4st + 6a (FN = 106), without evidence of sex chromosome heteromorphism or supernumerary chromosomes. The heterochromatin was detected at telomeric and centromeric positions in several chromosomal pairs. The Ag-nucleolar organizer regions were heteromorphic and located at terminal position on short arms of the 16th chromosomal pair, suggesting a positive association with heterochromatic regions. The inter-populational karyotypic differentiation reported indicates distinct evolutionary pathways within I. labrosus in the Tibagi River basin.     

Cytogenetic mapping of rRNAs and histone H3 genes in 14 species of Dichotomius (Coleoptera, Scarabaeidae, Scarabaeinae) beetles

Standard cytogenetic analyses and chromosomal mapping of the genes for 18S and 5S rRNAs and histone H3 were performed in 14 species of beetles of the genus Dichotomius (Coleoptera, Scarabaeidae, Scarabaeinae). Conserved karyotypes with 2n = 18 and biarmed chromosomes were observed in all species. Moreover, the presence of a large metacentric pair (pair 1) was characteristic in the studied species, evidencing a remarkable synapomorphy for this genus, which probably originated by an ancient fusion of 2 autosomes while the ancestral sex-chromosome pair remained conserved. FISH showed that the 5S rRNA and histone H3 genes are located in the proximal region of pair 2, with the 2 genes co-located. However, the major rDNA cluster probed by the 18S rRNA gene mapped to 1-3 bivalents, being exclusively autosomal, associated with sex elements, or both. In most species, the major rDNA cluster was observed in pair 3, and it was frequently (64.3%) located in the distal region regardless of the chromosome. The conserved number and position of the 5S rDNA/H3 histone cluster seems to be an ancient pattern shared by all of the studied species. In contrast, the major rDNA clusters apparently tolerate distinct patterns of diversification in the karyotypes of the species that could be associated with small inversions, ectopic recombination, and transposition. Moreover, we reinforced the association/co-localization between the 5S rRNA and histone H3 genes in this group contributing thus to the knowledge about the chromosomal organization and diversification patterns of multigene families in beetles and insects.     

Mapping five repetitive DNA classes in sympatric species of Hypostomus (Teleostei: Siluriformes: Loricariidae): analysis of chromosomal variability

Fish belonging to the genus Hypostomus are known for exhibiting a striking diversity in its karyotype structure, however the knowledge concerning the distribution patterns of heterochromatin and location of repetitive DNA sequences in the karyotypes is still limited. Aiming a better understanding of the chromosomal organization in this group, we analyzed three sympatric species of Hypostomus collected in the Hortelã stream, a component of the Paranapanema River basin, Botucatu/SP/Brazil. The analyses involved the cytogenetic characterization and chromosomal mapping of repetitive sequences and intra/interspecific comparisons using sequences of the cytochrome C oxidase subunit I. The results revealed that H. ancistroides presents a karyotype with 2n = 68 chromosomes, H. strigaticeps 2n = 72 chromosomes, and H. nigromaculatus 2n = 76 chromosomes. In addition to differences found in the diploid number, it was also observed variations in karyotypic formulae, amount of constitutive heterochromatin, and location of nucleolus organizer regions. The cytogenetic mapping of 5S and 18S rDNA, as well as of the H3 histone gene, disclosed a differential dispersion process among the three species. In some cases the Rex1 transposable element showed to be co-located with 5S rDNA sites. The molecular analyses support the cytogenetic data and represent an additional tool for the characterization of the analyzed species. The results evidenced that chromosomal variations are not restricted to differences in diploid number or karyotypic macrostructure in the genus Hypostomus, indicating that events such as transposition of heterochromatin and rDNA segments may participate in the differentiation process occurred in these species.     

纳特竖蟾(无尾目:滑背蟾科) 的细胞遗传、与相关属物种的核型相似性并兼论其分类地位

本文报道纳特竖蟾(Eupemphix nattereri Steindachner,1863)巴西3个产地标本的核型,均为2n=22,由1对端部和10对中或亚中部着丝点染色体组成.仅一对NORs位于No.11染色体对长臂端部,而其位置有别于常见的具有标志性意义的近着丝点位置.该NORs的位置为rDNA为探针的荧光原位杂交(FISH)所确认.各染色体对中,着丝点C-带明显,插入或端带偶见.某些标本的No.8同源染色体对间C-带的大小随异染色质化的程度不同而变化.居于较小的实验标本量,这种在3个产地的雌或雄性标本之一中观察到的C-带异型现象可能为种下细胞地理学变异,亦或为细胞学意义的性染体分化.3产地之一的标本核型的No.11着丝点C-带异染色质化的程度较高.CMA3染色检测到部分GC-rich区域,DAPI染色未显示任何AT-rich区.成功获得BrdU复制带,并将其与滑背蟾类动物(leiuperid)中近缘属及物种进行对比分析.比较结果表明,纳特竖蟾的核型与斑符泡蟾种组(Physalaemus signifer group)难以相互区别,而与肿肋蟾属(Pleurodema Tschudi,1838)极为相似.核型数据支持形态学上将纳特竖蟾、二光肿肋蟾[Pleurodema diplolister (Peters,1870)]和短头肿肋蟾[P.brachyops(Cope,1869)]划为同一分支的观点[动物学报 53(2):285-293,2007].     

Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH)

With the aim of characterizing the sex chromosomes of rainbow trout (Oncorhynchus mykiss) and to identify the sex chromosomes of coho salmon (O. kisutch), we used molecular markers OmyP9, 5S rDNA, and a growth hormone gene fragment (GH2), as FISH probes. Metaphase chromosomes were obtained from lymphocyte cultures from farm specimens of rainbow trout and coho salmon. Rainbow trout sex marker OmyP9 hybridizes on the sex chromosomes of rainbow trout, while in coho salmon, fluorescent signals were localized in the medial region of the long arm of one subtelocentric chromosome pair. This hybridization pattern together with the hybridization of a GH2 intron probe on a chromosome pair having the same morphology, suggests that a subtelocentric pair could be the sex chromosomes in this species. We confirm that in rainbow trout, one of the two loci for 5S rDNA genes is on the X chromosome. In males of this species that lack a heteromorphic sex pair (XX males), the 5S rDNA probe hybridized to both subtelocentrics This finding is discussed in relation to the hypothesis of intraspecific polymorphism of sex chromosomes in rainbow trout.     

Cytogenetics characterization of <Emphasis Type="Italic">Crenuchus spilurus</Emphasis> (Günther, 1863): a remarkable low diploid value within family Crenuchidae (Characiformes)

Conventional and molecular cytogenetic analyses were performed in specimens of the Neotropical Crenuchus spilurus freshwater fish species from a single location (Caeté River, Brazil). All specimens presented diploid values of 2n?=?38 chromosomes (12 m?+?4sm?+?2st?+?20a), the lowest reported for family Crenuchidae up to now. A single pair of nucleolar organizing regions (NORs) was detected in the subtelocentric chromosome pair no. 9 by silver-staining and fluorescence in situ hybridization (FISH) with 18S rDNA sequence-specific probe. Two pairs of 5S rRNA gene clusters were found either interstitial or terminally located in the long arms of the acrocentric chromosome pairs nos. 10 and 13. Heterochromatic regions were clearly observed in the short arms of the NOR-bearing chromosome pair and weakly-positive to the pericentromeric regions of most acrocentric chromosomes. Additionally, no sex chromosomes were identified in the surveyed specimens. Crenuchidae have signals of several mechanisms involved in karyotype diversification within this family: differential location of heterochromatin-rich regions, multiplication, and translocation of rDNA clusters, presence/absence of sex chromosomes, macrostructural changes in morphology and number of chromosomes. This variety of karyotype patterns reveals the importance of widening cytogenetic studies to more taxa for better know the chromosomal evolution occurred in this group.     

Sympatric occurrence of four cytotypes and one extra chromosome in Bryconamericus ecai (Characidae): 18S rDNA polymorphism and heterochromatin composition

In the present study, specimens of Bryconamericus ecai collected from the Forquetinha River/RS, were cytogenetically analyzed, disclosing a wide karyotypic diversity in this species. All individuals had 2n = 50, with different karyotypic formulae, resulting in four cytotypes and one B macrochromosome observed in cytotype III. Heterochromatin was distributed in the pericentromeric region of most chromosomes on the four cytotypes and also on a chromosome pair with interstitial markings in cytotype IV. Staining with CMA(3) and DAPI fluorochromes revealed a C-band region rich in AT base pairs in cytotypes I, II and III, and a pair with GC-rich heterochromatin in cytotypes II and III. Cytotype IV presented CMA(3) and DAPI positive heterochromatin. Silver nitrate impregnation, in situ hybridization, and fluorochrome staining showed a multiple system of AgNORs, 18S rDNA and CMA(3) sites in cytotypes I, III and IV, with both inter-and intraindividual variability in the number and location of these sites. Cytotype II had only one pair of NORs coincident with the 18S rDNA and CMA(3) sites, indicating a simple system. The chromosomal polymorphism observed among the specimens of B. ecai added to the literature data show that chromosomal rearrangements, especially pericentric inversions, play an important role in the karyotypic evolution of this group of fish. It can also be implied that more than one species of Bryconamericus is probably occurring, living in sympatry in the Forquetinha River/RS.     

Occurrence of two cytotypes in <Emphasis Type="Italic">Bryconamericus</Emphasis> aff. <Emphasis Type="Italic">iheringii</Emphasis> (Characidae): karyotype analysis by C- and G-banding and replication bands

Cytogenetic analyses of Bryconamericus aff. iheringii specimens from the upper Paraná River basin (State of Paraná, Brazil) are provided. They had 2n = 52 chromosomes and two cytotypes with variations in their karyotypic formulae: cytotype I with 12 metacentric, 18 submetacentric, 8 subtelocentric and 14 acrocentric chromosomes with a fundamental number (FN) of 90; cytotype II with 8 metacentric, 28 submetacentric, 6 subtelocentric and 10 acrocentric chromosomes with a fundamental number (FN) of 94. Differences in C- and G-band patterns between the cytotypes, distinguishing marker chromosomes for each karyotype, were reported. The R-band pattern by 5-bromodeoxyuridine incorporation was obtained in chromosomes of the cytotype II sample. In some metaphases, the second pair of submetacentric chromosomes is distinctive: its short arm is heterochromatic (positive C-band), corresponding to a late replication region. In the same cytotype, a G- and R-band size heteromorphism w as recorded in the long arm of pair 9 (submetacentric). These methodologies revealed an actual karyotypic differentiation in the B. aff. iheringii population analyzed. Morphometrical comparative analyses and a discussion of evolutionary aspects of chromosome diversification in species of this genus are provided as well.     

Chromosomal location of 18S and 5S rDNA sites in Triportheus fish species (Characiformes, Characidae)

The location of 18S and 5S rDNA sites was determined in eight species and populations of the fish genus Triportheus by using fluorescent in situ hybridization (FISH). The males and females of all species had 2n = 52 chromosomes and a ZZ/ZW sex chromosome system. A single 18S rDNA site that was roughly equivalent to an Ag-NOR was detected on the short arms of a submetacentric pair in nearly all species, and up to two additional sites were also observed in some species. In addition, another 18S rDNA cluster was identified in a distal region on the long arms of the W chromosome; this finding corroborated previous evidence that this cluster would be a shared feature amongst Triportheus species. In T. angulatus, a heterozygotic paracentric inversion involving the short arms of one homolog of a metacentric pair was associated with NORs. The 5S rDNA sites were located on the short arms of a single submetacentric chromosomal pair, close to the centromeres, except in T. auritus, which had up to ten 5S rDNA sites. The 18S and 5S rDNA sites were co-localized and adjacent on the short arms of a chromosomal pair in two populations of T. nematurus. Although all Triportheus species have a similar karyotypic macrostructure, the results of this work show that in some species ribosomal genes may serve as species-specific markers when used in conjunction with other putatively synapomorphic features.     

Characterization of yellow grouper Epinephelus awoara (Serranidae) karyotype by chromosome bandings and fluorescence in situ hybridization

The cytogenetics of yellow grouper Epinephelus awoara was studied using multiple cytogenetic markers [Giemsa staining, C-banding, Ag-NORs and fluorescence in situ hybridization (FISH)]. Giemsa staining results showed that the karyotypic formula of E. awoara was 2n = 48a, FN (fundamental number) = 48. Faint C-bandings were only detected at the centromeric regions of chromosome pair number 24, being almost indiscernible on the other chromosome pairs. After Ag-NOR staining, one pair of nucleolar organizer regions (NOR) was observed in the subcentromeric region of pair number 24. FISH results showed that 5S rDNA was located at a pair of medium-sized chromosomes, while 18S rDNA appeared at the same location in the subcentromeric region of pair number 24 where Ag-NORs were detected. The telomeric sequence (TTAGGG)(n) detected by FISH was located at both ends of each chromosome. The results suggested that E. awoara has retained general karyotypic structure stability during the evolutionary diversification process.     

Correction of karyotype of diploid Beesia calthifolia and discovery of a tetraploid cytotype

In this paper, the chromosomes of Beesia calthifolia were re-examined. In the 40 plant individuals of the population from Xinning County, Hunan Province, central China, one was found to be tetraploid with the karyotype formula of 2n＝4x=32=16m+8sm＋4st + 4t, and the remaining were all found to be diploids with the karyotype formula of 2n＝2x ＝16=8m+4sm+2st+2t. All the 10 individuals of the population from Cangshan Mountain, Dali City, Yunnan Province, southwestern China, were unexpectedly found to be tetraploids with the karyotype formula of 2n＝4x＝32=16m+8sm+4st+4t. Tetraploid cytotype was reported in this species for the first time. Based on the results and those previously reported, it is considered that there may exist some errors in the results of the karyotype analysis of this species previously reported by Shang(1985). He might have at least mistakenly recognized the centromere position of the fourth pair of chromosomes. This pair of chromosomes should have subterminal rather than median centromeres. Furthermore, the karyotypic differences of B. calthifolia and B. deltophylla were analyzed and the systematicposition of the genus Beesia was discussed in detail.     

Oogenesis and the Chromosomes of Twelve Bisexual Species of Heterodera (Nematoda: Heteroderidae)

Twelve bisexual species of Heterodera reproduced by amphimixis and had the same number of n=9 (2n=18) chromosomes in maturing oocytes. H. schachtii had slightly larger chromosomes than all other species. Only sperm nuclei with n=9 chromosomes were observed inside maturing oocytes and no specialized sex chromosomes were detected in any case. A "supernumerary" chromosome was observed occasionally in oocytes of H. schachtii and H. weissi and was transmitted regularly to one-half of the progeny of the nematodes that possessed it. Cytological characteristics were not very instructive in differentiating amphimictic tleterodera species. Such karyotypic uniformity indicates cytogenetic stability of the genus and close interrelationship among its members.     

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): comparative chromosome mapping of repetitive sequences

Erythrinus erythrinus presents extensive karyotypic diversity, with four karyomorphs (A–D) differing in the number of chromosomes, karyotype structure or sex chromosomes systems. Karyomorph A has 2n = 54 chromosomes in males and females without heteromorphic sex chromosomes, while karyomorph C has 2n = 52 chromosomes in females and 2n = 51 chromosomes in males, due a X₁X₁X₂X₂/X₁X₂Y sex chromosome system. Three allopatric populations of the karyomorph A and one population of the karyomorph C were now in deep investigated by molecular cytogenetic analyses, using repetitive DNAs as probes. The results reinforced the relatedness among populations of the karyomorph A, despite their large geographic distribution. Karyomorph C, however, showed a remarkably difference in the genomic constitution, especially concerning the amount and distribution of the 5S rDNA and Rex3 sequences on chromosomes. In addition, although karyomorphs C and D share several features, exclusive chromosomal markers show the derivative evolutionary pathway between them. Thus, besides the classical chromosomal rearrangements, the repetitive DNAs were useful tools to reveal the biodiversity, relatedness and differentiation of this fish group. The chromosomal set strongly corroborates that E. erythrinus corresponds to a species complex instead of a single biological entity.