Interregional cytogenetic comparisons in Halichoeres and Thalassoma wrasses (Labridae) of coastal and insular regions of the southwestern Atlantic.

The distribution patterns of marine biodiversity are complex, resulting from vicariant events and species dispersion, as well as local ecological and adaptive conditions. Furthermore, the wide geographic distribution of some species may be hindered by biogeographical barriers that can interfere in the gene flow. Cytogenetic analyses in marine fishes, especially those involving populations in small remote insular environments, remain scarce. In the Western Atlantic, species of wrasses from the genera Halichoeres and Thalassoma occur in biogeographic arrangements that make it possible to analyze cytogenetic patterns between coastal and widely separated island populations. Species of these genera were punctually analyzed in some Atlantic regions. In this study, we compared several chromosomal features, such as karyotype macrostructure, heterochromatic patterns, patterns of base-specific fluorochromes, Ag-NORs, and 18S and 5S ribosomal sites in Thalassoma noronhanum, Halichoeres poeyi, and Halichoeres radiatus individuals from distinct coastal or insular regions of Atlantic. Notably, all of them are characterized by multiple 18S and 5S rDNA sites with syntenic arrangements in some chromosome pairs. Individuals of T. noronhanum (between the insular regions of Rocas Atoll and Fernando de Noronha Archipelago - FNA) and H. poeyi (coastal areas from Northeastern Brazil) show no detectable differences among their cytogenetic patterns. On the other hand, H. radiatus from FNA and São Pedro and São Paulo Archipelago exhibit differences in the frequency of rDNA sites that could suggest some level of population structuring between these insular regions. Interregional cytogenetic inventories of marine species with wide geographic distribution need to be rapidly expanded. These data will allow a better understanding of the level of chromosomal stability between vast oceanic spaces, which may be less than previously thought.

Chromosomal evolution in large pelagic oceanic apex predators, the barracudas (Sphyraenidae, Percomorpha).

Sphyraena (barracudas) represents the only genus of the Sphyraenidae family and includes 27 species distributed into the tropical and subtropical oceanic regions. These pelagic predators can reach large sizes and, thus, attracting significant interest from commercial and sport fishing. Evolutionary data for this fish group, as well its chromosomal patterns, are very incipient. In the present study, the species Sphyraena guachancho, S. barracuda, and S. picudilla were analyzed under conventional (Giemsa staining, C-banding, and Ag-NOR) and molecular (CMA3 banding, and in situ hybridization with 18S rDNA, 5S rDNA, and telomeric probes) cytogenetic methods. The karyotypic patterns contrast with the current phylogenetic relationships proposed for this group, showing by themselves to be distinct among closely related species, and similar among less related ones. This indicates homoplasic characteristics, with similar karyotype patterns originating at least twice, independently. Although still cytogenetically poor investigated, our data were enough to put in evidence a variety of ancient conserved traits and evolutionary novelties for the Sphyraena genus. In this sense, it is fundamental that a larger number of Sphyraenidae species, as well as of other phylogenetically related families, be also investigated. This will solidify the knowledge of their karyotypic patterns, and the evolutionary path followed by the species of this particular fish family.

Highly conserved Z and molecularly diverged W chromosomes in the fish genus Triportheus (Characiformes, Triportheidae).

The main objectives of this study were to test: (1) whether the W-chromosome differentiation matches to species' evolutionary divergence (phylogenetic concordance) and (2) whether sex chromosomes share a common ancestor within a congeneric group. The monophyletic genus Triportheus (Characiformes, Triportheidae) was the model group for this study. All species in this genus so far analyzed have ZW sex chromosome system, where the Z is always the largest chromosome of the karyotype, whereas the W chromosome is highly variable ranging from almost homomorphic to highly heteromorphic. We applied conventional and molecular cytogenetic approaches including C-banding, ribosomal DNA mapping, comparative genomic hybridization (CGH) and cross-species whole chromosome painting (WCP) to test our questions. We developed Z- and W-chromosome paints from T. auritus for cross-species WCP and performed CGH in a representative species (T. signatus) to decipher level of homologies and rates of differentiation of W chromosomes. Our study revealed that the ZW sex chromosome system had a common origin, showing highly conserved Z chromosomes and remarkably divergent W chromosomes. Notably, the W chromosomes have evolved to different shapes and sequence contents within ~15-25 Myr of divergence time. Such differentiation highlights a dynamic process of W-chromosome evolution within congeneric species of Triportheus.

Comparative cytogenetic mapping of rRNA genes among naked catfishes: implications for genomic evolution in the Bagridae family.

In the present study, the karyotype and chromosomal characteristics of 9 species of the Bagridae fish family were investigated using conventional Giemsa staining as well as dual-color fluorescence in situ hybridization to detect the 18S and 5S rDNA sites. In addition to describing the karyotype of several Bagridae catfishes, we established molecular cytogenetic techniques to study this group. The 9 species contained a diploid chromosomal number, varying from 50 (Pseudomystus siamensis) to 62 (Hemibagrus wyckii), while none contained heteromorphic sex chromosomes. 18S rDNA sites were detected in only 1 chromosomal pair among all species evaluated. However, 3 different patterns were observed for the distribution of the 5S rDNA: 2 sites were found in the genus Mystus and in P. siamensis, multiple sites were observed in the genus Hemibagrus, and a syntenic condition for the 18S and 5S rDNA sites was identified in H. wyckii. The extensive variation in the number and chromosomal position of rDNA clusters observed among these Bagridae species may be related to the intense evolutionary dynamics of rDNA-repeated units, which generates divergent chromosomal distribution patterns even among closely related species. In summary, the distribution of repetitive DNA sequences provided novel, useful information regarding the evolutionary relationships between Bagridae fishes.

Evolutionary dynamics of rDNA genes on chromosomes of the Eucinostomus fishes: cytotaxonomic and karyoevolutive implications.

Several chromosomal features of Gerreidae fish have been found to be conserved. In this group, it is unclear whether the high degree of chromosomal stasis is maintained when analyzing more dynamic regions of chromosomes, such as rDNA sites that generally show a higher level of variability. Thus, cytogenetic analyses were performed on 3 Atlantic species of the genus Eucinostomus using conventional banding (C-banding, Ag-NOR), AT- and GC-specific fluorochromes, and fluorescence in situ hybridization mapping of telomeric sequences and 5S and 18S rDNA sites. The results showed that although the karyotypical macrostructure of these species is similar (2n = 48 chromosomes, simple Ag-NORs seemingly located on homeologous chromosomes and centromeric heterochromatin pattern), there are differences in the positions of rDNA subunits 5S and 18S. Thus, the ribosomal sites have demonstrated to be effective cytotaxonomic markers in Eucinostomus, presenting a different evolutionary dynamics in relation to other chromosomal regions and allowing access to important evolutionary changes in this group.

Chromosomal distribution of two multigene families and the unusual occurrence of an X1X1X2X2/X1X2Y sex chromosome system in the dolphinfish (Coryphaenidae): an evolutionary perspective.

Dolphinfishes (Coryphaenidae) are pelagic predators distributed throughout all tropical and subtropical oceans and are very important for commercial, traditional, and sport fishing. This small family contains the Coryphaena hippurus and Coryphaena equiselis species whose chromosomal aspects remain unknown, despite recent advances in cytogenetic data assimilation for Perciformes. In this study, both species were cytogenetically analyzed using different staining techniques (C-, Ag-, and CMA3 banding) and fluorescence in situ hybridization, to detect 18S rDNA and 5S rDNA. C. hippurus females exhibit 2n = 48 chromosomes, with 2m+4sm+42a (NF = 54). In C. equiselis, where both sexes could be analyzed, females displayed 2n = 48 chromosomes (2m+6sm+40a) and males exhibited 2n = 47 chromosomes (3m+6sm+38a) (NF = 56), indicating the presence of X1X1X2X2/X1X2Y multiple sex chromosomes. Sex-chromosome systems are rare in Perciformes, with this study demonstrating the first occurrence in a marine pelagic species. It remains unknown as to whether this system extends to other populations; however, these data are important with respect to evolutionary, phylogenetic, and speciation issues, as well as for elucidating the genesis of this unique sex system.

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): occurrence and distribution of B chromosomes.

Erythrinus erythrinus, a Neotropical fish species of the Erythrinidae family, has a wide distribution in South America. Previous cytogenetic analysis showed that this species presents extensive karyotype diversity, with 4 karyomorphs (A-D) described herein. This study investigated the karyotypic structure of 2 new populations of E. erythrinus from the Brazilian Pantanal region, in order to improve the knowledge of the chromosomal diversity in this species. Both populations showed typical characteristics of karyomorph A, with 2n=54 chromosomes (6m+2st+46a), without differentiation between males and females. In addition, identical supernumerary B chromosomes, appearing as double-minute chromosomes, were also found in both populations. These findings suggest the presence of mitotic instability in view of their high intra- and inter-individual numerical variation. The presence of these chromosomes is likely a basal characteristic for this group, since the same kind of Bs also occurs in some other populations and karyomorphs of E. erythrinus. As such, they are important markers of biodiversity found in this nominal species, which probably corresponds to a species complex.

Karyotype divergence and spreading of 5S rDNA sequences between genomes of two species: darter and emerald gobies ( Ctenogobius , Gobiidae).

Karyotype analyses of the cryptobenthic marine species Ctenogobius boleosoma and C. smaragdus were performed by means of classical and molecular cytogenetics, including physical mapping of the multigene 18S and 5S rDNA families. C. boleosoma has 2n = 44 chromosomes (2 submetacentrics + 42 acrocentrics; FN = 46) with a single chromosome pair each carrying 18S and 5S ribosomal sites; whereas C. smaragdus has 2n = 48 chromosomes (2 submetacentrics + 46 acrocentrics; FN = 50), also with a single pair bearing 18S rDNA, but an extensive increase in the number of GC-rich 5S rDNA sites in 21 chromosome pairs. The highly divergent karyotypes among Ctenogobius species contrast with observations in several other marine fish groups, demonstrating an accelerated rate of chromosomal evolution mediated by both chromosomal rearrangements and the extensive dispersion of 5S rDNA sequences in the genome.

Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes.

Approximately 90 species in the genus Leporinus (Characiformes, Anostomidae) are known, and most of them do not present differentiated sex chromosomes. However, there is a group of 7 species that share a heteromorphic ZW sex system. In all of these species, the W chromosome is the largest one in the karyotype and is mostly heterochromatic. We investigated the distribution of several microsatellites in the genome of 4 Leporinus species that possess ZW chromosomes. Our results showed a very large accumulation of mostly microsatellites on the W chromosomes. This finding supports the presence of an interconnection between heterochromatinization and the accumulation of repetitive sequences, which has been proposed for sex chromosome evolution, and suggests that heterochromatinization is the earlier of the 2 processes. In spite of the common origin that has been proposed for W chromosomes in all of the studied species, the microsatellites followed different evolutionary trajectories in each species, which indicates a high plasticity for sex chromosome differentiation.

Independent sex chromosome evolution in lower vertebrates: a molecular cytogenetic overview in the Erythrinidae fish family.

The Erythrinidae fish family is an excellent model for analyzing the evolution of sex chromosomes. Different stages of sex chromosome differentiation from homomorphic to highly differentiated ones can be found among the species of this family. Here, whole chromosome painting, together with the cytogenetic mapping of repetitive DNAs, highlighted the evolutionary relationships of the sex chromosomes among different erythrinid species and genera. It was demonstrated that the sex chromosomes can follow distinct evolutionary pathways inside this family. Reciprocal hybridizations with whole sex chromosome probes revealed that different autosomal pairs have evolved as the sex pair, even among closely related species. In addition, distinct origins and different patterns of differentiation were found for the same type of sex chromosome system. These features expose the high plasticity of the sex chromosome evolution in lower vertebrates, in contrast to that occurring in higher ones. A possible role of this sex chromosome turnover in the speciation processes is also discussed.

Transposable elements in fish chromosomes: a study in the marine cobia species.

Rachycentron canadum, a unique representative of the Rachycentridae family, has been the subject of considerable biotechnological interest due to its potential use in marine fish farming. This species has undergone extensive research concerning the location of genes and multigene families on its chromosomes. Although most of the genome of some organisms is composed of repeated DNA sequences, aspects of the origin and dispersion of these elements are still largely unknown. The physical mapping of repetitive sequences on the chromosomes of R. canadum proved to be relevant for evolutionary and applied purposes. Therefore, here, we present the mapping by fluorescence in situ hybridization of the transposable element (TE) Tol2, the non-LTR retrotransposons Rex1 and Rex3, together with the 18S and 5S rRNA genes in the chromosome of this species. The Tol2 TE, belonging to the family of hAT transposons, is homogeneously distributed in the euchromatic regions of the chromosomes but with huge colocalization with the 18S rDNA sites. The hybridization signals for Rex1 and Rex3 revealed a semi-arbitrary distribution pattern, presenting differentiated dispersion in euchromatic and heterochromatic regions. Rex1 elements are associated preferentially in heterochromatic regions, while Rex3 shows a scarce distribution in the euchromatic regions of the chromosomes. The colocalization of TEs with 18S and 5S rDNA revealed complex chromosomal regions of repetitive sequences. In addition, the nonpreferential distribution of Rex1 and Rex3 in all heterochromatic regions, as well as the preferential distribution of the Tol2 transposon associated with 18S rDNA sequences, reveals a distinct pattern of organization of TEs in the genome of this species. A heterogeneous chromosomal colonization of TEs may confer different evolutionary rates to the heterochromatic regions of this species.

Chromosomal distribution and evolution of repetitive DNAs in fish.

Fish exhibit the greatest diversity of all vertebrates, making this group extremely attractive for the study of a number of evolutionary questions. Fish genomes have intrinsic characteristics that may be responsible for the amazing diversity of fish species observed, but little is known about their structure and organization. A large amount of data from mapping of repetitive DNA sequences of several species has been generated, providing an important source of information for better understanding the involvement of repetitive DNA sequences in chromosomal organization. Almost all classes of repeated DNAs have been mapped in fishes, and all fish genomes analyzed contain at least one, mostly all types of repetitive DNAs. DNA sequence data combined with the chromosomal mapping of these repeated elements by means of cytogenetic techniques can provide a clearer picture of the genome, which is not yet clearly defined, even if already sequenced. In this chapter, we do not aim to analyze all available data on the chromosomal distribution of repetitive DNAs in fish species, but instead wish to draw attention to the impact of repetitive DNA sequences on fish karyotyping and genome evolution, with a particular focus on B chromosome origin and maintenance and on the differentiation of sex chromosomes. We also discuss the integration of chromosome analysis and genomic data, which represents a promising tool for fish cytogenomics.

Karyoevolutionary aspects of Atlantic hogfishes (Labridae-Bodianinae), with evidence of an atypical decondensed argentophilic heterochromatin.

Fish from the family Labridae elicit considerable ecological interest, especially due to their complex interactions with the reef environment. Different karyoevolutionary tendencies have been identified in the subfamilies Bodianinae, Corinae and Cheilinae. Chromosomal analyses conducted in the Atlantic species Bodianus rufus (2n=48; 6m+12sm+14st+16a, FN=80), Bodianus pulchellus (2n=48; 4m+12sm+14st+18a, FN=78) and Bodianus insularis (2n=48; 4m+12sm+14st+18a, FN=78) identified Ag-NOR/18SrDNA sites located only in the terminal region of the short arm (p) of the largest subtelocentric pair. The 5S rDNA genes were mapped in the terminal region of the long arm (q) of the largest acrocentric pair and the p arm of chromosome 19 in B. insularis. The karyotype of the three species shows an extensive heterochromatic and argentophilic region, exceptionally decondensed, located in the p arm of the second subtelocentric pair. This region does not correspond to a NOR site, since it is not hybridized with 18S rDNA probes, and is not GC-rich, as generally occurs with nucleolus organizer regions of lower invertebrates. Heterochromatin in the three species is reduced and distributed over the centromeric and pericentromeric regions of chromosomes. The elevated number of two-armed chromosomes in species of Bodianus, in relation to other Labridae, shows karyotype diversification based on pericentric inversions, differentiating them markedly in terms of evolutionary tendencies that occur in subfamilies Corinae and Cheilininae. Structural cytogenetic similarities between B. pulchellus and B. insularis, in addition to the conserved chromosomal location pattern of ribosomal multigenic families, indicate phylogenetic proximity of these species.

The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes.

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.

Chromosomal distribution of repetitive DNA sequences highlights the independent differentiation of multiple sex chromosomes in two closely related fish species.

The arrangement of 6 repetitive DNA sequences in the mitotic and meiotic sex chromosomes of 2 Erythrinidae fish, namely Hoplias malabaricus and Erythrinus erythrinus, both with a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y sex chromosome system, was analyzed using fluorescence in situ hybridization. The distribution patterns of the repetitive sequences were distinct for each species. While some DNA repeats were species-specific, others were present in the sex chromosomes of both species at different locations. These data, together with the different morphological types of sex chromosomes and the distinct chromosomal rearrangements associated with the formation of the neo-Y chromosomes, support the plasticity of sex chromosome differentiation in the Erythrinidae family. Our present data highlight that the sex chromosomes in fish species may follow diverse differentiation patterns, even in the same type of sex chromosome system present in cofamiliar species.

The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes.

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY - karyomorph B and a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y - karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.

Repetitive DNAs and differentiation of sex chromosomes in neotropical fishes.

The processes working on sex chromosome differentiation are still not completely understood. However, the accumulation of repetitive DNA sequences has been shown to be one of the first steps in the early stages of such differentiation. In addition, regions with suppressed or no recombination have a potential to accumulate these DNA sequences and, for this reason, the absence of recombination between the sex chromosomes favors, by itself, the accumulation of repetitive sequences on these chromosomes during evolution. The diversity of sex-determining mechanisms in fish, alongside with the absence of heteromorphic sex chromosomes in many species, makes this group a useful model to better understand evolutionary processes of sex chromosomes in vertebrates, considering that fish occupy the basal position in the phylogeny of this group. In this review we draw attention to a preferential accumulation and enrichment in repetitive DNAs in sex chromosomes of many neotropical fish species in comparison with autosomes. This phenomenon has been observed between both morphologically differentiated and nascent sex chromosome systems, which highlight the potential role of these sequences in the differentiation of fish sex chromosomes generating differences in morphology and size between them.

Differentiation of the XY sex chromosomes in the fish Hoplias malabaricus (Characiformes, Erythrinidae): unusual accumulation of repetitive sequences on the X chromosome.

The wolf fish Hoplias malabaricus (Erythrinidae) presents a high karyotypic diversity, with 7 karyomorphs identified. Karyomorph A is characterized by 2n = 42 chromosomes, without morphologically differentiated sex chromosomes. Karyomorph B also has 2n = 42 chromosomes for both sexes, but differs by a distinct heteromorphic XX/XY sex chromosome system. The cytogenetic mapping of 5 classes of repetitive DNA indicated similarities between both karyomorphs and the probable derivation of the XY chromosomes from pair No. 21 of karyomorph A. These chromosomes appear to be homeologous since the distribution of (GATA)(n) sequences, 18S rDNA and 5SHindIII-DNA sites supports their potential relatedness. Our data indicate that the differentiation of the long arms of the X chromosome occurred by accumulation of heterochromatin and 18S rDNA cistrons from the ancestral homomorphic pair No. 21 present in karyomorph A. These findings are further supported by the distribution of the Cot-1 DNA fraction. In addition, while the 18S rDNA cistrons were maintained and amplified on the X chromosomes, they were lost in the Y chromosome. The X chromosome was a clearly preferred site for the accumulation of DNA repeats, representing an unusual example of an X clustering more repetitive sequences than the Y during sex chromosome differentiation in fish.

Satellite DNA and chromosomes in Neotropical fishes: methods, applications and perspectives.

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C(0)t-1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII-DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.

Initial steps in XY chromosome differentiation in Hoplias malabaricus and the origin of an X(1)X(2)Y sex chromosome system in this fish group.

The neotropical fish, Hoplias malabaricus, is well known for its population-specific karyotypic diversity and the variation of its sex chromosomes. Seven karyomorphs (A to G) have been previously described with an XY, X(1)X(2)Y and XY(1)Y(2) sex chromosome system found in karyomorphs B, D and G, respectively. We compared the chromosomal characteristics of karyomorphs C and D using C-banding, staining with CMA(3) and DAPI, and by mapping the location of 18S rDNA, 5SHindIII-DNA and (TTAGGG)(n) repeat sequences. Our results show conserved karyotypes in both karyomorphs, a nascent XX/XY sex chromosome system in karyomorph C and the origin of neo-Y chromosome in karyomorph D. The X and Y chromosomes of karyomorph C differ only slightly because of the amplification of repetitive sequences on the X chromosome, resulting in a homomorphic condition in all females and a heteromorphic condition in all males examined. Our study showed that chromosomes X and 20 of karyomorph C have similar patterns to the X(1) and X(2) chromosomes of karyomorph D, and are probably homologous. We showed that the neo-Y chromosome of karyomorph D shares similar patterns to the chromosomes Y and 20 of karyomorph C, and probably evolved through tandem fusion between Ypter/20pter. An interstitial site of the satellite 5SHindIII-DNA on the neo-Y reinforces the hypothesized dicentric nature of this chromosome. Our study shows the initial steps in XY chromosome differentiation in H. malabaricus and, in a broader context, contributes to the understanding of the evolutionary pathway leading to a multiple X(1)X(2)Y sex chromosome system in fishes.