Karyotypic changes and diversification time in Epinephelidae groupers (Perciformes). Implications on reproductive isolation.

Groupers (Epinephelidae and Serranidae) have attracted special attention to fish farming, and their species offer good opportunities for successful hybridizations. Cytogenetic data allow a better understanding of the role of karyotypic diversification in the acquisition of post-zygotic reproductive isolation (RI). Thus, chromosomal analyses were performed on E. striatus (Caribbean Sea), E. coioides and E. tauvina (Indo-Pacific Region), using standard procedures and mapping of six repetitive DNA classes by the in situ hybridization. The three species have 2n=48 chromosomes. The karyotypes of E. coioides and E. striatus are composed only of acrocentric chromosomes (FN=48), while E. tauvina has 8 submetacentric chromosomes (FN=56). Heterochromatin has a preferential centromeric distribution, and the microsatellite repeats are dispersed throughout the chromosomes of all species. The 18S and 5S rDNA sites are unique but show a colocalization arrangement in E. tauvina and E. striatus. The chromosomal organization suggests that the three species still maintain a significant amount of syntenic regions. The range of the karyotype divergence and the RI levels showed low, but goes turn proportionally greater in relation to the divergence time between the parental species. The slow acquisition of postzygotic RI is consistent with the high karyotype homogeneity presented by Epinephelidae family.

Cytogenetics Meets Genomics: Cytotaxonomy and Genomic Relationships among Color Variants of the Asian Arowana Scleropages formosus.

Scleropages formosus (Osteoglossiformes, Teleostei) represents one of the most valued ornamental fishes, yet it is critically endangered due to overexploitation and habitat destruction. This species encompasses three major color groups that naturally occur in allopatric populations, but the evolutionary and taxonomic relationships of S. formosus color varieties remain uncertain. Here, we utilized a range of molecular cytogenetic techniques to characterize the karyotypes of five S. formosus color phenotypes, which correspond to naturally occurring variants: the red ones (Super Red); the golden ones (Golden Crossback and Highback Golden); the green ones (Asian Green and Yellow Tail Silver). Additionally, we describe the satellitome of S. formosus (Highback Golden) by applying a high-throughput sequencing technology. All color phenotypes possessed the same karyotype structure 2n = 50 (8m/sm + 42st/a) and distribution of SatDNAs, but different chromosomal locations of rDNAs, which were involved in a chromosome size polymorphism. Our results show indications of population genetic structure and microstructure differences in karyotypes of the color phenotypes. However, the findings do not clearly back up the hypothesis that there are discrete lineages or evolutionary units among the color phenotypes of S. formosus, but another case of interspecific chromosome stasis cannot be excluded.

The Genetic Differentiation of Pyrrhulina (Teleostei, Characiformes) Species is Likely Influenced by Both Geographical Distribution and Chromosomal Rearrangements.

Allopatry is generally considered to be one of the main contributors to the remarkable Neotropical biodiversity. However, the role of chromosomal rearrangements including neo-sex chromosomes for genetic diversity is still poorly investigated and understood. Here, we assess the genetic divergence in five Pyrrhulina species using population genomics and combined the results with previously obtained cytogenetic data, highlighting that molecular genetic diversity is consistent with their chromosomal features. The results of a principal coordinate analysis (PCoA) indicated a clear difference among all species while showing a closer relationship of the ones located in the same geographical region. This was also observed in genetic structure analyses that only grouped P. australis and P. marilynae, which were also recovered as sister species in a species tree analysis. We observed a contradictory result for the relationships among the three species from the Amazon basin, as the phylogenetic tree suggested P. obermulleri and P. semifasciata as sister species, while the PCoA showed a high genetic difference between P. semifasciata and all other species. These results suggest a potential role of sex-related chromosomal rearrangements as reproductive barriers between these species.

Integrating Cytogenetics and Population Genomics: Allopatry and Neo-Sex Chromosomes May Have Shaped the Genetic Divergence in the Erythrinus erythrinus Species Complex (Teleostei, Characiformes).

Diversity found in Neotropical freshwater fish is remarkable. It can even hinder a proper delimitation of many species, with the wolf fish Erythrinus erythrinus (Teleostei, Characiformes) being a notable example. This nominal species shows remarkable intra-specific variation, with extensive karyotype diversity found among populations in terms of different diploid chromosome numbers (2n), karyotype compositions and sex chromosome systems. Here, we analyzed three distinct populations (one of them cytogenetically investigated for the first time) that differed in terms of their chromosomal features (termed karyomorphs) and by the presence or absence of heteromorphic sex chromosomes. We combined cytogenetics with genomic approaches to investigate how the evolution of multiple sex chromosomes together with allopatry is linked to genetic diversity and speciation. The results indicated the presence of high genetic differentiation among populations both from cytogenetic and genomic aspects, with long-distance allopatry potentially being the main agent of genetic divergence. One population showed a neo-X1X2Y sexual chromosome system and we hypothesize that this system is associated with enhanced inter-population genetic differentiation which could have potentially accelerated speciation compared to the effect of allopatry alone.

Adding New Pieces to the Puzzle of Karyotype Evolution in Harttia (Siluriformes, Loricariidae): Investigation of Amazonian Species.

A remarkable morphological diversity and karyotype variability can be observed in the Neotropical armored catfish genus Harttia. These fishes offer a useful model to explore both the evolution of karyotypes and sex chromosomes, since many species possess male-heterogametic sex chromosome systems and a high rate of karyotype repatterning. Based on the karyotype organization, the chromosomal distribution of several repetitive DNA classes, and the rough estimates of genomic divergences at the intraspecific and interspecific levels via Comparative Genomic Hybridization, we identified shared diploid chromosome numbers (2n = 54) but different karyotype compositions in H. dissidens (20m + 26sm + 8a) and Harttia sp. 3 (16m + 18sm + 14st + 6a), and different 2n in H. guianensis (2n = 58; 20m + 26sm + 2st + 10a). All species further displayed similar patterns of chromosomal distribution concerning constitutive heterochromatin, 18S ribosomal DNA (rDNA) sites, and most of the surveyed microsatellite motifs. Furthermore, differences in the distribution of 5S rDNA sites and a subset of microsatellite sequences were identified. Heteromorphic sex chromosomes were lacking in H. dissidens and H. guianensis at the scale of our analysis. However, one single chromosome pair in Harttia sp. 3 males presented a remarkable accumulation of male genome-derived probe after CGH, pointing to a tentative region of early sex chromosome differentiation. Thus, our data support already previously outlined evidence that Harttia is a vital model for the investigation of teleost karyotype and sex chromosome dynamics.

Revisiting the Karyotypes of Alligators and Caimans (Crocodylia, Alligatoridae) after a Half-Century Delay: Bridging the Gap in the Chromosomal Evolution of Reptiles.

Although crocodilians have attracted enormous attention in other research fields, from the cytogenetic point of view, this group remains understudied. Here, we analyzed the karyotypes of eight species formally described from the Alligatoridae family using differential staining, fluorescence in situ hybridization with rDNA and repetitive motifs as a probe, whole chromosome painting (WCP), and comparative genome hybridization. All Caimaninae species have a diploid chromosome number (2n) 42 and karyotypes dominated by acrocentric chromosomes, in contrast to both species of Alligatorinae, which have 2n = 32 and karyotypes that are predominantly metacentric, suggesting fusion/fission rearrangements. Our WCP results supported this scenario by revealing the homeology of the largest metacentric pair present in both Alligator spp. with two smaller pairs of acrocentrics in Caimaninae species. The clusters of 18S rDNA were found on one chromosome pair in all species, except for Paleosuchus spp., which possessed three chromosome pairs bearing these sites. Similarly, comparative genomic hybridization demonstrated an advanced stage of sequence divergence among the caiman genomes, with Paleosuchus standing out as the most divergent. Thus, although Alligatoridae exhibited rather low species diversity and some level of karyotype stasis, their genomic content indicates that they are not as conserved as previously thought. These new data deepen the discussion of cytotaxonomy in this family.

Chromosomal Analysis of Ctenolucius hujeta Valenciennes, 1850 (Characiformes): A New Piece in the Chromosomal Evolution of the Ctenoluciidae.

Ctenoluciidae (Characiformes), a family of freshwater fishes, comprises 2 genera, Ctenolucius and Boulengerella, with 7 recognized species. Up to now, only species of the genus Boulengerella have been subjected to cytogenetic studies. Here, we investigated the karyotype and other cytogenetic features of pike characin, Ctenolucius hujeta, using conventional (Giemsa staining, C-banding, Ag-NOR staining) and molecular (rDNA, telomeric sequences, and fiber-FISH mapping) procedures. This species has a diploid chromosome number of 2n = 36, and a karyotype composed of 12m + 20sm + 4a and FN = 68, similar to that found in Boulengerella species. However, differences regarding the number and distribution of several chromosomal markers support a distinct generic status. Colocalization of the 18S and 5S rDNA genes is an exclusive characteristic of the C. hujeta genome, with an interspersed distribution in the chromosomal fiber, an unusual phenomenon among eukaryotes. Additionally, our results support the view that Ctenoluciidae and Lebiasinidae families are closely related.

High Genetic Diversity despite Conserved Karyotype Organization in the Giant Trahiras from Genus Hoplias (Characiformes, Erythrinidae).

In the fish genus Hoplias, two major general groups can be found, one of which is formed by the "common trahiras" (Hoplias malabaricus group) and the other by the "giant trahiras" (Hoplias lacerdae group, in addition to Hoplias aimara), which usually comprises specimens of larger body size. Previous investigations from the giant trahiras group recovered 2n = 50 meta/submetacentric chromosomes and no sex chromosome differentiation, indicating a probable conservative pattern for their karyotype organization. Here, we conducted comparative cytogenetic studies in six giant trahiras species, two of them for the first time. We employed standard and advanced molecular cytogenetics procedures, including comparative genomic hybridization (CGH), as well as genomic assessments of diversity levels and phylogenetic relationships among them. The results strongly suggest that the giant trahiras have a particular and differentiated evolutionary pathway inside the Hoplias genus. While these species share the same 2n and karyotypes, their congeneric species of the H. malabaricus group show a notable chromosomal diversity in number, morphology, and sex chromosome systems. However, at the same time, significant changes were characterized at their inner chromosomal level, as well as in their genetic diversity, highlighting their current relationships resulting from different evolutionary histories.

Chromosomes of Asian cyprinid fishes: Variable karyotype patterns and evolutionary trends in the genus Osteochilus (Cyprinidae, Labeoninae, "Osteochilini").

The Cyprinidae family is a highly diversified but demonstrably monophyletic lineage of cypriniform fishes. Among them, the genus Osteochilus contains 35 recognized valid species distributed from India, throughout Myanmar, Laos, Thailand, Malaysia, Indonesian archipelago to southern China. In this study, karyotypes and other chromosomal characteristics of five Osteochilus species occurring in Thailand, namely O. lini, O. melanopleura, O. microcephalus, O. vittatus and O. waandersii were examined using conventional and molecular cytogenetic protocols. Our results showed they possessed diploid chromosome number (2n) invariably 2n = 50, but the ratio of uni- and bi-armed chromosomes was highly variable among their karyotypes, indicating extensive chromosomal rearrangements. Only one chromosome pair bearing 5S rDNA sites occurred in most species, except O. melanopleura, where two sites were detected. In contrast, only one chromosomal pair bearing 18S rDNA sites were observed among their karyotypes, but in different positions. These cytogenetic patterns indicated that the cytogenomic divergence patterns of these Osteochilus species were largely corresponding to the inferred phylogenetic tree. Similarly, different patterns of the distributions of rDNAs and microsatellites across genomes of examined species as well as their different karyotype structures indicated significant evolutionary differentiation of Osteochilus genomes.

Chromosomal Evolution in Aspredinidae (Teleostei, Siluriformes): Insights on Intra- and Interspecific Relationships with Related Groups.

The family Aspredinidae comprises a clade of complex systematic relationships, both from molecular and morphological approaches. In this study, conventional and molecular cytogenetic studies coupled with nucleotide sequencing were performed in 6 Aspredininae species (Amaralia hypsiura, Bunocephalus cf. aloikae, Bunocephalus amaurus, Bunocephalus aff. coracoideus, Bunocephalus verrucosus, and Platystacus cotylephorus) from different locations of the Amazon hydrographic basin. Our results showed highly divergent diploid numbers (2n) among the species, ranging from 49 to 74, including the occurrence of an XX/X0 sex chromosome system. A neighbor-joining phylogram based on the cytochrome c oxidase I (COI) showed that Bunocephalus coracoideus is not a monophyletic clade, but closely related to B. verrucosus. The karyotypic data associated with COI suggest an ancestral karyotype for Aspredinidae with a reduced 2n, composed of bi-armed chromosomes and a trend toward chromosomal fissions resulting in higher diploid number karyotypes, mainly composed of acrocentric chromosomes. Evolutionary relationships were discussed under a phylogenetic context with related species from different Siluriformes families. The karyotype features and chromosomal diversity of Aspredinidae show an amazing differentiation, making this family a remarkable model for investigating the evolutionary dynamics in siluriforms as well as in fish as a whole.

Multiple Sex Chromosomes and Evolutionary Relationships in Amazonian Catfishes: The Outstanding Model of the Genus Harttia (Siluriformes: Loricariidae).

The armored Harttia catfishes present great species diversity and remarkable cytogenetic variation, including different sex chromosome systems. Here we analyzed three new species, H. duriventris, H. villasboas and H. rondoni, using both conventional and molecular cytogenetic techniques (Giemsa-staining and C-banding), including the mapping of repetitive DNAs using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) experiments. Both H. duriventris and H. villasboas have 2n = ♀56/♂55 chromosomes, and an X1X1X2X2 /X1X2Y sex chromosome system, while a proto or neo-XY system is proposed for H. rondoni (2n = 54♀♂). Single motifs of 5S and 18S rDNA occur in all three species, with the latter being also mapped in the sex chromosomes. The results confirm the general evolutionary trend that has been noticed for the genus: an extensive variation on their chromosome number, single sites of rDNA sequences and the occurrence of multiple sex chromosomes. Comparative genomic analyses with another congeneric species, H. punctata, reveal that the X1X2Y sex chromosomes of these species share the genomic contents, indicating a probable common origin. The remarkable karyotypic variation, including sex chromosomes systems, makes Harttia a suitable model for evolutionary studies focusing on karyotype differentiation and sex chromosome evolution among lower vertebrates.

An Insight into the Chromosomal Evolution of Lebiasinidae (Teleostei, Characiformes).

Lebiasinidae fishes have been historically neglected by cytogenetical studies. Here we present a genomic comparison in eleven Lebiasinidae species, in addition to a review of the ribosomal DNA sequences distribution in this family. With that, we develop ten sets of experiments in order to hybridize the genomic DNA of representative species from the genus Copeina, Copella, Nannostomus, and Pyrrhulina in metaphase plates of Lebiasina melanoguttata. Two major pathways on the chromosomal evolution of these species can be recognized: (i) conservation of 2n = 36 bi-armed chromosomes in Lebiasininae, as a basal condition, and (ii) high numeric and structural chromosomal rearrangements in Pyrrhulininae, with a notable tendency towards acrocentrization. The ribosomal DNA (rDNA) distribution also revealed a marked differentiation during the chromosomal evolution of Lebiasinidae, since both single and multiple sites, in addition to a wide range of chromosomal locations can be found. With some few exceptions, the terminal position of 18S rDNA appears as a common feature in Lebiasinidae-analyzed species. Altogether with Ctenoluciidae, this pattern can be considered a symplesiomorphism for both families. In addition to the specific repetitive DNA content that characterizes the genome of each particular species, Lebiasina also keeps inter-specific repetitive sequences, thus reinforcing its proposed basal condition in Lebiasinidae.

Molecular Cytogenetic Analysis in Freshwater Prawns of the Genus Macrobrachium (Crustacea: Decapoda: Palaemonidae).

Freshwater prawns of the genus Macrobrachium are one of the important components of circumtropical marine, estuarine, and freshwater environments. They have been extensively exploited for human consumption for many years. More than 250 species reflect the evolutionary success of this highly diversified group, with a complex and challenging taxonomy due to morphological variations and vast geographical distribution. Although genetic approaches have been used to clarify phylogenetic and taxonomic aspects of Macrobrachium species, cytogenetic information is still very scarce and mostly focused on chromosome number and morphology. Here, we present chromosome data for three species from the Neotropical region, M. carcinus, M. acanthurus, and M. amazonicum, and one species from the Oriental region, M. rosenbergii. Using conventional cytogenetic approaches and chromosome mapping of repetitive DNAs by fluorescence in situ hybridization (FISH), we identified numerical diversification of the diploid set, within and between both zoogeographic regions. These included M. acanthurus and M. amazonicum sharing diploid chromosomes of 98, while M. carcinus has 94, and M. rosenbergii has 118 chromosomes. Argentophilic sites are also variable in number, but they occur in a much higher number than 18S rDNA, representing two to 10 sites within the study species. Microsatellites repeat motifs are also abundant in the chromosomes, with a co-localization and uniform distribution along the chromosome arms, but completely absent in the AT-rich centromeric regions. As a whole, our study suggests that the 2n divergence was followed by a considerable rDNA diversification. The abundance of the exceptional amount of microsatellite sequences in the chromosomes also suggests that they are essential components of the Macrobrachium genome and, therefore, maintained as a shared feature by the species, the reason for which is yet unknown.

Comparative cytogenetic survey of the giant bonytongue Arapaima fish (Osteoglossiformes: Arapaimidae), across different Amazonian and Tocantins/Araguaia River basins

The South American giant fishes of the genus Arapaima, commonly known as pirarucu, are one of the most iconic among Osteoglossiformes. Previously cytogenetic studies have identified their karyotype characteristics; however, characterization of cytotaxonomic differentiation across their distribution range remains unknown. In this study, we compared chromosomal characteristics using conventional and molecular cytogenetic protocols in pirarucu populations from the Amazon and Tocantins-Araguaia river basins to verify if there is differentiation among representatives of this genus. Our data revealed that individuals from all populations present the same diploid chromosome number 2n=56 and karyotype composed of 14 pairs of meta- to submetacentric and 14 pairs of subtelo- to acrocentric chromosomes. The minor and major rDNA sites are in separate chromosomal pairs, in which major rDNA sites corresponds to large heterochromatic blocks. Comparative genomic hybridizations (CGH) showed that the genome of these populations shared a great portion of repetitive elements, due to a lack of substantial specific signals. Our comparative cytogenetic data analysis of pirarucu suggested that, although significant genetic differences occur among populations, their general karyotype patterns remain conserved.(AU)

Os peixes gigantes da América do Sul do gêneroArapaima, comumente conhecidos como pirarucus, são um dos mais icônicos de Osteoglossiformes. Estudos citogenéticos prévios identificaram suas características cariotípicas, entretanto a caracterização da diferenciação citotaxonômica através de suas distribuições geográficas ainda são desconhecidas. Nesse estudo, nós comparamos características cromossômicas utilizando técnicas de citogenética clássica e molecular em populações das bacias dos rios Amazonas e Tocantins-Araguaia, a fim de verificar se há alguma diferenciação entre representantes desse gênero. Nossos dados revelaram que indivíduos de todas as populações apresentam número diploide de 2n=56 cromossomos e que seus cariótipos são compostos de 14 pares de cromossomos meta- e submetacêntricos e 14 pares de subtelo- e acrocêntricos. Os sítios maiores e menores de rDNA estão localizados em pares cromossômicos separados, onde os sítios maiores de rDNA correspondem a grandes blocos heterocromáticos. Hibridizações genômicas comparativas (CGH) mostraram que o genoma dos espécimes dessas populações é amplamente compartilhado, devido à falta de sinais substanciais específicos. Nossos dados de citogenética comparativa do pirarucu sugerem que embora diferenças genéticas significativas ocorram entre populações, os padrões cariotípicos gerais se mantêm conservados.(AU)

Paracentric Inversions Differentiate the Conservative Karyotypes in Two Centropomus Species (Teleostei: Centropomidae).

Centropomus is the sole genus of the Centropomidae family (Teleostei), comprising 12 species widely distributed throughout the Western Atlantic and Eastern Pacific, with 6 of them occurring in the Western Atlantic in extensive sympatry. Their life history and phylogenetic relationships are well characterized; however, aspects of chromosomal evolution are still unknown. Here, cytogenetic analyses of 2 Centropomus species of great economic value (C. undecimalis and C. mexicanus) were performed using conventional (Giemsa, Ag-NOR, and fluorochrome staining, C- and replication banding) and molecular (chromosomal mapping of 18S and 5S rDNA, H2A-H2B and H3 hisDNA, and (TTAGGG)n repeats) approaches. The karyotypes of both species were composed of 48 solely acrocentric chromosomes (2n = 48; FN = 48), but the single ribosomal site was located in varying positions in the long arms of the second largest chromosome pair. Replication bands were generally similar, although conspicuous differences were observed in some chromosome regions. In both species, the histone H3 genes were located on 3 apparently homeologous chromosome pairs, but the exact position of these clusters differed slightly. Interspecific hisDNA and rDNA site displacements can indicate the occurrence of multiple paracentric inversions during the evolutionary diversification of the Centropomus genomes. Although the karyotypes remained similar in both species, our data demonstrate an unsuspected microstructural reorganization between them, driven most likely by a series of paracentric inversions.

Karyotype diversity and evolutionary trends in the Asian swamp eel Monopterus albus (Synbranchiformes, Synbranchidae): a case of chromosomal speciation?

BACKGROUND: Synbranchidae or swamp eels are fishes belonging to the order Synbranchiformes that occur in both freshwater and occasionally in brackish. They are worldwide distributed in tropical and subtropical rivers of four different continents. A large degree of chromosomal variation has been found in this family, mainly through the use of conventional cytogenetic investigations. Inside this group, a still almost unexplored species under the cytogenetic point of view is the Asian swamp eel Monopterus albus, a widely distributed species throughout Asia. Here, we tested the hypothesis of chromosomal speciation, where a case of sympatric speciation may occur as the primary consequence of chromosomal rearrangements. We performed a comparative chromosomal analysis of M. albus from 22 different localities in Thailand, using distinct staining methods (C-banding, Ag-NO3, and Chromomycin A3), and FISH with repetitive DNA probes (5S rDNA, 18S rDNA, Rex1 element and microsatellite repeats). RESULTS: This approach evidenced two contrasting karyotypes (named karyomorphs A and B) that varied concerning their 2n and repetitive DNAs distribution, where chromosomal fusions and pericentric inversions were involved in such differentiation. While the karyomorph A has 2n = 24 chromosomes, the karyomorph B has only 2n = 18, both with NF = 24. In addition, karyomorph A contains only acrocentric chromosomes, while karyomorph B contains three unique metacentric pairs. These features highlight that M. albus has already gone through a significant genomic divergence, and may include at least two cryptic species. CONCLUSIONS: This marked chromosomal differentiation, likely linked to the lifestyle of these fishes, point to the occurrence of a chromosomal speciation scenario, in which fusions and inversions had a prominent role. This highlights the biodiversity of M. albus and justifies its taxonomic revision, since this nominal species may constitute a species complex.

Differential hypomethylation of the repetitive Tol2/Alu-rich sequences in the genome of Bodianus species (Labriformes, Labridae).

Representatives of the order Labriformes show karyotypes of extreme conservatism together with others with high chromosomal diversification. However, the cytological characterization of epigenetic modifications remains unknown for the majority of the species. In the family Labridae, the most abundant fishes on tropical reefs, the genomes of the genus Bodianus Bloch, 1790 have been characterized by the occurrence of a peculiar chromosomal region, here denominated BOD. This region is exceptionally decondensed, heterochromatic, argentophilic, GC-neutral and, in contrast to classical secondary constrictions, shows no signals of hybridization with 18S rDNA probes. In order to characterize the BOD region, the methylation pattern, the distribution of Alu and Tol2 retrotransposons and of 18S and 5S rDNA sites, respectively, were analyzed by Fluorescence In Situ Hybridization (FISH) on metaphase chromosomes of two Bodianus species, B. insularis Gomon & Lubbock, 1980 and B. pulchellus (Poey, 1860). Immunolocalization of the 5-methylcytosine revealed hypermethylated chromosomal regions, dispersed along the entire length of the chromosomes of both species, while the BOD regions exhibited a hypomethylated pattern. Hypomethylation of the BOD region is associated with the precise co-location of Tol2 and Alu elements, suggesting their active participation in the regulatory epigenetic process. This evidence underscores a probable differential methylation action during the cell cycle, as well as the role of Tol2/Alu elements in functional processes of fish genomes.

Sex Chromosome Evolution and Genomic Divergence in the Fish Hoplias malabaricus (Characiformes, Erythrinidae).

The Erythrinidae family (Teleostei: Characiformes) is a small Neotropical fish group with a wide distribution throughout South America, where Hoplias malabaricus corresponds to the most widespread and cytogenetically studied taxon. This species possesses significant genetic variation, as well as huge karyotype diversity among populations, as reflected by its seven major karyotype forms (i.e., karyomorphs A-G) identified up to now. Although morphological differences in their bodies are not outstanding, H. malabaricus karyomorphs are easily identified by differences in 2n, morphology and size of chromosomes, as well as by distinct evolutionary steps of sex chromosomes development. Here, we performed comparative genomic hybridization (CGH) to analyse both the intra- and inter-genomic status in terms of repetitive DNA divergence among all but one (E) H. malabaricus karyomorphs. Our results indicated that they have close relationships, but with evolutionary divergences among their genomes, yielding a range of non-overlapping karyomorph-specific signals. Besides, male-specific regions were uncovered on the sex chromosomes, confirming their differential evolutionary trajectories. In conclusion, the hypothesis that H. malabaricus karyomorphs are result of speciation events was strengthened.

Genomic Organization of Repetitive DNAs and Differentiation of an XX/XY Sex Chromosome System in the Amazonian Puffer Fish, Colomesus asellus (Tetraodontiformes).

The genus Colomesus is the sole representative of the family Tetraodontidae in the Amazon region. Here, Colomesus asellus was analyzed using conventional and molecular cytogenetic protocols. Its diploid chromosome number is 2n = 46 with 12 meta-, 10 submeta-, 16 subtelo-, and 8 acrocentric chromosomes and a fundamental number of FN = 84. An XX/XY sex chromosome system was identified. Mapping of 18S rDNA correlated with the nucleolus organizer regions (Ag-NORs) in the short arms of the 2 X chromosomes in females and in the Y chromosome in males. C-banding revealed heterochromatin in the centromeric regions of all chromosomes, except for pair 3. Prominent sex chromosome-specific heterochromatin amplification was observed, covering the short arms of the Y chromosome almost entirely. FISH with telomeric and tropomyosin (tpm1) sequences, respectively, revealed terminal signals in all chromosomes. The analysis of extended DNA fibers confirmed the colocalization and the interspersed pattern of the telomeric and tpm1 sequences. Thus, this study highlights the remarkable evolutionary dynamism presented by the Amazonian puffer fish regarding the differentiation of a heteromorphic XY sex chromosome system and a particular sex-specific amplification of rDNA sites. This is the first record of such an association in the Tetraodontidae family.

The Bunocephalus coracoideus Species Complex (Siluriformes, Aspredinidae). Signs of a Speciation Process through Chromosomal, Genetic and Ecological Diversity.

Bunocephalus is the most species-rich Aspredinidae genus, corresponding to a monophyletic clade with 13 valid species. However, many species have their classification put in question. Here, we analyzed individuals from four Amazonian populations of Bunocephalus coracoideus by cytogenetic and molecular procedures. The geographic distribution, genetic distances and karyotype data indicate that each population represents an Evolutionary Significant Unit (ESU). Cytogenetic markers showed distinct 2n and karyotype formulas, as well as different numbers and locations of the rDNA sites among ESUs. One of such populations (ESU-D) highlighted an extensive polymorphic condition, with several cytotypes probably due to chromosomal rearrangements and meiotic non-disjunctions. This resulted in several aneuploid karyotypes, which was also supported by the mapping of telomeric sequences. Phylograms based on Maximum Likelihood (ML) and Neighbor Joining (NJ) analyses grouped each ESU on particular highly supported clades, with the estimation of evolutionary divergence indicating values being higher than 3.8-12.3% among them. Our study reveals a huge degree of chromosomal and genetic diversity in B. coracoideus and highly points to the existence of four ESUs in allopatric and sympatric speciation processes. In fact, the high divergences found among the ESUs allowed us to delimitate lineages with taxonomic uncertainties in this nominal species.