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.

Comparative Cytogenetics of Four Sea Turtle Species (Cheloniidae): G-Banding Pattern and in situ Localization of Repetitive DNA Units.

Sea turtles are considered flagship species for marine biodiversity conservation and are considered to be at varying risk of extinction globally. Cases of hybridism have been reported in sea turtles, but chromosomal analyses are limited to classical karyotype descriptions and a few molecular cytogenetic studies. In order to compare karyotypes and understand evolutive mechanisms related to chromosome dif-ferentiation in this group, Chelonia mydas, Caretta caretta, Eretmochelys imbricata, and Lepidochelys olivacea were cytogenetically characterized in the present study. When the obtained cytogenetic data were compared with the putative ancestral Cryptodira karyotype, the studied species showed the same diploid number (2n) of 56 chromosomes, with some variations in chromosomal morphology (karyotypic formula) and minor changes in longitudinal band locations. In situ localization using a 18S ribosomal DNA probe indicated a homeologous microchromosome pair bearing a 45S ribosomal DNA locus and size heteromorphism in all 4 species. Interstitial telomeric sites were identified in a microchromosome pair in C. mydas and C. caretta. The data showed that interspecific variations occurred in chromosomal sets among the Cheloniidae species, in addition to other Cryptodira karyotypes. These variations generated lineage-specific karyotypic diversification in sea turtles, which will have considerable implications for hybrid recognition and for the study, the biology, ecology, and evolutionary history of regional and global populations. Furthermore, we demonstrated that some chromosome rearrangements occurred in sea turtle species, which is in conflict with the hypothesis of conserved karyotypes in this group.

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.

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.

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.

Structurally Complex Organization of Repetitive DNAs in the Genome of Cobia (Rachycentron canadum).

Repetitive DNAs comprise the largest fraction of the eukaryotic genome. They include microsatellites or simple sequence repeats (SSRs), which play an important role in the chromosome differentiation among fishes. Rachycentron canadum is the only representative of the family Rachycentridae. This species has been focused on several multidisciplinary studies in view of its important potential for marine fish farming. In the present study, distinct classes of repetitive DNAs, with emphasis on SSRs, were mapped in the chromosomes of this species to improve the knowledge of its genome organization. Microsatellites exhibited a diversified distribution, both dispersed in euchromatin and clustered in the heterochromatin. The multilocus location of SSRs strengthened the heterochromatin heterogeneity in this species, as suggested by some previous studies. The colocalization of SSRs with retrotransposons and transposons pointed to a close evolutionary relationship between these repetitive sequences. A number of heterochromatic regions highlighted a greater complex organization than previously supposed, harboring a diversity of repetitive elements. In this sense, there was also evidence of colocalization of active genetic regions and different classes of repetitive DNAs in a common heterochromatic region, which offers a potential opportunity for further researches regarding the interaction of these distinct fractions in fish genomes.

Unusual dispersion of histone repeats on the whole chromosomal complement and their colocalization with ribosomal genes in Rachycentron canadum (Rachycentridae, Perciformes).

Rachycentron canadum, the only representative of the family Rachycentridae, has been the focus of biotechnological interest due to its significant potential in marine fish farming. The chromosome set of this species has been widely investigated with respect to the location of genes and multigene families. A FISH analysis was performed using 4 multigene families as probes, represented by 5S and 18S ribosomal genes and histones H2B-H2A and H3. Earlier data suggested that differential replication of heterochromatin could be partially associated with functional genes. Indeed, our results showed that the DNA contained in heterochromatic regions of R. canadum contains 5S and 18S ribosomal genes as well as the gene sequences of histones H2B-H2A and H3, which were colocalized. The distribution of H3 sequences in all heterochromatic regions, except in 13q, could indicate an important evolutionary role for this class of repetitive sequences. Besides, the presence of chromosome regions bearing multifunctional repetitive sequences formed by H2B-H2A/H3/18S rDNA and H2B-H2A/H3/5S rDNA clusters was demonstrated for the first time in fishes. The implications of differential histone gene extension and its functionality in the karyotype of R. canadum remain unknown.