Genomic Organization of Repetitive DNA Elements and Its Implications for the Chromosomal Evolution of Channid Fishes (Actinopterygii,Perciformes)

Channid fishes, commonly referred to as “snakeheads”, are currently very important in Asian fishery and aquaculture due to the substantial decline in natural populations because of overexploitation. A large degree of chromosomal variation has been found in this family, mainly through the use of conventional cytogenetic investigations. In this study, we analyzed the karyotype structure and the distribution of 7 repetitive DNA sequences in several Channa species from different Thailand river basins. The aim of this study was to investigate the chromosomal differentiation among species and populations to improve upon the knowledge of its biodiversity and evolutionary history. Rearrangements, such as pericentric inversions, fusions and polyploidization, appear to be important events during the karyotypic evolution of this genus, resulting in the chromosomal diversity observed among the distinct species and even among populations of the same species. In addition, such variability is also increased by the genomic dynamism of repetitive elements, particularly by the differential distribution and accumulation of rDNA sequences on chromosomes. This marked diversity is likely linked to the lifestyle of the snakehead fishes and their population fragmentation, as already identified for other fish species. The karyotypic features highlight the biodiversity of the channid fishes and justify a taxonomic revision of the genus Channa, as well as of the Channidae family as a whole, as some nominal species may actually constitute species complexes.     

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.     

Chromosomal differentiation and speciation in sister-species of Grammatidae (Perciformes) from the Western Atlantic

In the tropical Atlantic, the ichthyofauna between the coast of Brazil and the Caribbean regions, divided by the Amazon barrier, is very similar presenting several geminate species, including Gramma brasiliensis, endemic in Brazil, and its Caribbean counterpart Gramma loreto. Morphological and molecular studies have helped establish evolutionary patterns that sister-species of these two marine habitats are subjected to. However, their chromosomal characteristics are only beginning to be better characterized. Accordingly, a comparative cytogenetic analysis was carried out in G. brasiliensis and G. loreto, seeking evidence of cytotaxonomic markers implicated in the karyotypic diversification of these species and likely associated with speciation events. Heterochromatic regions and their affinity to fluorochromes GC- or AT-specific were identified, as well as the distribution of ribosomal DNA sites in chromosomes, either by silver nitrate impregnation (Ag-NORs) or dual-color FISH mapping with 18S and 5S rDNA probes. While displaying the same diploid number, 2n?=?48 chromosomes, considered basal for Perciformes, the two species differed in karyotype structure, showing karyotypic formulas and species-specific heterochromatin pattern. The cytological characters found support the differentiating status of these species, possibly achieved under the conditions of allopatry due to the Amazon/Orinoco barrier, showing chromosomal peculiarities in Grammatidae species when compared to other groups of Perciformes.     

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 markers in Parodontidae: an analysis of new and reviewed data with phylogenetic inferences

The taxonomy of the family Parodontidae is confused, with many open questions regarding the most appropriate generic groupings. Studies on the organization, structure, composition, and in situ location of chromosomal features have led to consistent advances in the understanding of genome evolution. Among the species of Parodontidae, the consistent chromosomal divergences can be helpful in taxonomic classification, such as heteromorphic chromosome sex, karyotypic formulae, and number/location of the repetitive DNAs. Molecular analysis of repetitive sequences of satellite DNA and their physical mapping in the chromosomes of different species in a single group may be used to infer evolutionary divergence and cladistic grouping. In the present study, rDNA and the satellite DNA pPh2004 were mapped by fluorescent in situ hybridization on the chromosomes of some species of Parodontidae. These results were analyzed and reviewed together with other chromosomal markers and previously published data, to formulate inferences about the diversification of the genomes and propose a clustering of some Parodontidae species. This analysis indicated that the species Apareiodon affinis, Parodon moreirai, Parodon hilarii, Parodon nasus, and Parodon pongoensis have an apomorphic state for satellite DNA pPh2004 in Parodontidae in relation to previously studied species of Apareiodon.     

Chromosomal diversity in three species of electric fish (Apteronotidae,Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.     

Stable karyotypes: a general rule for the fish of the family Prochilodontidae?

Cytogenetic studies involving the family Prochilodontidae have shown that these fish can be characterized by a constant diploid number and a conserved karyotypic macrostructure. This study focused on comparative physical chromosomal mapping using 18S and 5S rDNA to compare the species Semaprochilodus insignis and S. taeniurus. Our results indicated the conservation of large number of conventional chromosomal markers. The molecular cytogenetic analyses of the location of the 18S rDNA indicated the maintenance of a chromosome pair bearing these sites in both species analyzed, and it appears to be a conserved character among the majority of the species of this family. The stability of the number of 5S ribosomal DNA sites and their chromosomal localization as has been reported for the Prochilodontidae was not, however, confirmed for S. insignis and S. taeniurus, as these species showed multiple specific rDNA 5S sites. As such, and in spite of the fact that a number of studies indicate that the family Prochilodontidae has a conserved karyotypic structure, the utilization of molecular tools that use chromosomal segments as markers revealed that this presumed stability cannot be extended to the genome level for the species S. insignis and S. taeniurus.     

Molecular cytogenetic analysis of Haemulidae fish (Perciformes): Evidence of evolutionary conservation

Several fish species belonging to the family Haemulidae present a karyotype consisting of 48 acrocentric chromosomes (FN = 48), and apparently similar chromosomal microstructure, especially in genus Haemulon, representing a striking example of intrafamiliar chromosomal conservation. In this study, a more detailed cytogenetic analysis of the species Conodon nobilis and Pomadasys corvinaeformis was performed using C-banding, Ag-NOR, DAPI/CMA₃ staining, in situ digestion by distinct endonucleases and double-FISH to map the 18S and 5S ribosomal genes. Both species showed a similar karyotypic macrostructure with 2n = 48 acrocentric chromosomes and active ribosomal sites at interstitial position on long arms of chromosomal pair 18 and 24 in P. corvinaeformis and C. nobilis, respectively. These sites were the only CMA₃⁺/DAPI^-regions in the karyotype. Digestion with restriction enzymes revealed a low number of digestion sites in the heterochromatic segments of both species. The data indicate some degree of interspecific evolutionary diversification At the microstructural level, incorporated in a general pattern of extensive karyotypic conservatism. Thus, the interspecific reproductive isolation leading to phyletic diversification apparently occurred without the contribution of conspicuous karyotypic changes.     

Isolation and characterization of microsatellite markers for the ornamental discus fish Symphysodon discus and cross-species amplification in other Heroini cichlid species

The discus fishes (Symphysodon spp.) are economically important ornamental species. Thirteen microsatellite markers were developed from a CT(12) - and CA(12) -enriched whole genomic DNA library of Symphysodon discus. Allelic variability was tested on 44 individuals of two species (S. discus and S. aequifasciatus). Allelic richness ranged from two to 11 alleles per locus and observed heterozygosities from 0.083 to 0.998. All loci were at Hardy-Weinberg equilibrium, and no pair of loci showed linkage disequilibrium within a species. Cross-species amplification was also successfully performed in the Neotropical cichlids Uaru amphiacanthoides, Hoplarchus psittacus, Hypselecara coryphaenoides, Pterophyllum sp., Mesonauta sp. and Heros sp.     

Differentiated evolutionary pathways in Haemulidae (Perciformes): karyotype stasis versus morphological differentiation

Extensive phenotypic diversity (size, colors and shapes) among species of Haemulidae is practically dissociated from the conservative cytogenetic pattern observed in this family. Detailed analyses indicate that karyotypic stasis is maintained even under the scrutiny of different chromosome investigation methods. Chromosomal banding patterns (endophenotype) of five Atlantic species are presented here: Conodon nobilis, Pomadasys corvinaeformis, Haemulon aurolineatum, H. plumierii and H. steindachneri, obtained by incorporating the base analog 5-BrdU, C-banding and staining with base-specific fluorochromes. Despite a few chromosomal specificities, relative karyotypic conservation was confirmed, corroborating earlier studies on this family. On the other hand, phenotypic patterns (exophenotype), identified by geometric morphometrics contrast visibly with the chromosomal conservation of this group. As such, the evolutionary rates of chromosomes and body morphology demonstrate clear asynchrony. Possible causes of karyotypic stasis in Haemulidae are discussed as well as the sharing of this condition with other Perciformes.     

Chromosome mapping of repetitive sequences in four Serrasalmidae species (Characiformes)

The Serrasalmidae family is composed of a number of commercially interesting species, mainly in the Amazon region where most of these fishes occur. In the present study, we investigated the genomic organization of the 18S and 5S rDNA and telomeric sequences in mitotic chromosomes of four species from the basal clade of the Serrasalmidae family: Colossoma macropomum, Mylossoma aureum, M. duriventre, and Piaractus mesopotamicus, in order to understand the chromosomal evolution in the family. All the species studied had diploid numbers 2n = 54 and exclusively biarmed chromosomes, but variations of the karyotypic formulas were observed. C-banding resulted in similar patterns among the analyzed species, with heterochromatic blocks mainly present in centromeric regions. The 18S rDNA mapping of C. macropomum and P. mesopotamicus revealed multiple sites of this gene; 5S rDNA sites were detected in two chromosome pairs in all species, although not all of them were homeologs. Hybridization with a telomeric probe revealed signals in the terminal portions of chromosomes in all the species and an interstitial signal was observed in one pair of C. macropomum.     

Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH)

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species.     

Examination of hemiplasy, homoplasy and phylogenetic discordance in chromosomal evolution of the Bovidae

Robertsonian chromosomal fusions predominate in shaping the genomes of many species of Bovidae. These and other cytogenetic data (from 52 taxa representing 51 species and 9 tribes of Bovidae) were (i) examined for usefulness in defining phylogenetic relationships and (ii) subsequently mapped to a consensus tree based on mitochondrial and nuclear DNA gene sequences with divergence dates of the corresponding species calculated from cytochrome b sequences. This permitted persistence time estimates for the various rearrangements. The chromosomal data resulted in an unsupported higher-level topology, but with recognition of the monophyly of some genera and tribes within Bovidae. The distribution and temporal spread of character states on the species tree is suggestive of a restricted role for hemiplasy (the retention of an ancestral chromosomal polymorphism through multiple speciation events) and for introgression (resulting from secondary contact among taxa), processes that can potentially lead to phylogenetic discordance. We conclude that the most probable interpretation for these data is that genuine karyotypic homoplasy predominates, but that hemiplasy (and/or introgression) is a realistic hypothesis for the observed patterns of several shared characters in Bovidae.     

Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation

Fish of the genus Selene, known as lookdowns or moonfish, are one of the most morphologically derived groups of the family Carangidae, whose phylogenetic relationships are still largely unknown. In this study, we discuss karyoevolutionary aspects of three representatives of this genus from the Western Atlantic: Selene brownii (2n = 48; FN = 48), Selene setapinnis (2n = 46; FN = 48), and Selene vomer (2n = 48; FN = 50). Their body patterns were also investigated and compared to one another and in relation to two other species of different genera. Two mechanisms of karyotypic evolution seem to have acted in the diversification of this genus, namely pericentric inversions and centric fusions. Mapping of rDNA sequences showed that chromosome pairs bearing 5S rDNA sites are similar, whereas those bearing 18 rDNA sites are morphologically distinct while apparently also exhibiting interspecies synteny. Although the nucleolar organizer-bearing chromosomes are extremely efficient cytotaxonomic markers among Selene species, others cytogenetic patterns of these species are relatively conserved. Hybridization with telomeric probes (TTAGGG)_n did not exhibit interstitial telomeric sites (ITS), especially in S. setapinnis, where, along with a reduction in diploid number, a large metacentric pair derived from centric fusion is present. Data obtained by geometric morphometrics enable a clear morphological distinction among the three species, as well as in relation to two other species of the genus Caranx and Oligoplites. Data obtained suggest that morphologic evolution in Selene species was primarily dissociated from visible changes that occurred at the chromosomal level.     

Inferring haplotypes and parental genotypes in larger full sib-ships and other pedigrees with missing or erroneous genotype data

Background

For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped.

Results

Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA.

Conclusions

Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others.     

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.     

Chromosomal diversification of diploid number,heterochromatin and rDNAs in two species of Phanaeus beetles (Scarabaeidae,Scarabaeinae)

The genus Phanaeus is included in the tribe Phanaeini, one of the most diverse tribes within the subfamily Scarabaeinae in terms of chromosomal characteristics. However, so far the species of this genus were not studied with differential cytogenetic techniques, limiting any inference of the probable mechanisms responsible for this diversity. In this work, several techniques were applied with the aim of cytogenetically characterizing two Phanaeus species. The karyotype found for Phanaeus (Notiophanaeus) chalcomelas was 2n = 12, neo-XY, and that of P. (N.) splendidulus was 2n = 20, Xy_p, considered primitive for the family Scarabaeidae. The chromosomes of both species showed a high amount of constitutive heterochromatin (CH), with blocks rich in base pairs GC (CMA₃⁺). Moreover, in P. (N.) chalcomelas the marks revealed by C-banding and fluorochrome staining were different in size, showing CH variability. Sites of 18S ribosomal DNA (rDNA) were identified in one autosomal pair of P. (N.) chalcomelas and in five autosomal pairs of P. (N.) splendidulus. On the other hand, only one autosomal pair exhibited 5S rDNA sequences in these species. The results suggest that the karyotype differentiation of the Phanaeus species studied here involved pericentric inversions and centric fusions, as well as mechanisms related to amplification and dispersion of CH and rDNA sequences.     

Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): dispersive location, association and co-localization in the genome

Repetitive DNA sequences constitute a great portion of the genome of eukaryotes and are considered key components to comprehend evolutionary mechanisms and karyotypic differentiation. Aiming to contribute to the knowledge of chromosome structure and organization of some repetitive DNA classes in the fish genome, chromosomes of two allopatric populations of Astyanax bockmanni were analyzed using classic cytogenetics techniques and fluorescent in situ hybridization, with probes for ribosomal DNA sequences, histone DNA and transposable elements. These Astyanax populations showed the same diploid number (2n = 50), however with differences in chromosome morphology, distribution of constitutive heterochromatin, and location of 18S rDNA and retroelement Rex3 sites. In contrast, sites for 5S rDNA and H1, H3 and H4 histones showed to be co-located and highly conserved. Our results indicate that dispersion and variability of 18S rDNA and heterochromatin sites are not associated with macro rearrangements in the chromosome structure of these populations. Similarly, distinct evolutionary mechanisms would act upon histone genes and 5S rDNA, contributing to chromosomal association and co-location of these sequences. Data obtained indicate that distinct mechanisms drive the spreading of repetitive DNAs in the genome of A. bockmanni. Also, mobile elements may account for the polymorphism of the major rDNA sites and heterochromatin in this genus.     

Extensive chromosome conservatism in Atlantic butterflyfishes,genus Chaetodon Linnaeus, 1758: Implications for the high hybridization success

Chaetodontidae is a family of marine butterflyfishes phylogenetically derived within the Perciformes, whose representatives are important members of coral reef ecosystems worldwide. Biological aspects of Chaetodontidae have been intensively studied, except for chromosomal analyses. Although previous reports indicate a conserved perciform-like karyotype in butterflyfishes, it remains unclear if this pattern extends to the chromosomal microstructure. New cytogenetic data are presented for two Chaetodontidae species (Chaetodon striatus and Chaetodon ocellatus) from the Western Atlantic, including karyotyping, C-banding, Ag-NOR, CMA₃/DAPI staining, and two-color-FISH for mapping of 18S and 5S ribosomal genes. All populations of both species shared a karyotype with 2n = 48 acrocentric chromosomes, with pericentromeric C-positive heterochromatin and 5S and 18S rDNA located in the same region on the long arms of pairs 10 and 21, respectively. The cytogenetic similarities within and between both Chaetodon species reinforce their remarkable stability also in the chromosome microstructure. Therefore, speciation in this genus was not followed by significant karyotypic changes. Both ecological and chromosomal properties, combined with recent diversification, might be responsible for the apparent karyotype stasis and high hybridization levels found in marine butterflyfishes.