Débora Diniz

The key role of repeated DNAs in sex chromosome evolution in two fish species with ZW sex chromosome system

Despite substantial progress, there are still several gaps in our knowledge about the process of sex chromosome differentiation. The degeneration of sex-specific chromosome in some species is well documented, but it is not clear if all species follow the same evolutionary pathway. The accumulation of repetitive DNA sequences, however, is a common feature. To better understand this involvement, fish species emerge as excellent models because they exhibit a wide variety of sex chromosome and sex determining systems. Besides, they have much younger sex chromosomes compared to higher vertebrates, making it possible to follow early steps of differentiation. Here, we analyzed the arrangement of 9 repetitive DNA sequences in the W chromosomes of 2 fish species, namely Leporinus reinhardti and Triportheus auritus, which present well-differentiated ZZ/ZW sex system, but differ in respect to the size of the sex-specific chromosome. Both W chromosomes are almost fully heterochromatic, with accumulation of repeated DNAs in their heterochromatic regions. We found that microsatellites have strongly accumulated on the large W chromosome of L. reinhardti but not on the reduced-size W chromosome of T. auritus and are therefore important players of the W chromosome expansion. The present data highlight that the evolution of the sex chromosomes can diverge even in the same type of sex system, with and without the degeneration of the specific-sex chromosome, being more dynamic than traditionally appreciated.

Microdissection and whole chromosome painting. Improving sex chromosome analysis in Triportheus (Teleostei, Characiformes)

Triportheus fish species present 2n = 52 chromosomes. The karyotypes show similar macrostructure and a ZZ/ZW sex chromosome system, which probably represents synapomorphy for the genus. A probe of the Z chromosome was obtained from T. nematurus through microdissection, followed by unspecific amplification via DOP-PCR. This probe was used for WCP (whole chromosome painting) through fluorescent in situ hybridization (FISH) in several other Triportheus species, to analyze the differentiation of the ZZ/ZW system. The homology between this probe and chromosomes of species from other genera, putatively related to Triportheus, was also examined to search for evidence about evolution of their sex chromosomes. Complete homology was found among the Z chromosomes of all Triportheus species, while only small positive signals were found on the W chromosomes. Hybridization signals were absent in species from other genera. The present results reinforce both the conservative nature of Z chromosomes and the hypothesis that the ZZ/ZW sex chromosome system is a synapomorphic feature of Triportheus. On the other hand, besides reduction of size, W chromosomes have undergone accentuated composition changes in relation to Z chromosomes, since only a small region, usually located in the short arm, kept homology with the Z chromosomes.

Karyotypic studies on Hoplerythrinus unitaeniatus (Pisces, Erythrinidae) populations. A biodiversity analysis

Abstract Three populations of Hoplerythrinus unitaeniatus from two distinct Brazilian hydrographic basins were cytogenetically analyzed. Karyotypical variations were detected among them. The populations from Upper Paraná basin presented 2n=48, FN=96 and karyotype formula 44M/SM+4ST whereas that from São Francisco basin presented 2n=52, FN=100 and 44M/SM+4ST+4A. Constitutive heterochromatin showed a same pattern for all populations. Multiple Ag-NORs were found, ranging from four to six, and located on interstitial regions of the chromosomes, as observed in 2n=48 populations, and also in telomeric regions, as in 2n=52 specimens. Fluorescence in situ hybridizations (FISH) with 18S rDNA probe were in agreement with Ag-NORs, further revealing a size heteromorphism between NOR sites as well as additional regions than those detected by the AgNO3 staining. 5S rRNA genes were present on pericentromeric regions of M/SM chromosomes in the three populations, displaying numerical variations specific to each locality. The present study adds new data about the karyotypic diversity of H. unitaeniatus and corroborates the divergent chromosomal evolution observed within the family Erythrinidae.

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

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

Comparative chromosomal mapping in Triportheus fish species. Analysis of synteny between ribosomal genes

All Triportheus species show the conserved diploid number of 52 chromosomes and a ZZ/ZW sex chromosome system. Previous studies conducted on Triportheus nematurus reported a syntenical location of 18S and 5S sites on this species, in addition to some indications that this condition could be shared by other Triportheus species, possibly constituting a synapomorphy for this genus. In the present study, fluorescence in situ hybridization (FISH) experiments were performed in seven Triportheus species in view of a comparative analysis of the distribution of the 18S and 5S ribosomal DNAs on the chromosomes. The double-FISH experiments have showed that the synteny of the 18S and 5S rDNA genes is not a synapomorphy for the genus, since it is not present in all the species investigated, although it is present in most of them. The findings suggest that the syntenical location of the ribosomal genes is an ancestral trait in Triportheus, which was changed during the course of evolution of this group.

A Unique Sex Chromosome System in the Knifefish Gymnotus bahianus with Inferences About Chromosomal Evolution of Gymnotidae

Cytogenetic studies in Neotropical electric knifefish of genus Gymnotus have shown a remarkable interspecific variability, including distinct sex chromosome systems. In this study, we present the first chromosomal data in Gymnotus bahianus from Contas River basin, northeastern South America. Based on extensive analyses, the modal diploid values were 2n = 36 (30m/sm + 6st) for females and 2n = 37 (32m/sm + 5st) for males. Therefore, a novel XX/XY1Y2 sex chromosome system is described for the genus. Single nucleolar organizer regions (NORs) interspersed to GC-rich sites were detected on a subtelocentric pair (7th) for both sexes and confirmed by fluorescent in situ hybridization with 18S rDNA probes. Heterochromatin was detected at pericentromeric regions of all chromosomes and interspersed to NORs on pair 7 and 5S rDNA cistrons on pair 9. The highly differentiated karyotype of Gymnoytus bahianus, with low diploid numbers and a unique XX/XY1Y2 system, reinforces the independent origin of sex chromosomes in Gymnotiformes and seems to reflect the particular evolutionary history of this species in a small and isolated drainage system. Moreover, in spite of morphological similarities, the present results indicate a remarkable chromosomal divergence in relation to closely related species such as G. sylvius and G. carapo.

Intra- and inter-individual chromosome variation in Hoplerythrinus unitaeniatus (Pisces, Erythrinidae). A population from the Brazilian São Francisco river basin

Chromosomal studies were carried out on a population of the fish Hoplerythrinus unitaeniatus (jeju) from Prata river in the Sao Francisco river basin, Minas Gerais, Brazil using conventional Giemsa staining, C-banding, silver nitrate nucleolar organizer region (Ag-NOR) staining and fluorescent in situ hybridization (FISH) with 18S and 5S rDNA probes. We found a high degree of inter- and intra-individual variability with the identification of 2n = 50, 2n = 51 and 2n = 52 karyotypes at nearly the same frequency within the population. Intra-individual variation in chromosomal morphology and, consequently, karyotype formulae was also observed, chiefly in the specimens with 2n = 50 and 2n = 52 chromosomes. Ag-NORs and 18S rDNA sites also showed numerical and chromosomal variation similar to that found for the 5S rDNA sites. Some putative hypotheses are considered in order to explain these results.

ZZ/ZW sex chromosome system in the endangered fish Lignobrycon myersi Miranda-Ribeiro, 1956 (Teleostei, Characiformes, Triportheidae).

Abstract Lignobrycon myersi is an endemic fish species from a few coastal rivers in northeastern Brazil. Based on molecular evidence, Lignobrycon myersi and genera Triportheus Cope, 1872, Agoniates Müller & Troschel, 1845, Clupeacharax Pearson, 1924 and Engraulisoma Castro, 1981 were placed in the family Triportheidae. In the present work, we report the first cytogenetic data for Lignobrycon myersi to test the hypothesis that Lignobrycon and Triportheus are closely related. Studied specimens presented 2n=52 with 28 metacentric (m), 18 submetacentric (sm) and six subtelocentric (st) chromosomes for males and 27 m, 19 sm and 6 st for females, characterizing a ZZ/ZW sex chromosome system. The Z chromosome corresponds to the largest chromosome in karyotype while the W is about 50% smaller than the Z and largely heterochromatic. Terminal nucleolus organizer regions, GC-rich sites and 18S rDNA signals were detected on pair 14. However, additional 18S rDNA sites were observed in the W chromosome. The 5S rDNA was mainly detected on long arms of pair 7. The apparent synapomorphic chromosomal traits of Triportheus and Lignobrycon myersi reinforce their close phylogenetic relationship, suggesting that the ZZ/ZW chromosome system in both genera has arisen before cladogenic events.

Whole chromosome painting of B chromosomes of the red-eye tetra Moenkhausia sanctaefilomenae (Teleostei, Characidae).

Abstract B chromosomes are dispensable genomic elements found in different groups of animals and plants. In the present study, a whole chromosome probe was generated from a specific heterochromatic B chromosome occurring in cells of the characidae fish Moenkhausia sanctaefilomenae (Steindachner, 1907). The chromosome painting probes were used in fluorescence in situ hybridization (FISH) experiments for the assessment of metaphase chromosomes obtained from individuals from three populations of Moenkhausia sanctaefilomenae. The results revealed that DNA sequences were shared between a specific B chromosome and many chromosomes of the A complement in all populations analyzed, suggesting a possible intra-specific origin of these B chromosomes. However, no hybridization signals were observed in other B chromosomes found in the same individuals, implying a possible independent origin of B chromosome variants in this species. FISH experiments using 18S rDNA probes revealed the presence of non-active ribosomal genes in some B chromosomes and in some chromosomes of the A complement, suggesting that at least two types of B chromosomes had an independent origin. The role of heterochromatic segments and ribosomal sequences in the origin of B chromosomes were discussed.

Possible interspecific origin of the B chromosome of Hypsiboas albopunctatus (Spix, 1824) (Anura, Hylidae), revealed by microdissection, chromosome painting, and reverse hybridisation

Abstract The B chromosome in the hylid Hypsiboas albopunctatus (2n = 22 + B) is small, almost entirely composed of C-positive heterochromatin, and does not pair with any chromosome of the A complement. B probe, obtained by microdissection and DOP-PCR amplification, was used to search for homology between the B and regular chromosomes of H. albopunctatus and of the related species H. raniceps (Cope, 1862). Reverse hybridisation was also carried out in the investigation. The B probe exclusively painted the supernumerary, not hybridising any other chromosomes in H. albopunctatus, but all H. raniceps chromosomes showed small labelling signals. This result might be an indication that differences exist between the repetitive sequences of A and B chromosomes of H. albopunctatus, and that the chromosomes of H. raniceps and the heterochromatin of the B chromosome of H. albopunctatus are enriched with the same type of repetitive DNA. In meiotic preparations, the B labelled about 30% of scored spermatids, revealing a non-mendelian inheritance, and the painted B in micronucleus suggests that the supernumerary is eliminated from germ line cells. Although our results could suggest an interespecific origin of the B at first sight, further analysis on its repetitive sequences is still necessary. Nevertheless, the accumulation of repetitive sequences, detected in another species, even though closely related, remains an intriguing question.