Marcelo Ricardo Vicari

Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis

The heterochromatin composition and loca- tion in the genome of the fish Astyanax janeiroensis was investigated using Chromomycin A3 and DAPI fluorochromes and fluorescence in situ hybridization (FISH) with 18S rDNA and As51 satellite DNA probes, respectively. Distinct repetitive DNA classes were found, namely: (1) C-positive centromeric/telomeric heterochromatin, (2) NOR-associated GC-rich heterochromatin (18S+/GC+) and (3) As51+/18S+ heterochromatin colocalized on 14 distinct heterochromatic domains with attenuated fluorescence of DAPI staining (As51+/18S+/DAPI attenuated signal).Besides these fourteen associated repetitive DNAs, another eight sites with only 18S rDNA were also found, comprising altogether 22 18S rDNA sites in the genome of the species under study. Up to seven 18S rDNA sites were found to be active, i.e., were characterized as positive after silver staining (Ag-NORs). It was noteworthy that in all As51+/18S+ domains the 18S rDNA were not found to be active sites due to the silencing of these genes when associated with the As51 satellite DNA in the same heterochromatic domain. The dispersion of the As51 sites in the genome of the species is hypothesized to probably originate from a transposable element. Several chromosomal and karyotype markers are similar between A. janeiroensis and A. scabripinnis, indicating a close relationship between these species.

Astyanax aff. fasciatus Cuvier, 1819 (Teleostei; Characidae): evidences of a species complex in the upper rio Tibagi basin (Paraná, Brazil)

Four populations of Astyanax aff. fasciatus of the upper rio Tibagi (municipal district of Ponta Grossa, Parana State, Brazil), had their karyotypes and morphometry analyzed. The cytogenetic data show the occurrence of distinct karyotypes (cytotypes), here named cytotype A, with 2n=48 chromosomes (6m+18sm+14st+10a), cytotype B, with 2n=50 chromosomes (8m+18sm+14st+10a) and cytotype C, with 2n=50 chromosomes (8m+18sm+14st+10a). The distribution pattern of the constitutive heterochromatin was very similar between cytotypes A and B, but diverged in relation to cytotype C. Distinct cytotypes may occur in sympatry in the upper rio Tibagi region, with the exception of the Furna 2 sample, which presents cytotype A exclusively. In addition, a specimen with 2n=49 chromosomes (7m+18sm+14st+10a) was also found and, by the characteristics presented, may be a consequence of a rare hybridization event between cytotypes A and B. The morphometric analyses of canonical variates indicate a consistent isolation of the Furna 2 sample, while the other samples seem to be superimposed, indicating a possible gene flow or even a recent isolation event. This model points to a probable complex of cryptic species in the studied region.

Comparative cytogenetics of Hoplias malabaricus (Pisces, Erythrinidae): a population analysis in adjacent hydrographic basins

The chromosomes of specimens from four Hoplias malabaricus populations from headwaters of adjacent river basins at Ponta Grossa, southern Brazil, were investigated using differential staining techniques (C-banding, AgNO3 and CMA3) and fluorescent in situ hybridization (FISH) with an 18S rDNA probe. The diploid chromosome number in representatives of all four populations was invariably 2n = 42, with karyotypes composed of 12 pairs of metacentrics and 9 pairs of submetacentrics, without heteromorphic sex chromosomes. This kind of karyotype represents cytotype A in regard to cytotypes identified previously in H. malabaricus, exhibiting however, at the same time, some differences in the distribution of constitutive heterochromatin segments and in the locations of nucleolus organizer regions (NORs). The apparent karyotype similarity strongly suggests a close kinship among the studied populations, but the small differences detected in the examined chromosomal markers indicate some evolutionary divergence due to gene flow restriction among them.

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.

Physical Mapping of 5S rDNA in Two Species of Knifefishes: Gymnotus pantanal and Gymnotus paraguensis (Gymnotiformes)

Physical mapping of 5S rDNA in 2 species of knifefishes, Gymnotuspantanal and G. paraguensis (Gymnotiformes), was performed using fluorescence in situ hybridization with a 5S rDNA probe. The 5S rDNA PCR product from the genomes of both species was also sequenced and aligned to determine non-transcribed spacer sequences (NTS). Both species under study had different patterns of 5S rDNA gene cluster distribution. While in the karyotype of G. pantanal two 5S rDNA-bearing pairs were observed, the karyotype of G. paraguensis possessed as many as 19 such pairs. Such multiplication of 5S rDNA gene clusters might be caused by the involvement of transposable elements because the NTS of G. paraguensis was 400 bp long with high identity (90%) with a mobile transposable element called Tc1-like transposon, described from the cyprinid fish Labeo rohita.

Heterochromatin polymorphism associated with 18S rDNA: a differential pathway among Hoplias malabaricus fish populations

The distribution of constitutive heterochromatin has showed a relevant association with karyotypic evolution in several animal groups. In the present paper, a polymorphic condition for a heterochromatic segment overlapping a nucleolar organizer region in the fish species Hoplias malabaricus from the Iguaçu river, Southern Brazil, was analyzed. In accordance with previous studies, this population belongs to cytotype A, showing 2n = 42 meta-submetacentric chromosomes, without differentiation of sex chromosomes. Three phenotypes were established with regard to the heterochromatin located near the centromere of chromosome pair 16, and named rr for homomorphic reduced bands, dd for homomorphic duplicated bands and rd for the heteromorphic condition. The rr phenotype was predominant (70.6%), followed by rd (23.5%) and dd (5.9%), showing Hardy-Weinberg equilibrium in the population. In addition, the 18S rDNA sites also show the polymorphic condition. A possible role for this NOR/heterochromatin segment in the evolution of sex chromosomes in other H. malabaricus cytotypes is discussed.

Basic and molecular cytogenetics in freshwater Cichlidae (Osteichthyes, Perciformes). Karyotypic conservationism and divergence

Abstract The chromosomes of one Cichlasoma facetum and three Geopbagus brasiliensis populations from the headwaters of adjacent river basins (Paraná State, southern Brazil), were investigated using differential staining techniques (C-banding, Ag-NORs, DAPI and CMA3) and fluorescent in situ hybridization (FISH) with 18S rDNA and 5S rDNA probes. The diploid chromosome number (2n) was invariably 48 in the four populations, with karyotypes composed of 3 submetacentric and 21 subtelo/acrocentric chromosome pairs in G. brasiliensis and 5 submetacentric and 19 subtelo/acrocentric chromosome pairs in C. facetum, with no heteromorphic sex chromosomes. The differences detected in the FN numbers between G. brasiliensis (FN =54) and C. facetum (FN =58) indicate that pericentric inversion is the probable rearrangement that led to the cytogenetic divergence between these species. The overall karyotype similarity strongly suggests a close kinship among the three G. brasiliensis populations, despite a distinct C-banding pattern showed by the Jaguariaíva river population. The data suggest that dispersion events were responsible for the present fish distribution in the river basins analyzed. The divergence in the C-banding pattern is probably due to gene flow restriction between populations during their geological/evolutionary history.

ZZ/ZW sex chromosome system in an undescribed species of the genus Apareiodon (Characiformes, Parodontidae)

The chromosomes of an undescribed species of the genus Apareiodon (Characiformes, Parodontidae) from the Verde River, a headwater affluent of the Tibagi River (Paraná State, Brazil), were investigated using conventional Giemsa and Ag stainings, C-banding, CMA3 fluorescence and fluorescent in situ hybridization (FISH) using 18S and 5S rDNA probes. The diploid chromosome number was 2n = 54, with the karyotype composed of 48 meta/submetacentric and six subtelocentric chromosomes in males, and 47 meta/submetacentric + seven subtelocentric chromosomes in females. The difference is hypothesized to be due to a ZZ/ZW heteromorphic sex chromosome system, a cytotaxonomic characteristic previously observed only in some species of the genus Parodon (family Parodontidae). The presence of similar and/or identical heteromorphic sex chromosome systems might suggest that species of the genera Parodon and Apareiodon bearing ZZ/ZW heteromorphic sex chromosomes likely constitute a monophyletic group, a hypothesis to be tested by a robust phylogeny of the family.

Cytogenetic and comparative morphology of two allopatric populations of Astyanax altiparanae Garutti & Britski, 2000 (Teleostei: Characidae) from upper rio Paraná basin

The recently described fish species, Astyanax altiparanae (tetra) is common in the upper rio Parana basin, and has been reported in the Iguacu basin. However, its natural origin in the rio Iguacu is questionable. In the present work, karyotypical and morphological features of two populations of Astyanax altiparanae from the upper rio Tibagi and upper rio Iguacu were compared. Both populations showed 2n=50 chromosomes and differences in their karyotype formula, NOR-bearing chromosomes and location of heterochromatin. Morphometric data from both populations were analyzed through free-size canonical variables. Cytogenetic and morphological results were mostly coincident showing exclusive markers that reflect their degree of populational isolation. In addition to other geographic, morphological and molecular data for A. altiparanae populations from the lower rio Iguacu and rio Parana tributaries upstream from the Itaipu Dam (South Brazil), the present results indicate that the two populations analyzed in this study belong to different stocks. The presence of this species along the rio Iguacu basin would be a consequence of a complex and poorly understood evolutionary history.

Cytogenetics and biogeography: considerations about the natural origin of Hoplias malabaricus ( Characiformes, Erythrinidae ) on the Iguaçu river

Hoplias malabaricus (trahyra) is a widespread fish species over the Neotropical region with diversified inter-populational karyotypes (cytotypes), which may correspond to a species complex. Despite the wide distribution in the South American basins, some authors have questioned its natural origin in the Iguacu river, an important Brazilian river basin which is characterized by several endemic fish species. We have analyzed the karyotype of H. malabaricus from different collection sites of this river, by conventional and banding methods. Our results, in addition to our previous data concerning geographic distribution of the cytotypes, contribute to better understand the origin of H. malabaricus on the Iguacu river, reinforcing the proposition that it is a natural fish species in this river basin.