No abstract

The mapping of repetitive DNA sites by fluorescence in situ hybridization has been widely used for karyotype studies in different species of fish, especially when dealing with related species or even genera presenting high chromosome variability. This study analyzed three populations of Bryconamericus, with diploid number preserved, but with different karyotype formulae. Bryconamericus ecai, from the Forquetinha river/RS, presented three new cytotypes, increasing the number of karyotype forms to seven in this population. Other two populations of Bryconamericus sp. from the Vermelho stream/PR and Cambuta river/PR exhibited interpopulation variation. The chromosome mapping of rDNA sites revealed unique markings among the three populations, showing inter- and intrapopulation variability located in the terminal region. The molecular analysis using DNA barcoding complementing the cytogenetic analysis also showed differentiation among the three populations. The U2 small nuclear DNA repetitive sequence exhibited conserved features, being located in the interstitial region of a single chromosome pair. This is the first report on its occurrence in the genus Bryconamericus. Data obtained revealed a karyotype variability already assigned to the genus, along with polymorphism of ribosomal sites, demonstrating that this group of fish can be undergoing a divergent evolutionary process, constituting a substantive model for studies of chromosomal evolution.

Rhamdia quelen, a species of Heptapteridae, is considered to be a complex because of taxonomic and phylogenetic inconsistencies. Determining the physical location of repetitive DNA sequences on the chromosomes and the DNA barcode might increase our understanding of these inconsistencies within different groups of fish. To this end, we analyzed R. quelen populations from two river basins in Brazil, Paraguay and Parana, using DNA barcoding and different chromosomal markers, including U2 snDNA, which has never been analyzed for any Rhamdia species. Cytochrome c oxidase I gene sequence analysis revealed a significant differentiation among populations from the Miranda and Quexada rivers, with genetic distances compatible to those found among different species in neotropical fishes. Our results, in general, revealed a conservative chromosomal evolution in R. quelen and a differential distribution of some markers, such as 5S rDNA and U2 snDNA, in different populations. We suggest that R. quelen must undergo a major revision in its morphological, genetic, and cytogenetic molecular and taxonomic structure to elucidate possible operational taxonomic units.

The Pseudopimelodidae family comprises 35 species however, cytogenetic studies have been performed in only six species. This study uncovered karyotypic data on Pseudopimelodus pulcher and Microglanis cottoides. Both species possessed 2n = 54, with 20m + 16sm + 10st + 8a and FN = 100 for P. pulcher and 30m + 14sm + 6st + 4a and FN = 104 for M. cottoides. A female of M. cottoides with 45m + 21sm + 9st + 6a (2n = 81) plus two extra small chromosomes was found, indicating a natural triploidy with supernumerary chromosomes. The formation of the polyploid individual seems to have come from a diploid female gamete, due to the presence of a marker chromosome pair partially heterochromatic presents only in females and common to that exemplar. This triploid female showed three chromosomes with nitrate staining (AgNOR), 18S rDNA probe and chromomycin A3 (CMA3) staining. AgNORs were observed on pairs 12 and 23 in P. pulcher and pair 24 in M. cottoides, results that were confirmed with an 18S rDNA probe and CMA3 fluorochrome. These are the first chromosomal data for P. pulcher and provide the first description of natural triploidy with the presence of supernumerary chromosomes in this family and emphasizing well the chromosomal rearrangements diversification between this species.

Physical mapping of repetitive DNA families in the karyotypes of fish is important to understand the organization and evolution of different orders, families, genera, or species. Fish in the genus Imparfinis show diverse karyotypes with various diploid numbers and ribosomal DNA (rDNA) locations. Here we isolated and characterized Tc1-mariner nucleotide sequences from Imparfinis schubarti, and mapped their locations together with 18S rDNA, 5S rDNA, and microsatellite probes in Imparfinis borodini and I. schubarti chromosomes. The physical mapping of Tc1/Mariner on chromosomes revealed dispersed signals in heterochromatin blocks with small accumulations in the terminal and interstitial regions of I. borodini and I. schubarti. Tc1/Mariner was coincident with rDNA chromosomes sites in both species, suggesting that this transposable element may have participated in the dispersion and evolution of these sequences in the fish genome. Our analysis suggests that different transposons and microsatellites have accumulated in the I. borodini and I. schubarti genomes and that the distribution patterns of these elements may be related to karyotype evolution within Imparfinis.

In the present work, six curimatid species were analyzed: Cyphocharax voga (Hensel, 1870), Cyphocharax spilotus (Vari, 1987), Cyphocharax saladensis (Meinken, 1933), Cyphocharax modestus (Fernández-Yépez, 1948), Steindachnerina biornata (Braga & Azpelicueta, 1987) and Steindachnerina insculpta (Fernández-Yépez, 1948) collected from two hydrographic basins. All samples presented 2n=54 meta-submetacentric (m-sm) chromosomes and FN equal to 108, and 1 or 2 B microchromosomes in the mitotic and meiotic cells of the six sampled populations showing inter-and intraindividual variation. The analysis of the meiotic cells in Cyphocharax saladensis, Cyphocharax spilotus, and Cyphocharax voga showed a modal number of 54 chromosomes in the spermatogonial metaphases and 27 bivalents in the pachytene, diplotene, diakinesis and in metaphase I stages, and 27 chromosomes in metaphase II; in Cyphocharax modestus, Steindachnerina biornata, and Steindachnerina insculpta, spermatogonial metaphases with 54 chromosomes and pachytene and metaphase I with 27 bivalents were observed. The B microchromosome was observed as univalent in the spermatogonial metaphase of Cyphocharax spilotus, in the pachytene stage in the other species, with the exception of Cyphocharax saladensis, and Steindachnerina biornata in metaphase I. New occurrences of the B microchromosome in Cyphocharax voga, Cyphocharax saladensis and Steindachnerina biornata were observed, confirming that the presence of this type of chromosome is a striking characteristic of this group of fish.

B chromosomes are additional elements to standard karyotypes observed in different species of fishes, especially in Curimatidae. However, despite studies demonstrating the occurrence of Bs, little is known about their origin and evolution. To better understand the genomic composition and evolutionary processes involving B chromosomes, microdissection of B microchromosomes in Steindachnerina insculpta was conducted. Chromosome painting revealed the totally hybridized B and markings on A chromosomes both in S. in sculpta and in Cyphocharax spilotus, demonstrating a strong homology between these different species. In specimens of C. modestus, which do not have Bs, the signals were observed on A chromosomes. Cloning and sequencing of some B fragments revealed that the B microchromosome in S. insculpta is composed of repetitive elements, homologous to the DIRS-4 LTR retrotransposon of Xenopus (Silurana) tropicalis. FISH with clone pSi48 with the DIRS-4 retroelement revealed signals on all A chromosomes in the 2 species and also on the B, suggesting the insertion of repetitive elements in these species.

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter-and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished.Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.