Repetitive DNAs and chromosome evolution in Megaleporinus obtusidens and M. reinhardti (Characiformes: Anostomidae).

The high dynamism of repetitive DNAs is a major driver of chromosome evolution. In particular, the accumulation of repetitive DNA sequences has been reported as part of the differentiation of sex-specific chromosomes. In turn, the fish species of the genus Megaleporinus are a monophyletic clade in which the presence of differentiated ZZ/ZW sex chromosomes represents a synapomorphic condition, thus serving as a suitable model to evaluate the dynamic evolution of repetitive DNA classes. Therefore, transposable elements (TEs) and in tandem repeats were isolated and located on chromosomes of Megaleporinus obtusidens and M. reinhardti to infer their role in chromosome differentiation with emphasis on sex chromosome systems. Despite the conserved karyotype features of both species, the location of repetitive sequences - Rex 1, Rex 3, (TTAGGG)n, (GATA)n, (GA)n, (CA)n, and (A)n - varied both intra and interspecifically, being mainly accumulated in Z and W chromosomes. The physical mapping of repetitive sequences confirmed the remarkable dynamics of repetitive DNA classes on sex chromosomes that might have promoted chromosome diversification and reproductive isolation in Megaleporinus species.

Comparative cytogenetics among Boana species (Anura, Hylidae): focus on evolutionary variability of repetitive DNA.

Boana comprises a diverse genus of Neotropical treefrogs, currently rearranged into seven taxonomic species groups. Although cytogenetic studies have demonstrated diversity in its representatives, the chromosomal mapping of repetitive DNA sequences is still scarce. In this study, Boana albopunctata, Boana faber, and Boana prasina were subjected to in situ localization of different repetitive DNA units to evaluate trends of chromosomal evolution in this genus. Boana faber and B. prasina had 2n=24 chromosomes, while B. albopunctata has 2n=22 and an intra-individual variation related to the presence/absence of one B chromosome. The location of 45S rDNA sites was different in the analyzed karyotypes, corroborating with what was found in the distinct phylogenetic groups of Boana. We presented the first description of 5S rDNA in a Boana species, which showed markings resulting from transposition/translocation mechanisms. In situ localization of microsatellite loci proved to be a helpful marker for karyotype comparison in Boana, commonly with cis accumulation in the heterochromatin. On the other hand, genomic dispersion of microsatellites may be associated with hitchhiking effects during the spreading of transposable elements. The obtained results corroborated the independent diversification of these lineages of species from three distinct phylogenetic groups of Boana.

Karyotypic Diversification in Two Megaleporinus Species (Characiformes, Anostomidae) Inferred from In Situ Localization of Repetitive DNA Sequences.

Anostomidae species have conserved diploid numbers (2n = 54), although comparative cytogenetic studies have demonstrated chromosomal rearrangements occurrence among them, especially in repetitive DNA rich regions. The location and distribution of ribosomal DNA (rDNA) and small nuclear RNAs (snRNAs) multigene families are highly dynamic in the genomes of several organisms. In this study, we in situ located the rDNA and snRNA sites in two populations of Megaleporinus obtusidens and a sample of Megaleporinus reinhardti to infer their chromosomal changes in the evolutionary lineages. Both species of Megaleporinus shared 2n = 54 chromosomes with the presence of ZZ/ZW sex chromosome system, but they diverged in relationship to the location of 5S and 45S rDNAs as well as the distribution of snRNAs sites. The characterization of the analyzed sequences revealed the presence of complete rDNA and snRNAs sequences as well as snRNAs containing transposable elements (TEs) and microsatellite repeats. After chromosomal mapping, the sequences encompassing TEs proved to be dispersed through autosomes and accumulated on sex chromosomes. The data demonstrate that intra- and interspecific chromosomal changes occurred involving the multigene family's sites in Megaleporinus karyotypes. Furthermore, we detected TE-like sequences in the differentiation of sex chromosome systems in M. obtusidens and M. reinhardti.

Comparative cytogenetic analysis of four species of Dendropsophus (Hylinae) from the Brazilian Atlantic forest.

We conducted a cytogenetic study of four hyline frog species (Dendropsophus elegans, D. microps, D. minutus and D. werneri) from southern Brazil. All species had 2n = 30 chromosomes, with interspecific and intraspecific variation in the numbers of metacentric, submetacentric, subtelocentric and telocentric chromosomes. C-banding and fluorochrome staining revealed conservative GC-rich heterochromatin localized in the pericentromeric regions of all species. The location of the nucleolus organizer regions, as confirmed by fluorescent in situ hybridization, differed between species. Telomeric probes detected sites that were restricted to the terminal regions of all chromosomes and no interstitial or centromeric signals were observed. Our study corroborates the generic synapomorphy of 2n = 30 chromosomes for Dendropsophus and adds data that may become useful for future taxonomic revisions and a broader understanding of chromosomal evolution among hylids.

Comparative cytogenetic analysis of marine needlefishes (Beloniformes) from southern Brazil.

Cytogenetic studies have assisted in the taxonomic classification of organisms, especially those involving species with highly similar morphologic characteristics, or so-called cryptic species. Strongylura marina and Strongylura timucu collected from Paranaguá Bay, Paraná Coast in Southern Brazil are considered cryptic species, and the identification of interspecific variations based on the number and/or morphology of its chromosomes may serve as differentiating cytotaxonomic markers. Chromosomes of the two species were subjected to different banding and staining methods (C-, Ag-, and DAPI-CMA3), as well as chromosomal mapping of major rDNA (45S), revealed with an 18S probe by fluorescence in situ hybridization (FISH). The pattern of distribution of constitutive heterochromatin showed distinct features involving the pericentromeric and telomeric bands in both species. In S. marina, chromosome 1 represents the main species-specific marker, appearing almost entirely heterochromatic. In both species, the 45S rDNA is located at terminal region of the short arm of the chromosome 6, as detected by silver nitrate staining and FISH. Despite the apparent conserved diploid number of 48 chromosomes, data on the karyotype microstructure characterize the cytogenetic profile of the genus and may allow the establishment of cytotaxonomic and evolutionary inferences for these fishes.

Comparative cytogenetics among populations of Hollandichthys multifasciatus (Teleostei: Characidae).

Two populations of Hollandichthys multifasciatus from headwaters of adjacent river coastal basins in Paraná state, southern Brazil, were cytogenetically studied in order to evaluate intraspecific divergences. Both populations presented 2n = 50 chromosomes, divided into 14m + 18sm + 18st in the sample from Antonina and 14m + 20sm + 16st for the population from Guaraqueçaba. Analyses of active nucleolar organizer regions (Ag-NORs) and fluorescent in situ hybridization (FISH) with 18S rDNA probes revealed a single metacentric pair bearing marks at proximal positions on the short arms. The location of the 5S rDNA and GC-rich sites showed chromosomal divergence between both populations. Therefore, several population markers were detected, reflecting a population differentiation, possibly driven by the formation of Paranaguá Bay and Serra do Mar mountain range. Since the existence of a species complex in H. multifasciatus has been previously characterized by morphological, molecular, and karyotype population differentiation, this species needs to be taxonomically studied in detail.

A comparative study of two marine catfish (Siluriformes, Ariidae): Cytogenetic tools for determining cytotaxonomy and karyotype evolution.

The family Ariidae comprises approximately 130 catfish species on both warm-temperate and tropical continental shelves around the world. The systematics of the group is problematic, with several misidentification problems. In order to better understand the evolutionary relationships in the family, the present study used a cytogenetic approach to characterize two populations of Genidens genidens and two populations of Aspistor luniscutis from the southern coast of Brazil using conventional techniques and fluorescent in situ hybridization with 18S rDNA probes. The two species had the same diploid number (2n = 56), high fundamental numbers and similar banding patterns, thereby corroborating the karyotypic homogeneity proposed for the group. Single nucleolus organizer regions (NORs) were found in the genus Genidens and multiple NORs were found in Aspistor, which are considered an important cytotaxonomic marker for this genus. Karyotypic evolution trends were hypothesized, providing a better understanding of the karyotype diversity and chromosome evolution processes.

Genome size evaluation in Tetraodontiform fishes from the Neotropical region.

Smooth pufferfish of the family Tetraodontidae had become pure genomic models because of the remarkable compaction of their genome. This trait seems to be the result of DNA loss following its divergence from the sister family Diodontidae, which possess larger genomes. In this study, flow cytometry was used for estimate the genome size of four pufferfish species from the Neotropical region. Cytogenetic data and confocal microscopy were also used attempting to confirm relationships between DNA content and cytological parameters. The haploid genome size was 0.71 + or - 0.03 pg for Sphoeroides greeleyi, 0.34 + or - 0.01 pg for Sphoeroides spengleri, 0.82 + or - 0.03 pg for Sphoeroides testudineus (all Tetraodontidae), and 1.00 + or - 0.03 pg for Chilomycterus spinosus (Diodontidae). These differences are not related with ploidy level, because 46 chromosomes are considered basal for both families. The value for S. spengleri represents the smallest vertebrate genome reported to date. Since erythrocyte cell and nuclear sizes are strongly correlated with genome size, the variation in this last is considered under both adaptive and evolutionary perspectives.

Chromosomal studies of five species of the marine fishes from the Paranaguá Bay and the karyotypic diversity in the marine teleostei of the Brazilian coast

In this study, five species of marine fishes from the Paranaguá Bay in the Brazilian coast were evaluated. Eucinostomus argenteus and Diapterus rhombeus (Gerreidae) presented 48 chromosomes, all of which more acrocentric (FN = 48); Strongylura timucu and S. marina (Belonidae) also presented 48 chromosomes, but with a higher karyotypic complexity than the Gerreidae, 10M+2SM+36A (FN = 60) and 4M+44A (FN = 52), respectively. The fifth species, Mugil curema (Mugilidae), different than the others, presented only 28 chromosomes 20M+4ST+4A (FN = 48). The species presented diversity in the karyotypic macro-structure, which should be relevant for the cytotaxonomy and the evolution of this group of the vertebrate.

Nas últimas décadas tem ocorrido no Brasil um incremento de estudos cariotípicos em peixes marinhos. Atualmente são conhecidos os cariótipos de 118 espécies, distribuídas em 43 famílias e 80 gêneros. Foram estudadas cinco espécies de peixes marinhos do complexo estuarino da Baía de Paranaguá na costa brasileira. Eucinostomus argenteus e Diapterus rhombeus (Gerreidae), apresentaram 48 cromossomos todos acrocêntricos (NF = 48); Strongylura timucu e S. marina (Belonidae) apresentaram 48 cromossomos, porém com complexidade cariotípica maior do que apresentada pelos gerreídeos, 10M+2SM+36A (NF = 60) e 4M+44A (NF = 52), respectivamente. A quinta espécie, Mugil curema (Mugilidae), ao contrário das outras quatro espécies aqui analisadas, apresentou apenas 28 cromossomos 20M+4ST+4A (NF = 48). Apesar da tendência em se verificar um cariótipo constituído por 48 cromossomos em teleósteos marinhos, as espécies aqui analisadas apresentam uma diversidade para a macroestrutura cariotípica a ser considerada para a citotaxonomia e evolução desse grupo de vertebrados.

Comparative cytogenetics among species of the Astyanax scabripinnis complex: evolutionary and biogeographical inferences

Karyotype data are presented for distinct species of the genus Astyanax from four rivers belonging to three different hydrographic basins of the State of Paraná, Brazil (Verde River - Tibagi basin, Açungui River - Ribeira basin, and Santo Antônio and Jaguariaíva Rivers - Jaguariaíva basin). Three karyotypic forms were identified, here denominated karyotype A (2n = 50 chromosomes, with 8m+18sm+10st+14a, and thirteen 18S rDNA sites); karyotype B (2n = 50 chromosomes, with 8m+18sm+10st+1f4a, and four 18S rDNA sites); and karyotype C (2n = 48 chromosomes, with 10m+16sm+10st+12a, and eight 18S rDNA sites). The pattern of constitutive heterochromatin was similar among the three karyotypic forms, with few differences. The 5S rDNA corresponds to a synapomorphic character regarding its number and chromosomal localization. The karyotypic form A occurs in the distribution center of the type locality of A. paranae, in the proximities of the town of Castro (Tibagi basin), and may have reached the headwaters of the Ribeira River by the breakdown of geographical barriers. The karyotypic forms B and C are sympatric and syntopic, occurring solely in the Jaguariaíva River basin. Our hypothesis is that the karyotypic form A corresponds to the species A. paranae and forms B and C correspond to other species of the A. scabripinnis complex.

Population analysis of a chromosome polymorphism in Astyanax (Teleostei, Characiformes) species endemic to the Iguaçu River

Chromosomal analyses were performed in the fish Astyanax sp.D collected from three different points: two streams from the right bank and one from the left bank of the Upper Iguaçu River, Paraná State, Brazil. The individuals from all localities possess 2n = 50 chromosomes and a FN = 84 (4m+24sm+6st+16a). The C-banding pattern was similar in all populations. However, within each population, an interindividual variation concerning the number and localization of heterochromatic bands was observed. Some of these variations were quantified in each population, and the results indicate that the samples were not different when studying the variable frequencies. Considering that Astyanax sp.D is typical in the headwaters of the Iguaçu River, these results were not expected. The data indicate that gene flow is occurring and that the Iguaçu River is not an ecological barrier among the Astyanax sp. D populations.

Cytotaxonomy, heterochromatic polymorphism and natural triploidy of a species of Astyanax (Pisces, Characidae) endemic to the Iguaçu river basin

Cytogenetic analysis with Astyanax sp. D revealed a karyotype of 2n=50 with 2M+26SM+6ST+16A, besides a triploid specimen showing 2n=75 chromosomes (3M+39SM+9ST+24A). C-banding strongly stained the terminal regions of several SM-ST-A chromossomes. Two pairs of acrocentric chromosomes presented interstitial heterochromatin, this state being polymorphic and occuring due to possible paracentric inversions. The results obtained with the AluI restriction enzyme and A3 chromomycin were similar to the C-banding. Relationships were proposed between Astyanax sp. D and A. scabripinnis, as well as considerations for a possible origin of the triploid specimen (2n=3x=75). When comparing the present results with cytogenetic features of other endemic Astyanax species in the Iguaçu river (A. sp. B and C), a clear differentiation was observed between them, indicating cytogenetics as an important cytotaxonomic tool.

Análises citogenéticas em Astyanax sp. D evidenciaram 2n=50 cromossomos e um cariótipo com 2M+26SM+6ST+16A. O bandamento C destacou as regiões teloméricas de diversos cromossomos SM-ST-A. Dois pares de cromossomos acrocêntricos possuem heterocromatina intersticial, sendo este estado polimórfico decorrente de prováveis inversões paracêntricas. O resultados obtidos com a enzima de restrição AluI e a Cromomicina A3 foram semelhantes aos do bandamento C. São propostas relações de parentesco entre Astyanax sp. D e Astyanax scabripinnis, bem como considerações sobre a possível origem do exemplar triplóide (2n=3x=75). Ao comparar os resultados deste trabalho com outras espécies de Astyanax do Rio Iguaçu, estas espécies se tornam claramente distinguíveis, evidenciando a citogenética como uma importante ferramenta taxonômica.

Physical mapping of 5S and 45S rDNA loci in pufferfishes (Tetraodontiformes).

Chromosomal features, location and variation of the major and minor rDNA genes cluster were studied in three pufferfish species: Sphoeroides greeleyi and Sphoeroides testudineus (Tetraodontidae) and Cyclichthys spinosus (Diodontidae). The location of the major rDNA was revealed with an 18S probe in two loci for all species. The minor rDNA loci (5S rDNA) was found in one chromosome pair in tetraodontid fishes and four sites located on two distinct chromosomal pairs in C. spinosus. A syntenical organization was not observed among the ribosomal genes. Signal homogeneity for GC/AT-DNA specific fluorochromes was observed in diodontid fish except in the NORs regions, which were CMA3-positive. Giemsa karyotypes of tetraodontid species presents 2n=46, having the same diploid value of other Sphoeroides species that have been investigated. On the other hand, the karyotype of C. spinosus, described for the first time, shows 2n=50 chromosomes (4m+18sm+12st+16a). The foreknowledge of the karyotypic structure of this group and also the physical mapping of certain genes could be very helpful for further DNA sequence analysis.

Karyotypic characterization of Hydromedusa tectifera (Testudines, Pleurodira) from the upper Iguaçu River in the Brazilian state of Paraná

We present the karyotypic characterization of 26 specimens of the side-necked turtle Hydromedusa tectifera collected in the upper Iguaçu River, Paraná state, Brazil. The turtles were cytogenetically analyzed using Giemsa staining and other banding techniques (C, G, Ag-NOR and CMA3) as well as fluorescence in situ hybridization (FISH) with a rDNA 18S probe. All the specimens showed a diploid number of 58 composed of 22 macro and 36 microchromosomes. The Ag-NOR, CMA3 and FISH techniques permitted the identification and characterization of the chromosome pairs bearing nucleolus organizer regions (NORs), while G-banding facilitated a better recognition and pairing of macrochromosomes. These data agree with some information available in the literature and should be very useful for further cytotaxonomic and cytosystematic studies.