Cytomolecular investigations using repetitive DNA probes contribute to the identification and characterization of Characidium sp. aff. C. vidali (Teleostei: Characiformes)

Characidium sp. aff. C. vidali is a species found in coastal streams in southeastern Brazil, which has karyotypic explanatory elements as the occurrence of microstructural variations, keeping the chromosomal macrostructure of the genus. The objective of this study was to apply cytomolecular tools in the chromosomes of Characidium sp. aff. C. vidali to identify characteristics in their karyotype contributing to cytogenetic definition of this species, adding information about the evolution of the chromosomal structure of the group. The species showed 2n = 50 chromosomes and from 1 to 4 additional B microchromosomes. FISH technique showed histone H3 and H4 genes in the short arm of pair 10, and microsatellites (CA)15, (CG)15, (GA)15 and (TTA)10 clustered in the subtelomeric portions of all A chromosomes, with total accumulation by supernumerary. The telomeric probe marked terminal regions of all chromosomes, in addition to the interstitial portion of four pairs, called ITS sites, with these markings being duplicated in two pairs, hence the double-ITS classification. C-banding revealed that supernumerary chromosomes are completely heterochromatic, that ITS sites are C-banding positive, but double-ITS sites are C-banding negative. So, throughout the evolution to Characidium, genomic events are occurring and restructuring chromosomes in populations.(AU)

Characidium sp. aff. C. vidali é uma espécie encontrada em riachos costeiros do sudeste do Brasil, que apresenta elementos cariotípicos elucidativos quanto à ocorrência de variações microestruturais, conservando a macroestrutura cromossômica do gênero. O objetivo deste estudo foi aplicar ferramentas citomoleculares para identificar características no cariótipo de Characidium sp. aff. C. vidali, que contribuam para a definição citogenética desta espécie, agregando informações quanto à evolução da estruturação cromossômica do grupo. A espécie apresentou 2n = 50 cromossomos, além de 1 a 4 microcromossomos B por célula. A FISH mostrou os genes de histona H3 e H4 sintênicos no braço curto do par 10, e os microssatélites (CA)15, (CG)15, (GA)15 e (TTA)10 clusterizados nas porções subteloméricas de todos os cromossomos do complemento A, com grande acúmulo nos supranumerários. A sonda telomérica identificou marcações terminais em todos os cromossomos, além de quatro pares marcados intersticialmente, chamados de sítios ITS, e dois pares com duas marcações intersticiais, chamados de double-ITS. O bandamento C revelou que os cromossomos supranumerários são completamente heterocromáticos, que os sítios ITS são banda C positivos, mas os sítios double-ITS são banda C negativos. Então, ao longo da evolução de Characidium, eventos genômicos estão ocorrendo e reestruturando cromossomos nas populações.(AU)

Occurrence of euchromatic B chromosomes in natural populations of Moenkhausia bonita and M. forestii (Pisces: Characidae)

Moenkhausia is a highly specious genus among the Characidae, composed of 96 valid species. Only twelve species have a known karyotype. Thus, here are presented the first cytogenetic data of two allopatric populations of Moenkhausia bonita and one of M. forestii, both belonging to the upper Paraná River basin (PR) with discussion on the evolutionary and cytotaxonomic aspects of the genus. The two species presented 2n = 50 chromosomes but different karyotype formulas and occurrence of 1-2 B chromosomes. These elements are small metacentrics in M. bonita and small acrocentrics in M. forestii. In both species, B chromosomes were euchromatic. Ag-NOR sites were found in pair 3 (metacentric), coinciding with fluorescent in situ hybridization (FISH) by the 18S rDNA probe in both species. However, the species differed in terms of the number and position of 5S rDNA sites. Heterochromatic blocks, mapped in M. bonita showed the least amount of heterochromatin in the terminal and pericentromeric regions, while the M. forestii karyotype revealed a greater amount of interstitial heterochromatic blocks. The karyotype distinctions between the two species, including the morphology of B chromosomes, may contribute as a reference in the taxonomic studies in this group.(AU)

Moenkhausia é um gênero altamente especioso dentre os Characidae, composto por 96 espécies válidas, mas apenas doze espécies têm seus cariótipos conhecidos. Portanto, são apresentados aqui os primeiros dados citogenéticos de duas populações alopátricas de Moenkhausia bonita e uma de M. forestii, ambas pertencentes à bacia do alto rio Paraná (PR), com uma ampla discussão sobre os aspectos evolutivos e citotaxonômicos do gênero. As duas espécies apresentaram 2n = 50 cromossomos, mas diferentes fórmulas cariotípicas e ocorrência de 1-2 cromossomos B. Esses elementos são pequenos metacêntricos em M. bonita e acrocêntricos pequenos em M. forestii. Em ambas as espécies, os cromossomos B apresentaram-se eucromáticos. Sítios Ag-NOR foram encontrados no par 3 (metacêntrico), coincidindo com a hibridização fluorescente in situ (FISH) pela sonda 18S rDNA em ambas as espécies. No entanto, as espécies diferiram em termos de número e posição dos sítios de 5S rDNA. Blocos heterocromáticos mapeados em M. bonita revelaram pequena quantidade de heterocromatina nas regiões terminal e pericentromérica, enquanto o cariótipo de M. forestii revelou uma maior quantidade de blocos heterocromáticos intersticiais. As distinções cariotípicas entre as duas espécies, incluindo a morfologia dos cromossomos B, podem contribuir como uma referência em estudos taxonômicos neste grupo.(AU)

First chromosomal analysis of Gymnorhamphichthys britskii: the remarkable lowest diploid value within the family Rhamphichthyidae (Gymnotiformes)

Gymnorhamphichthys britskii is a Neotropical electric fish of family Rhamphichthyidae described from the Paraná-Paraguay system. This study reports the first karyotypic description of G. britskii collected from the upper Paraná river basin, which presented 2n=38 chromosomes, karyotype composed of 14 metacentric, 8 submetacentric, 2 subtelocentric and 14 acrocentric chromosomes, and fundamental number as 62 for both sexes. Heteromorphic sex chromosomes were absent. A single pair of nucleolar organizing regions (NORs) was detected in the submetacentric chromosome pair number 9 by silver staining and confirmed by the 18S rDNA probe. The 5S rDNA was located in a single chromosome pair. Heterochromatic regions were clearly observed in the short arms of the NOR-bearing chromosome pair and in the telomeric positions of most acrocentric chromosomes. Besides the present data are valuable to help in understanding karyotypic evolution in Rhamphichthyidae, data from NORs confirmed the tendency of this family in presenting simple NORs sites, similar to the other Gymnotiformes clades. Yet, the presence of a large heterochromatic block in the NOR-bearing chromosome can be used as cytogenetic markers for G. britskii, and that centric fusions appear to be an important mechanism in the karyotype evolution and differentiation among Gymnotiformes species.(AU)

Gymnorhamphichthys britskii é um peixe neotropical da família Rhamphichthyidae descrita no sistema Paraná-Paraguai. Este estudo relata a primeira descrição cariotípica de G. britskii coletado na bacia do alto rio Paraná, que apresentou 2n = 38 cromossomos, cariótipo composto por 14 metacêntricos, 8 submetacêntricos, 2 subtelocêntricos e 14 acrocêntricos, e número fundamental 62 para ambos sexos. Cromossomos sexuais heteromórficos estavam ausentes. Um único par de regiões organizadoras de nucléolos (RONs) foi detectado no par de cromossomos submetacêntricos número 9 por coloração com prata e confirmado pela sonda DNAr 18S. O DNAr 5S foi localizado em um único par cromossômico. Regiões heterocromáticas foram claramente observadas nos braços curtos do par de cromossomos que carrega a RON e nas posições teloméricas da maioria dos cromossomos acrocêntricos. Além dos dados presentes serem valiosos para auxiliar na compreensão da evolução cariotípica em Rhamphichthyidae, dados de RONs confirmaram a tendência desta família em apresentar sítios simples de RONs, semelhantes aos demais clados de Gymnotiformes. No entanto, a presença de um grande bloco heterocromático no cromossomo portador da RON, pode ser usado como marcador citogenético para G. britskii e as fusões cêntricas parecem ser um mecanismo importante na evolução e diferenciação cariotípica entre as espécies de Gymnotiformes.(AU)

Molecular and cytogenetic analyses of cryptic species within the Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) grouping: species delimitations, karyotypic evolution and intraspecific diversification

The fish species Synbranchus marmoratus has been reported to exist as a species complex due to high intraspecific karyotypic variability in spite of the difficulty or impossibility to distinguish them using morphological traits alone. The goal of this work was to use cytogenetic and molecular methods to determine the species delimitations and understand the karyoevolution of S. marmoratus using samples collected from distinct Brazilian localities. Among the analyzed specimens, a large degree of cytogenetic variation related to diploid numbers and karyotype structure was observed, with karyotypes showing 2n=42, 44 and 46 chromosomes. In addition, using sequences of three mitochondrial genes, the phylogenetic relationships between every sample with a known karyotype were determined, which revealed significant nucleotide divergence among the karyomorphs. Also, the analyses indicate that chromosomal rearrangements occurred independently within the distinct lineages of S. marmoratus complex, which resulted in the appearance of distinct karyotypic variants in a non-linear fashion related to diploid numbers and in the appearance of similar non-homologous chromosomes. Finally, the integration of both molecular cytogenetic and phylogenetic approaches allowed the determination of specific chromosomes possibly involved in rearrangements and a better understanding about the evolutionary processes involved in the differentiation of Synbranchus genus.

A espécie de peixe Synbranchus marmoratus tem sido reportada como um complexo de espécies devido à elevada variabilidade cariotípica intraespecífica a despeito da dificuldade ou impossibilidade de distingui-las usando apenas caracteres morfológicos. O objetivo deste trabalho foi utilizar métodos citogenéticos e moleculares para determinar a delimitação das espécies e compreender a carioevolução de S. marmoratus utilizando amostras coletadas em distintas localidades brasileiras. Dentre os espécimes analisados, um alto grau de variação citogenética relativo aos números diploides e estrutura cariotípica foi observado, com cariótipos mostrando 2n=42, 44 e 46 cromossomos. Adicionalmente, utilizando sequências de três genes mitocondriais, as relações filogenéticas entre cada amostra com cariótipo conhecido foram determinadas, revelando uma divergência nucleotídica significativa entre os cariomorfos. Além disso, as análises indicam que rearranjos cromossômicos ocorreram independentemente nas distintas linhagens do complexo S. marmoratus, o que resultou no aparecimento de distintas variantes cariotípicas de forma não linear em relação aos números diploides e no surgimento de cromossomos similares e não homólogos. Finalmente, a integração de uma abordagem citogenética molecular e filogenética permitiu a determinação de cromossomos específicos que, possivelmente, estão envolvidos em rearranjos e um melhor entendimento sobre os processos evolutivos envolvidos na diferenciação do gênero Synbranchus.

Chromosome polymorphism of heterochromatin and nucleolar regions in two populations of the fish Astyanax bockmanni (Teleostei: Characiformes)

Karyotype and other chromosomal markers as revealed by C-banding and silver (Ag) impregnation in two Astyanax bockmanni populations (Barra Seca Stream and Campo Novo River) were examined. The diploid chromosome number 2n = 50 and nearly identical karyotypes were documented. C-banding revealed heterochromatic blocks on the terminal regions of some chromosomes, with high frequencies of polymorphisms. The Ag-impregnation showed that the nucleolus organizer regions (NORs) varied in number, location and organization. Astyanax bockmanni revealed chromosome characteristics similar those of the species complex "A. scabripinnis". Mechanisms that may be responsible for the high degree of polymorphism are discussed.

Cariótipo e outros marcadores cromossômicos revelados pelo bandamento C e impregnação da prata (Ag) em duas populações de Astyanax bockmanni (córrego Barra Seca e rio Campo Novo) foram examinados. O número cromossômico diploide 2n = 50 e cariótipo quase idênticos são documentados. Bandamento C revelou blocos heterocromáticos na região terminal de alguns cromossomos, com um elevado nível de polimorfismos. A impregnação da prata mostrou variabilidade do número, posição e organização para as regiões organizadoras de nucléolo (RONs). Astyanax bockmanni revelou características cromossômicas similares ao complexo "A. scabripinnis" e os mecanismos responsáveis pelo alto nível do polimorfismo foram discutidos.

Comparative cytogenetics among populations of Astyanax altiparanae (Characiformes, Characidae, Incertae sedis)

Cytogenetic data are presented for Astyanax altiparanae populations from three Brazilian hydrographic systems. The chromosomal data obtained in A. altiparanae support the hypothesis of diploid number conservation. However, small differences in the karyotype formula and number of nucleolar organizer regions were observed in these populations. The apparent karyotypical similarity among the studied populations strongly suggests a close relationship among them with some chromosomal divergences due to gene flow restriction.

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.

Cytogenetical studies in five Atlantic Anguilliformes fishes

The order Anguilliformes comprises 15 families, 141 genera and 791 fish species. Eight families had at least one karyotyped species, with a prevalence of 2n = 38 chromosomes and high fundamental numbers (FN). The only exception to this pattern is the family Muraenidae, in which the eight species analyzed presented 2n = 42 chromosomes. Despite of the large number of Anguilliformes species, karyotypic reports are available for only a few representatives. In the present work, a species of Ophichthidae, Myrichthys ocellatus (2n = 38; 8m+14sm+10st+6a; FN = 70) and four species of Muraenidae, Enchelycore nigricans (2n = 42; 6m+8sm+12st+16a; FN = 68), Gymnothorax miliaris (2n = 42; 14m+18sm+10st; FN = 84), G. vicinus (2n = 42; 8m+6sm+28a; FN = 56) and Muraena pavonina (2n = 42; 6m+4sm+32a; FN = 52), collected along the Northeastern coast of Brazil and around the St Peter and St Paul Archipelago were analyzed. Typical large metacentric chromosomes were observed in all species. Conspicuous polymorphic heterochromatic regions were observed at the centromeres of most chromosomes and at single ribosomal sites. The data obtained for Ophichthidae corroborate the hypothesis of a karyotypic diversification mainly due to pericentric inversions and Robertsonian rearrangements, while the identification of constant chromosome numbers in Muraenidae (2n = 42) suggests a karyotype diversification through pericentric inversions and heterochromatin processes.

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

The genetic monitoring of interspecific hybrids involves the application of methodologies able to provide an easy and indubitable genetic characterization of both parental and hybrid individuals. In the present work, cytogenetic techniques were used to identify a hybrid lineage of "Piaupara" in order to caracterize them in relation to the parental species, Leporinus macrocephalus (piauçu) and L. elongatus (piapara). The cytogenetic analysis revealed that L. macrocephalus presented 2n = 54 chromosomes and a nucleolar organizer regions (NOR) at the telomere of the long arm of the submetacentric chromosome pair 2. Analysis of constitutive heterochromatin (C-banding) revealed a conspicuous block at the pericentromeric region on the long arm of a submetacentric chromosome pair. L. elongatus presented the same diploid number, 2n = 54, and a karyotypic formula similar to that of L. macrocephalus. The NORs were also at the telomere of the long arm of the submetacentric pair 2, which was morphologically different from that of L. macrocephalus. Heterochromatic blocks were observed at both telomeres of a submetacentric chromosome pair. The hybrid "Piaupara" presented the same diploid number (2n = 54) and karyotypic formula as the parental species and there were no visible differences between parental and hybrid individuals. Differently from the Giemsa staining, NOR- and C-banding analysis showed marked differences which allowed the identification of the hybrids by the different morphology and/or size of the chromosomes carrying the NORs and patterns of heterochromatin distribution in their chromosomes. Such genetic studies are important for fish culture since they can provide tools for monitoring natural and artificial hybridization. They are also useful in biological conservation programmes and in the proper management of natural and reared fish stocks.

Comparative cytogenetics of Carnegiella marthae and Carnegiella strigata (Characiformes, Gasteropelecidae) and description of a ZZ/ZW sex chromosome system

Comparative cytogenetic analyses of hatchetfishes Carnegiella marthae and Carnegiella strigata (Gasteropelecidae) from the Rio Negro basin were performed using conventional Giemsa staining, silver (Ag) -staining and C-banding. The diploid chromosome numbers of both species equaled 2n = 50 but their karyotypes were distinct. We found evidence for sex chromosomes in C. marthae since karyotype of males presented 20 M + 12 SM + 4 ST + 14 A and ZZ ST chromosomes while the females presented 20 M + 12 SM + 4 ST + 14 A and ZW ST chromosomes of distinct size. Conversely, C. strigata presented 4 M + 4 SM + 2 ST + 40 A chromosomes without sex chromosome heteromorphism. Karyotypes of both species had two NOR-bearing SM chromosomes of distinct size indicating the presence of multiple NOR phenotypes. The sex chromosome pair had specific C-banding pattern allowing identification of both Z and W. This heteromorphic system has previously been described for the gasteropelecids.

First report of a B chromosome in a natural population of Astyanax altiparanae (Characiformes, Characidae)

Several species of the genus Astyanax have already been genetically studied, and B-chromosomes have been considered to be an interesting feature in some species of this group. In the present paper we report, for the first time, the occurrence of a B microchromosome in a natural population of A. altiparanae. This additional genomic element was identified as an acrocentric chromosome, similar in size to the smallest chromosomal pairs of the standard karyotype. Analysis of the constitutive heterochromatin pattern by C-banding evidenced heterochromatic blocks located on centromeric, pericentromeric, and interstitial regions of some chromosomes, and also positive marks in a subtelocentric chromosomal pair that presented the short arms entirely heterochromatic. The application of this methodology also revealed a heterochromatic pattern in the extra chromosome, a typical feature of supernumerary chromosomes.

Cytogenetic analysis in western Atlantic snappers (Perciformes, Lutjanidae)

The Lutjanidae or snappers are a family of perciform fishes, mainly marine but with some members living in estuaries and entering fresh water to feed. Some are important food fish. Cytogenetic data for Lutjanidae are scarce. In the present work, we cytogenetically characterized through conventional Giemsa staining techniques, Ag-NOR and C-banding the species Ocyurus chrysurus, Lutjanus analis, L. alexandrei, L. cyanopterus, L. jocu and L. synagris, all found along the Brazilian coast. Karyotype analysis of all six species showed a modal value of 2n = 48 acrocentric chromosomes. Single NORs were found at pericentromeric position on the long arms of the 2nd pair in O. chrysurus, L. alexandrei and L. cyanopterus, on the 5th pair in L. analis and on the 23rd pair in L. synagris. The species L. jocu presented multiple NORs located on the 2nd pair at a pericentromeric region and on the 5th pair at a telomeric region. Heterochromatic blocks were identified at the centromeric region of all chromosomes of the studied species. These results indicate that, despite of the chromosomal stability of this family, a relative structural diversification seems to have occurred in the chromosome evolution of the group. Such diversification was evidenced by divergent number and location of ribosomal sites among species. The NOR-bearing pairs represented an efficient cytotaxonomic marker for most of the analyzed species. The data suggest that the presence of interstitially located single NORs on a large acrocentric pair should represent a basal condition for lutjanids.

Cytogenetic studies in Brazilian marine Sciaenidae and Sparidae fishes (Perciformes)

Fishes from the families Sciaenidae and Sparidae, the former comprising coastal species associated with shallow waters on the continental shelf and the latter composed of typically marine species, are of significant economic value. Karyotypic data are available for about 20% of the total number of species in these groups. In the present study, cytogenetic analyses were carried out in three Sciaenidae species, Menticirrhus americanus, Ophioscion punctatissimus and Pareques acuminatus, as well as in the sparid fish, Archosargus probatocephalus, using conventional staining (Giemsa) and Ag-nucleolar organizer regions (NORs) and C-banding techniques. The diploid values (2n) and number of chromosome arms were equal to 48 in all species analyzed. NORs were located at pericentromeric positions, equivalent to large heterochromatic blocks, in M. americanus (1st pair), O. punctatissimus (10th pair), P. acuminatus (2nd pair), and A. probatocephalus (3rd pair). Heterochromatin was detected at the centromeric position in most chromosome pairs, being more conspicuous among Scianidae members. The remarkable karyotypic conservativeness detected in these species is similar to that observed in other perciform groups previously studied, regarding both the number of acrocentric chromosomes and NOR location. However, unusual events of heterochromatinization seem to have taken place along the karyotypic evolution of members of the family Sciaenidae. For the family Sparidae, distinct cytotypes between samples of Northeast Brazil and those previously analyzed on the southeastern coast were identified, suggesting that putative biogeographic barriers could be present throughout both regions on South Atlantic coast.

Cytotaxonomic and evolutionary considerations about karyotipic data of fishes from the Iguaçu River Basin in South of Brazil

The cytogenetic data available in the literature about the ichthyofauna of the Iguaçu River basin were analyzed in this review. The ichthyofauna was characterized by the high level of endemism and by the low diversity of species. Twenty-four of the eighty-one species were already karyotyped; six Characiformes, fourteen Siluriformes and four Perciformes. The chromosomal data showed the taxonomic and systematic complexity of the groups. Hypothesis related to the evolution of some Characiformes and Siluriformes groups from the Iguaçu River are proposed, as well as the utilization of karyotypic data for cytotaxonomy.

Nesta revisão são analisados os dados citogenéticos disponíveis na literatura relativos à ictiofauna da bacia do Rio Iguaçu, a qual é caracterizada pelo alto grau de endemismo e pela baixa diversidade de espécies. Das oitenta e uma espécies conhecidas, vinte e quatro já foram cariotipadas sendo 6 Characiformes, 14 Siluriformes e 4 Perciformes. Os dados cromossômicos evidenciam a complexidade taxonômica e sistemática dos grupos. São propostas hipóteses relacionadas à evolução de alguns grupos de Characiformes e Siluriformes do Rio Iguaçu, assim como o aproveitamento de dados cariotípicos para a citotaxonomia.