Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

Despite several difficulties in chromosomal analyses of small-sized fishes, the cytogenetics of the Lebiasinidae was largely improved in the last years, showing differential patterns in the chromosomal evolution inside the family. In this context, it has been shown that genus Lebiasina preserves its karyotypic macrostructure, composed of 2n = 36 chromosomes, whereas the other genera generally present higher 2n. This study focused on the comparative cytogenetics of three Lebiasina species, one of them analyzed here for the first time, using conventional and molecular procedures. The results reinforced the differentiated evolutionary path of the genus Lebiasina while, at the same time, highlighted the genomic particularities that have accompanied the evolution of each species. In this sense, the repetitive components of the genome played a significant role in the differentiation of each species. It is also notable that L. minuta and L. melanoguttata, the two species that occur exclusively in the Brazilian territory, show greater chromosomal similarities to each other than to the trans-Andean sister species, L. bimaculata.(AU)

Apesar das dificuldades encontradas em se realizar análises cromossômicas em peixes de pequeno porte, os estudos citogenéticos em Lebiasinidae vêm crescendo nos últimos anos e demonstrando padrões diferenciados na evolução cromossômica entre os membros da família. Nesse contexto, o gênero Lebiasina tem mostrado preservar sua macroestrutura cariotípica, composta por 2n = 36 cromossomos, enquanto os demais gêneros geralmente apresentam 2n maiores. Este estudo tem como foco a citogenética comparativa de três espécies de Lebiasina, sendo uma delas analisada pela primeira vez aqui, através do emprego de técnicas convencionais e moleculares. Os resultados obtidos reforçam a trajetória evolutiva diferenciada do gênero Lebiasina, ao mesmo tempo em que evidenciam as particularidades genômicas que acompanham a evolução de cada uma das espécies. Neste contexto, os componentes repetitivos do genoma tiveram um papel importante na caracterização particular de cada uma das espécies. Também, é notável que L. minuta e L. melanoguttata, duas espécies que ocorrem exclusivamente no território brasileiro, apresentam maior proximidade citogenética entre elas do que com a espécie irmã transandina, L. bimaculata.(AU)

Molecular cytogenetics insights in two pelagic big-game fishes in the Atlantic, the tarpon, Megalops atlanticus (Elopiformes: Megalopidae), and the sailfish, Istiophorus platypterus (Istiophoriformes: Istiophoridae)

Some pelagic and usually large sized fishes are preferential targets for sport and commercial fishing. Despite their economic importance, cytogenetic data on their evolutionary processes and management are very deficient, especially due to logistical difficulties. Here, information for two of such charismatic species, the tarpon, Megalops atlanticus (Elopiformes: Megalopidae), and the sailfish, Istiophorus platypterus (Istiophoriformes: Istiophoridae), both with a wide Atlantic distribution, were provided. Cytogenetic data were obtained using conventional methods (Giemsa staining, Ag-NORs technique, and C-banding), base-specific fluorochrome staining and fluorescence in situ hybridization (FISH) with rDNA probes. Megalops atlanticus has 2n = 50 chromosomes, all acrocentric ones (NF = 50), while Istiophorus platypterus has 2n = 48 chromosomes, 2m + 2st + 44a (NF = 52). Megalops atlanticus populations from the South Atlantic and Caribbean share identical karyotypic patterns, likely associated with gene flow between them. In turn, I. platypterus presents karyotype similarities with phylogenetically close groups, such as Carangidae. The chromosomal characteristics of these species highlight their independent evolutionary paths. Additionally, the current data contribute to knowledge of new aspects of pelagic fish fauna and will support further comparative studies with congeneric species, clarifying evolutionary karyotype trends of these fish groups.(AU)

Alguns peixes pelágicos de grande porte são alvos preferenciais para a pesca esportiva e comercial. Apesar de sua importância econômica, os dados citogenéticos sobre seus processos evolutivos e de manejo são muito deficientes, principalmente devido às dificuldades logísticas. Aqui são apresentadas informações cromossômicas de duas espécies carismáticas, o tarpão, Megalops atlanticus (Elopiformes: Megalopidae), e o agulhão-vela, Istiophorus platypterus (Istiophoriformes: Istiophoridae), ambos com ampla distribuição no oceano Atlântico. Os dados citogenéticos foram obtidos usando métodos convencionais (coloração em Giemsa, técnica de Ag-NORs e bandamento C), coloração com fluorocromos específicos e hibridização fluorescente in situ (FISH) com sondas DNAr. Megalops atlanticus possui 2n = 50 cromossomos, todos acrocêntricos (NF = 50), enquanto Istiophorus platypterus possui 2n = 48 cromossomos, 2m + 2st + 44a (NF = 52). Populações de M. atlanticus do Atlântico Sul e Caribe compartilham padrões cariotípicos idênticos, provavelmente associados ao fluxo gênico entre regiões. Por sua vez, I. platypterus apresenta semelhanças cariotípicas micro e macroestruturais com grupos filogeneticamente próximos, como Carangidae. As características cromossômicas destas espécies destacam seus caminhos evolutivos independentes. Adicionalmente, os dados apresentados contribuem com novos aspectos da fauna pelágica e apoiarão futuros estudos comparativos com espécies congenéricas, esclarecendo as tendências evolutivas do cariótipo destes grupos de peixes.(AU)

Karyotype differentiation and cytotaxonomic considerations in species of Serrasalmidae (Characiformes) from the Amazon basin

Six species of Serrasalmidae from the central Amazon, representatives of the genera Serrasalmus (S. elongatus, S. maculatus, S. cf. rhombeus, and S. rhombeus), Pygocentrus (P. nattereri), and Colossoma (C. macropomum), were analyzed regarding the distribution of the Ag-NORs, C-positive heterochromatin and 18S and 5S rRNA genes on the chromosomes. All specimens had 2n = 60 chromosomes, except S. cf. rhombeus, with 2n = 58, and C. macropomum with 2n = 54 chromosomes. The Ag-NORs were multiple and located on the short arms of subtelo-acrocentric chromosomes in all Serrasalmus species and in P. nattereri, but were found on metacentric chromosomes in C. macropomum. The 18S rDNA sites were usually coincident with Ag-NORs, although some species had a higher number and/or a distinct localization of these sites. C-positive heterochromatin was preferentially situated in centromeric regions, remarkably on metacentric pair number 7 in all Serrasalmus species and number 3 in P. nattereri, which beared a conspicuous proximal C-band on the long arms. The 5S rDNA sites were detected in a single chromosomal pair in all species. In Serrasalmus and P. nattereri, this pair was the number 7 and 3, respectively, thereby revealing its co-localization with the conspicuous heterochromatic band. However, in C. macropomum, only one homologue (probably belonging to pair number 12) exhibited 5S rDNA sites on the short arms, close to the centromere. The present data revealed reliable cytotaxonomic markers, enabling the evaluation of karyotype differentiation and interrelationships among Serrasalmidae, as well as the probable occurrence of a species complex in S. rhombeus.

Seis espécies de Serrasalmidae da Amazônia central, representantes dos gêneros Serrasalmus (S. elongatus, S. maculatus, S. cf. rhombeus e S. rhombeus ), Pygocentrus (P. nattereri) e Colossoma (C. macropomum), foram analisadas quanto à distribuição das Ag-RONs, heterocromatina C-positiva e dos genes de RNAr 18S e 5S nos cromossomos. Todos os espécimes apresentaram 2n = 60 cromossomos, exceto S. cf. rhombeus, com 2n = 58, e C. macropomum com 2n = 54 cromossomos. As Ag-RONs foram múltiplas e localizadas nos braços curtos de cromossomos subtelo-acrocêntricos em todas as espécies de Serrasalmus e em P. nattereri, mas foram encontrados em cromossomos metacêntricos em C. macropomum. Os sítios de DNAr 18S, foram geralmente coincidentes com as Ag-RONs, embora algumas espécies tenham apresentado um maior número e/ou uma localização distinta desses sítios. A heterocromatina C-positiva foi preferencialmente encontrada como uma conspícua banda proximal no par metacêntrico número 7 em todas as espécies de Serrasalmus e número 3 em P. nattereri. Os sítios de DNAr 5S foram detectados em um único par cromossômico nas seis espécies sendo que nas espécies de Serrasalmus, este par foi o de número 7 e em P. nattereri o de número 3, colocalizados com bandas heterocromáticas conspícuas. No entanto, em C. macropomum, apenas um homólogo (provavelmente pertencente ao par número 12) apresentou sítios de DNAr 5S nos braços curtos, próximos ao centrômero. Os dados apresentados revelaram confiáveis marcadores citotaxonômicos, permitindo a avaliação da diferenciação cariotípica, e as inter-relações entre Serrasalmidae, bem como a provável ocorrência de um complexo de espécies em S. rhombeus.

Karyotypic diversity between allopatric populations of the group Hoplias malabaricus (Characiformes: Erythrinidae): evolutionary and biogeographic considerations

Three populations of the group Hoplias malabaricus from the hydrographic basins of the São Francisco, Araguaia/Tocantins and Xingu Rivers in Brazil were analyzed using classic cytogenetic methods (Giemsa staining, C-banding and Ag-NORs) and molecular methods (fluorescent in situ hybridization with 18S rDNA, 5S rDNA and 5SHindIII satellite DNA probes). The chromosome markers allowed the characterization of these populations as belonging to karyomorph A and the detection of inter-population divergences. These differences likely stem from different evolutionary histories resulting from geographic isolation between populations associated to the dispersive mode of these organisms, reinforcing genetic diversity in the group Hoplias malabaricus.

Três populações do grupo Hoplias malabaricus das bacias hidrográficas dos rios São Francisco, Araguaia/Tocantins e Xingu foram analisadas citogeneticamente utilizando-se métodos clássicos (coloração com Giemsa, bandamento-C e Ag-RONs) e moleculares (hibridização in situ fluorescente com sondas de rDNA 18S, rDNA 5S e DNA satélite 5SHindIII). Os marcadores cromossômicos foram fundamentais para a caracterização destas populações como pertencentes ao cariomorfo A e para detecção de claras divergências interpopulacionais. Estas diferenças são provavelmente oriundas de diferentes histórias evolutivas do isolamento geográfico entre as populações associado ao modo dispersivo destes organismos, reiterando a diversidade genética do grupo Hoplias malabaricus.

Chromosome polymorphism in Ancistrus cuiabae Knaack, 1999 (Siluriformes: Loricariidae: Ancistrini)

Cytogenetic and FISH analyses were performed in 30 Ancistrus cuiabae specimens from a bay near the town of Poconé, in the Pantanal of Mato Grosso, Brazil. The observed diploid number was 2n = 34 chromosomes for both sexes and three distinct katyotypic formulae were found, namely cytotype A (20m, 8sm, 6st, Fundamental Number/FN = 68; 6 males and 11 females), cytotype B (19m, 8sm, 6st, 1a, FN = 67; 8 males and 4 females) and cytotype C (18m, 8sm, 6st, 2a, FN = 66; a single male). NORs's analyses showed that these regions were located in distinct sites on the NOR-bearing chromosome pair, according to cytotypes. Thus, in cytotype A, NORs were located in the terminal region of the short arm of the second metacentric chromosome pair; in cytotype B, they were detected in the short arm of the metacentric chromosome and interstitially on the acrocentric chromosome and, in cytotype C, NORs were observed in the interstitial region of the acrocentric chromosome pair. C-positive heterochromatic bands were adjacent to the rDNA sites in the corresponding chromosomes. Thus, the chromosomal polymorphism of A. cuiabae was probably originated through a pericentric inversion in chromosome pair nº 2 involving the NOR sites, which represents a novelty in the Ancistrini tribe. The results also broaden the knowledge of the chromosomal evolution in Ancistrus, the most derived genus of the Ancistrini tribe.(AU)

Foram analisados, com técnicas convencionais de citogenética e FISH, 30 exemplares da espécie Ancistrus cuiabae da baía Arrombado, próximo a Poconé, Pantanal do Mato Grosso. Foram observadas metáfases com número diploide 2n = 34 cromossomos para ambos os sexos e três fórmulas cariotípicas distintas, aqui denominadas de citótipo A, verificado em 06 machos e 11 fêmeas (20m, 8sm, 6st, Número Fundamental, NF = 68); citótipo B, em 08 machos e 04 fêmeas (19m, 8sm, 6st, 1a, NF = 67) e citótipo C em apenas 01 macho (18m, 8sm, 6st, 2a, NF = 66). As NORs confirmaram os distintos citótipos verificados, além de evidenciar que os cromossomos portadores de rDNA são os que representam o polimorfismo na espécie Ancistrus cuiabae. No citótipo A, as NORs foram verificadas na região terminal do braço curto do segundo par de cromossomos metacêntricos; no citótipo B, foram evidenciadas no segundo par, heteromórfico, no braço curto do cromossomo metacêntrico e intersticial no seu homólogo acrocêntrico; no citótipo C as NORs foram observadas na região intersticial num par de cromossomos acrocêntricos. A análise da heterocromatina constitutiva evidenciou blocos discretos adjacentes ao rDNA no segundo par de cromossomos de ambos os citótipos. Uma provável inversão pericêntrica é a hipótese proposta para a origem deste polimorfismo na espécie Ancistrus cuiabae. Estes resultados ampliam o conhecimento sobre o gênero Ancistrus, o mais derivado da tribo, contribuem para o conhecimento sobre este grupo de peixes e para inferir sobre a evolução cromossômica dos Ancistrini(AU)

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 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.

Physical mapping of the 18S and 5S ribosomal genes in nine Characidae species (Teleostei, Characiformes)

Characidae is one of the largest fish families of the Neotropical region, and presenting a pronounced morphological variability, certainly does not constitute a monophyletic group. The cytogenetical data also show a large chromosomal variation and can provide important information for a better understanding of the relationships between the species of this group. 18S and 5S rDNA probes were used in the present study for the chromosomal mapping in different Characidae species from the São Francisco River (Astyanax lacustris, Astyanax scabripinnis, Hasemania nana, Piabina argentea, Orthospinus franciscensis, Serrapinnus heterodon, Serrapinnus piaba and Myleus micans) and Alto Paraná (Astyanax altiparanae) basins. Species with a single pair of chromosomes bearing the nucleolar organizing regions (NORs) were identified, as well as species with multiple NORs, up to a maximum of seven 18S rDNA sites. The number of 5S rDNA site was also not constant, varying from two to eight. The mapping of the ribosomal genes was useful for the characterization and differentiation of the analyzed species.

Satellite DNA sites in four species of the genus Astyanax (Teleostei, Characiformes)

Cytogenetic data about satellite DNA distribution in four Astyanax species (Characidae) from the Paraitinga river, Paraíba do Sul river basin, Brazil, are presented. In order to characterize the constitutive heterochromatin, C-banding, chromomycin A3 and DAPI fluorescence staining, as well as fluorescence in situ hybridization (FISH) with the satellite DNA As-51 probe were performed. A. scabripinnis and A. parahybae presented 2n = 50 and 2n = 48 chromosomes, respectively. The heterochromatin was located in the pericentromeric and terminal regions of many chromosomes, corresponding to GC-positive regions and to the As-51 satellite DNA in terminal regions. A. intermedius and A. giton, both with 2n = 50 chromosomes, showed little heterochromatin, mostly restricted to the terminal and pericentromeric regions of a few chromosomes. No GC-positive regions, neither any correspondence between the scarce heterochromatin of these species and the As-51 satellite DNA was observed. AT-positive blocks were not detected in any of the species studied. Based on these and other available data, the hypothesis that Astyanax represents a polyphyletic group is discussed.

Chromosomal differentiation between populations of Oligosarcus hepsetus (Teleostei, Characidae) from small tributaries at opposite margins of the Paraíba do Sul river (Brazil)

Cytogenetic analyses were carried out in two populations of Oligosarcus hepsetus from tributaries at opposite margins of the Paraíba do Sul river. The same diploid number was observed in both populations (2n=50), but they showed remarkable differences related to karyotype formula and distribution of rDNA sites as revealed by silver nitrate staining and in situ hybridization with 18S probes. The results suggested that the main channel of the Paraíba do Sul river acted as barrier to gene flow between populations.

Análises citogenéticas foram realizadas em duas populações de Oligosarcus hepsetus provenientes de riachos de margens opostas do rio Paraíba do Sul. O número diplóide foi o mesmo para ambas as populações (2n=50), porém diferenças significativas foram observadas com relação à fórmula cariotípica e distribuição dos sítios de DNAr detectados pela impregnação por prata e pela hibridação "in situ" com sonda 18S. Estes resultados sugerem que a calha principal do rio Paraíba do Sul estaria atuando como uma barreira para o fluxo gênico entre essas populações.

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

Hoplias malabaricus (traíra) é uma espécie de peixe com ampla distribuição pela região Neotropical e alta diversidade cariotípica interpopulacional (citótipos), que podem corresponder a um complexo de espécies. Apesar da sua ampla distribuição pelas bacias hidrográficas da América do Sul, alguns autores têm questionado sua origem natural no rio Iguaçu, uma importante bacia hidrográfica brasileira que é caracterizada pela presença de várias espécies de peixes endêmicas. Assim, nós temos analisado o cariótipo de H. malabaricus de diferentes locais de coleta neste rio por métodos convencionais e bandamentos cromossômicos. Nossos resultados, em adição a dados prévios a respeito da distribuição geográfica dos citótipos contribuem para um melhor entendimento da origem de H. malabaricus no rio Iguaçu, reforçando a proposta que esta é uma espécie de peixe natural desta bacia hidrográfica.

Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA in the fish Prochilodus lineatus (Characiformes, Prochilodontidae)

We used differential staining techniques (BSG, GTG, AgNO3, DAPI and CMA3 banding) and fluorescent in situ hybridization (FISH) with 5S and 18S probes to investigated the karyotypic and cytogenetic chracteristics of Prochilodus lineatus specimens from a population in Vila Velha state park (Parque Estadual de Vila Velha, Ponta Grossa, Paraná state, southern Brazil). The specimens studied showed the same karyotype as that found in other P. lineatus populations, i.e. 2n = 54 biarmed chromosomes (40m + 14 sm) and c-positive heterochromatin preferentially located pericentromerically in all chromosomes. The presence of partial or totally heterochromatic supernumerary chromosomes with numeric intra-individual variation was confirmed in the analyzed population. The nucleolar organizing regions (NORs) were interstitially situated on the long arm of chromosome pair 4 directly beneath the centromere. The differential banding techniques and FISH revealed NOR size polymorphism due to structural events such as breaks and duplication of the larger rDNA site cluster. We also observed syntenic localization of the 5S ribosomal genes in the distal segment of the 45S cluster.

Comparative cytogenetics of Hoplias malabaricus (Pisces, Erythrinidae): apopulation 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.

Population structure, fluctuating asymmetry and genetic variability in an endemic and highly isolated Astyanax fish population (Characidae)

Morphological and chromosomal markers were used to infer the structure and genetic variability of a population of fish of the genus Astyanax, geographically isolated at sinkhole 2 of Vila Velha State Park, Paraná, Brazil. Two morphotypes types were observed, the standard phenotype I and phenotype II which showed an anatomical alteration probably due to an inbreeding process. Fluctuating asymmetry (FA) analysis of different characters showed low levels of morphological variation among the population from sinkhole 2 and in another population from the Tibagi river (Paraná, Brazil). The Astyanax karyotype was characterized in terms of chromosomal morphology, constitutive heterochromatin and nucleolar organizer regions. Males and females presented similar karyotypes (2n=48, 6M+18SM+14ST+10A) with no evidence of a sex chromosome system. One female from sinkhole 2 was a natural triploid with 2n=3x=72 chromosomes (9M+27SM+21ST+15A). The data are discussed regarding the maintenance of population structure and their evolutionary importance, our data suggesting that Astyanax from the Vila Velha State Park sinkhole 2 is a recently isolated population.