Chromosomal Diversity in Two Allopatric Populations of Farlowella hahni Meinken 1937 (Teleostei: Siluriformes): Cytogenetics and Cytochrome b Analyses.

Farlowella is the second richest genus in Loricariinae, broadly distributed in freshwater streams and rivers of South America. In this article, we aimed to expand on the cytogenetic and molecular data available for two allopatric populations of Farlowella hahni. Both populations had diploid chromosome number 58, but with karyotype differences, indicative of chromosomal rearrangements. C-banding showed large heterochromatic blocks at telomeric regions in acrocentric chromosomes in both populations. Fluorescence in situ hybridization (FISH) revealed a single 18S rDNA site in both populations and a single 5S rDNA site for individuals from lower Paraná River basin (native region) and multiple 5S rDNA sites for individuals from upper Paraná River basin (non-native region). Mitochondrial sequence analyses did not separate the two F. hahni populations. The cytogenetic and molecular data obtained are relevant in a preliminary study and suggested the existence of cryptic diversity and the hypothesis that at least two Farlowella lineages may coexist in the Paraná basin.

Unrevealing the Karyotypic Evolution and Cytotaxonomy of Armored Catfishes (Loricariinae) with Emphasis in Sturisoma, Loricariichthys, Loricaria, Proloricaria, Pyxiloricaria, and Rineloricaria.

This study provides new insight into the chromosomal diversification in Loricariinae. We analyzed nine species from different Brazilian hydrographic basins, using conventional and molecular cytogenetic methods, aiming to understand the karyotypic diversification, and contribute with cytotaxonomic markers in this group considered one of the most diverse of Loricariidae. Our results evidenced a high karyotypic variability in diploid number (2n) ranging from 2n = 54 (Loricariichthys platymetopon and Loricariichthys anus), 2n = 60 (Rineloricaria reisi and Rineloricaria parva), 2n = 62 (Proloricaria prolixa), 2n = 64 (Loricaria cataphracta complex species), 2n = 66 (Sturisoma barbatum), and 2n = 68 (Pyxiloricaria menezesi). Different patterns of 18S and 5S ribosomal DNA (rDNA) were also identified, while slight divergences in heterochromatin distribution were observed. This high variability is probably related with independent events of Robertsonian translocations, pericentric inversions, and different mechanisms of rDNA sites dispersion (nonreciprocal translocation and transposable element [TEs] co-localization). In addition, our study provides a set of efficient chromosomal markers for the characterization of all analyzed species, and certainly, in future analyzes, will contribute as a useful cytotaxonomic tool in groups where the traditional taxonomy based on morphological data are not sufficient to clarify their relationship.

Chromosomal mapping of repetitive sequences in Hyphessobrycon eques (Characiformes, Characidae): a special case of the spreading of 5S rDNA clusters in a genome.

Cytogenetic data showed a variation in diploid chromosome number in the genus Hyphessobrycon ranging from 2n = 46 to 52, and studies involving repetitive DNA sequences are scarce in representatives of this genus. The purpose of this paper was the chromosomal mapping of repetitive sequences (rDNA, histone genes, U snDNA and microsatellites) and investigation of the amplification of 5S rDNA clusters in the Hyphessobrycon eques genome. Two H. eques populations displayed 2n = 52 chromosomes, with the acrocentric pair No. 24 bearing Ag-NORs corresponding with CMA3+/DAPI-. FISH with a 18S rDNA probe identified the NORs on the short (p) arms of the acrocentric pairs Nos. 22 and 24. The 5S rDNA probe visualized signals on almost all chromosomes in genomes of individuals from both populations (40 signals); FISH with H3 histone probe identified two chromosome pairs, with the pericentromeric location of signals; FISH with a U2 snDNA probe identified one chromosome pair bearing signals, on the interstitial chromosomal region. The mononucleotide (A), dinucleotide (CA) and tetranucleotide (GATA) repeats were observed on the centromeric/pericentromeric and/or terminal positions of all chromosomes, while the trinucleotide (CAG) repeat showed signals on few chromosomes. Molecular analysis of 5S rDNA and non-transcribed spacers (NTS) showed microsatellites (GATA and A repeats) and a fragment of retrotransposon (SINE3/5S-Sauria) inside the sequences. This study expanded the available cytogenetic data for H. eques and demonstrated to the dispersion of the 5S rDNA sequences on almost all chromosomes.

Chromosomal analysis of Centromochlus heckelii (Siluriformes: Auchenipteridae), with a contribution to Centromochlus definition

Historically, there are divergences in the species allocation between Centromochlus and Tatia. This study aimed to generate the first cytogenetic data about Centromochlus and, by analyzing a population of Centromochlus heckelii from the Amazon River basin, to contribute as evidence to a historical taxonomic dilemma. Diploid number of 46 chromosomes and a heteromorphic pair was found in the female karyotypes, thus characterizing a ZZ/ZW sex chromosome system. Pale blocks of heterochromatin were located in centromeric regions of some chromosomes; however, the exclusive female chromosome (W) is almost entirely heterochromatic. AgNORs were detected in terminal position on the short arms of one acrocentric pair in males and two chromosome pairs in females, the acrocentric plus the sex chromosome pair. Notable differences between Centromochlus heckelii and previous data about species of Tatia are: lower diploid number, presence of a sex chromosome system and multiple AgNORs in Centromochlus, while species of Tatia have simple AgNORs and the absence of acrocentric chromosomes. Results in this study show that chromosomal markers could contribute as evidence to taxonomic delimitation studies.(AU)

Historicamente, há divergências na alocação de espécies entre Centromochlus e Tatia. Este estudo teve como objetivo gerar os primeiros dados citogenéticos para Centromochlus e, através da análise de uma população de Centromochlus heckelii da bacia do rio Amazonas, contribuir como evidência para o dilema histórico taxonômico. Foi encontrado o número diploide de 46 cromossomos e um par heteromórfico nos cariótipos das fêmeas, o que caracteriza um sistema sexual ZZ/ZW. Blocos pálidos de heterocromatina foram localizados na região centromérica de alguns cromossomos; no entanto, o cromossomo exclusivo das fêmeas (W) se apresenta quase todo heterocromático. As AgRONs foram detectadas na posição terminal do braço curto de um par acrocêntrico nos machos e em dois pares cromossômicos nas fêmeas, um par de cromossomos acrocêntricos e o par sexual. Notáveis diferenças entre os dados cromossômicos de Centromochlus heckelii e os dados anteriores das espécies de Tatia são: menor número diploide, presença de sistema de cromossomos sexuais e AgRONs múltiplas em Centromochlus, enquanto espécies de Tatia apresentam AgRON simples e ausência de cromossomos acrocêntricos. Resultados deste estudo mostram que marcadores cromossômicos podem contribuir como evidência para estudos de delimitação taxonômica.(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)

Cytogenetic markers as tools in delimiting species of the highly diverse Neotropical fish Bryconamericus (Characiformes: Characidae)

Bryconamericus is a highly diverse group of characid fishes, being cytogenetic a valuable tool for the delimitation of species. Bryconamericus aff. iheringii (Upper Uruguay/Lower Paraná), B. coeruleus (Upper Paraná), B. cf. ecai e B. cf. eigenmanni (Upper Uruguay) were studied cytogenetically, and presented 2n=52 chromosomes, with interpopulational/interspecific variation of karyotype and fundamental number. Heterochromatin was evidenced in pericentromeric, telomeric and interstitial regions, and it was shown to be an important cytogenetic marker. Single nucleolar organizing regions (NORs) were found in B. cf. eigenmanni, B. cf. ecai and B. aff. iheringii (Lower Paraná), and multiple in B. aff. iheringii (Upper Uruguay) and B. coeruleus, with occurrence of two patterns for the first species, and three for the second. The 5S/18S rDNA-FISH confirmed the location of the NORs and showed single 5S rDNA cistrons only in B. aff. iheringii (Lower Paraná), evidencing the dispersion of both genes, often co-located, in the karyotype of the others species. The data of this work contribute for the delimitation of the species of the genus. Co-localization of ribosomal genes may represent a plesiomorphic condition for the group, and their dispersion suggest the occurrence of duplication, pseudogeneization and transposition events mediated by mobile genetic elements.(AU)

Bryconamericus é um grupo altamente diverso de caracídeos, sendo a citogenética uma valiosa ferramenta para a delimitação de espécies. Bryconamericus aff. iheringii (Alto Uruguai/Baixo Paraná), B. coeruleus (Alto Paraná), B. cf. ecai e B. cf. eigenmanni (Alto Uruguai) foram estudados citogeneticamente, e apresentaram 2n=52 cromossomos, com variação interpopulacional/interespecífica de cariótipo e número fundamental (NF). Heterocromatinas foram evidenciadas nas regiões pericentromérica, telomérica e intersticial, e mostrou-se um importante marcador citogenético. Regiões organizadores de nuclcéolos (RONs) simples foram encontradas em B. cf. eigenmanni, B. cf. ecai e B. aff. iheringii (Baixo Paraná), e múltiplas em B. aff. iheringii (Alto Uruguai) e em B. coeruleus, com a ocorrência de dois padrões de localização para a primeira espécie, e três para a segunda. A FISH-DNAr 5S/18S confirmou a localização das RONs e mostrou cístrons simples de DNAr 5S apenas em B. aff. iheringii (Baixo Paraná), evidenciando a dispersão de ambos os genes, muitas vezes co-localizados, no cariótipo das demais espécies. Os dados deste trabalho contribuem para a delimitação das espécies do gênero. A co-localização dos genes ribossomais pode representar uma condição plesiomórfica para o grupo, e sua dispersão sugere a ocorrência de eventos de duplicação, pseudogenização e transposição mediada por elementos genéticos móveis.(AU)

Comparative Cytogenetics Analysis on Five Genera of Thorny Catfish (Siluriformes, Doradidae): Chromosome Review in the Family and Inferences About Chromosomal Evolution Integrated with Phylogenetic Proposals.

Doradidae has been a target of phylogenetic studies over the last few years, but chromosomal information about the family is still scarce. Therefore, new cytogenetic data are provided herein and they are correlated with phylogenetic proposals to contribute to the knowledge of chromosomal evolution within doradids. Cytogenetic studies were performed on Trachydoras paraguayensis, Anadoras sp. "araguaia," Ossancora eigenmanni, Platydoras armatulus, and Rhinodoras dorbignyi. O. eigenmanni, P. armatulus, and R. dorbignyi had 2n = 58 chromosomes as found for most doradids, but T. paraguayensis and Anadoras sp. "araguaia" had 2n = 56 chromosomes, probably caused by a chromosomal reduction. There is a great maintenance of 2n = 58 verified in doradids, but karyotype formulas are diverse. Moreover, other markers (i.e., nucleolar organizer regions, heterochromatin distribution, and 5S and 18S rDNA) showed a great diversity among the analyzed species. Contrasting the variability in the chromosomal markers with the maintenance of diploid number, it is likely that inversions and translocations played an important role in chromosome differentiation in Doradidae. Herein, we created an integrative discussion linking cytogenetic data to phylogenetic proposals, based on morphological and genetic features, enabling us to identify possible cytogenetic traits, as well as probable chromosomal plesiomorphy and apomorphy of Doradidae species.

Comparative Cytogenetics of the Black Ghost Knifefish (Gymnotiformes: Apteronotidae): Evidence of Chromosomal Fusion and Pericentric Inversions in Karyotypes of Two Apteronotus Species.

The karyotype and chromosomal characteristics of Apteronotus albifrons and Apteronotus caudimaculosus collected from populations of two different large Brazilian river basins were analyzed by conventional and molecular cytogenetic techniques, to contribute to the differentiation and identification of the species in this genus. The diploid chromosome number was 2n = 24 for A. albifrons, but with difference in the karyotype structure and fundamental number values between two populations under study. In A. caudimaculosus, the diploid chromosome number was 2n = 26, which was classified as 22 metacentric (m), 2 submetacentric (sm), and 2 acrocentric (a) chromosomes. Heterochromatins were preferentially located in pericentromeric regions for both species. However, there are more C-banded chromosomes in A. caudimaculosus than A. albifrons. The sites of 18S DNA as revealed by fluorescence in situ hybridization (FISH) in the karyotypes of both species corresponded to sites revealed by Ag impregnation, although some additional 18S rDNA sites were observed in the genome of A. caudimaculosus. FISH with 5S rDNA-probe revealed interstitial sites on the m pair No. 1 for individuals of both A. albifrons populations, and in pericentromeric regions on the long arm of pair Nos. 5 and 9 in those of A. caudimaculosus. The karyotypes of A. albifrons and A. caudimaculosus indicated a reduction of 2n resulting from chromosomal fusion, as could be hypothesized from the presence of an interstitial telomere sequence in two chromosome pairs in karyotype of A. caudimaculosus. Thus, the present study demonstrated species-specific cytogenetic markers of otherwise morphologically very similar species A. albifrons and A. caudimaculosus.

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae (Siluriformes): a report case in Corydoras carlae Nijssen & Isbrücker, 1983.

Corydoras Lacepède, 1803 is the most specious genus of Corydoradinae subfamily and many of its species are still unknown in relation to molecular cytogenetic markers. However, the diploid number and karyotypic formula were recorded for many species of this group. In current study, we provided the first cytogenetic information of Corydoras carlae Nijssen & Isbrücker, 1983, an endemic fish species from Iguassu River basin, Paraná State, Brazil. The individuals were collected in Florido River, a tributary of Iguassu River and analysed with respect to diploid number, heterochromatin distribution pattern, Ag-NORs and mapping of 5S and 18S ribosomal genes. The karyotype of this species comprises 46 chromosomes arranged in 22m+22sm+2st. The heterochromatin is distributed in centromeric and pericentromeric positions in most of the chromosomes, and also associated with NORs. The Ag-NORs were detected in the terminal position on the long arm of the metacentric pair 6. The double-FISH technique showed that 5S rDNA and 18S rDNA were co-localized in the terminal portion on the long arm of the metacentric pair 6. This condition of co-localization of ribosomal genes in Corydoras carlae seems to represent a marker for this species.

Basic cytogenetics and physical mapping of ribosomal genes in four Astyanax species (Characiformes, Characidae) collected in Middle Paraná River, Iguassu National Park: considerations on taxonomy and systematics of the genus.

Karyotypes and chromosomal characteristics of both minor and major rDNAs in four fish species known popularly as "lambaris", namely Astyanaxabramis (Jenyns, 1842), Astyanaxasuncionensis Géry, 1972, Astyanaxcorrentinus (Holmberg, 1891) and Astyanax sp. collected from downstream of the Iguassu Falls (Middle Paraná River basin), preservation area of the Iguassu National Park, were analyzed by conventional and molecular protocols. Astyanaxabramis had diploid chromosome number 2n=50 (4m+30sm+8st+8a) and single AgNORs (pair 22), Astyanaxasuncionensis had 2n=50 (8m+24sm+6st+12a) and single AgNORs (pair 20), Astyanax sp. had 2n=50 (4m+26sm+8st+12a) and single AgNORs (pair 25), and Astyanaxcorrentinus had 2n=36 (12m+16sm+2st+6a) and multiple AgNORs (pairs 12, 15, 16, 17). FISH with 18S rDNA showed a single site for Astyanaxabramis, Astyanaxasuncionensis and Astyanax sp. and multiple for Astyanaxcorrentinus (14 sites). FISH with 5S rDNA showed single 5S-bearing loci chromosome pair only for Astyanaxasuncionensis and multiple for Astyanaxabramis (four sites), Astyanaxcorrentinus (five sites) and Astyanax sp. (four sites). Distinct distribution patterns of heterochromatin were observed for karyotypes of all species, with the exception of the first acrocentric chromosome pair characterized by centromeric, interstitial-proximal and telomeric blocks of heterochromatin on the long arm, which may represent homeology between karyotypes of Astyanaxabramis and Astyanaxasuncionensis. Our study showed species-specific characteristics which can serve in diagnosis and differentiation between Astyanaxabramis and Astyanaxasuncionensis, considered cryptic species, as well as strengthening the occurrence of a species of Astyanax not yet described taxonomically. In addition, the data obtained from first cytogenetic studies in Astyanaxcorrentinus suggest a high similarity with Astyanaxschubarti Britski, 1964, suggesting that these species may belong to the same morphological group and that can be phylogenetically related.

Basic cytogenetics and physical mapping of 5S and 18S ribosomal genes in Hoplias malabaricus (Osteichthyes, Characiformes, Erythrinidae) from isolated natural lagoons: a conserved karyomorph along the Iguaçu river basin.

Erythrinidae include Neotropical teleost fish that are widely distributed in South America. Hoplias Gill, 1903 include two large groups: H. malabaricus Bloch, 1794 and H. lacerdae Miranda Ribeiro, 1908. Hoplias malabaricus is characterized by remarkable karyotype diversity, with some karyomorphs widely distributed geographically while others are more restricted to certain river basins. Cytogenetic analyzes were performed in a population of Hoplias malabaricus from the Wildlife Refuge of Campos de Palmas, the Iguaçu River basin. The specimens showed diploid number of 42 chromosomes (24m+18sm) without differentiated sex chromosomes system. The impregnation by silver nitrate showed multiple AgNORs. Seven pairs (4, 7, 10, 13, 16, 20 and 21) carrying 18S rDNA were detected by FISH. Heterochromatin was verified in the centromeric and pericentromeric region of most chromosomes and the terminal region of some pairs. FISH with 5S rDNA probes showed two chromosome pairs carrying these sites in the interstitial region (8 and 14). The data obtained in this study are similar to those found for two other populations of H. malabaricus already studied in the basin of the Iguaçu River, confirming the hypothesis that this species is natural, not having been introduced, as well as having an intrinsic characteristic, such as the largest number of sites of 18S rDNA.

Heterochromatin polymorphism and physical mapping of 5S and 18S ribosomal DNA in four populations of Hypostomus strigaticeps (Regan, 1907) from the Paraná River basin, Brazil: evolutionary and environmental correlation.

A cytogenetic analysis was performed on four populations of Hypostomus strigaticeps from the Paraná River basin, Brazil. Two populations were collected from the large channel river at the Itaipu reservoir area and the other two were from the upper stretches of tributaries of the Paraná River. All populations showed 2n=72 chromosomes (12m+12sm+18st+30a), intra- and interpopulation 18S rDNA site polymorphisms (two to three acrocentric chromosome pairs), and multiple 5S rDNA sites in three chromosome pairs (4, 21, and 28). C-banding revealed heterochromatin located in the centromere and pericentromere regions of most chromosome; however, large heterochromatic blocks (CMA3(-)/DAPI(+)) on the long arm of acrocentric chromosomes identified intra- and interpopulation polymorphism. The amount and distribution of heterochromatin seem to be correlated to biogeographical characteristics of H. strigaticeps along the Paraná River. Morphometric results also showed diversity among the populations, suggesting phenotypic plasticity of this species. Evolutionary, taxonomy, and biogeographical approaches with regard to H. strigaticeps and interrelationships in Hypostomus are discussed.