Multiple NORs Research Articles

Comparative Cytogenetics of Hoplerythrinus unitaeniatus (Agassiz, 1829) (Characiformes, Erythrinidae) Species Complex from Different Brazilian Hydrographic Basins

Chromosomal characteristics of Hoplerythrinus unitaeniatus populations from 5 Brazilian river basins, namely Arinos (Amazonas basin), Araguaia, Paraguai, Alto Paraná, and São Francisco were analyzed by conventional Giemsa staining, C-banding, silver nitrate impregnation, and fluorescence in situ hybridization (FISH) with 18S and 5S rDNA and telomeric sequence (TTAGGG)_n probes. The diploid chromosome number was 2n = 48 in representatives of the populations from Paraguai and Alto Paraná River basins and 2n = 52 for those from the Arinos and Araguaia River basins. The São Francisco population had individuals with 2n = 50 and 52 occurring in sympatry. C-banding showed heterochromatic blocks mainly located at interstitial and pericentromeric positions in most of the chromosomes. Silver nitrate impregnation demonstrated simple NORs for representatives from Arinos and Araguaia River populations and multiple NORs for specimens from Paraguai, Alto Paraná, and São Francisco River populations. FISH with 18S and 5S rDNA probes revealed many chromosomes carrying these cistrons, with up to 21 chromosomes bearing 18S rDNA sites (Alto Rio Paraná basin) and up to 12 chromosomes with 5S rDNA sites (Paraguai basin), besides the occurrence of colocalization in all populations. FISH with telomeric sequence (TTAGGG)_n detected sites in the terminal portion of the chromosomes in all populations. These data reinforce the idea that H. unitaeniatus is a species complex. Evolutionary and biogeographical aspects of the group in the Neotropical region are discussed.

Cytogenetic Insights into the Evolution of Chromosomes and Sex Determination Reveal Striking Homology of Turtle Sex Chromosomes to Amphibian Autosomes

Turtle karyotypes are highly conserved compared to other vertebrates; yet, variation in diploid number (2n = 26-68) reflects profound genomic reorganization, which correlates with evolutionary turnovers in sex determination. We evaluate the published literature and newly collected comparative cytogenetic data (G- and C-banding, 18S-NOR, and telomere-FISH mapping) from 13 species spanning 2n = 28-68 to revisit turtle genome evolution and sex determination. Interstitial telomeric sites were detected in multiple lineages that underwent diploid number and sex determination turnovers, suggesting chromosomal rearrangements. C-banding revealed potential interspecific variation in centromere composition and interstitial heterochromatin at secondary constrictions. 18S-NORs were detected in secondary constrictions in a single chromosomal pair per species, refuting previous reports of multiple NORs in turtles. 18S-NORs are linked to ZW chromosomes in Apalone and Pelodiscus and to X (not Y) in Staurotypus. Notably, comparative genomics across amniotes revealed that the sex chromosomes of several turtles, as well as mammals and some lizards, are homologous to components of Xenopus tropicalis XTR1 (carrying Dmrt1). Other turtle sex chromosomes are homologous to XTR4 (carrying Wt1). Interestingly, all known turtle sex chromosomes, except in Trionychidae, evolved via inversions around Dmrt1 or Wt1. Thus, XTR1 appears to represent an amniote proto-sex chromosome (perhaps linked ancestrally to XTR4) that gave rise to turtle and other amniote sex chromosomes.

Karyotype structure of Hypostomus cf. plecostomus (Linnaeus, 1758) from Tapajós River basin, Southern Amazon: occurrence of sex chromosomes (ZZ/ZW) and their evolutionary implications.

Hypostomus is a group of fish with numerical and struc-tural karyotypic variability. Among them, only six species, three of which belong to the Amazon basin, show a sex chromosome. In this study, we present the karyotype structure of Hypostomus cf. plecos-tomus from the Teles Pires river basin in the municipality of Alta Flo-resta, MT. The species has 2n = 68 and the karyotype formula 14m+ 24sm+ 14st+ 16a [fundamental number (FN) = 120] in males and 15m+ 24sm+14st+15a (FN = 121) in females and sex chromosomes ZZ/ZW. Argyrophilic nucleolar organizer regions (AgNORs) were identified in two pairs of chromosomes at different positions: short arm of the pair 21and long arm of the pair 27, matching the signals displayed by 18S FISH and indicating multiple NORs. Analysis of band C detected few blocks of constitutive heterochromatin in the pericentromeric regions of most chromosomes and the telomeric regions of some pairs, includ-ing the nucleolar pair 21. However, large blocks on the long arm of the nucleolar pair 27 still stood out. GC-rich heterochromatin (CMA3) was visualized only coincidently with nucleolar sites. Mapping of 5S rDNA sites with FISH revealed markings in eight chromosomes, demonstrat-ing synteny between the 18S and 5S sites. The data obtained for H. cf. plecostomus are important for taxonomic studies of this Amazon com-plex "H. plecostomus group". The occurrence of sex chromosomes in Amazon species of Hypostomus suggests an evolutionary event that is independent of other species in the group.

Karyotypic diversity in a population of Bryconamericus aff. iheringii (Characidae).

Bryconamericus comprises 56 species distributed into three groups, on the basis of the position and shape of the maxillary teeth: B. exodon, B. microcephalus and B. iheringii groups. Few cytogenetic data are available for this genus, but the diploid number of 52 chromosomes is quite common, although the karyotypic variability is extensive. This study aimed to characterize a population of B. aff. iheringii and thus contribute more cytogenetic information and better understanding of the structure and karyotypic evolution of this genus. We found 6 cytotypes with different NOR patterns: cytotype I showed a karyotype formula of 12m+10sm+16st+14a (FN = 90) and single NORs; cytotype II with 18m+14sm+10st+10a (FN = 94) and cytotype III with 20m+18sm+4st+10a (FN = 94), showing both single and multiple NORs; cytotype IV with 20m+14sm+12st+6a (NF = 98), cytotype V with 22m+18sm+8st+4a (FN = 100) and cytotype VI with 18m+24sm+6st+4a (FN = 100), all with multiple NORs. Cytotype I is the most different in relation to FN and NOR pattern, and can be regarded as belonging to another species of the genus Bryconamericus, living in sympatry in Três Bocas Stream. The remaining cytotypes may have been generated by crosses between them and by pericentric inversions. Meiotic cells were also analyzed and showed that despite the high karyotypic variability, chromosome pairing occurred normally. The great variability found in B. aff. iheringii may be related to a high degree of polymorphism; nevertheless, the possibility of occurrence of more than one species in this location is not ruled out, demonstrating the need for conservation of the Três Bocas Stream.

Cytotaxonomic Studies in Four Species of Genus Puntius (Hamilton, 1822) from Central India

The cytotaxonomic analyses of four species of genus Puntius, viz. P. chola, P. conchonius, P. sophore and P. ticto from central India was carried out for the first time. The metaphase chromosomes were prepared from kidney and gill tissues. The diploid chromosome number and karyotype formula (KF) were found to be 50 & 2m + 4sm + 2st + 42t in P. chola; 50 & 14m + 28sm + 8st in P. conchonius; 48 & 4m + 2st + 42t in P. sophore and 50 & 14m + 24sm + 8st + 4t in P. ticto. Based on KF, the fundamental arm number were determined as 56, 92, 52 and 88, respectively, for these species. The karyomorphological features indicated that P. conchonius and P. ticto are closely related and the same holds true for P. chola and P. sophore. The chromosomes of all the four species exhibited constitutive heterochromatic blocks at their centromeric position, as detected by Cbanding technique. Variations in number of NORs were observed with presence of single pair of NORs in P. chola and P. conchonius, whereas in P. sophore and P. ticto multiple NORs were observed. Thus, based on the karyological features it can be hypothesized that P. conchonius and P. ticto may be in advanced stage of karyo-evolution.

Are NORs Always Located on Homeologous Chromosomes? A FISH Investigation with rDNA and Whole Chromosome Probes in Gymnotus Fishes (Gymnotiformes)

Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. Cytogenetic studies in Gymnotus demonstrate a huge karyotypic diversity for this genus, with diploid numbers ranging from 34 to 54. The NOR are also variable in this genus, with both single and multiple NORs described. A common interpretation is that the single NOR pair is a primitive trait while multiple NORs are derivative. However this hypothesis has never been fully tested. In this report we checked if the NOR-bearing chromosome and the rDNA site are homeologous in different species of the genus Gymnotus: G. carapo (2n = 40, 42, 54), G. mamiraua (2n = 54), G. arapaima (2n = 44), G. sylvius (2n = 40), G. inaequilabiatus (2n = 54) and G. capanema (2n = 34), from the monophyletic group G. carapo (Gymnotidae-Gymnotiformes), as well as G. jonasi (2n = 52), belonging to the G1 group. They were analyzed with Fluorescence in situ hybridization (FISH) using 18S rDNA and whole chromosome probes of the NOR-bearing chromosome 20 (GCA20) of G. carapo (cytotype 2n = 42), obtained by Fluorescence Activated Cell Sorting. All species of the monophyletic G. carapo group show the NOR in the same single pair, confirmed by hybridization with CGA20 whole chromosome probe. In G. jonasi the NORs are multiple, and located on pairs 9, 10 and 11. In G. jonasi the GCA20 chromosome probe paints the distal half of the long arm of pair 7, which is not a NOR-bearing chromosome. Thus these rDNA sequences are not always in the homeologous chromosomes in different species thus giving no support to the hypothesis that single NOR pairs are primitive traits while multiple NORs are derived. The separation of groups of species in the genus Gymnotus proposed by phylogenies with morphologic and molecular data is supported by our cytogenetic data.

Cytogenetic analysis of Phyllomedusa distincta Lutz, 1950 (2n = 2x = 26), P. tetraploidea Pombal and Haddad, 1992 (2n = 4x = 52), and their natural triploid hybrids (2n = 3x = 39) (Anura, Hylidae, Phyllomedusinae)

BackgroundNatural polyploidy has played an important role during the speciation and evolution of vertebrates, including anurans, with more than 55 described cases. The species of the Phyllomedusa burmeisteri group are mostly characterized by having 26 chromosomes, but a karyotype with 52 chromosomes was described in P. tetraploidea. This species was found in sintopy with P. distincta in two localities of São Paulo State (Brazil), where triploid animals also occur, as consequence of natural hybridisation. We analyse the chromosomes of P. distincta, P. tetraploidea, and their triploid hybrids, to enlighten the origin of polyploidy and to obtain some evidence on diploidisation of tetraploid karyotype.ResultsPhyllomedusa distincta was 2n = 2x = 26, whereas P. tetraploidea was 2n = 4x = 52, and the hybrid individuals was 2n = 3x = 39. In meiotic phases, bivalents were observed in the diploid males, whereas both bivalents and tetravalents were observed in the tetraploid males. Univalents, bivalents or trivalents; metaphase II cells carrying variable number of chromosomes; and spermatids were detected in the testis preparations of the triploid males, indicating that the triploids were not completely sterile. In natural and experimental conditions, the triploids cross with the parental species, producing abnormal egg clutches and tadpoles with malformations. The embryos and tadpoles exhibited intraindividual karyotype variability and all of the metaphases contained abnormal constitutions. Multiple NORs, detected by Ag-impregnation and FISH with an rDNA probe, were observed on chromosome 1 in the three karyotypic forms; and, additionally, on chromosome 9 in the diploids, mostly on chromosome 8 in the tetraploids, and on both chromosome 8 and 9 in the triploids. Nevertheless, NOR-bearing chromosome 9 was detected in the tetraploids, and chromosome 9 carried active or inactive NORs in the triploids. C-banding, base-specific fluorochrome stainings with CMA3 and DAPI, FISH with a telomeric probe, and BrdU incorporation in DNA showed nearly equivalent patterns in the karyotypes of P. distincta, P. tetraploidea, and the triploid hybrids.ConclusionsAll the used cytogenetic techniques have provided strong evidence that the process of diploidisation, an essential step for stabilising the selective advantages produced by polyploidisation, is under way in distinct quartets of the tetraploid karyotype.

Cytogenetic comparison between two allopatric populations of Astyanax altiparanae Garutti & Britski, 2000 (Teleostei, Characidae), with emphasis on the localization of 18S and 5S rDNA

Two populations of Astyanax altiparanae (Garutti & Britski, 2000) of the Água dos Patos stream/SP and lake Igapó/PR were analyzed. All individuals showed 2n = 50, however, different karyotypic formulae were observed. The population of the Água dos Patos stream showed 8m +24sm+6st+12a (NF=88) and the population of lake Igapó, 8m+28sm+4st+10a (NF=90). Nucleolus organizing regions (AgNORs) were observed in the terminal position on the short and long arm of different chromosomes of both populations, showing a variation from 3 to 4 chromosomes. Fluorescent in situ hybridization (FISH) using 18S rDNA probes revealed only one pair of chromosomes with fluorescent signals in the terminal site on the short arm in the Igapó lake population, while the population of Água dos Patos stream showed 4 fluorescence terminal signals, characterizing a system of simple and multiple NORs, respectively. 5S rDNA fluorescent signals were detected in the interstitial position of a pair of chromosomes in the two studied populations. Some AgNOR sites revealed to be GC-rich when stained with Chromomycin A3 (CMA3), however, AT positive regions were not observed. The data obtained show that, despite the conservation of the diploid number and location of 5S DNAr, differences in both the distribution of 18S rDNA and karyotypic formula among the populations were found, thus corroborating the existing data on chromosome variability in Astyanax altiparanae that can be significant for cytotaxonomy in this group.

Karyotype variability in neotropical catfishes of the family Pimelodidae (Teleostei: Siluriformes)

Karyotypic data are presented for four species of fish belonging to the Pimelodidae family. These species show a conserved diploid number, 2n = 56 chromosomes, with different karyotypic formulae. The analyzed species showed little amount of heterochromatin located preferentially in the centromeric and telomeric regions of some chromosomes. The nucleolus organizer regions activity (Ag-NORs) and the chromosomal location of ribosomal genes by fluorescent in situ hybridization (FISH), with 18S and 5S probes, showing only one chromosome pair marked bearer of ribosomal genes, the only exception was Pimelodus britskii that presented multiple NORs and syntenic location of the 18S and 5S probes. Non-Robertsonian events, as pericentric inversion and NORs duplication are requested to explain the karyotype diversification in Pseudoplatystoma from the rio Paraguay (MS), Pimelodus from the rio Iguaçu (PR), Sorubim from the rio Paraguay (MS) and Steindachneridion from the rio Paraíba do Sul (SP). The obtained data for the karyotype macrostructure of these species corroborates a conserved pattern observed in Pimelodidae. On the other hand, interspecific variations detected by molecular cytogenetics markers made possible cytotaxonomic inferences and differentiation of the species here analyzed.

Derived cytogenetic traits, multiple NORs and B chromosomes in the compact karyotype of Canthigaster figueiredoi (Tetraodontiformes)

The Tetraodontiformes are one of the main irradiation lineages of Teleosts representing a highly specialized group. Among its families, Tetraodontidae has the lowest DNA/cell content of the vertebrates, and for this reason has been used as a model group for genomic evolution studies. Seeking to widen the cytogenetic database of this family, we performed a chromosomal analysis of the species Canthigaster figueiredoi using conventional staining, Ag-NORs, C-banding, base-specific fluorochromes DAPI-CMA 3, and in situ hybridization with 18 S rDNA probe. This species has 2n = 36 (10 m + 6sm + 20a; FN = 52). Multiple Ag-NORs (CMA 3 +) sites were detected on the four chromosome pairs. Heterochromatic blocks (CMA 3 +) were present in the centromeric regions of most of the pairs, extending on the short arm of some chromosomes. A GC-positive polymorphic heterochromatic region was identified in some of the individuals, in one or both of the homologs of the 6th pair. The presence of heteromorphic B microchromosomes was detected in the karyotype of one female, exhibiting intra-individual variation of 0–3 Bs. The occurrence of heterochromatic polymorphisms, multiple NORs, and B chromosomes in C. figueiredoi are very infrequent events in marine fish. They are probably associated to the accentuated restructuring and genomic reduction suffered by this family.

Cytogenetic survey of species of two distinct genera of Iberian nases (Cyprinidae, Leuciscinae) that hybridize extensively in nature. I. Evidence of a similar and conserved chromosome pattern with some few species-specific markers at macro-structural level

Pseudochondrostoma duriense and Pseudochondrostoma polylepis hybridize extensively with Achondrostoma oligolepis in natural populations. In this first survey, karyotypes were comparatively analyzed by C-, AgNOR- and CMA(3)-banding procedures in pure (non-introgressed) fish specimens. Leuciscinae pattern was evidenced in the three species: metacentrics and submetacentrics dominance, a big subtelo/acrocentric (marker) chromosome pair and a 2n = 50; small macro-structural differences were observed. Heterochromatin was centromere-associated. Exceptions were found at sm1 and st/a1 long arms and at m1, sm3 and sm6 short arms. The st/a1 band was telomeric in the straight-mouth nases and sub-terminal in A. oligolepis. Multiple NORs of heterochromatic nature were found in sm pairs of the three species. Signals were telomeric except for one pair in A. oligolepis. Two to four structural and two functional NORs were found in P. duriense and P. polylepis, and four to six structural and four functional NORs in A. oligolepis. Species-specific markers will prove useful in hybrid zones' cytogenetic characterization and for in-depth studies of genome compatibility-related issues in future studies.

Karyotype and genome size of Iberochondrostoma almacai (Teleostei, Cyprinidae) and comparison with the sister-species I. lusitanicum

This study aimed to define the karyotype of the recently described Iberian endemic Iberochondrostoma almacai, to revisit the previously documented chromosome polymorphisms of its sister species I.lusitanicum using C-, Ag-/CMA3 and RE-banding, and to compare the two species genome sizes. A 2n = 50 karyotype (with the exception of a triploid I.lusitanicum specimen) and a corresponding haploid chromosome formula of 7M:15SM:3A (FN = 94) were found. Multiple NORs were observed in both species (in two submetacentric chromosome pairs, one of them clearly homologous) and a higher intra and interpopulational variability was evidenced in I.lusitanicum. Flow cytometry measurements of nuclear DNA content showed some significant differences in genome size both between and within species: the genome of I. almacai was smaller than that of I.lusitanicum (mean values 2.61 and 2.93 pg, respectively), which presented a clear interpopulational variability (mean values ranging from 2.72 to 3.00 pg). These data allowed the distinction of both taxa and confirmed the existence of two well differentiated groups within I. lusitanicum: one that includes the populations from the right bank of the Tejo and Samarra drainages, and another that reunites the southern populations. The peculiar differences between the two species, presently listed as “Critically Endangered”, reinforced the importance of this study for future conservation plans.

Karyotype Analysis and Mapping of the 18S and 5S Ribosomal Genes in Astyanax fasciatus (Teleostei, Characiformes) from Paranapanema River Basin

Astyanax fasciatus from Parapanema River basin was studied with respect to the karyotype structure, nucleolar organizer regions, 18S and 5S rRNA genes and C-banding pattern. The specimens presented 2n=46 chromosomes, with multiple NORs detected by silver nitrate staining (Ag-NORs) and confirmed by fluorescence in situ hybridization (FISH). Ten 18S rDNA sites were found, located in the telomeric regions of 4 distinct chromosome pairs, with bitelomeric locations in one of them. The 5S rDNA sites were detected in the interstitial regions of 4 chromosomes, 2 metacentric and 2 acrocentric ones. The C-positive heterochromatin was located in the telomeric regions of some submetacentric, subtelocentric and acrocentric chromosomes. Variations in the karyotypic structure, C-positive heterochromatin pattern and location 18S rDNA sites are discussed.

A ZZ–ZW sex chromosome system in the finless eel Dalophis imberbis (Anguilliformes, Ophichtidae)

The karyotype of the finless eel Dalophis imberbis was investigated by conventional (C and replication banding, CMA(3) fluorescence) and molecular (FISH with 45S rDNA probe) techniques. The diploid chromosome number was 2n = 46 and the karyotype consisted of four pairs of meta-submetacentric and 18 pairs of subtelo-acrocentric autosomes and a pair of sex chromosomes heteromorphic in females, composed by a large subtelocentric Z, and a medium sized acrocentric W chromosome; in males this pair was composed by two large subtelocentric ZZ chromosomes. Multiple NORs have been localized by FISH and have been found to be CMA(3) positive; one of the NORs was located on the W chromosome and it was found transcriptionally active by silver staining. The Z chromosome contained distinct intercalar C-positive heterochromatin and W was almost completely heterochromatic. Our report contributes to the knowledge to the cytotaxonomy of Anguilliformes where differentiated sex chromosomes are reported in about a quarter of the species: both male and female heterogamety as well as a multiple chromosome system have been reported. Moreover, this is the first anguilliform species in which multiple NORs have been reported.

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.

Chromosomal rearrangements associated with pelagic larval duration in Labridae (Perciformes)

The family Labridae is one of the largest and most important groups of reef fishes in the Southern Atlantic. There is a remarkable ecologic interest in this family because of their complex interactions in the reef environment. Predictions of genetic variability in fish based on biological patterns have often been contradictory. The present work aimed to increase the cytogenetic data about the family and verify the possible correlation between larval pelagic phase and chromosomal rearrangements based on the putative basal Perciformes karyotype (2n = 48a). Therefore, cytogenetic analyses were performed in the species Halichoeres brasiliensis (2n = 48, 48a, FN = 48); Halichoeres radiatus (2n = 48, 48a, FN = 48) and in three populations of Halichoeres poeyi (2n = 48, 4m + 44st-a, FN = 52) from Brazilian coastline. A conserved diploid number was observed in all species and populations. Single NORs were identified in H. brasiliensis and in two populations of H. poeyi (BA and RJ), while multiple NORs were observed in H. radiatus and in H. poeyi from Rio Grande do Norte. The constitutive heterochromatin is reduced and distributed over centromeric and pericentromeric regions. The ribosomal sites allowed differentiating two groups of H. poeyi along the Brazilian coast; one of them comprising the population from RN, bearing multiple NORs, and another representing the populations from BA and RJ, bearing single NORs. The recently separated species, H. brasiliensis and H. radiatus, although presenting similar diploid numbers and chromosomal formulae, could be distinguished by the number of NOR-bearing chromosomes. The results revealed an evolutionary pattern chiefly derived from pericentric inversions. The correlation between larval pelagic phase and cytogenetic data on Labridae indicates that the degree of karyotypic diversification reported within this family, ranging from a highly conserved to a derived pattern, is probably influenced by the species-specific duration of larval pelagic phase.

Cytogenetic studies in three species of the genus Oligosarcus

Karyotypic studies were carried out on three species of the genus Oligosarcus: O. paranensis, O. pintoi, and O. longisrostris. All of them showed a diploid number of 50 chromosomes and the same karyotypic formulae (4M + 10SM + 16ST + 20A). Silver nitrate staining revealed single NORs in O. longirostris and multiple NORs in O. paranensis and O. pintoi. Heterochromatin seemed to play an important role in the chromosomal diversification of these species. Based on cytogenetical data, speciation hypotheses within this group were proposed, reinforcing the importance of chromosomal studies for a better understanding of evolution within the genus Oligosarcus, as well as within the family Characidae.

Chromosomes of three freshwater stingrays (Rajiformes Potamotrygonidae) from the Rio Negro basin, Amazon, Brazil

Potamotrygonidae is the representative family of South American freshwater elasmobranchs. It is a monophyletic group containing 20 species grouped into three genera. Three species belonging to two genera of this family were collected from the middle Negro River, Amazonas, Brazil, and studied cytogenetically: Paratrygon aiereba, Potamotrygon motoro and Potamotrygon orbignyi. Paratrygon aiereba presented 2n = 90 chromosomes and 4M+2SM+10ST+74A. Both species of Potamotrygon presented 2n = 66 chromosomes and differed in their chromosomal formulas: P. motoro had 18M+12SM+10ST+26A and P. orbignyi had 22M+10SM+8ST+26A. No sex heteromorphism was detected. The Fundamental Number (FN) was 106 for the three species. A system of multiple NORs was found in the three species, but with interspecific differences in terms of location and position of the active Ag-NORs sites. Paratrygon aiereba presented only four sites on the short arms of two chromosomal pairs, both in terminal regions. Potamotrygon motoro presented seven sites, on the long and short arms, all in terminal regions of non-homologous chromosomes; P. orbignyi presented eight sites on the long arms, all in terminal regions, of non-homologous chromosomes. The constitutive heterochromatin was in pericentromeric regions of all chromosomes, and no significant interspecific difference was found in relation to this marker.

Multiple nucleolus organizer regions in Leptodactylus mystacinus (Amphibia, Anura) and comments on its systematic position in the L. fuscus group based on cytogenetic and molecular analyses

Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA(3) some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy.

Multiple Nors in Bryconamericus aff. exodon (Osteichthyes, Characidae, Tetragonopterinae).

Fifteen examples of Bryconamericus aff. exodon from the Três Bocas stream in the basin of the Tibagi river (Parana, Brazil) were analyzed. They presented a diploid number of 52 chromosomes. Multiple NORS were detected by silver nitrate impregnation in the telomeric region on the short or long arm of different chromosomes, showing a variation from 2 to 5 NOR-bearing chromosomes. Treatment with chromomycin A3 (CMA3) showed regions rich in GC in different chromosomes, probably coincident with the AgNORS. In situ hybridization by fluorescence (FISH) with 18S rDNA probe showed 8 hybridization signs in the telomeric regions of the chromosomes, proving that this B. aff. exodon population has a greater number of ribosomal cistrons than were detected by Ag-banding.