Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes.

Silver nitrate staining to evidence the location of nucleolar organizer regions (Ag-NORs) in chromosomes is widely used as a classical method in plant cytogenetics. Here, we present the most used procedures and highlight some aspects in terms of their replicability by plant cytogeneticists. Some technical features described are materials and methods used, procedures, protocol modifications, and precautions in order to obtain positive signals. The methods to obtain Ag-NOR signals have different degrees of replicability, but do not require any sophisticated technology or equipment for their application.

Cytogenetic Analysis of Panaqolus tankei Cramer & Sousa, 2016 (Siluriformes, Loricariidae), an Ornamental Fish Endemic to Xingu River, Brazil.

Despite conservation of the diploid number, a huge diversity in karyotype formulae is found in the Ancistrini tribe (Loricariidae, Hypostominae). However, the lack of cytogenetic data for many groups impairs a comprehensive understanding of the chromosomal relationships and the impact of chromosomal changes on their evolutionary history. Here, we present for the first time the karyotype of Panaqolus tankei Cramer & Sousa, 2016. We focused on the chromosomal characterization, using conventional and molecular cytogenetic techniques to unravel the evolutionary trends of this tribe. P. tankei, as most species of its sister group Pterygoplichthini, also possessess a conserved diploid number of 52 chromosomes. We observed heterochromatin regions in the centromeres of many chromosomes; pairs 5 and 6 presented interstitial heterochromatin regions, whereas pairs 23 and 24 showed extensive heterochromatin regions in their q arms. In situ localization of 18S rDNA showed hybridization signals correlating with the nucleolus organizer regions, which are located in the q arms of pair 5. However, the 5S rDNA was detected in the centromeric and terminal regions of the q arms of pair 8. (TTAGGG)n hybridized only in the terminal regions of all chromosomes. Microsatellite in situ localization showed divergent patterns, (GA)15 repeated sequences were restricted to the terminal regions of some chromosomes, whereas (AC)15 and (GT)15 showed a scattered hybridization pattern throughout the genome. Intraspecific comparative genomic hybridization was performed on the chromosomes of P. tankei to verify the existence of sex-specific regions. The results revealed only a limited number of overlapping hybridization signals, coinciding with the heterochromatin in centromeric regions without any sex-specific signals in both males and females. Our study provides a karyotype description of P. tankei, highlighting extensive differences in the karyotype formula, the heterochromatin regions, and sites of 5S and 18S rDNA, as compared with data available for the genus.

Chromosomal Diversification in Two Species of Pimelodus (Siluriformes: Pimelodidae): Comparative Cytogenetic Mapping of Multigene Families.

Pimelodidae has a high number of species, but cytogenetic studies are generally restricted to classical chromosomal characterization and in situ localization of ribosomal DNA (rDNA) genes. This study was developed to compare Pimelodus microstoma and Pimelodus pohli focusing on chromosomal diversification provided by the transposition of DNA sequences containing multigene families. Both species share 56 chromosomes, with centromeric and terminal heterochromatic blocks. The silver nucleolus organizer regions (Ag-NORs)/45S rDNA was located in the chromosome pair 24 for both species. The 5S rDNA sites were evidenced in the pair 8 of P. microstoma, and in the pairs 1, 17, and 18 in P. pohli. The U1 small nuclear RNA (snRNA) was located at terminal site in the first subtelocentric pair in both species. The U2 snRNA site was syntenic to 5S rDNA in non-homeologue chromosomes between analyzed species. The histones H3 and H4 were clustered in chromosome pairs 19 and 23 in P. microstoma, and 21 and 22 in P. pohli. Our study proposes that the movement of DNA sequences carrying multigene families has been driven on the chromosomal diversification of Pimelodidae. These multigene location in the genomes can explain most of the visualized chromosomal rearrangements in Pimelodidae and it is useful to understand the chromosomal changes and their distinctive karyotype formulae.

Sex Chromosomes and Internal Telomeric Sequences in Dormitator latifrons (Richardson 1844) (Eleotridae: Eleotrinae): An Insight into their Origin in the Genus.

The freshwater fish species Dormitator latifrons, commonly named the Pacific fat sleeper, is an important food resource in CentralSouth America, yet almost no genetic information on it is available. A cytogenetic analysis of this species was undertaken by standard and molecular techniques (chromosomal mapping of 18S rDNA, 5S rDNA, and telomeric repeats), aiming to describe the karyotype features, verify the presence of sex chromosomes described in congeneric species, and make inferences on chromosome evolution in the genus. The karyotype (2n = 46) is mainly composed of metacentric and submetacentic chromosomes, with nucleolar organizer regions (NORs) localized on the short arms of submetacentric pair 10. The presence of XX/XY sex chromosomes was observed, with the X chromosome carrying the 5S rDNA sequences. These heterochromosomes likely appeared before 1 million years ago, since they are shared with another derived Dormitator species (Dormitator maculatus) distributed in the Western Atlantic. Telomeric repeats hybridize to the terminal portions of almost all chromosomes; additional interstitial sites are present in the centromeric region, suggesting pericentromeric inversions as the main rearrangement mechanisms that has driven karyotypic evolution in the genus. The data provided here contribute to improving the cytogenetics knowledge of D. latifrons, offering basic information that could be useful in aquaculture farming of this neotropical fish.

Chromosome evolution in Lophyohylini (Amphibia, Anura, Hylinae).

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi, P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution.

Chromosome evolution in Cophomantini (Amphibia, Anura, Hylinae).

The hylid tribe Cophomantini is a diverse clade of Neotropical treefrogs composed of the genera Aplastodiscus, Boana, Bokermannohyla, Hyloscirtus, and Myersiohyla. The phylogenetic relationships of Cophomantini have been comprehensively reviewed in the literature, providing a suitable framework for the study of chromosome evolution. Employing different banding techniques, we studied the chromosomes of 25 species of Boana and 3 of Hyloscirtus; thus providing, for the first time, data for Hyloscirtus and for 15 species of Boana. Most species showed karyotypes with 2n = 2x = 24 chromosomes; some species of the B. albopunctata group have 2n = 2x = 22, and H. alytolylax has 2n = 2x = 20. Karyotypes are all bi-armed in most species presented, with the exception of H. larinopygion (FN = 46) and H. alytolylax (FN = 38), with karyotypes that have a single pair of small telocentric chromosomes. In most species of Boana, NORs are observed in a single pair of chromosomes, mostly in the small chromosomes, although in some species of the B. albopunctata, B. pulchella, and B. semilineata groups, this marker occurs on the larger pairs 8, 1, and 7, respectively. In Hyloscirtus, NOR position differs in the three studied species: H. alytolylax (4p), H. palmeri (4q), and H. larinopygion (1p). Heterochromatin is a variable marker that could provide valuable evidence, but it would be necesserary to understand the molecular composition of the C-bands that are observed in different species in order to test its putative homology. In H. alytolylax, a centromeric DAPI+ band was observed on one homologue of chromosome pair 2. The band was present in males but absent in females, providing evidence for an XX/XY sex determining system in this species. We review and discuss the importance of the different chromosome markers (NOR position, C-bands, and DAPI/CMA3 patterns) for their impact on the taxonomy and karyotype evolution in Cophomantini.

Chromosome Banding in Amphibia. XXXV. Highly Mobile Nucleolus Organizing Regions in Craugastor fitzingeri (Anura, Craugastoridae).

A 7-year cytogenetic study on the leaf litter frog Craugastor fitzingeri from Costa Rica and Panama revealed the existence of highly mobile nucleolus organizing regions (NORs) in their genomes. Silver (Ag)-staining of the active NORs demonstrated an exceptional interindividual pattern of NOR distribution at the telomeres of the chromosomes. All individuals examined showed a different and specific NOR location in their karyotypes. Furthermore, intraindividual variation in the NOR sites was found. This observation suggested the existence of mobile NORs in C. fitzingeri. Confirmation of this phenomenon was possible by systematic FISH analysis using an 18S + 28S rDNA probe. The extremely variable number and position of the NORs in C. fitzingeri is best explained by highly mobile NORs that move freely between the telomeres of the chromosomes. These transpositions must occur preferentially in premeiotic, meiotic, or postmeiotic stages, but also at a lower incidence in the somatic tissues of the animals. It is hypothesized that transposable (mobile) elements are closely linked to the NORs or are inserted into the major 18S + 28S rDNA spacers of C. fitzingeri. When such transposable elements spread by transpositions, they can carry with them complete or partial NORs. The present study provides detailed information on various differential chromosome banding techniques, in situ hybridization experiments, chromosomal hypermethylation patterns, determination of the genome size, and analyses of restriction fragment length polymorphisms of the DNA.

A New Approach to Chromosomal Evolution in the Giant Water Bug (Heteroptera: Belostomatidae).

The genus Belostoma, known colloquially as "giant water bugs," presents striking cytogenetic diversity and extensive chromosome variability. Notwithstanding, its karyotype evolution is not well understood. We analyzed 8 species of Belostoma (77 samples). The meiotic analysis revealed 2n = 14 + XY for Belostoma horvathi and Belostoma candidulum; 2n = 22 + XY for Belostoma cummings; 2n = 26 + X1X2Y for Belostoma dentatum, Belostoma elongatum, and Belostoma discretum; and 2n = 26 + X1X2X3Y for Belostoma testacopallidum and Belostoma dilatatum. All species showed holokinetic chromosomes. Based on heterochromatin distribution patterns and 18S rDNA, the species of the genus Belostoma were separated into four groups. The analysis of C0t-1 DNA showed that the repetitive DNA, partly composed of microsatellite DNA, was absent on the Y chromosome. Fluorescent in situ hybridization (FISH) using a microdissected X chromosome in species with simple sex system presents uniform hybridization in the nuclear region corresponding to the X chromosome. Species with multiple systems revealed discrete markings. The present data in conjunction with the existing literature led us to propose a new evolutionary hypothesis for the group, with an ancestral karyotype with a low diploid number, simple sex determination system, and nucleolus organizer regions (NORs) on the sex chromosomes. That karyotype would have originated other karyotypes through agmatoploidy, simploidy, heterochromatinization, and movement of the 18S rDNA.

FISH and AgNor mapping of the 45S and 5S rRNA genes in wild and cultivated species of Capsicum (Solananceae).

Chromosome number and position of rDNA were studied in 12 wild and cultivated species of the genus Capsicum with chromosome numbers x = 12 and x = 13 (22 samples). For the first time in these species, the 5S and 45S rRNA loci were localized and physically mapped using two-color fluorescence in situ hybridization and AgNOR banding. We focused on the comparison of the results obtained with both methods with the aim of accurately revealing the real functional rRNA genes. The analyzes were based on a previous work that reported that the 18S-5.8S-25S loci mostly coincide with GC-rich heterochromatic regions and likely have given rise to satellite DNAs, which are not active genes. These data show the variability of rDNA within karyotypes of the genus Capsicum, providing anchor points for (comparative) genetic maps. In addition, the obtained information might be useful for studies on evolution of repetitive DNA.

Closely Related Syntopic Cytotypes of Astyanax taeniatus (Jenyns, 1842) from the Upper Piranga River, Upper Doce Basin in Southeastern Brazil.

Astyanax taeniatus occurs in coastal areas of southeastern Brazil, and it is very abundant in the Upper Doce River Basin. Our objective was to study C-, argyrophilic nucleolar organizer region (Ag-NOR) and fluorescent in situ hybridization (FISH) banding patterns using 5S, 18S, CA(15), and GA(15) repetitive DNA probes on a population of A. taeniatus present in the Piranga River, in the Doce Basin. Two syntopic cytotypes were found, both with 2n = 50: cytotype A (14m + 12sm + 16st + 8t) and cytotype B (10m + 14sm + 18st + 8t). In both cytotypes, heterochromatic blocks occurred in all the chromosomes; Ag-NOR sites were multiple, ranging from four to eight. The 5S rDNA probe marked eight chromosomes in both cytotypes, a unique condition within Astyanax, suggesting a recent divergence between these cytotypes. The 18S rDNA probe differed between the cytotypes, marking 10 and 8 chromosomes in cytotypes A and B, respectively. CA(15) and GA(15) FISH patterns were mainly subtelomeric, but CA(15) showed centromeric markings that were diagnostic for each cytotype. Although overall cytogenetic evidence suggests that these cytotypes are closely related, morphological and molecular data in progress will provide further hypothesis test on their phylogenetic relationship.

Intra-generic and interspecific karyotype patterns of Leptodactylus and Adenomera (Anura, Leptodactylidae) with inclusion of five species from Central Amazonia.

The genera Leptodactylus and Adenomera comprise 92 species distributed throughout the Neotropical region. These species have a modal diploid chromosome number 2n = 22. However, chromosome rearrangements are evident in the differentiation of five intra-generic groups in the genus Leptodactylus (L. fuscus, L. latrans, L. marmoratus (formally composed by the species of the genus Adenomera), L. melanonotus, L. pentadactylus), yet it is not clear if there is a karyotype pattern for each group. Aiming to understand the intra-generic and interspecific karyotype patterns of Leptodactylus and Adenomera, cytogenetic analyses were performed in A. andreae, L. macrosternum, L. pentadactylus, L. petersii, and L. riveroi using conventional staining, C-banding, nucleolus organizer region (NOR) and hybridization in situ fluorescent (FISH). The karyotype of Leptodactylus riveroi was described for the first time. Adenomera andreae had 2n = 26, while the remaining species 2n = 22. The NOR was found on pair No. 8 of A. andreae, L. macrosternum, L. pentadactylus, and L. riveroi, whereas L. petersii had it on pairs Nos. 6 and 10. These locations were confirmed by the FISH with 18S rDNA probe, except for pair No. 10 of L. petersii. The C-banding pattern was evident at the centromeres of chromosomes of all species and some interspecific variations were also observed. 2n = 22 was observed in the species of the L. latrans group, as well as in the intra-generic groups L. fuscus and L. pentadactylus; in the L. melanonotus group there were three diploid chromosome numbers 2n = 20, 22 and 24; and a larger variation in 2n was also evident in the L. marmoratus group.

A unique sex chromosome system in the knifefish Gymnotus bahianus with inferences about chromosomal evolution of Gymnotidae.

Cytogenetic studies in Neotropical electric knifefish of genus Gymnotus have shown a remarkable interspecific variability, including distinct sex chromosome systems. In this study, we present the first chromosomal data in Gymnotus bahianus from Contas River basin, northeastern South America. Based on extensive analyses, the modal diploid values were 2n = 36 (30m/sm + 6st) for females and 2n = 37 (32m/sm + 5st) for males. Therefore, a novel XX/XY1Y2 sex chromosome system is described for the genus. Single nucleolar organizer regions (NORs) interspersed to GC-rich sites were detected on a subtelocentric pair (7th) for both sexes and confirmed by fluorescent in situ hybridization with 18S rDNA probes. Heterochromatin was detected at pericentromeric regions of all chromosomes and interspersed to NORs on pair 7 and 5S rDNA cistrons on pair 9. The highly differentiated karyotype of Gymnoytus bahianus, with low diploid numbers and a unique XX/XY1Y2 system, reinforces the independent origin of sex chromosomes in Gymnotiformes and seems to reflect the particular evolutionary history of this species in a small and isolated drainage system. Moreover, in spite of morphological similarities, the present results indicate a remarkable chromosomal divergence in relation to closely related species such as G. sylvius and G. carapo.

Sequential steps of chromosomal differentiation in Atlantic surgeonfishes: evolutionary inferences.

Surgeonfishes are a species-rich group and a major biomass on coral reefs. Three species are commonly found throughout South Atlantic, Acanthurus bahianus, A. chirurgus, and A. coeruleus. In this paper, we present the first cytogenetic data of these species, revealing a sequential chromosomal diversification. A. coeruleus was characterized by a relatively conserved karyotype evolved by pericentric inversions of some pairs (2n = 48, 2sm + 4st + 42a). In contrast, the karyotypes of A. bahianus (2n = 36) and A. chirurgus (2n = 34) were highly differentiated by the presence of six large metacentric pairs in A. bahianus (12m + 2sm + 4st + 18a) and A. chirurgus (12m + 2sm + 4st +1 6a) probably derived by chromosomal fusions that corroborate their closer relationship. A discernible in tandem fusion represents an autapomorphic character to A. chirurgus. In spite of macrostructure variation, single nucleolar organizer regions (NORs) on short arms of a subtelocentric pair and similar distribution of C-bands were observed in the three species. Overlapping of chromosomal data with molecular phylogeny indicated pericentric inversions which took place nearly at 19 Ma while centric fusions are as recent as 5 Ma. A physical mapping of coding and noncoding sequences in Acanthurus could clarify the role of additional rearrangements during their chromosomal evolution.

Karyotypic diversity in seven Amazonian anurans in the genus Hypsiboas (family Hylidae).

BACKGROUND: Hypsiboas species have been divided into seven groups using morphological and genetic characters, but for most of the species, there is no cytogenetic information available. A cytogenetic analysis using conventional staining, C-banding, silver staining, and fluorescence in situ hybridization (FISH) with telomeric sequence probes were used to investigate the karyotype of seven Amazon species of the genus Hypsiboas belonging to the following intrageneric groups: H. punctatus (H. cinerascens), H. semilineatus (H. boans, H. geographicus, and H. wavrini), and H. albopunctatus (H. lanciformis, H. multifasciatus, and H. raniceps). The aim was to differentiate between the karyotypes and use the chromosomal markers to distinguish between the Hypsiboas groups. The data were compared with a previous phylogenetic proposal for these anurans. In addition, H. lanciformis, H. boans, and H. wavrini are described here for the first time, and we characterize the diploid numbers for H. cinerascens, H. geographicus, H. multifasciatus, and H. raniceps. RESULTS: The diploid number for all of the species analyzed was 24, with the exception of Hypsiboas lanciformis, which had 2n = 22 chromosomes. The constitutive heterochromatin distribution, nucleolar organizer region locations, and interstitial telomeric sites differed between the species. A hypothesis that the heterochromatic patterns are evolving is proposed, with the divergence of the groups probably involving events such as an increase in the heterochromatin in the species of the H. semilineatus group. The FISH conducted with the telomeric probes detected sites in the terminal regions of all of the chromosomes of all species. Interstitial telomeric sites were detected in three species belonging to the H. semilineatus group: H. boans, H. geographicus, and H. wavrini. CONCLUSION: The results of this study reinforce the complexity previously observed within the genus Hypsiboas and in the different groups that compose this taxon. More studies are needed focusing on this group and covering larger sampling areas, especially in the Brazilian Amazon, to improve our understanding of this fascinating and complex group.

Activity patterns of nucleolar organizer region during spermatogenesis of different curimatid species (Characiformes: Curimatidae).

The nucleolus is an important nuclear structure where transcription of ribosomal DNA (rDNA) takes place. During mitotic division, the nucleolus passes through different processes that inactivate rDNA transcription; in meiosis, its reassembly takes place during telophase II. The objective of this study was to identify the activity patterns and localization of nucleolar organizer regions (NORs) during meiotic division in fish species of the family Curimatidae. For this analysis, the meiotic division in five curimatid species was studied using silver nitrate impregnation, fluorescent in situ hybridization (FISH), and base-specific fluorochrome staining. Silver nitrate staining indicated the presence of a nucleolus in interphase nuclei, one chromosome pair in the spermatogonial metaphases, and one bivalent at the pachytene stage. No Ag-NORs were identified for cells at the diplotene, diakinesis, metaphase I, or metaphase II stages; however, FISH confirmed the presence of Ag-NORs in the nuclei, in spermatogonia, and at the pachytene phase. FISH identified this region during the other stages of meiosis, as did fluorochrome CMA3 staining, which revealed fluorescent marks corresponding to NORs during all stages of meiosis analyzed. The gene activity and localization of this ribosomal sequence during the different stages involved will also be discussed.

Polymorphisms of the nucleolus organizing regions in Loricaria cataphracta (Siluriformes, Loricariidae) of the upper Paraguay River basin indicate an association with transposable elements.

A cytogenetic analysis of Loricaria cataphracta revealed a diploid number of 2n = 64 chromosomes, distributed as 12 metacentric + 8 submetacentric + 2 subtelocentric + 42 acrocentric, with a fundamental number of 86. Analysis of the nucleolus organizing region (NOR) using silver nitrate impregnation and fluorescence in situ hybridization (18S rDNA probe) techniques showed intra-population chromosomal polymorphism that could be classified into five different patterns (I to V), involving four pairs of chromosomes (8, 9, 12, and 13). In pattern I, the NOR was located in pair 12, whereas in pattern II, the NOR was detected in pair 8; these two patterns were characterized as a simple-NOR system. A multiple NOR system was evident in the other patterns (III, IV, and V). In pattern III, the NOR was located in only one of the homologs of pairs 12 and 8, and in patterns IV and V, the NOR was observed in pair 12 and in only one of the homologs of pairs 9 and 13, respectively. In addition, C-band analysis also showed this pattern of variation, and characterized a polymorphism in relation to the constitutive heterochromatin; the composition of this region was GC-rich (positive CMA3) and 4',6-diamidino-2-phenylindole negative. Transposition of NOR sites for mobile elements is suggested to explain this polymorphism.

Chromosome evolution in dendropsophini (Amphibia, Anura, Hylinae).

Dendropsophini is the most species-rich tribe within Hylidae with 234 described species. Although cytogenetic information is sparse, chromosome numbers and morphology have been considered as an important character system for systematic inferences in this group. Using a diversity of standard and molecular techniques, we describe the previously unknown karyotypes of the genera Xenohyla, Scarthyla and Sphaenorhynchus and provide new information on Dendropsophus and Lysapsus. Our results reveal significant karyotype diversity among Dendropsophini, with diploid chromosome numbers ranging from 2n = 22 in S. goinorum, 2n = 24 in Lysapsus, Scinax, Xenohyla, and almost all species of Sphaenorhynchus and Pseudis, 2n = 26 in S. carneus, 2n = 28 in P. cardosoi, to 2n = 30 in all known Dendropsophus species. Although nucleolar organizer regions (NORs) and C-banding patterns show a high degree of variability, NOR positions in 2n = 22, 24 and 28 karyotypes and C-banding patterns in Lysapsus and Pseudis are informative cytological markers. Interstitial telomeric sequences reveal a diploid number reduction from 24 to 22 in Scarthyla by a chromosome fusion event. The diploid number of X. truncata corroborates the character state of 2n = 30 as a synapomorphy of Dendropsophus.

Molecular cytogenetics of nucleolar organizer regions in Phyllomedusa and Phasmahyla species (Hylidae, Phyllomedusinae): a cytotaxonomic contribution.

Chromosome numbers, morphology, and nucleolus organizer region (NOR) locations are useful cytological characters for taxonomic and evolutionary studies. In this study, we provide the first cytogenetic analysis of Phyllomedusa bahiana and Phasmahyla spectabilis, and report new cytogenetic data on variation in NOR numbers and positions in Phyllomedusa rohdei and Phyllomedusa nordestina using conventional staining, AgNOR-banding, and 45S rDNA fluorescence in situ hybridization. All 4 species showed 2n = 26 chromosomes. P. spectabilis and P. bahiana had only 1 pair of NOR-carrying chromosomes. P. bahiana showed an NOR length polymorphism, and a rare homomorphic self-compatibility for both NOR lengths in anurans. Variation in the number of NOR-bearing chromosomes was found between the sampled populations of P. nordestina, ranging from 3 to 4. This study also clarified previous conflicting results concerning the occurrence of inter- and intra-population NOR variation in P. rohdei. The variation, confirmed by 45S rDNA fluorescence in situ hybridization analysis, was congruent with results obtained from AgNOR-banding in all species.

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.

Chromosomal diversity in Hypostomus (Siluriformes, Loricariidae) with emphasis on physical mapping of 18S and 5S rDNA sites.

We examined chromosomes of three species of the genus Hypostomus, in order to contribute to the understanding of the karyotype evolution of this group. Specimens of H. ancistroides and H. nigromaculatus displayed differences in karyotype formulas, distribution and location of heterochromatin and nucleolus organizer regions when compared to other populations of the same species. We made the first cytogenetic characterization of H. tapijara, an endemic species in the Ribeira de Iguape River. These specimens had 2n = 66 chromosomes, while H. ancistroides showed 2n = 68 and H. nigromaculatus 2n = 76 chromosomes. Physical mapping of 18S and 5S rDNA sites of the three species showed simple, multiple and syntenic clusters. Synteny of ribosomal sites was found in H. ancistroides and H. tapijara, and an interspersed pattern between these sites in all chromosomes bearing the synteny was observed. We conclude that the genus Hypostomus has a high chromosome complexity that is accompanied by great morphological variation. It is evident that this group comprises an interesting model for understanding the chromosome evolution of Neotropical ichthyofauna.