NOR Site Research Articles

Insights into the Karyotype Evolution of Charinidae, the Early-Diverging Clade of Whip Spiders (Arachnida: Amblypygi).

Simple SummaryWhip spiders (Amblypygi) are spectacular arachnids characterized by powerful raptorial pedipalps and exceptionally elongated forelegs. Although cytogenetic data from amblypygids might be, given their phylogenetic position, important for the reconstruction of arachnid ka-ryotype evolution, cytogenetics of this order is still largely understudied. Here, we applied conventional and molecular cytogenetics to describe the karyotype patterns in Charinidae—the family placed almost at the base of amblypygid phylogeny, thereby providing insights into the ancestral amblypygid karyotype traits. We surveyed four Charinus and five Sarax species and found a wide range of diploid chromosome numbers (2n) in both genera. Representatives with lower 2n possessed generally a higher proportion of biarmed (metacentric and submetacentric) chromosomes. Our results indicate the important roles of chromosome fusions and pericentric inversions in the karyotype differentiation of Charinidae, similarly to that suggested previously for neoamblypygids. Our data, gathered from C-banding, fluorescent banding, and chromosomal mapping of ribosomal DNA and telomeric repeats, bring evidence for the action of these rearrangements and suggest the general trajectory towards 2n reduction from ancestral high chromosome counts. However, we cannot rule out the contribution of chromosome fissions. Whip spiders (Amblypygi) represent an ancient order of tetrapulmonate arachnids with a low diversity. Their cytogenetic data are confined to only a few reports. Here, we analyzed the family Charinidae, a lineage almost at the base of the amblypygids, providing an insight into the ancestral traits and basic trajectories of amblypygid karyotype evolution. We performed Giemsa staining, selected banding techniques, and detected 18S ribosomal DNA and telomeric repeats by fluorescence in situ hybridization in four Charinus and five Sarax species. Both genera exhibit a wide range of diploid chromosome numbers (2n = 42–76 and 22–74 for Charinus and Sarax, respectively). The 2n reduction was accompanied by an increase of proportion of biarmed elements. We further revealed a single NOR site (probably an ancestral condition for charinids), the presence of a (TTAGG)n telomeric motif localized mostly at the chromosome ends, and an absence of heteromorphic sex chromosomes. Our data collectively suggest a high pace of karyotype repatterning in amblypygids, with probably a high ancestral 2n and its subsequent gradual reduction by fusions, and the action of pericentric inversions, similarly to what has been proposed for neoamblypygids. The possible contribution of fissions to charinid karyotype repatterning, however, cannot be fully ruled out.

Karyomorphological delineation and linear differentiation of microsatellite patterns, and meiosis in giant Asian river frog (Limnonectes blyhii) from Thailand

The present investigation provides information on linear chromosomal differentiation and microsatellite patterns [d(CA)15, d(CGG)10, d(TA)15] in the genome of giant Asian river frog (Limnonectes blyhii). Five male and female samples each collected from southern Thailand, were used for the study. The metaphase chromosome preparations were prepared from the bone marrow using the standard protocol. Results show that both male and female have the diploid chromosome number 2n = 24, and the fundamental number NF = 48. The karyotypes compose of 4 large metacentric, 4 large submetacentric, 2 medium metacentric, 8 small metacentric and 6 small submetacentric chromosomes. The Ag-NOR, FISH as well as hybridization with the microsatellite d(CGG)10 confirm the location of NOR site in the subcentromeric region on chromosome pair 11. The in situ localization pattern of d(CA)15 microsatellite was positive on chromosome pairs 1 and 6, while microsatellite d(TA)15 show its location in the long arm of chromosome 5,8 and 10. The information provided has value as species specific marker for this species.

Chromosome spreading of the (TTAGGG)n repeats in the Pipa carvalhoi Miranda-Ribeiro, 1937 (Pipidae, Anura) karyotype.

Pipidae is a clade of Anura that diverged relatively early from other frogs in the phylogeny of the group. Pipids have a unique combination of morphological features, some of which appear to represent a mix of adaptations to aquatic life and plesiomorphic characters of Anura. The present study describes the karyotype of Pipa carvalhoi Miranda-Ribeiro, 1937, including morphology, heterochromatin distribution, and location of the NOR site. The diploid number of P. carvalhoi is 2n=20, including three metacentric pairs (1, 4, 8), two submetacentric (2 and 7), three subtelocentric (3, 5, 6), and two telocentric pairs (9 and 10). C-banding detected centromeric blocks of heterochromatin in all chromosome pairs and the NOR detected in chromosome pair 9, as confirmed by FISH using the rDNA 28S probe. The telomeric probes indicated the presence of interstitial telomeric sequences (ITSs), primarily in the centromeric region of the chromosomes, frequently associated with heterochromatin, suggesting that these repeats are a significant component of this region. The findings of the present study provide important insights for the understanding of the mechanisms of chromosomal evolution in the genus Pipa, and the diversification of the Pipidae as a whole.

Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size.

A detailed cytogenetic analysis of the aspidogastrean fluke Aspidogaster limacoides revealed a karyotype consisting of six medium-sized chromosome pairs. The first and the last pairs were two-armed while four remaining were one-armed; 2n = 12, n = 1 m + 1 m - sm + 4a. Fluorescence in situ hybridization with 18S ribosomal DNA (rDNA) probe detected a single cluster of ribosomal genes (NOR) located in pericentromeric regions of the long arms of the third chromosome pair in a site of secondary constriction apparent in meiotic prophase, especially in diplotene. The silver nitrate staining showed only a single active NOR site on one of homologous chromosomes in the majority of spermatogonia and spermatocyte divisions. A course of meiosis corresponded to standard schemes. The nucleolus was apparent in early meiotic spermatocytes and disintegrated by the end of pachytene. For the first time in Aspidogastrea, the genome size was determined. The flow cytometry showed 1.21 pg DNA per haploid nucleus in A. limacoides which is in accordance with relatively low genome sizes of other flukes and tapeworms (Neodermata). A comparison of cytogenetic data available to date in the fluke sister groups Aspidogastrea and Digenea suggests that the lower chromosome number of Aspidogastrea might represent an ancestral condition and their split might have been accompanied by an increase in chromosome number via either chromosome fissions or paleopolyploidy.

Karyoevolutionary aspects of Atlantic hogfishes (Labridae–Bodianinae), with evidence of an atypical decondensed argentophilic heterochromatin

Fish from the family Labridae elicit considerable ecological interest, especially due to their complex interactions with the reef environment. Different karyoevolutionary tendencies have been identified in the subfamilies Bodianinae, Corinae and Cheilinae. Chromosomal analyses conducted in the Atlantic species Bodianus rufus (2n=48; 6m+12sm+14st+16a, FN=80), Bodianus pulchellus (2n=48; 4m+12sm+14st+18a, FN=78) and Bodianus insularis (2n=48; 4m+12sm+14st+18a, FN=78) identified Ag-NOR/18SrDNA sites located only in the terminal region of the short arm (p) of the largest subtelocentric pair. The 5S rDNA genes were mapped in the terminal region of the long arm (q) of the largest acrocentric pair and the p arm of chromosome 19 in B. insularis. The karyotype of the three species shows an extensive heterochromatic and argentophilic region, exceptionally decondensed, located in the p arm of the second subtelocentric pair. This region does not correspond to a NOR site, since it is not hybridized with 18S rDNA probes, and is not GC-rich, as generally occurs with nucleolus organizer regions of lower invertebrates. Heterochromatin in the three species is reduced and distributed over the centromeric and pericentromeric regions of chromosomes. The elevated number of two-armed chromosomes in species of Bodianus, in relation to other Labridae, shows karyotype diversification based on pericentric inversions, differentiating them markedly in terms of evolutionary tendencies that occur in subfamilies Corinae and Cheilininae. Structural cytogenetic similarities between B. pulchellus and B. insularis, in addition to the conserved chromosomal location pattern of ribosomal multigenic families, indicate phylogenetic proximity of these species.

Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P. saltica

Taxonomic changes have frequently occurred in the anuran genus Pseudopaludicola as a consequence of high morphological similarity among its species. The present work reports karyotypic analysis of three Pseudopaludicola species sampled in their type locality and four Pseudopaludicola populations from distinct localities, aiming at contributing to the systematics of this genus. Chromosomes were stained with Giemsa or submitted to the silver staining (Ag-NOR) and C-banding techniques. The karyotype was 2n=22 in P. mineira, Pseudopaludicola sp. and two populations of P. saltica. The chromosome pair 8 was heteromorphic in P. saltica, characterizing a XX/XY sex-determination system with telocentric X and submetacentric Y. Highly similar karyotypes with 2n=18 chromosomes were observed in P. canga, P. aff. canga from Barreirinhas, State of Maranhão, Uberlândia, State of Minas Gerais and Icém, State São Paulo. The high similarity among the karyotypes 2n=18 suggested that the populations of P. aff. canga belong to the group 'pusilla', the same group of P. canga. The data demonstrated also that P. aff. canga from Barreirinhas (northeast region) is cytogenetically identical to P. canga with regarding the NOR site position in pair 3 and the presence of a heterochromatic block in the pair 2, whereas P. aff. canga from Uberlândia and Icém (southeast) had the NOR in the pair 9. Moreover, the cytogenetic data discriminated P. mineira and Pseudopaludicola sp. from the previously analyzed species with 22 chromosomes, and suggested that Pseudopaludicola sp. is an undescribed species. Sexual heteromorphic chromosomes are firstly reported in Pseudopaludicola and the data indicated the need of an extensive taxonomic review in this genus.

Karyotypic similarity among Barycholos ternetzi and five species of the genus Eleutherodactylus from southeastern Brazil (Anura, Brachycephalidae)

Comparative cytogenetic analyses were carried out in six species of Brachycephalidae from southeastern Brazil. Barycholos ternetzi, Eleutherodactylus binotatus, Eleutherodactylus guentheri, Eleutherodactylus juipoca, Eleutherodactylus parvus and Eleutherodactylus sp. have 2 n = 22 karyotypes with a marked variation in the morphology of chromosome pairs 8, 10 and 11, which are of telocentric or metacentric types, resulting in FN = 38, 40 and 44. Eleutherodactylus have a single chromosome pair bearing Ag-NOR, i.e. pair 1 in E. binotatus, pair 6 in E. guentheri and E. parvus, and pair 11 in E. juipoca and Eleutherodactylus sp. In contrast, B. ternetzi showed Ag-positive sites in the chromosome pairs 1, 4, 5, 9 and 11, and only one to three labelings per metaphase in each individual. Nevertheless, the main chromosome pair with Ag-NOR in the species seems to be the 11th, like in E. juipoca and Eleutherodactylus sp. The NOR site was confirmed by fluorescence in situ hybridization (FISH) technique in E. binotatus and in B. ternetzi, bearing 1p1p and 9p11p11p Ag-NOR pattern, respectively. All the species exhibited predominantly centromeric C-banding pattern, but interstitial bands have also been observed in some cases. In E. binotatus, there is an indication of geographical difference in the distribution of the interstitial C-bands. The fluorochromes GC-specific chromomycin A 3 (CMA 3) and AT-specific 4′,6-diamidino-2-phenylindole (DAPI), with distamycin A (DA) counterstaining, provided the molecular content of some repetitive regions in the karyotypes of the species. One male of E. binotatus presented an extensive heteromorphism, involving at least five different pairs, probably as a consequence of multiple reciprocal translocations. Such rearrangements might be responsible for the multivalent chain seen in the meiosis of this specimen, as well as in another male, although not exhibiting chromosome heteromorphism. The remaining males and those belonging to the other species have always shown 11 bivalents in diplotene and metaphase I cells. In all male specimens, metaphases II presented 11 chromosomes. Despite the observed discrepancies, the five species of Eleutherodactylus have a great uniformity in the 2 n = 22 karyotypes, suggesting an assemblage of species from southeastern and southern Brazil, in contrast to northern and northeastern assemblage which is characterized by higher diploid numbers. Undoubtedly, B. ternetzi could be included in that proposed assemblage, due to its karyotypic similarity with the Eleutherodactylus species, as evidenced in the present study. This fact strongly supports the close relationships of both genera, previously inferred on the basis of several characters shared by their species.

Chromosomal Study of Colostethus brunneus from the Type Locality and Two Related Species (Anura – Dendrobatidae)

In this paper, we report a chromosomal study of three Brazilian species of Colostethus, C. brunneus from the type locality, Colostethus sp. (aff. trilineatus), and Colostethus sp., which is morphologically similar to C. brunneus. The diploid number for C. brunneus was 2n = 24 chromosomes, in agreement with that previously described for specimens from Peru. Colostethus sp. (aff. trilineatus) and Colostethus sp. showed a very similar karyotype with 22 chromosomes. The NOR was located on pair 3 in C. brunneus, on pair 4 in Colostethus sp. (aff. trilineatus), and on pair 2 in Colostethus sp. In one specimen of Colostethus sp., an additional NOR site was located on pair 7 in only one of the homologs. This extra Ag-NOR site was confirmed by FISH using an rDNA probe. In addition to the NOR location, the C-banding pattern was also species-specific, despite the similar chromosomal morphology of the species. These results indicate that although these species may be closely related, there is a clear dichotomy in their chromosome number.

Chrosomal banding inParodon hilarii(Pisces Parodontidae), with emphassis on constitutive heterochromatin

Abstract Several banding methods were employed to analyze the chromosomes of the fish Parodon hilarii, i.e., C-banding, digestion with the restriction endonuclease Hae III, G-banding (trypsin), incorporation of 5-BrdU, and base-specific fluorochrome staining, with emphasis on the constitutive heterochromatin. The pattern obtained after the chromosomal cleavage with the restriction enzyme Hae III was similar to that of the C-banding. The incorporation of 5-bromodeoxiuridin (5-BrdU) in the chromosomes was able to detect several heterochromatin segments of late replication, with exception of some C-banded sites which were not evidenced after the 5-BrdU treatment. The terminal segment of the long arm of the 15th chromosome pair, which corresponds to a NOR site, seems to be composed by AT-rich heterochromatic regions interspersed with CG-rich regions. Besides, the long arm of the W chromosome shows a structural differentiation concerning the distribution of the heterochromatin.

Polymorphism of major ribosomal gene chromosomal sites (NOR-phenotypes) in the hybridogenetic fish Squalius alburnoides complex (Cyprinidae) assessed through crossing experiments

Chromosomal locations of major ribosomal sites, i.e. NOR-phenotypes, were assigned in Squalius alburnoides complex using sequential chromomycin A3 (CMA3)- and silver (Ag)-staining. This hybridogenetic Iberian minnow comprises diploid, triploid and tetraploid forms that arose by interspecific hybridisation between S. pyrenaicus and an unknown species. Inheritance of NOR patterns was studied by means of crossing experiments involving most diploid-polyploid forms of the S. alburnoides complex with identified specific genotype constitution. In all the specimens studied, the NORs were localised in the short arms of submetacentric chromosomes. Although S. pyrenaicus presented only one pair of NOR-bearing chromosomes, the data from experimental crosses evidenced that S. alburnoides complex was characterised by a multiple NOR phenotype composed of one chromosome pair with stable NORs and two chromosome pairs with NOR site polymorphism of presence/absence type. These data suggest that the karyotype of the unknown parental species of the S. alburnoides complex should have a multiple NOR pattern and emphasised the role of the all-male diploid linage in the dynamics and evolutionary potential of the S. alburnoides complex allowing the preservation of the missing ancestor genome. Cross-analyses evidenced that in spite of the high polymorphic nature of NORs in this fish complex, we have no reason to reject the hypothesis that their inheritance patterns were in accordance with Mendelian segregation.

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes, Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the São Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n = 54 and FN = 108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.

In Situ Restriction Endonucleases Digestion by on the Nucleolar Organizer Region(NOR) in Neuroblast Mitotic Chromosomes of Bradysia hygida(Diptera: Sciaridae).

In this work we analyzed the in situ restriction endonucleases digestion on the nucleolar organizer region, NOR, localized in C chromosomes of Bradysia hygida. Neuroblast chromosomes in situ digestion with PstI revealed evident chromatin extraction at the NOR region on both homologous C chromosomes, while the digestion with EcoRI or HindIII removed only one of the C chromosomes NOR chromatin. The same results were observed when in situ digestion with HindIII+PstI and HindIII+EcoRI were processed. These data are correlated with the fragments size by hybridization of purified genomic DNA with Rhynchosciara americana rDNA. The cleavage with EcoRI or PstI produced fragments of high molecular weight, while the single digestion with HindIII and combined with EcoRI or PstI only showed fragments of low molecular weight. In conclusion, no correlation between the size of the fragment and the in situ digestion was observed. The accessibility of the enzyme at the NOR site can be facilitated when the chromatin is an open state and the region presents transcriptional activity, allowing the digestion and removal of the chromatin out of the chromosome.

A new karyotype in Callicebus torquatus (Cebidae, Primates).

We describe a new karyotype of Callicebus torquatus using conventional staining, G-banding with Wright Stain, CBG, Ag-NOR staining and fluorescence in situ hybridization (FISH) with human telomere probes and comparative analysis with the previously reported karyotype of C. torquatus torquatus (2n = 20). We studied a female specimen maintained in captivity at the Centro Nacional de Primatas (Para, Brazil). This titi monkey presented 2n = 22, with four large biarmed and six acrocentric autosome pairs; the X chromosome is a medium submetacentric. C-bands were revealed at the centromeric region of all acrocentrics and X chromosome; punctual C-bands also are visualized at the centromeric region in the large biarmed pairs. The NOR site was located at the long arm of pair 4, at the position of a conspicuous secondary constriction. Hybridization signals were detected exclusively at the terminal region of all chromosomes. The karyotype described here has one acrocentric pair more than that found in the literature and also differs by amount and distribution of constitutive heterochromatin. Our data support the notion that the torquatus group may be composed of distinct species, each with its own karyotype.

Chromosome comparison between two species of Phyllostomus (Chiroptera - Phyllostomidae) from Eastern Amazonia, with some phylogenetic insights

The karyotypes of Phyllostomus discolor and P. hastatus from Eastern Amazonia were studied by G-, C-, G/C sequential and Ag-NOR techniques. Both species presented 2n = 32, with the autosome complement composed of 30 bi-armed in P. discolor and 28 bi-armed plus 1 acrocentric in P. hastatus. In both species, the X chromosome is medium submetacentric while the Y is minute acrocentric. The present study found only one difference between the karyotypes of P. discolor and P. hastatus: the smallest autosome (pair 15) is bi-armed in discolor and acrocentric in hastatus, a result best explained by pericentric inversion. The C-banding revealed constitutive heterochromatin only at the centromeric regions of all chromosomes, with the NOR site located at the distal region of short arm of pair 15, in both species. The taxon P. discolor is considered primitive for genus Phyllostomus and the bi-armed form of pair 15 is the assumed primitive condition which, rearranged by a pericentric inversion originated the acrocentric from found in P. hastatus.

Genome characterization and relationships between two species of the genusLobelia (Campanulaceae) determined by repeated DNA sequences

Three repeated DNA sequences (rDNA 5S, 18S-5.8S-26S and telomeric repeats) were localised in the genomes ofLobelia brasiliensis andL. imperialis var.kanitzii (subg.Tupa), both with 2n = 28, by fluorescence in situ hybridization (FISH). The results were used to analyse the genomic relationship between the species. With probe pTa71, the karyotypes of these species showed only one NOR site. Probe pTa794, which contains 5S rDNA, demonstrated differences between the species. Telomeric sequences, studied with probe pLT11, were not detected in ectopic sites, but different telomeres showed signals of varying intensity. Based on the results obtained, considerations are made on karyotype evolution inLobelia.

Analysis of the nucleolar organizing regions in the ant Tapinoma nigerrimum (Hymenoptera, Formicidae).

This study analyses the NORs of Tapinoma nigerrimum, a species that, as known from previous studies, has various chromosomes which carry a NOR site. The analysis was made by a combination of three methods: silver nitrate staining, in situ hybridization with fluorescein-or digoxigenin-labelled probes, and staining with the CG-specific fluorochrome chromomycin A3. The silver staining technique showed an Ag-positive region on chromosome 6 and on various other chromosomes. However, the application of in situ hybridization techniques showed only one positive signal in the proximal region of the short arm of chromosome 6 of T. nigerrimum. Similar results were observed by CMA banding. The absence of rDNA genes or the presence of only a small number of these, not detectable with the above probes, might explain the absence of hybridization signal in the remaining chromosomes.

Analysis of the nucleolar organizing regions in the ant Tapinoma nigerrimum (Hymenoptera, Formicidae)

This study analyses the NORs of Tapinoma nigerrimum, a species that, as known from previous studies, has various chromosomes which carry a NOR site. The analysis was made by a combination of three methods: silver nitrate staining, in situ hybridization with fluorescein- or digoxigenin-labelled probes, and staining with the CG-specific fluorochrome chromomycin A3. The silver staining technique showed an Ag-positive region on chromosome 6 and on various other chromosomes. However, the application of in situ hybridization techniques showed only one positive signal in the proximal region of the short arm of chromosome 6 of T. nigerrimum. Similar results were observed by CMA banding. The absence of rDNA genes or the presence of only a small number of these, not detectable with the above probes, might explain the absence of hybridization signal in the remaining chromosomes.

Localization of the nucleolar organizer and heterochromatin evolution in three European brown frogs: Rana dalmatina, Rana graeca, Rana iberica

AbstractThis study investigates the karyotypes, genome sizc, constitutive heterochromatin, and NOR localization in three European brown frog species: Rana iberica, R. dalmatina and R. graeca. The three species all possess a karyotype of 26 chromosomes. The genome size is respectively 6.9, 9.7 and 11.3 pg/N in R. iberica, R. dalmatina and R. graeca. Constitutive heterochromatin is 1.7% in R. iberica, 3.1 % in R. dalmatina and 7.0% in R. graeca. The NOR is located on the long arm of the 10th chromosome in R. iberica and R. graeca, whereas it is pericentromerically localized on the short arm of the 3rd chromosome in R. dalmatina. A single NOR extrasite is found in R. graeca. Results suggest that European brown frogs have evolved independently from North American and Asian groups. Moreover, the variation observed in constitutive heterochromatin is informative of different amplification levels of satellite-like DNA. Data also suggest that in R. dalmatina NOR localization on the 3rd chromosome may be derivcd by amplification of a pre-existing NOR site.