rDNA Clusters Research Articles

First Cytogenetic Study of a Member of the Harvestman Family Neogoveidae (Opiliones: Cyphophthalmi: Sternophthalmi) from Cameroon with Description of a New SpeciesParogovia parasironoidessp. nov.

The harvestman family Neogoveidae (suborder Cyphophthalmi) is distributed across Neotropics, south-eastern USA and Western and Central Africa. Nevertheless, the core of its known diversity lies in Americas. The sole genus known from Africa is Parogovia, with only three species described so far. Here we describe a new species, Parogovia parasironoides sp. nov. from Cameroon, which is morphologically close to P. sironoides. In addition to the analyses of external morphology, spermatopositor and ovipositor, we also described the karyotype (2n=32), a course of the male mitosis and meiosis, and the distribution of major ribosomal RNA genes using fluorescence in situ hybridization (FISH) with 18S rDNA probe. In the karyotype, the one-armed chromosomes predominated and we detected one pair of the 18S rDNA cluster close to centromere region on long arms of subtelocentric chromosomes. Our study represents the first cytogenetic data in infraorder Sternophthalmi and plays an important role in the reconstruction of the karyotype evolution of Cyphophthalmi.

Karyotype Characterization of Nine Periwinkle Species (Gastropoda, Littorinidae).

Periwinkles of the family Littorinidae (Children, 1834) are common members of seashore littoral communities worldwide. Although the family is composed of more than 200 species belonging to 18 genera, chromosome numbers have been described in only eleven of them. A molecular cytogenetic analysis of nine periwinkle species, the rough periwinkles Littorina arcana, L. saxatilis, and L. compressa, the flat periwinkles L. obtusata and L. fabalis, the common periwinkle L. littorea, the mangrove periwinkle Littoraria angulifera, the beaded periwinkle Cenchritis muricatus, and the small periwinkle Melarhaphe neritoides was performed. All species showed diploid chromosome numbers of 2n = 34, and karyotypes were mostly composed of metacentric and submetacentric chromosome pairs. None of the periwinkle species showed chromosomal differences between male and female specimens. The chromosomal mapping of major and minor rDNA and H3 histone gene clusters by fluorescent in situ hybridization demonstrated that the patterns of distribution of these DNA sequences were conserved among closely related species and differed among less related ones. All signals occupied separated loci on different chromosome pairs without any evidence of co-localization in any of the species.

Cytb as a New Genetic Marker for Differentiation of Prototheca Species.

Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by Protothecawickerhamii, followed by Protothecazopfii and, sporadically, by Protothecacutis and Protothecamiyajii In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of Prototheca spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae.

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3′OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3′OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

Cytogenetic studies in Trachymyrmex holmgreni Wheeler, 1925 (Formicidae: Myrmicinae) by conventional and molecular methods

Over the past several decades, ant cytogenetic studies have focused on chromosome number and morphology; however, recently, additional information concerning heterochromatin composition and 45S rDNA location has become accessible. The fungus-growing ants are a peculiar ant group that cultivates fungus for food, and Trachymyrmex is suspected to be the sister group of leafcutter ants. Cytogenetic data are so far available for sixn Trachymyrmex species. The present study aimed to increase the knowledge about Trachymyrmex cytogenetics by the chromosomal characterization of Trachymyrmex holmgreni including the karyotyping, fluorochromes staining, 18S rDNA, and microsatellite (GA)15 fluorescence in situ hybridization (FISH). Karyotyped samples from four ant colonies showed 2n = 20 metacentric chromosomes. Centromeric heterochromatin rich in GC base pairs was detected in all chromosomes. FISH revealed the presence of rDNA clusters on the fourth chromosome pair, and an intense spreading of the microsatellite (GA)15 including exclusively euchromatic areas of the chromosomes. The GC-rich heterochromatin observed in different ant species may have a common origin and, thus, phylogenetic implication that needs to be further investigated. To the best of our knowledge, this study is the first report of the use of chromosomal physical location of repetitive DNA sequences by means of microsatellite probes in Formicidae.

Redescription, molecular features, and neotype deposition of Rhipicephalus pusillus Gil Collado and Ixodes ventalloi Gil Collado (Acari, Ixodidae).

Two species of ticks, Rhipicephalus pusillus Gil-Collado and Ixodes ventalloi Gil-Collado are redescribed, their molecular features (16S rDNA) compared with near species, and neotypes named and deposited. The male of R. pusillus is characterized by a smooth dorsal surface, with relatively short marginal grooves, not reaching the eyes and reaching the first festoon, longer than wide adanal plates, internally concave and widely rounded posteriorly, without internal spurs, accessory adanal plates not projecting over the cuticle, and a narrow, long and straight process on the spiracular plate. The female has very small porose areas, separated by 2.5-3 diameters, and a large triangular spur on the ventro-internal side of the palpal article 1. The nymph has lateral processes curved anteriorly in the ventral aspect of the basis capituli, long auriculae, and internal spurs on the four coxae. The larva of R. pusillus is separated by the presence of spurs on the ventral surface of palpal segment I, together with the rounded apices of the palpi. Features of 16S rDNA cluster this species near the R. sanguineus s.l. complex of species. The distinctive feature of the female of I. ventalloi is the long and curved auriculae, which are absent in every other species of Ixodes reported in the Western Palaearctic. The male is characterized by relatively long conscutal setae and deep and well defined cervical fields. The nymph is unique by having slightly apparent lateral carinae and small cornua, very abundant and long alloscutal setae, about six times longer than those on the scutum and internal spur of coxa I reaching coxa II. The larva of I. ventalloi has Md1-Md3 setae of the same size as the rest of the conscutal setae, which are only slightly longer than the scutal setae, cornua present, and anal groove clearly divergent. In addition to the unique combination of morphological features, the molecular 16rDNA sequence clearly separates I. ventalloi from other species of the I. ricinus group. We named and deposited in an internationally accessible collection the neotypes of both species, since the holotypes were lost.

Conserved number of U2 snDNA sites in Piabina argentea, Piabarchus stramineus and two Bryconamericus species (Characidae, Stevardiinae)

ABSTRACT The chromosomal location of 5S rRNA and U2 snRNA genes of Piabina argentea, Piabarchus stramineus and two Bryconamericus species from two different Brazilian river basins were investigated, in order to contribute to the understanding of evolutionary characteristics of these repetitive DNAs in the subfamily Stevardiinae. The diploid chromosome number was 2n = 52 for Bryconamericus cf. iheringii, Bryconamericus turiuba, Piabarchus stramineus and Piabina argentea. The 5S rDNA clusters were located on one chromosome pair in P. stramineus and B. cf. iheringii, and on two pairs in B. turiuba and P. argentea. The U2 snDNA clusters were located on the one pair in all species. Two-color FISH experiments showed that the co-localization between 5S rDNA and U2 snDNA in P. stramineus can represent a marker for this species. Thus, the present study demonstrated that the number of U2 snDNA clusters observed for the four species was conserved, but particular characteristics can be found in the genome of each species.

FISH-aimed karyotype analysis in Aconitum subgen. Aconitum reveals excessive rDNA sites in tetraploid taxa

The location of 5S and 35S rDNA sequences in chromosomes of four Aconitum subsp. Aconitum species was analyzed after fluorescence in situ hybridization (FISH). Both in diploids (2n = 2x = 16; Aconitum variegatum, A. degenii) and tetraploids (2n = 4× = 32; A. firmum, A. plicatum), rDNA repeats were localized exclusively on the shorter arms of chromosomes, in subterminal or pericentromeric sites. All analyzed species showed similar basal genome size (Cx = 5.31–5.71 pg). The most striking features of tetraploid karyotypes were the conservation of diploid rDNA loci and emergence of many additional 5S rDNA clusters. Chromosomal distribution of excessive ribosomal sites suggests their role in the secondary diploidization of tetraploid karyotypes.

A Comparative Chromosome Mapping Study in Japanese Podismini Grasshoppers (Orthoptera: Acrididae: Melanoplinae)

In the present paper, karyotypes of 7 Japanese Podismini species, Anapodisma beybienkoi, Fruhstorferiola okinawaensis, Parapodisma caelestis, P. mikado, P. setouchiensis, P. tenryuensis, and Sinopodisma punctata (2n♂ = 21, all acrocentric), are described and compared on the basis of conventional (C-banding, DAPI/CMA₃-staining, Ag-NOR) and molecular (FISH with 18S rDNA and telomeric probes) cytogenetic staining methods. This is the first study to report karyotypes of A. beybienkoi and P. caelestis. Differential staining techniques showed karyotypic diversity in these species. The number of 18S rDNA signals ranged from 2 to 6, and the signals were located on the autosomes or sex chromosomes. In all species, clusters of rDNA coincided with Ag-NORs. Telomeric signals occurred at the chromosome ends at the pachytene stage and seldom at other stages of meiosis. Paracentromeric and some distal and interstitial blocks of constitutive heterochromatin were detected in the chromosomes of Anapodisma, Fruhstorferiola, and Parapodisma species. Staining with DAPI and CMA₃ revealed 2 groups of heterochromatin composition. In addition, intraspecific differences in the number of rDNA clusters and C-bands were observed within Parapodisma species. Based on the evidence of cytogenetic characteristics, the monophyly of Tonkinacridina cannot be supported.

Chromosome Painting in Tyrant Flycatchers Confirms a Set of Inversions Shared by Oscines and Suboscines (Aves, Passeriformes)

Tyrannidae is the largest family of Passeriformes in the Neotropical region. However, despite an interesting chromosomal diversity, there are only few cytogenetic studies of this family, and most of these are based on conventional cytogenetics. Hence, we analyzed here the chromosomal diversity and karyotypical evolution of this group by chromosome painting in 3 different species - Pitangus sulphuratus, Serpophaga subcristata, and Satrapa icterophrys - and make comparisons with previous data. In addition to chromosome painting with Gallus gallus (GGA) and Leucopternis albicollis (LAL) probes, karyotypes were analyzed by conventional staining, C-banding, and FISH with 18S rDNA and telomeric probes. Although this family is characterized by extensive chromosomal variation, we found similar karyotypes and diploid numbers ranging from 2n = 80 in P. sulphuratus to 2n = 82 in S. subcristata and S. icterophrys. Constitutive heterochromatin was located centromerically in all 3 species. Clusters of 18S rDNA were present in 1 pair of microchromosomes, except in S. subcristata, where 2 pairs of microchromosomes were labeled. No interstitial telomeric sequences were detected. GGA and LAL whole-chromosome probes revealed the occurrence of fissions and both paracentric and pericentric inversions commonly seen in other Passeriformes. In general terms, tyrants show the typical karyotype found in Passeriformes, suggesting that the observed rearrangements occurred before the division of the suborders Oscines and Suboscines.

A karyotype comparison between Largus fasciatus and L. rufipennis (Heteroptera, Insecta) by conventional, chromosome banding and rDNA-FISH

The most important economic impact of Largus fasciatus and L. rufipennis is the feeding effect on potato, carrot, tobacco, and bean crops in Argentine. Largidae have a low diploid chromosome number, a sex chromosome system of X0/XX (male/female), except to one species, and a pair of m chromosomes, excluding the genus Largus. Cytogenetic and heterochromatin characterization, and the analysis of the location of rDNA genes between both species were performed. They possess a same diploid number 2n=12+X0/XX and very large chromosomes. The C-banding patterns showed discrete C-positive bands terminally located in all autosomes and the X chromosomes, which were observed in all stages of mitosis and meiosis. The main cytogenetic difference was detected in the location of the rDNA clusters. In L. fasciatus the hybridization signals were located at subterminal position in an autosomal bivalent, whereas in L. rufipennis a cluster of rDNA genes was located at one end of the X chromosome. As a result, the rDNA clusters are very useful tools for the study of the karyotype structure and chromosome evolution in groups with holokinetic chromosomes due to it can contribute to understand the karyotype evolution and taxonomic relationships among several taxa. On the other hand, the absence of m chromosomes and the presence of sex chromosome system X0 in species of Larginae could be considered as derived characters, which arose during karyotype evolution. We suggest that within this subfamily, the m chromosome pair could have lost its particular meiotic behaviour and became a regular autosomal pair, or fused to an autosomal pair, contributing thus to the increase in the chromosome size and, also, to the decrease in the chromosome number of the species. Therefore, chromosome studies are of great importance for understanding the evolutionary history of different insects groups because the data obtained, together with existing for other species, showed that different chromosome changes are involved in the evolution of the several species in the same family and even in Heteroptera.

Chromosomal dynamics, molecular organization and evolutionary patterns of major ribosomal DNA in the Grasshopper Abracris flavolineata (Acrididae)

In the grasshopper Abracris flavolineata it was documented huge intrapopulation variation for 18S rDNA in individuals collected in six populations, but causes for this pattern was not understand yet. In this work we aimed to elucidate the molecular structure, evolutionary patterns of the rDNA 18S distribution and its causes in the genome of A. flavolineata. It was used a combination of classical cytogenetic techniques, mapping of18S rDNA through Fluorescence in situ hybridization (FISH) and genomics analyses from entire sequenced genome by Illumina hiseq 2000. It was analyzed 48 individuals collected in six distantly sampling places from Brazil and Argentina. FISH analyses confirmed the variability of 18S rDNA for all six populations that presented between 6-11 chomosomes labeled. Signals were observed in chromosomes 1, 3, 6 and 9 in all individuals and also it was noticed variation of cluster sizes. It was not found a fixed pattern at intra- or interpopulation levels in population from the same geographic region or between distant ones. Silver nitrate staning revealed Nocleolar Organizer Regions (NORs) in chromosome 9 of all individuals, indicating that the expression of the rDNA cluster in this chromosome could be under selection to be expressed, and also could represent ancestral placement of this sequence. It is a pattern also shared with congeneric species. In some individuals chromosome 10 presented NORs, however for some of them FISH signals in this bivalent was not observed, indicating cryptic loci in accordance with ‘dispersion-amplification-deletion’ model, suggested also for rDNA dispersion for other grasshoppers. Finally the reconstruction of entire 45S rDNA presented different coverages in the conserved genes (18S, 5.8S e 25S) and in the internal transcripts (ITS 1 e ITS 2) reveling that huge part of the copies are pseudogenes. The transposons Nimb and R2 are present in 45S rDNA, although apparently they are not able to transpose sequences. All data together suggests that the high amount of rDNA in A. flavolineata genome is caused by occurrence of pseudogene sequences that were moved random between chromosomes caused most parsimonialy by ectopic recombination, extra-chromosomal DNA or even by transposons not localized in the 45S cluster

In Situ Localization of Ribosomal Sites in Peckoltia and Ancistomus (Loricariidae: Hypostominae) from the Amazon Basin.

Loricariidae is a diverse group of fish from the neotropical region, occupying a wide variety of freshwater environments. Cytogenetic data have brought important insights into Loricariidae diversity because they help validate undescribed species as well as our understanding of inter- and intraspecific diversity. However, conventional cytogenetic approaches are limited in their ability to detect variability in some lineages, as seen in the Peckoltia clade, owing to their apparent conserved karyotype. Thus, the aim of this work was to map 5S and 18S ribosomal (rDNA) sites in five species of Peckoltia and one species of Ancistomus from the Amazon basin, and discusses the mechanisms of organization and diversification of these clusters. The species analyzed were found to have 2n = 52 and share KF = 38 m-sm +14st-a chromosomes, except Peckoltia vittata with KF = 34 m-sm +18st-a. Extensive variations in the number and location of 5S and 18S rDNA sites were observed among species. These data indicate that inversions are not the most important events in karyotype evolution in this group, and should prove useful in identifying the species studied here. In addition to inversions, transpositions are important evolutionary events that are involved at least in rDNA clusters spreading in Peckoltia and probably in other species of Hypostominae.

Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish

Co-located 5S rDNA genes and interstitial telomeric sites (ITS) revealed the involvement of multiple 5S rDNA clusters in chromosome rearrangements of Loricariidae. Interstitial (TTAGGG)n vestiges, in addition to telomeric sites, can coincide with locations of chromosomal rearrangements, and they are considered to be hotspots for chromosome breaks. This study aimed the molecular characterization of 5S rDNA in two Rineloricaria latirostris populations and examination of roles of 5S rDNA in breakpoint sites and its in situ localization. Rineloricaria latirostris from Brazil's Das Pedras river (2n = 46 chromosomes) presented five pairs identified using a 5S rDNA probe, in addition to a pair bearing a co-located ITS/5S rDNA. Rineloricaria latirostris from the Piumhi river (2n = 48 chromosomes) revealed two pairs containing 5S rDNA, without ITS. A 702-bp amplified sequence, using 5S rDNA primers, revealed an insertion of the hAT transposable element (TE), referred to as a degenerate 5S rDNA. Double-FISH (fluorescence in situ hybridization) demonstrated co-localization of 5S rDNA/degenerate 5S rDNA, 5S rDNA/hAT and ITS/5S rDNA from the Das Pedras river population. Piumhi river isolates possessed only 5S rDNA sites. We suggest that the degenerate 5S rDNA was generated by unequal crossing over, which was driven by invasion of hAT, establishing a breakpoint region susceptible to chromosome breakage, non-homologous recombination and Robertsonian (Rb) fusion. Furthermore, the presence of clusters of 5S rDNA at fusion points in other armored catfish species suggests its re-use and that these regions represent hotspots for evolutionary rearrangements within Loricariidae genomes.

Cytogenetic Characterization of Eight Odonata Species Originating from the Curonian Spit (the Baltic Sea, Russia) Using C-Banding and FISH with 18S rDNA and Telomeric (TTAGG)n Probes

We studied the karyotypes of 8 dragonfly species originating from the Curonian Spit (the Baltic Sea, Russia) using C-banding and FISH with 18S rDNA and “insect” telomeric (TTAGG)_n probes. Our results show that Leucorrhinia rubicunda, Libellula depressa, L. quadrimaculata, Orthetrum cancellatum, Sympetrum danae, and S. vulgatum from the family Libellulidae, as well as Cordulia aenea and Epitheca bimaculata from the family Corduliidae share 2n = 25 (24 + X) in males, with a minute pair of m-chromosomes being present in every karyotype except for that of C. aenea. Major rDNA clusters are located on one of the large pairs of autosomes in all the species. No hybridization signals were obtained by FISH with the (TTAGG)_n probe in the examined species with the only exception of S. vulgatum. In this species, clear signals were detected at the ends of almost all chromosomes. This finding raises the possibility that in Odonata the canonical “insect” (TTAGG)_n telomeric repeat is in fact present but in very low copy number and is consequently difficult to detect by in situ hybridization. We conclude that more work needs to be done to answer questions about the organization of telomeres in this very ancient and thus phylogenetically important insect order.

Application of cytogenetic markers in the taxonomy of flat rock scorpions (Scorpiones: Hormuridae), with the description of Hadogenes weygoldti sp. n.

In the present study, we performed the first comparative cytogenetic study in Hadogenes species using both standard and molecular cytogenetic approaches. Information about the diploid set, number and distribution of 18S rDNA and telomeric sequences was obtained from three South African species, Hadogenes trichiurus (Gervais, 1843), H. zuluanus Lawrence, 1937 and H. weygoldti sp. n.. All species analysed differ considerably in the number of chromosomes (H. trichiurus 2n=48, H. zuluanus 2n=80, H. weygoldti sp. n. 2n=113). In contrast, the number of 18S rDNA clusters and distribution of telomeric sequences represent rather stable cytogenetic characters in Hadogenes. Within all karyotypes, we identified one pair of 18S rDNA clusters. The telomeric signals were exclusively on the terminal chromosomal regions. Interestingly, the chromosomal location of 18S rDNA clusters varied from terminal to interstitial in species karyotypes, indicating the presence of hidden structural chromosomal changes. Additionally, the present comparative study is complemented by the description of a new species, H. weygoldti sp. n., based on specific karyotype features and morphological characters. Finally, our cytogenetic results are compared with known chromosomal data of other Hadogenes species, and the use of cytogenetic approaches in the taxonomy of scorpions is discussed.

Repetitive DNAs and shrink genomes: A chromosomal analysis in nine Columbidae species (Aves, Columbiformes).

An extensive karyotype variation is found among species belonging to the Columbidae family of birds (Columbiformes), both in diploid number and chromosomal morphology. Although clusters of repetitive DNA sequences play an important role in chromosomal instability, and therefore in chromosomal rearrangements, little is known about their distribution and amount in avian genomes. The aim of this study was to analyze the distribution of 11 distinct microsatellite sequences, as well as clusters of 18S rDNA, in nine different Columbidae species, correlating their distribution with the occurrence of chromosomal rearrangements. We found 2n values ranging from 76 to 86 and nine out of 11 microsatellite sequences showed distinct hybridization signals among the analyzed species. The accumulation of microsatellite repeats was found preferentially in the centromeric region of macro and microchromosomes, and in the W chromosome. Additionally, pair 2 showed the accumulation of several microsatellites in different combinations and locations in the distinct species, suggesting the occurrence of intrachromosomal rearrangements, as well as a possible fission of this pair in Geotrygon species. Therefore, although birds have a smaller amount of repetitive sequences when compared to other Tetrapoda, these seem to play an important role in the karyotype evolution of these species.

Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa (Pleuronectiformes: Cynoglossidae)

AbstractThe eukaryotic ribosomal DNA (rDNA) cluster consists of multiple copies of three genes (18S, 5.8S, and 28S rDNA) and two internal transcribed spacers (ITS1 and ITS2). In recent years, an increasing number of rDNA sequence polymorphisms have been identified in numerous species. In the present study, we provide 33 complete ITS (ITS1-5.8S-ITS2) sequences from twoSymphurus plagiusaindividuals. To the best of our knowledge, these sequences are the first detailed information on ITS sequences in Pleuronectiformes. Here, two divergent types (Type A and B) of the ITS1-5.8S-ITS2 rDNA sequence were found, which mainly differ in sequence length, GC content, nucleotide diversity (π), secondary structure and minimum free energy. The ITS1-5.8S-ITS2 rDNA sequence of Type B was speculated to be a putative pseudogene according to pseudogene identification criteria. Cluster analysis showed that sequences from the same type clustered into one group and two major groups were formed. The high degree of ITS1-5.8S-ITS2 sequence polymorphism at the intra-specific level indicated that theS. plagiusagenome has evolved in a non-concerted evolutionary manner. These results not only provide useful data for ribosomal pseudogene identification, but also further contribute to the study of rDNA evolution in teleostean genomes.

Cytogenetics characterization of Crenuchus spilurus (Günther, 1863): a remarkable low diploid value within family Crenuchidae (Characiformes)

Conventional and molecular cytogenetic analyses were performed in specimens of the Neotropical Crenuchus spilurus freshwater fish species from a single location (Caete River, Brazil). All specimens presented diploid values of 2n = 38 chromosomes (12 m + 4sm + 2st + 20a), the lowest reported for family Crenuchidae up to now. A single pair of nucleolar organizing regions (NORs) was detected in the subtelocentric chromosome pair no. 9 by silver-staining and fluorescence in situ hybridization (FISH) with 18S rDNA sequence-specific probe. Two pairs of 5S rRNA gene clusters were found either interstitial or terminally located in the long arms of the acrocentric chromosome pairs nos. 10 and 13. Heterochromatic regions were clearly observed in the short arms of the NOR-bearing chromosome pair and weakly-positive to the pericentromeric regions of most acrocentric chromosomes. Additionally, no sex chromosomes were identified in the surveyed specimens. Crenuchidae have signals of several mechanisms involved in karyotype diversification within this family: differential location of heterochromatin-rich regions, multiplication, and translocation of rDNA clusters, presence/absence of sex chromosomes, macrostructural changes in morphology and number of chromosomes. This variety of karyotype patterns reveals the importance of widening cytogenetic studies to more taxa for better know the chromosomal evolution occurred in this group.

Uncovering the molecular organization of unusual highly scattered 5S rDNA: The case of Chariesterus armatus (Heteroptera)

One cluster of 5S rDNA per haploid genome is the most common pattern among Heteroptera. However, in Chariesterus armatus, highly scattered signals were noticed. We isolated and characterized the entire 5S rDNA unit of C. armatus aiming to a deeper knowledge of molecular organization of the 5S rDNA among Heteroptera and to understand possible causes and consequences of 5S rDNA chromosomal spreading. For a comparative analysis, we performed the same approach in Holymenia histrio with 5S rDNA restricted to one bivalent. Multiple 5S rDNA variants were observed in both species, though they were more variable in C. armatus, with some of variants corresponding to pseudogenes. These pseudogenes suggest birth-and-death mechanism, though homogenization was also observed (concerted evolution), indicating evolution through mixed model. Association between transposable elements and 5S rDNA was not observed, suggesting spreading of 5S rDNA through other mechanisms, like ectopic recombination. Scattered organization is a rare example for 5S rDNA, and such organization in C. armatus genome could have led to the high diversification of sequences favoring their pseudogenization.