5S rDNA Sites Research Articles

Cytogenetic Analysis and Chromosomal Mapping of Repetitive DNA in Melipona Species (Hymenoptera, Meliponini)

Stingless bees of the genus Melipona are subdivided into 4 subgenera called Eomelipona, Melikerria, Melipona sensu stricto, and Michmelia according to species morphology. Cytogenetically, the species of the genus Melipona show variation in the amount and distribution of heterochromatin along their chromosomes and can be separated into 2 groups: the first with low content of heterochromatin and the second with high content of heterochromatin. These heterochromatin patterns and the number of chromosomes are characteristics exclusive to Melipona karyotypes that distinguish them from the other genera of the Meliponini. To better understand the karyotype organization in Melipona and the relationship among the subgenera, we mapped repetitive sequences and analyzed previously reported cytogenetic data with the aim to identify cytogenetic markers to be used for investigating the phylogenetic relationships and chromosome evolution in the genus. In general, Melipona species have 2n = 18 chromosomes, and the species of each subgenus share the same characteristics in relation to heterochromatin regions, DAPI/CMA₃ fluorophores, and the number and distribution of 18S rDNA sites. Microsatellites were observed only in euchromatin regions, whereas the (TTAGG)₆ repeats were found at telomeric sites in both groups. Our data indicate that in addition to the chromosome number, the karyotypes in Melipona could be separated into 2 groups that are characterized by conserved cytogenetic features and patterns that generally are shared by species within each subgenus, which may reflect evolutionary constraints. Our results agree with the morphological separation of the Melipona into 4 subgenera, suggesting that they must be independent evolutionary lineages.

Genomic Organization of Repetitive DNA Elements and Extensive Karyotype Diversity of Silurid Catfishes (Teleostei: Siluriformes): A Comparative Cytogenetic Approach.

The catfish family Siluridae contains 107 described species distributed in Asia, but with some distributed in Europe. In this study, karyotypes and other chromosomal characteristics of 15 species from eight genera were examined using conventional and molecular cytogenetic protocols. Our results showed the diploid number (2n) to be highly divergent among species, ranging from 2n = 40 to 92, with the modal frequency comprising 56 to 64 chromosomes. Accordingly, the ratio of uni- and bi-armed chromosomes is also highly variable, thus suggesting extensive chromosomal rearrangements. Only one chromosome pair bearing major rDNA sites occurs in most species, except for Wallago micropogon, Ompok siluroides, and Kryptoterus giminus with two; and Silurichthys phaiosoma with five such pairs. In contrast, chromosomes bearing 5S rDNA sites range from one to as high as nine pairs among the species. Comparative genomic hybridization (CGH) experiments evidenced large genomic divergence, even between congeneric species. As a whole, we conclude that karyotype features and chromosomal diversity of the silurid catfishes are unusually extensive, but parallel some other catfish lineages and primary freshwater fish groups, thus making silurids an important model for investigating the evolutionary dynamics of fish chromosomes.

Evolution of Bird Sex Chromosomes Narrated by Repetitive Sequences: Unusual W Chromosome Enlargement in Gallinula melanops (Aves: Gruiformes: Rallidae)

Among birds, species with the ZZ/ZW sex determination system generally show significant differences in morphology and size between the Z and W chromosomes (with the W usually being smaller than the Z). In the present study, we report for the first time the karyotype of the spot-flanked gallinule (Gallinula melanops) by means of classical and molecular cytogenetics. The spot-flanked gallinule has 2n = 80 (11 pairs of macrochromosomes and 29 pairs of microchromosomes) with an unusual W chromosome that is larger than the Z. Besides being totally heterochromatic, it has a secondary constriction in its long arm corresponding to the nucleolar organizer region, as confirmed by both silver staining and mapping of 18S rDNA probes. This is an unprecedented fact among birds. Additionally, 18S rDNA sites were also observed in 6 microchromosomes, while 5S rDNA was found in just 1 microchromosomal pair. Seven out of the 11 used microsatellite sequences were found to be accumulated in microchromosomes, and 6 microsatellite sequences were found in the W chromosome. In addition to the involvement of heterochromatin and repetitive DNAs in the differentiation of the large W chromosome, the results also show an alternative scenario that highlights the plasticity that shapes the evolutionary history of bird sex chromosomes.

Cytogenetics of tuna in Argentina (two forms of Opuntia ficus-indica (L.) Mill. and O. robusta J. C. Wendl., Cactaceae)

Opuntia ficus-indica (L.) Mill. f. ficus-indica (yellow tuna, cordobesa orange tuna, italiana orange tuna, saltena tuna) and f. amyclaea (white tuna, reddish tuna) and O. robusta J. C. Wendl. (cuaresma tuna) are extensively cultivated cacti in Argentina, mainly used as fodder and human food. Despite the importance of these resources, no studies of molecular cytogenetics considering the morphological characteristics of the varieties have been carried out. The main goal of this contribution is to determine the chromosome number, physical localization of ribosomal genes and amount of DNA in these taxa, including several populations with different fruit colors. The f. ficus-indica samples were octoploid, f. amyclaea hexaploid, and O. rubusta tetraploid. All taxa exhibited small similar-sized symmetrical chromosomes. Karyotypes were symmetrical with slight variations within each species. The 18S–5.8S–26S sites in all taxa were located on the secondary constrictions and the adjacent satellites at telomeric positions. The f. ficus-indica presented four 18S–5.8S–26S rDNA sites and the f. amyclaea three, whereas O. robusta had two. There were eight and six 5S rDNA sites in f. ficus-indica and f. amyclaea, respectively, and four in O. robusta. They were always proximally located on short arms of different chromosome pairs, always in a pericentromeric position. Both ribosomal genes were always asynthenic. The nuclear DNA in the octoploid f. ficus-indica presented a lower value (1Cx = 0.91 pg) than the hexaploid f.amyclaea (1Cx = 1.06 pg), whereas the tetraploid O. robusta had 1Cx = 0.87 pg. This cytogenetic evaluation is important for developing more efficient and faster selection strategies, contributing to obtaining new and improved varieties.

Karyological traits related to phylogenetic signal and environmental conditions within the Hymenaea clade (Leguminosae, Detarioideae)

The Hymenaea clade is a lineage of the subfamily Detarioideae of Leguminosae. The clade is currently composed of three genera: Guibourtia, Hymenaea, and Peltogyne. The genera Guibourtia and Hymenaea present an Amphi-Atlantic distribution, whereas Peltogyne is exclusively found in the Neotropics. Previous studies have focused on the phylogeny of the group but there are no studies that demonstrate mechanisms that could be involved in the evolution of this Amphi-Atlantic clade. The present work is the largest cytogenetic analysis for the group published so far. We aimed to provide a study of karyotypic characteristics of the Hymenaea clade, interpreted using the plastidial trnK phylogenetic reconstruction as background. We analyzed karyological characteristics such as chromosome number, chromosome size, centromeric position, CMA/DAPI patterns, and banding for 5S and 45S rDNA. With the present data available, multivariate analyses (PCA) and Phylogenetic Eigenvector Regression (PVR) demonstrated that centromeric asymmetry and number of 5S rDNA sites may present a strong phylogenetic signal, whereas number of CMA+ sites is associated with geography/climate characteristics. The association of CMA bands and geography has been reported for other legumes of the Caesalpinia group. This study demonstrates that the use of statistical tools for analyzing karyotypic characteristics can result in more precise interpretation of evolutionary processes related to structural changes in chromosomes and how these changes can also be related to environmental conditions.

Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae).

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3+ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3+) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3+ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3+, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.

Cytogenetic Characterization of Two Metynnis Species (Characiformes, Serrasalmidae) Reveals B Chromosomes Restricted to the Females

Karyotypes and chromosomal characteristics with focus on B chromosomes of 2 species of the serrasalmid genus Metynnis, namely M. lippincottianus and M. maculatus, were examined using conventional (C-banding) and molecular (FISH mapping of minor and major rDNAs and Rex1, Rex3, and Rex6 retrotransposable elements) protocols. Both species possessed a diploid chromosome number of 2n = 62 and karyotypes composed of 32 metacentric + 28 submetacentric + 2 subtelocentric and 32 metacentric + 26 submetacentric + 4 subtelocentric, respectively; one small B element was found in the female genome of M. lippincottianus. C-banding revealed heterochromatin in the pericentromeric and terminal portions of all chromosomes of both species; the B chromosome was entirely heterochromatic. FISH showed 18S rDNA sites in 2 chromosome pairs in both species (pairs 19 and 22), and a large block in the B chromosome, while 5S rDNA signals were detected in the first pair of subtelocentric chromosomes in both species, moreover in M. maculatus an additional labeled pair 4 was observed. Mapping of the Rex1, Rex3, and Rex6 retrotransposable elements in the genomes of M. lippincottianus and M. maculatus indicated that they were dispersed throughout nearly all the chromosomes of the complement, except for the B chromosome of M. lippincottianus.

Cytogenetics, genomics and biodiversity of the South American and African Arapaimidae fish family (Teleostei, Osteoglossiformes).

Osteoglossiformes represents one of the most ancestral teleost lineages, currently widespread over almost all continents, except for Antarctica. However, data involving advanced molecular cytogenetics or comparative genomics are yet largely limited for this fish group. Therefore, the present investigations focus on the osteoglossiform family Arapaimidae, studying a unique fish model group with advanced molecular cytogenetic genomic tools. The aim is to better explore and clarify certain events and factors that had impact on evolutionary history of this fish group. For that, both South American and African representatives of Arapaimidae, namely Arapaima gigas and Heterotis niloticus, were examined. Both species differed markedly by diploid chromosome numbers, with 2n = 56 found in A. gigas and 2n = 40 exhibited by H. niloticus. Conventional cytogenetics along with fluorescence in situ hybridization revealed some general trends shared by most osteoglossiform species analyzed thus far, such as the presence of only one chromosome pair bearing 18S and 5S rDNA sites and karyotypes dominated by acrocentric chromosomes, resembling thus the patterns of hypothetical ancestral teleost karyotype. Furthermore, the genomes of A. gigas and H. niloticus display remarkable divergence in terms of repetitive DNA content and distribution, as revealed by comparative genomic hybridization (CGH). On the other hand, genomic diversity of single copy sequences studied through principal component analyses (PCA) based on SNP alleles genotyped by the DArT seq procedure demonstrated a very low genetic distance between the South American and African Arapaimidae species; this pattern contrasts sharply with the scenario found in other osteoglossiform species. Underlying evolutionary mechanisms potentially explaining the obtained data have been suggested and discussed.

Karyotypic characterization of melon accessions

Cucumis melo (melon) is a species from the Iberian Peninsula and is included in the family Cucurbitaceae. Despite the knowledge about the physical structure of melon chromosomes, little is known about the intraspecific karyotype diversity of the species. To study karyotype diversity in eight melon accessions, the following methods were used: Giemsa 3% staining; application of CMA 3 /DAPI fluorochromes; and location of 45S and 5S rDNA sequences by fluorescence in situ hybridization. Conventional staining analysis revealed stability in the chromo­some number, with accessions presenting 2 n = 24. There were significant variations in the mean chromosome size between accessions, ranging from 0.98 μm to 1.46 μm for accessions A26 and A18, respectively. Scott-Knott clustering distributed the accessions into two groups. GC-rich heterochromatic blocks (CMA 3 + /DAPI - ) were observed in pericentromeric regions of all chromosomes in the complement. CMA 3 + /DAPI - blocks were also lo­cated in terminal regions, being specific to satellite regions. Hybridization sites of 45S rDNA probes revealed the presence of a chromosome pair with this locus . In addition, 5S rDNA sites revealed a labeled chromosome pair. No quantitative variation was observed in rDNA sites between the accessions analyzed, indicating these markers as ideal for the verification of karyotypic stability in C . melo .

Cytogenetic and agronomic characterization of intergeneric hybrids between Saccharum spp. hybrid and Erianthus arundinaceus

In sugarcane (Saccharum spp. hybrid) breeding, introgression of useful genes via intergeneric hybridization is a powerful strategy for improving the crop productivity. Erianthus arundinaceus shows great potential in terms of useful traits; however, little is known about the cytogenetic and agronomic characteristics of intergeneric hybrids between these two species. Here, we examine the cytogenetic and agronomic characteristics, and relationships between the two in intergeneric F1 hybrids between modern sugarcane cultivar and E. arundinaceus identified by amplification of 5S rDNA markers and morphological characteristics. The nuclear DNA content of the hybrids varied from 6.07 to 8.94 pg/2C, with intra-clonal variation in DNA content and 5S rDNA sites. Genomic in situ hybridization revealed 53 to 82 chromosomes in the hybrids, with 53 to 56 derived from sugarcane and 1 to 29 from E. arundinaceus. There were significant positive correlations between the number of E. arundinaceus chromosomes and dry matter yield, millable stalk weight, single stalk weight, and stalk diameter, but not sucrose content, reducing sugar content, sucrose/reducing sugar ratio or fiber content. This detailed information on intergeneric F1 hybrids between modern sugarcane cultivar and E. arundinaceus will contribute to effective utilization of E. arundinaceus in sugarcane breeding.

Comparative analysis of rDNA distribution in 29 species of Begonia sect. Coelocentrum Irmsch.

The rDNA sites are useful chromosome landmarks and can provide valuable information for species identification and species relationships. In this study, we investigated the distribution of 5S and 45S rDNA sites in 29 species of Begonia sect. Coelocentrum Irmsch. using a two-colour fluorescence in situ hybridization (FISH) technique. This is the first report of chromosomal rDNA mapping in Begonia species. The analyzed species showed considerable diversity in rDNA distribution patterns. The 45S rDNA signals are always located in terminal regions on 1−4 chromosomes, while 5S rDNA signals are mainly located at proximal regions on 2−8 chromosomes, varying from specific major signals to highly dispersed minor signals. Based on rDNA FISH patterns, most of the investigated species could be distinguished from each other and species relationships were identified. In addition, the results provided clear proof that B. huangii is of hybrid origin and the triploid B. longgangensis was allotriploid rather than autotriploid as suggested before. The data will provide a useful reference for evaluation, conservation and utilization of the natural resources of the mega-diverse genus Begonia.

Karyotype analysis of eight cultivated Allium species

The karyotypes of Allium, a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA3/DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers (x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species (A. moly, A. oreophilum, A. karataviense, A. nigrum, A. sphaerocephalon, A. porrum). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A. sphaerocephalon L.) or four chromosomes (tetraploid A. porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA3+ bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A. fistulosum L. in which positive CMA3+ bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa.

Relationship between epigenetic marks and the behavior of 45S rDNA sites in chromosomes and interphase nuclei of Lolium-Festuca complex.

The grasses of the Lolium-Festuca complex show a prominent role in world agricultural scenario. Several studies have demonstrated that the plasticity of 45S rDNA sites has been recently associated with the possible fragility of the loci. Often, these fragile sites were observed as extended sites and gaps in metaphases. This organization can be evaluated in relation to their transcriptional activity/accessibility through epigenetic changes. Thus, this study aimed to investigate the relationship of the 5-methylcytosine and histone H3 lysine-9 dimethylation in different conformations of 45S rDNA sites in interphase nuclei and in metaphase chromosomes of L. perenne, L. multiflorum and F. arundinacea. The FISH technique using 45S rDNA probes was performed sequentially after the immunolocalization. The sites showed predominantly the following characteristics in the interphase nuclei: intra- and perinucleolar position, decondensed or partially condensed and hypomethylated and hyper/hypomethylated status. Extranucleolar sites were mainly hypermethylated for both epigenetic marks. The 45S rDNA sites with gaps identified in metaphases were always hypomethylated, which justifies it decondensed and transcriptional state. The frequency of sites with hypermethylated gaps was very low. The structural differences observed in these sites are directly related to the assessed epigenetic marks, justifying the different conformations throughout the cell cycle.

45S rDNA sites in meiosis of Lolium multiflorum Lam.: variability, non-homologous associations and lack of fragility

In this study, we evaluated the behavior of 45S ribosomal DNA (rDNA) sites during the meiosis of Lolium multiflorum. The reason to study it in this species is that 45S rDNA sites are usually visualized as gaps in mitotic metaphase chromosomes and were initially denominated fragile sites (FSs). In different species, FSs were related to rearrangements that alter the karyotype and affect the chromosome pairing in meiosis. However, our findings show that the chromosome pairing in L. multiflorum is regular and, as in mitosis, the number of sites is variable. In diakinesis with five sites, one of the bivalents was in hemizygous state while, in diakinesis with seven sites, one of the bivalents had three conspicuous signals, two in syntheny in one of the homologous. Only four cells had gaps in the region of the 45S rDNA. Owing to the lower number of signals observed at the initial stages of meiosis, it is assumed that they are involved both in homologous and non-homologous associations and that they might assist the chromosome pairing. Regarding segregation, only meiocytes with five and six 45S rDNA signals were observed, and they were characterized by the segregation of 2/3 signals in the poles of anaphases I up to metaphases II; 2/2 and 3/3 in anaphases II and telophases II; and also 2/2 and 4/4 in the nuclei of tetrads, unlike the number of 45S signals expected. The numerical non-equivalence of sites among nuclei at later stages of meiosis is explained by the presence of chromosomes with hemizygous sites.

Origin and parental genome characterization of the allotetraploid Stylosanthes scabra Vogel (Papilionoideae, Leguminosae), an important legume pasture crop.

The genus Stylosanthes includes nitrogen-fixing and drought-tolerant species of considerable economic importance for perennial pasture, green manure and land recovery. Stylosanthes scabra is adapted to variable soil conditions, being cultivated to improve pastures and soils worldwide. Previous studies have proposed S. scabra as an allotetraploid species (2n = 40) with a putative diploid A genome progenitor S. hamata or S. seabrana (2n = 20) and the B genome progenitor S. viscosa (2n = 20). We aimed to provide conclusive evidence for the origin of S. scabra. We performed fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) experiments and Illumina paired-end sequencing of S. scabra, S. hamata and S. viscosa genomic DNA, to assemble and compare complete ribosomal DNA (rDNA) units and chloroplast genomes. Plastome- and genome-wide single nucleotide variation detection was also performed. GISH and phylogenetic analyses of plastid DNA and rDNA sequences support that S. scabra is an allotetraploid formed from 0.63 to 0.52 million years ago (Mya), from progenitors with a similar genome structure to the maternal donor S. hamata and the paternal donor S. viscosa. FISH revealed a non-additive number of 35S rDNA sites in S. scabra compared with its progenitors, indicating the loss of one locus from A genome origin. In S. scabra, most 5S rDNA units were similar to S. viscosa, while one 5S rDNA site of reduced size most probably came from an A genome species as revealed by GISH and in silico analysis. Our approach combined whole-plastome and rDNA assembly with additional cytogenetic analysis to shed light successfully on the allotetraploid origin of S. scabra. We propose a Middle Pleistocene origin for S. scabra involving species with maternal A and paternal B genomes. Our data also suggest that variation found in rDNA units in S. scabra and its progenitors reveals differences that can be explained by homogenization, deletion and amplification processes that have occurred since its origin.

Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems

ABSTRACT Pimelodidae harbors several species and is widely distributed throughout the Neotropical region. Pimelodus is the genus with the largest number of species, however it is a polyphyletic group. Cytogenetic analyzes of the valid species still covers less than half of them. Herein, seven Pimelodus species from three Brazilian hydrographic systems were analyzed through basic (Giemsa, AgNORs and C banding) and molecular (5S and 18S rDNA-FISH) cytogenetic methods. All species had 2n=56 chromosomes with different karyotype formulas observed among the species. AgNORs were corresponding to 18S rDNA and localized on long arm of one chromosome pair in all species. Heterochromatin distribution follows the pattern commonly verified in the family and allows to identify each one of the studied species. 5S rDNA marker was interspecifically variable in number and position of cistrons. Pimelodus ortmanni had B chromosomes varying intra and inter-individually. We performed a discussion on our own and available cytogenetic data for Pimelodidae, and the associating of them with available phylogeny enable us identifying features that distinguish subgroups within Pimelodidae, such as NORs location (terminal/long arm for species belonging to “Iheringichthys-Parapimelodus” and “Pimelodus maculatus” subclades) and location of 5S rDNA sites (pericentromeric/interstitial/ long arm for species belonging to Pimelodus group).

Inter- and intraspecific hypervariability in interstitial telomeric-like repeats (TTTAGGG)n in Anacyclus (Asteraceae).

Interstitial telomeric repeat (ITR) sites, consisting of tandem repeats of telomeric motifs localized at intrachromosomal sites, have been reported in a few unrelated organisms including plants. However, the causes for the occurrence of ITRs outside of the chromosomal termini are not fully understood. One possible explanation are the chromosomal rearrangements involving telomeric sites, which could also affect the location of other structural genome elements, such as the 45S rDNA. Taking advantage of the high dynamism in 45S rDNA loci previously found in Anacyclus (Asteraceae, Anthemideae), the occurrence and patterns of variation of ITRs were explored in this genus with the aim of finding common underlying causes. In total, 132 individuals from 44 populations of nine species were analysed by fluorescence in situ hybridization using an Arabidopsis-type telomeric sequence as a probe. Variable presence of ITR sites was detected in six out of nine species of Anacyclus, ranging from two to 45 sites and showing contrasting chromosomal locations and a differential presence of the ITR site on homologous chromosome pairs. At the intraspecific level, the ranges were as large as 0-12 ITR sites. Although only 26 % of the total observed ITR sites were localized in chromosomes bearing 45S rDNA loci, all cases of interstitial 45S rDNA reported in a previous work co-occurred with ITRs in close proximity in the same chromosome arms. High levels of ITR polymorphism within a single species have not been previously reported in plants and suggest that this pattern might have been overlooked due to insufficient sampling. Although ancient Robertsonian translocations or the amplification of terminal 45S rDNA sites cannot, on their own, explain all of the levels of variability in ITRs reported here, there are suggestions that they may have been involved in the evolutionary history of this genus or its ancestors in Anthemideae.

Molecular cytogenetic of the Amoy croaker, Argyrosomus amoyensis (Teleostei, Sciaenidae)

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fluorescence in situ hybridization (FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18S and 5S rDNA probes, and a self-genomic in situ hybridization procedure (Self-GISH). The karyotype of A. amoyensis comprised 2n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions (NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation (AgNO3) staining and denaturation-propidium iodide (DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole (DAPI) staining, and was confirmed by FISH with 18S rDNA probes. The 5S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with different intensities, but internal telomeric sequences (ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specific chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18S rDNA are conserved, the distribution of 5S rDNA varies dynamically among sciaenid species. Thus, the 5S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be effective cytotaxonomic markers in Sciaenidae.

Chromosome investigations in annual Medicago species (Fabaceae) with emphasis on the origin of the polyploid Medicago rugosa and M. scutellata

Chromosome number variations play an important role in the genus Medicago. In addition to polyploidy there are cases of dysploidy as evidenced by two basic numbers, x = 8 and x = 7, the latter limited to five annual species having 2n = 14. Annuals are diploid with the exception of Medicago scutellata and Medicago rugosa which have 2n = 30 and are considered the result of crosses between the 2n = 16 and 2n = 14 species. However, this hypothesis has never been tested. This study was carried out to investigate the 2n = 14 and 2n = 30 karyotypes and verify the allopolyploid origin of M. scutellata and M. rugosa. Fluorescence in situ hybridization (FISH) of rDNA probes and genomic in situ hybridization (GISH) were performed. FISH showed that all five diploids with 2n = 14 have one pair of 45S and one pair of 5S rDNA sites. M. scutellata displayed four sites of 45S and four sites of 5S rDNA, while in M. rugosa only one pair of each of these sites was found. GISH did not produce signals useful to identify the presumed progenitors with 14 chromosomes. This result suggests alternative evolutionary pathways, such as the formation of tetraploids (2n = 32) and subsequent dysploidy events leading to the chromosome number reduction.

Karyotype diversity and 2C DNA content in species of the Caesalpinia group

BackgroundThe Leguminosae family is the third-largest family of angiosperms, and Caesalpinioideae is its second-largest subfamily. A great number of species (approximately 205) are found in the Caesalpinia group within this subfamily; together with these species’ phenotypic plasticity and the similarities in their morphological descriptors, make this a complex group for taxonomic and phylogenetic studies. The objective of the present work was to evaluate the karyotypic diversity and the 2C DNA content variation in 10 species of the Caesalpinia group, representing six genera: Paubrasilia, Caesalpinia, Cenostigma, Poincianella, Erythrostemon and Libidibia. The GC-rich heterochromatin and 45S rDNA sites (which are used as chromosome markers) were located to evaluate the karyotype diversity in the clade. The variation in the 2C DNA content was determined through flow cytometry.ResultsThe fluorochrome banding indicated that the chromomycin A3+/4′,6-diamidino-2-phenylindole− blocks were exclusively in the terminal regions of the chromosomes, coinciding with 45S rDNA sites in all analyzed species. Physical mapping of the species (through fluorescence in situ hybridization) revealed variation in the size of the hybridization signals and in the number and distribution of the 45S rDNA sites. All hybridization sites were in the terminal regions of the chromosomes. In addition, all species had a hybridization site in the fourth chromosome pair. The 2C DNA content ranged from 1.54 pg in Erythrostemon calycina to 2.82 pg in the Paubrasilia echinata large-leaf variant. The Pa. echinata small-leaf variant was isolated from the other leaf variants through Scoot-Knott clustering.ConclusionsThe chromosome diversity and the variation in the 2C DNA content reinforce that the actual taxonomy and clustering of the analyzed taxa requires more genera that were previously proposed. This fact indicates that taxonomy, phylogeny and cytoevolutionary inference related to the complex Caesalpinia group have to be done through integrative evaluation.