5S rDNA Probes Research Articles

Chromosome number reduction in the sister clade of Carica papaya with concomitant genome size doubling.

Caricaceae include six genera and 34 species, among them papaya, a model species in plant sex chromosome research. The family was held to have a conserved karyotype with 2n = 18 chromosomes, an assumption based on few counts. We examined the karyotypes and genome size of species from all genera to test for possible cytogenetic variation. We used fluorescent in situ hybridization using standard telomere, 5S, and 45S rDNA probes. New and published data were combined with a phylogeny, molecular clock dating, and C values (available for ∼50% of the species) to reconstruct genome evolution. The African genus Cylicomorpha, which is sister to the remaining Caricaceae (all neotropical), has 2n = 18, as do the species in two other genera. A Mexican clade of five species that includes papaya, however, has 2n = 18 (papaya), 2n = 16 (Horovitzia cnidoscoloides), and 2n = 14 (Jarilla caudata and J. heterophylla; third Jarilla not counted), with the phylogeny indicating that the dysploidy events occurred ∼16.6 and ∼5.5 million years ago and that Jarilla underwent genome size doubling (∼450 to 830-920 Mbp/haploid genome). Pericentromeric interstitial telomere repeats occur in both Jarilla adjacent to 5S rDNA sites, and the variability of 5S rDNA sites across all genera is high. On the basis of outgroup comparison, 2n = 18 is the ancestral number, and repeated chromosomal fusions with simultaneous genome size increase as a result of repetitive elements accumulating near centromeres characterize the papaya clade. These results have implications for ongoing genome assemblies in Caricaceae.

Fragile sites of 45S rDNA of Lolium multiflorum are not hotspots for chromosomal breakages induced by X-ray.

Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50Gy X-ray and evaluated oneday following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages.

A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n=9), L. heterotoma (Thomson, 1862) (n=10), L. victoriae Nordlander, 1980 (n=10) and Ganaspis xanthopoda (Ashmead, 1896) (n=9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed.

Chromosomal Diversification Higher Than Molecular Variation in Astyanax aff. fasciatus (Teleostei, Characidae).

Comprising a large number of species, the genus Astyanax has been intensively studied by several approaches to elucidate its evolutionary relationships. Such studies have demonstrated that many nominal species are artificial clusters where distinct taxa are grouped under the same denomination. Astyanax aff. fasciatus stands out due to its high karyotypic diversity, since cytogenetic studies have reported three standard cytotypes (2n = 46, 48, and 50), as well as cases of sympatry between cytotypes, variant cytotypes, and B chromosomes. In this study, we attempted to evaluate the reliability of the chromosomal differences in relation to the analysis of the ATPase6/8 mitochondrial DNA (mtDNA) sequence, thereby providing subsidies to the evolutionary reconstruction of this group. Nine populations from four distinct hydrographic basins along Southeastern Brazil were analyzed. These are the first cytogenetic data collected for four of them. Fluorescent in situ hybridization with 5S rDNA probe evidenced the presence of a standard phenotype for the group and the existence of a new arrangement in the individuals from Ribeira de Iguape River. Besides the karyotypic variation, the genetic distance was low among the studied populations and some aspects of the evolutionary relationships among distinct cytotypes/populations could be ascertained by phylogeographic studies. The incipient molecular structuring of certain cytotypes in different hydrographic basins indicates the role of different evolutionary processes on the diversification of the group.

Chromosomal features ofFosterellaspecies (Bromeliaceae, Pitcairnioideae)

Fosterella (Bromeliaceae) comprises 31 species with rosulate leaves and mostly small, whitish flowers. Previous karyological studies were restricted to chromosome counts. In the present study, chromosomal variation in Fosterella was analysed using CMA3/DAPI staining and/or fluorescence in situ hybridization (FISH) using 45S and 5S rDNA probes, generating data for nine taxa with either 2n = 50 or 100 chromosomes. A single chromosome pair containing one CMA+/DAPI− band was identified in all diploid species. In the tetraploid Fosterella hatschbachii one pair had a CMA+/DAPI− band, whereas the other tetraploid studied, Fosterella yuvinkae, had two pairs with proximal bands. The presence of two CMA+/DAPI− pairs in F. yuvinkae may indicate a recent polyploidization event. This paper also reports the application of FISH in Bromeliaceae. FISH using 45S rDNA as the probe revealed one pair of terminal sites in most species and a co-localization with CMA+/DAPI− bands in all analysed species. The 5S rDNA sites were terminal in the tetraploid F. hatschbachii and proximal in all other species studied. Our data indicate that Fosterella species have little heterochromatin and it is largely restricted to the vicinity of the nucleolus organizer region. The data also indicate that hybridization (sometimes associated with polyploidy) has probably played an important role in the evolution of Fosterella.

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

In the present study, conventional staining and NORs banding as well as Fluorescence in situ hybridizat ion (FISH) using the 18S rDNA and telomeric (TTA GGG)n probes were applied to stain the chromosomes of crocodile catfish, Bagariussuchus (Siluriformes, Sisoridae) fro m the Chao Praya River, Thailand. Kidney cells of 6 male and 6 female crocodile catfishes were used as a sample. The mitotic chro mosome preparations were done directly fro m kidney cells. The results showed that the diploid chro mosome number of B. suchus was 2n=56, the fundamental numbers (NF) were 102 in both male and female. The karyotype comprises 17m+17sm+12a+10t. The nucleo lar organizer reg ions (NORs) were detected by Ag -NORs banding and 18S rDNA probe mapping. The 18S rDNA are terminally located on the short arm adjacent to the telomere of the single pair of the 1st chromosome pair whereas NOR-bearing chro mosome is only one chromosome of the 1st chromosome pair (1a 1b, poly morphic characteristic) at the subtelomeric region of the short arm. Moreover, FISH with telomeric probe showed hybridizat ion signals on each telomere of all chro mosomes and interstitial telomeric sites were not detected. There were variations in signals of FISH and their position in the karyotype along with variat ion in DNA sequences. These markers are useful for future discrimination of population of closely related species and their poly morphis m.

Evolutionary trends in the family Curimatidae (Characiformes): inferences from chromosome banding

The family Curimatidae is a fish group usually considered chromosomally conserved in their diploid number. However, some studies show small changes in the karyotype microstructure, and the presence of B chromosomes, indicating a chromosomal diversification within the group, even if structural changes in the karyotypes are not visible. Few studies associate this trait with an evolutionary pattern within the family. This study aimed to characterize the karyotype, nucleolus organizer regions (NORs), and heterochromatin distribution of six species of Curimatidae of the genera Cyphocharax Fowler, 1906 and Steindachnerina Fowler, 1906: Cyphocharax voga (Hensel, 1870), Cyphocharax spilotus (Vari, 1987), Cyphocharax saladensis (Meinken, 1933), Cyphocharax modestus (Fernández-Yépez, 1948), Steindachnerina biornata (Braga et Azpelicueta, 1987) and Steindachnerina insculpta (Fernández-Yépez, 1948) and contribute data to a better understanding of the mechanisms involved in the chromosomal evolution of this group of fish. All specimens had 2n=54, m-sm, and B microchromosomes. Five species exhibited single NORs, except for Steindachnerina biornata, which showed a multiple pattern of ribosomal sites. NORs were chromomycin A3 positive (CMA3+) and 4’-6-diamino-2-phenylindole (DAPI-) negative, exhibiting differences in the pair and chromosomal location of each individual of the species. FISH with 5S rDNA probe revealed sites in the pericentrometic position of a pair of chromosomes of five species. However, another site was detected on a metacentric chromosome of Cyphocharax spilotus. Heterochromatin distributed both in the pericentromeric and some terminal regions was revealed to be CMA3+/DAPI-. These data associated with the previously existing ones confirm that, although Curimatidae have a very conservative karyotype macrostructure, NORs and heterochromatin variability are caused by mechanisms of chromosome alterations, such as translocations and/or inversions, leading to the evolution and diversification of this group of fish.

CMA band variability and physical mapping of 5S and 45S rDNA sites in Brazilian Cactaceae: Pereskioideae and Opuntioideae

Representatives of the Cactaceae subfamilies Pereskioideae and Opuntioideae from northeastern Brazil were studied using banding with the fluorochromes, CMA3 and DAPI, as well as with fluorescent in situ hybridization using 45S and 5S rDNA probes to identify the distributions of their heterochromatin and rDNA sites. Pereskia aculeata, P. bahiensis, P. grandifolia (Pereskioideae), Brasilopuntia brasiliensis, Tacinga funalis, and T. palmadora showed 2n = 22, while Opuntia dillenii showed 2n = 44, and O. ficus-indica 2n = 88. The karyotypes of all of the species were symmetric, with average chromosome lengths varying from 1.94 µm in O. dillenii to 3.17 µm in P. aculeata. One pair of terminal CMA+ bands corresponding to NORs occurred in all of the diploid cytotypes (except O. ficus-indica, which has two pairs of terminal CMA+ bands) as well as in O. dillenii (tetraploid). CMA+ bands were also observed in the interstitial region of the long arm of a chromosome pair in B. brasiliensis, while a number of variable proximal bands were observed on three chromosome pairs in O. dillenii and on most of the chromosomes of O. ficus-indica. The 45S rDNA sites corresponded to the terminal CMA+ bands, while the 5S rDNA sites were located in the interstitial regions of the long arms of the chromosome pairs of P. aculeata, P. bahiensis, P. grandifolia, and B. brasiliensis. Our data, and earlier publications, suggest that the subfamily Opuntioideae can be characterized as having proximal/interstitial CMA+ heterochromatin in at least one chromosome pair (except in Tacinga). The absence of proximal heterochromatic bands, however, appears to be a synapomorphy of the basal lineages of Cactaceae (subfamily Pereskioideae + Maihuenioideae), suggesting that karyotypes with heterochromatin restricted to the terminal region of a chromosome pair (45S rDNA) represent a plesiomorphic character of the family.

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

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

Comparative karyotype investigations in the white-clawed crayfish Austropotamobius pallipes (Lereboullet, 1858) species complex and stone crayfish A. torrentium (Schrank, 1803) (Decapoda: Astacidae)

We report for the first time the chromosome number and karyological characteristics of the endangered European freshwater crayfish genus Austropotamobius (Decapoda, Astacidae). The karyotypes of the white-clawed crayfish ( Austropotamobius pallipes species complex) and of the stone crayfish ( A. torrentium ) consisted of 2 n = 176 chromosomes arranged into 76 metacentric, 11 metacentric-submetacentric, and one heteromorphic acrocentric/metacentric chromosome pair. Fluorochrome staining with 4,6-diamino-2-phenylindole (DAPI) revealed distinctive centromeric/pericentromeric as well as interstitial AT-rich blocks in A. pallipes and A. torrentium . Centromeric/pericentromeric AT-rich blocks were observed on all chromosomes, whereas interstitial AT-rich blocks were observed on some chromosomes, mostly on the larger ones. Fluorescence in situ hybridization (FISH) with the 45S rDNA probe on mitotic metaphases of A. pallipes and A. torrentium have revealed four major Texas-red signals, corresponding to four sites of 45S rDNA. One of the two 45S rDNA-bearing chromosome pairs was highly heteromorphic, considering the length of chromosomes as well as the size and intensity of 45S rDNA FISH signal. We speculate that the heteromorphic chromosome pair might represents X and Y sex chromosomes, suggesting the presence of XX-XY sex determination system in the A. pallipes species complex and A. torrentium .

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

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

Meiotic analysis and FISH with rDNA and rice BAC probes of the Thai KPS 01-01-25 sugarcane cultivar

The interspecific sugarcane hybrid “KPS 01-01-25” is one of Thailand’s most successful cultivars, but its genetics and genomic constitution are greatly complicated due to the highly polyploid nature of this crop. Here we analyzed the crop’s karyotype, studied chromosome pairing at meiosis I and performed fluorescent in situ hybridization (FISH) of rDNA and rice BACs as probes. Mitotic cell complements in early tapetal cells showed 110 well-spread chromosomes. Pollen mother cells at pachytene clearly demonstrated fully paired configurations of the long chromosomes, in which few pairing partner exchanges and loops could be discerned. In diakinesis cells we observed 74.1 % bivalents, 18.6 % univalents, 2.9 % trivalents and 4.4 % quadrivalents. During this stage secondary associations were abundant, but these threadlike structures disappear after metaphase I. FISH with 5S rDNA produced up to ten fluorescent spots, of which 2–5 are often very small, whereas the 45S rDNA probe showed foci on 8–9 chromosomes. Based on our results and that of related studies we estimate that this sugarcane is composed of 11–12 genomes. We also selected rice BACs containing chromosome 5 genes controlling economically important genes. Eight BACs with relatively strong dot-blot hybridization on genomic sugarcane DNA were selected for FISH on sugarcane chromosome slides. These cross-species chromosome BAC FISH revealed weak signals, and most of the foci were distributed over parts of the chromosomes. Based on these experiments we concluded that cross-species FISH in sugarcane can better be carried out with sorghum BACs as probes.

Karyotypic variation of Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae) along the Iguazu river basin.

The Iguazu river is a tributary of the left margin of the Paraná river, isolated from this basin about 22 million years ago with the appearance of the Iguazu Falls. The Iguazu river is characterized by high endemism due to two factors: its rugged topography and the old isolation caused by formation of the Iguazu Falls. This study analyzed cytogenetically a population of Glanidium ribeiroi collected in a region at the final stretch of this basin, by Giemsa staining, C-banding, impregnation by silver nitrate, and FISH with probes of 5S rDNA, 18S rDNA, telomeric sequence [TTAGGG]n, and [GATA]n repeats. The diploid number was equal to 58 chromosomes. The heterochromatin was present in the terminal region of almost all chromosomes. The Ag-NORs were simple and presented interstitially on the short arm of the submetacentric pair 14, which was confirmed by FISH with 18S rDNA probe. The 5S rDNA-FISH marked only the submetacentric pair 16 on the long arm in interstitial position. The FISH with [TTAGGG]n probe presented all telomeres labeled as expected, with an absence of Interstitial Telomeric Sequence (ITS). The repetitive [GATA]n sequence was dispersed throughout the genome, with preferential location in the terminal region of all chromosomes. The data obtained are discussed herein with other species of Auchenipteridae, and other previously analyzed populations of G. ribeiroi from the Iguazu river, verifying differences among these populations, which should be mainly related to the rugged topography of this basin.

First chromosomal study of Mantophasmatodea: Karyotype of Karoophasma biedouwense (Austrophasmatidae)

We have investigated for the first time the chromosomes of Karoophasma biedouwense, a species belonging to the Mantophasmatodea, a recently discovered order of carnivorous insects. Our study has revealed that males of this species display testes with numerous seminal tubes (follicles), as in other Polyneoptera, and short tubular seminal vesicles embedded in a utricular gland. The karyotype consists of 2n = 12A + X and biarmed, meta/submetacentric chromosomes (fundamental number of arms: FN = 26) with blocks of heterochromatin around centromeres. The autosomes are classified into two size groups, one represented by a single, very large pair of autosomes, the other by five smaller pairs which constitute a continuous series gradually decreasing in size. Among monocentric orders of Polyneoptera, K. biedouwense shares its low chromosome number, 2n = 13, as also found with some Orthoptera (Acridoidea, Grylloidea, Gryllacridoidea). Male meiosis is of the classical pre-reductional type and the X(0) sex determination system is probably an ancestral state. Use of FISH along with an 18S rDNA probe revealed multiple ribosomal clusters, which most likely represent an apomorphic condition. We identified the ancestral insect telomeric sequence (TTAGG)n in the terminal areas of the chromosomes. Currently available data on the polyneopteran orders putatively related to Mantophasmatodea showed a wide variability of cytogenetic characteristics within and between them. The only character allowing some tentative inference to be made on the ancestry of K. biedouwense is its low chromosome number, the karyotypic pattern so far unreported for the Polyneoptera except in certain Orthoptera.

Cytogenetic analyses of five amazon lizard species of the subfamilies Teiinae and Tupinambinae and review of karyotyped diversity the family Teiidae.

Lizards of the family Teiidae (infraorder Scincomorpha) were formerly known as Macroteiidae. There are 13 species of such lizards in the Amazon, in the genera Ameiva (Meyer, 1795), Cnemidophorus (Wagler, 1830), Crocodilurus (Spix, 1825), Dracaena (Daudin, 1801), Kentropyx (Spix, 1825) and Tupinambis (Daudin, 1802). Cytogenetic studies of this group are restricted to karyotype macrostructure. Here we give a compilation of cytogenetic data of the family Teiidae, including classic and molecular cytogenetic analysis of Ameiva ameiva (Linnaeus, 1758), Cnemidophorus sp.1, Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868) and Tupinambis teguixin (Linnaeus, 1758) collected in the state of Amazonas, Brazil. Ameiva ameiva, Kentropyx calcarata and Kentropyx pelviceps have 2n=50 chromosomes classified by a gradual series of acrocentric chromosomes. Cnemidophorus sp.1 has 2n=48 chromosomes with 2 biarmed chromosomes, 24 uniarmed chromosomes and 22 microchromosomes. Tupinambis teguixin has 2n=36 chromosomes, including 12 macrochromosomes and 24 microchromosomes. Constitutive heterochromatin was distributed in the centromeric and terminal regions in most chromosomes. The nucleolus organizer region was simple, varying in its position among the species, as evidenced both by AgNO3 impregnation and by hybridization with 18S rDNA probes. The data reveal a karyotype variation with respect to the diploid number, fundamental number and karyotype formula, which reinforces the importance of increasing chromosomal analyses in the Teiidae.

Whole chromosome painting of B chromosomes of the red-eye tetra Moenkhausia sanctaefilomenae (Teleostei, Characidae).

B chromosomes are dispensable genomic elements found in different groups of animals and plants. In the present study, a whole chromosome probe was generated from a specific heterochromatic B chromosome occurring in cells of the characidae fish Moenkhausia sanctaefilomenae (Steindachner, 1907). The chromosome painting probes were used in fluorescence in situ hybridization (FISH) experiments for the assessment of metaphase chromosomes obtained from individuals from three populations of Moenkhausia sanctaefilomenae. The results revealed that DNA sequences were shared between a specific B chromosome and many chromosomes of the A complement in all populations analyzed, suggesting a possible intra-specific origin of these B chromosomes. However, no hybridization signals were observed in other B chromosomes found in the same individuals, implying a possible independent origin of B chromosome variants in this species. FISH experiments using 18S rDNA probes revealed the presence of non-active ribosomal genes in some B chromosomes and in some chromosomes of the A complement, suggesting that at least two types of B chromosomes had an independent origin. The role of heterochromatic segments and ribosomal sequences in the origin of B chromosomes were discussed.

Karyotype analysis of Lablab purpureus (L.) Sweet using fluorochrome banding and fluorescence in situ hybridisation with rDNA probes

She Ch.-W., Jiang X.-H. (2015): Karyotype analysis of Lablab purpureus (L.) Sweet using fluorochrome banding and fluorescence in situ hybridisation with rDNA probes. Czech J. Genet. Plant Breed., 51: 110–116. The mitotic chromosomes of Lablab purpureus (L.) Sweet were characterised using sequential combined pro pidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) (CPD) staining and fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. The detailed karyotype of this species was established using prometaphase chromosomes. After CPD staining, CPD and DAPI + bands were shown simultaneously. CPD bands occurred in the proximal regions of the long arms of all chromosome pairs and at all 45S rDNA sites, while the DAPI + bands appeared in all centromeres. FISH with rDNA probes revealed one 5S locus and eight 45S loci. The single 5S locus was interstitially located on the long arms of the shortest chromosome pair. Among the 45S loci, two large loci were located in the secondary constrictions of the short arms of two chromosome pairs; six small or minimal loci were proximally located on the short or long arms of six chromosome pairs. Each prometaphase chromosome pair could be identified using the CPD and DAPI + bands, the rDNA-FISH signals in combination with the chromosome measurements and condensation patterns. The karyotype was formulated as 2 n = 2x = 22 = 14m (2SAT) + 6sm + 2st (2SAT), and the asymmetry indices, CI, A 1, A2, As K%, AI and the Stebbins category were 38.23 ± 7.06, 0.36, 0.31, 61.99, 5.68 and 2B, respectively.

Chromosomal characterization in two species of an Astyanax bimaculatus complex (Characidae, Characiformes) using different techniques of chromosome banding.

Astyanax has been the subject of extensive cytogenetic studies due to its wide karyotypic diversity. This genus comprises species complexes, namely groups of fish of difficult morphological differentiation, such as the bimaculatus complex, which includes the characids with a rounded humeral spot. Thence, the present study proposed to accomplish a cytogenetic characterization of two species of this complex: A. asuncionensis and A. altiparanae, aiming to find chromosomal markers that differentiate these species, as well as achieve a better understanding of the karyotype evolution in the genus. For this we used different techniques of chromosome banding as C-banding, impregnation by silver nitrate, fluorochrome staining and FISH with 18S rDNA probe. This is the first cytogenetic study in A. asuncionensis, from Miranda river, which presented 2n=50 and 18m+22sm+6st+4a (FN=96) and single NORs. The populations of A. altiparanae also presented 2n=50, but with different karyotypic formulae: the population of the Quexada river presented 16m+24sm+4st+6a (FN=94) and the Esperança stream and Jacutinga river showed 16m+20sm+4st+10a (FN=90). All analyzed populations showed an interindividual variation in the number and location of the nucleolar organizer regions (NORs). Single and multiple NORs were detected either by impregnation with silver nitrate or by FISH with 18S rDNA probe. After C-banding, the two species differed in relation to the composition and heterochromatin distribution. The meiotic cells of A. altiparanae male individuals were also analyzed, showing that, despite the high karyotype variability, chromosome pairing occurs normally. The data show that A. altiparanae and A. asuncionensis share some characteristics with other species of the bimaculatus complex, suggesting a close phylogenetic relationship among those species. However, some features can be used as differentiation chromosomal markers in altiparanae/asuncionensis morphotypes, which could result from a natural speciation process.

Cytogenetic data on the threatened leafcutter ant Atta robusta Borgmeier, 1939 (Formicidae: Myrmicinae: Attini)

The karyotype of the threatened ant species Atta robusta is described so as to establish the evolutionary relationships of this taxon with other leafcutter ants. Standard Giemsa staining, C-banding, NOR banding, fluorochromes CMA3/DAPI, Hsc-FA technique and Fluorescence in situ Hybridization (FISH) using 18S rDNA probe were conducted on a population from Aracruz, state of Espírito Santo, Brazil, allowing for comparisons with data available on Atta and other fungus-growing ant species. The diploid chromosome number observed for A. robusta was 2n=22, and the karyotypic formula was 18m+2sm+2st. Heterochromatic blocks were observed in the centromeric region of most chromosomes, where one pair of metacentric chromosomes is characterized by a GC-rich heterochromatic band in the interstitial region of its long arm. The detection of 18S rDNA using FISH confirmed the presence of single NOR for A. robusta. This is the first report of rDNA 18S detection using FISH for leafcutter ants. The cytogenetic results of this study confirm the information available for Atta and allow us to confirm the conserved chromosome number, morphology and banding pattern within the genus for the taxa studied to date, which included species from three out of the four groups of Atta indicated by molecular data. The accumulation of cytogenetic data on fungus-growing ants enhances the understanding of the genomic evolutionary patterns of Atta, since it belongs to a group of recent origin between the most well studied ants. Cytogenetic data does not indicate restrictions in relocation or reintroduction in areas where populations were extinct due to the conserved karyotype. This study allows for cytogenetic comparison of A. robusta with other ants of Atta, emphasizing the importance of chromosomal information for species conservation.

The evolution of genome size and rDNA in diploid species ofChenopodium s.l.(Amaranthaceae)

The evolution of genome size and ribosomal DNA (rDNA) locus organization was analysed in 23 diploid species of Chenopodium s.l., all of which share the same base chromosome number of x = 9. Phylogenetic relationships among these species were inferred from plastid and nuclear ribosomal internal transcribed spacer (nrITS) DNA sequences. The molecular phylogenetic analyses assigned all analysed species of Chenopodium s.l. to six evolutionary lineages, corresponding to the recent new generic taxonomic treatment of Chenopodium s.l. The distribution of rDNA loci for four species is presented here for the first time using fluorescence in situ hybridization (FISH) with 5S and 35S rDNA probes. Most of the 23 analysed diploid Chenopodium spp. possessed a single subterminally located 35S rDNA locus, except for three species which possessed two 35S rDNA loci. One or two 5S rDNA loci were typically localized subterminally on chromosomes, rarely interstitially. Analyses of rDNA locus numbers in a phylogenetic context resulted in the reconstruction of one locus each of 35S rDNA and 5S rDNA, both in subterminal positions, as the ancestral state. Genome sizes determined using flow cytometry were relatively small (2C value < 2.8 pg), ranging from 0.734 pg in C. schraderianum to 2.721 pg in C. californicum (nearly four-fold difference), and were often conserved within major phylogenetic lineages, suggesting an adaptive value. The reconstructed ancestral genome size was small for all evolutionary lineages, and changes have probably coincided with the divergence of major lineages. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 218–235.