Comparative karyology of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata (Phyllostomidae, Chiroptera): banding patterns, base-specific fluorochromes and FISH of ribosomal genes

This paper provides new data on chromosomes of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata. These species were analyzed by GTG, CBG- and CB-DAPI banding, AgNO3/CMA3 sequential staining, base-specific fluorochrome dyes and in situ hybridization with 18S rDNA probe. C-banding (CBG) revealed constitutive heterochromatin in the pericentromeric regions in all autosomes and the X and Y chromosomes appeared entirely heterochromatic in both species. CB-DAPI revealed a coincident banding pattern to that obtained by CBG. Triple staining CMA3/DA/DAPI revealed an R-banding and a weak G-banding pattern in the karyotypes. Sequential AgNO3/CMA3 staining showed a NOR located interstitially on the long arm of pair 8 in D. rotundus and on the short arm of pair 13 in D. ecaudata. FISH with a rDNA probe confirmed the location and number of NORs; a difference neither in intensity nor in size of hybridization signal was detected between homologues for both species.     

Cytogenetics of the darkling beetles Zophobas aff. confusus and Nyctobates gigas (Coleoptera, Tenebrionidae)

Males of Zophobas aff. confusus and Nyctobates gigas (Tenebrionidae) collected in the State of Pernambuco, Brazil, were studied through conventional staining, C-banding, silver nitrate impregnation (AgNO(3)), and the base specific fluorochromes CMA(3) and DAPI. Z. aff. confusus was found to have 2n = 20 (9+Xyp) while N. gigas exhibited 2n = 18 (8+neoXY). Large pericentromeric blocks of constitutive heterochromatin (CH) were detected throughout the autosomal complement of the two species, except in one autosomal pair of N. gigas in which no heterochromatic block was observed. The sex chromosomes of both species were almost totally heterochromatic. Double staining with CMA(3)/DA (distamycin) and DAPI/DA marked CH in Z. aff. confusus. However, DAPI staining was more intense. N. gigas was found to possess blocks of CH-positive CMA(3) and homogeneous DAPI. AgNO(3) staining also revealed differences between the two species. In Z. confusus an NOR was observed in the sexual bivalent Xyp and N. gigas was found to have an autosomal NOR.     

Karyotypic characterization of representatives from Melolonthinae (Coleoptera: Scarabaeidae): karyotypic analysis, banding and fluorescent in situ hybridization (FISH)

Meiotic chromosomes of Phyllophaga (Phytalus) vestita, Phyllophaga (Phyllophaga) aff capillata and Lyogenys fuscus (Melolonthinae) were analyzed by conventional staining, C-banding, fluorochromes, silver nitrate and FISH. The three species had a diploid number of 2n=20 and a sex mechanism of the (Xyp; XYp) parachute type. P. (Phytalus) vestita,P. (Phyllophaga) aff capillata and Lyogenys fuscus showed pericentromeric constitutive heterochromatin (CH) in all autosomal bivalents and on X chromosomes. Staining with CMA3 and DAPI fluorochromes showed that the CH of P. (Phytalus) vestita is not specifically rich in AT and GC-base pairs, whereas in P. (Phyllophaga) aff capillata the sex bivalent and one autosomal pair were found to be enriched in GC base pairs with CMA3, and in Lyogenys fuscus CH was positive for DAPI. Silver nitrate staining revealed nucleolar remnants in all three species. However, FISH obtained a precise identification of nucleolar organizing regions with an rDNA 18S and 25S probe. A signal of hybridization was seen in each species, being detected in the X chromosome of P. (Phytalus) vestita and Lyogenys fuscus, and in a small autosomal bivalent of P. (Phyllophaga) aff capillata.     

Cytogenetic analysis of the neotropical spider Nephilengys cruentata (Araneomorphae, Tetragnathidae): standard staining nors, C-bands and base-specific fluorochromes.

The aim of this work is to characterize Nephilengys cruentata in relation to the diploid number, chromosome morphology, type of sex determination chromosome system, chromosomes bearing the Nucleolar Organizer Regions (NORs), C-banding pattern, and AT or GC repetitive sequences. The chromosome preparations were submitted to standard staining (Giemsa), NOR silver impregnation, C-banding technique, and base-specific fluorochrome staining. The analysis of the cells showed 2n = 24 and 2n = 26 chromosomes in the embryos, and 2n = 26 in the ovarian cells, being all the chromosomes acrocentric. The long arm of the pairs 1, 2 and 3 showed an extensive negative heteropycnotic area when the mitotic metaphases were stained with Giemsa. The sexual chromosomes did not show differential characteristics that allowed to distinguish them from the other chromosomes of the complement. Considering the diploid numbers found in N. cruentata and the prevalence of X1X2 sex determination chromosome system in Tetragnathidae, N. cruentata seems to possess 2n = 24 = 22 + X1X2 in the males, and 2n = 26 = 22 + X1X1X2X2 in the females. The pairs 1, 2 and 3 showed NORs which are coincident with the negative heteropycnotic patterns. Using the C-banding technique, the pericentromeric region of the chromosomes revealed small quantity or even absence of constitutive heterochromatin, differing of the C-banding pattern described in other species of spiders. In N. cruentata the fluorochromes DAPI/DA, DAPI/MM and CMA3/DA revealed that the constitutive heterochromatin is rich in AT bases and the NORs possess repetitive sequences of GC bases.     

Comparative cytogenetic analysis of two grasshopper species of the tribe Abracrini (Ommatolampinae, Acrididae)

The grasshopper species Orthoscapheus rufipes and Eujivarus fusiformis were analyzed using several cytogenetic techniques. The karyotype of O. rufipes, described here for the first time, had a diploid number of 2n = 23, whereas E. fusiformis had a karyotype with 2n = 21. The two species showed the same mechanism of sex determination (XO type) but differed in chromosome morphology. Pericentromeric blocks of constitutive heterochromatin (CH) were detected in the chromosome complement of both species. CMA(3)/DA/DAPI staining revealed CMA(3)-positive blocks in CH regions in four autosomal bivalents of O. rufipes and in two of E. fusiformis. The location of active NORs differed between the two species, occurring in bivalents M(6) and S(9) of O. rufipes and M(6) and M(7) of E. fusiformsi. The rDNA sites revealed by FISH coincided with the number and position of the active NORs detected by AgNO(3) staining. The variability in chromosomal markers accounted for the karyotype differentiation observed in the tribe Abracrini.     

Comparative cytogenetic analysis between Lonchorhina aurita and Trachops cirrhosus (Chiroptera, Phyllostomidae)

Phyllostomidae comprises the most diverse family of neotropical bats, its wide range of morphological features leading to uncertainty regarding phylogenetic relationships. Seeing that cytogenetics is one of the fields capable of providing support for currently adopted classifications through the use of several markers, a comparative analysis between two Phyllostomidae species was undertaken in the present study, with a view to supplying datasets for the further establishment of Phyllostomidae evolutionary relationships. Karyotypes of Lonchorhina aurita (2n = 32; FN = 60) and Trachops cirrhosus (2n = 30; FN = 56) were analyzed by G- and C-banding, silver nitrate staining (Ag-NOR) and base-specific fluorochromes. Chromosomal data obtained for both species are in agreement with those previously described, except for X chromosome morphology in T. cirrhosus, hence indicating chromosomal geographical variation in this species. A comparison of G-banding permitted the identification of homeologies in nearly all the chromosomes. Furthermore, C-banding and Ag-NOR patterns were comparable to what has already been observed in the family. In both species CMA(3) /DA/DAPI staining revealed an R-banding-like pattern with CMA (3) , whereas DAPI showed uniform staining in all the chromosomes. Fluorochrome staining patterns for pericentromeric constitutive heterochromatin (CH) regions, as well as for nucleolar organizing regions (NORs), indicated heterogeneity regarding these sequences among Phyllostomidae species.     

Nature and distribution of constitutive heterochromatin and NOR location in the grasshopper Phaeoparia megacephala (Romaleidae: Orthoptera)

The constitutive heterochromatin (CH) of Phaeoparia megacephala was studied using C-banding and fluorochrome staining (CMA3, DAPI and acridine orange). The nucleolar organizer regions (NOR) were identified with silver staining. The chromosome complement of this species was 2n = 23, XO in males, and 2n = 24, XX in females. The CH was pericentromeric in all chromosomes. L1, L2, L3 and X chromosomes showed large blocks of CH, while the medium and small chromosomes had small blocks. The staining procedure with acridine orange revealed the same pattern. All the pericentromeric regions showed small blocks of CMA3-positive constitutive heterochromatin (GC-rich regions), while only part of the large C-band positive chromosome segments (L1, L2, L3 and X) were CMA3 positive. This character demonstrates an uncommon heterogeneity of constitutive heterochromatin in P. megacephala. The fluorochrome DAPI did not reveal DAPI-positive regions (AT-rich regions). Silver staining revealed only one pair of medium chromosomes with NOR.     

Chromosomal characteristics of ribosomal DNA in two extant species of North American mudminnows Umbra pygmaea and U. limi (Euteleostei: Umbridae)

The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.     

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xy(p) type in D. nisus and D. semisquamosus and of the Xy (r) type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group.     

Description of the karyotype of Rhagomys rufescens Thomas, 1886 (Rodentia, Sigmodontinae) from Southern Brazil Atlantic forest

Rhagomys rufescens (Rodentia: Sigmodontinae) is an endemic species of the Atlantic forest from Southern and Southeastern Brazil. Some authors consider Rhagomys as part of the tribe Thomasomyini; but its phylogenetic relationships remain unclear. Chromosomal studies on eight specimens of Rhagomys rufescens revealed a diploid number of 2n = 36 and a number of autosome arms FN = 50. GTG, CBG and Ag-NOR banding and CMA(3) /DAPI staining were performed on metaphase chromosomes. Eight biarmed and nine acrocentric pairs were found in the karyotype of this species. The X and Y chromosomes were both acrocentric. Most of the autosomes and the sex chromosomes showed positive C-bands in the pericentromeric region. The X chromosome showed an additional heterochromatic block in the proximal region of the long arm. Nucleolus organizer regions (NORs) were located in the pericentromeric region of three biarmed autosomes (pairs 4, 6 and 8) and in the telomeric region of the short arm of three acrocentrics (pairs 10, 12 and 17). CMA (3) /DAPI staining produced fluorescent signals in many autosomes, especially in pairs 4, 6, and 8. This study presents cytogenetic data of Rhagomys rufescens for the first time.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Erpetoichthys calabaricus</Emphasis> (Osteichthyes,Polypteridae)

Polypterids are a group of Osteichthyan fish whose evolutionary relationships with closer basal ray-finned and lobe-finned fish have been disputed since their discovery. Very little is known about the evolutive karyology in the whole Polypteriformes group. In order to fill this gap, a cytogenetic analysis of Erpetoichthys calabaricus species was performed, using both classical and molecular techniques. Karyotype structure (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG)n and ribosomal 5S and 18S rRNA genes were examined in twenty specimens of E. calabaricus by using Ag-NOR, classical C-banding, sequential CMA3/4',6-diaminidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization (FISH). CMA3 marked all centromerical and some (no. 1 and no. 15) telomeric regions. Staining with Ag-NOR and CMA3 showed the presence of two NORs on the p arm of the chromosome pair no. 1. Hybridization with telomeric probes (TTAGGG)n showed signals at the end of all chromosomes. 5S rDNA was cloned and sequenced. After the alignment, the 5S rRNA sequences revealed an organization made up of two different classes of tandem arrays (type I and type II). FISH with 5S rDNA marked the telomeric regions of the small chromosome pair no. 15, while FISH with 18S rDNA marked the telomeric region of the pair no. 1. The results obtained were compared with cariological data on closer species now available in literature.     

Comparative cytogenetics of giant trahiras Hoplias aimara and H. intermedius (Characiformes, Erythrinidae): chromosomal characteristics of minor and major ribosomal DNA and cross-species repetitive centromeric sequences mapping differ among morphologically identical karyotypes

Karyotype and cytogenetic characteristics of 2 species of giant trahiras, Hopliasintermedius, S?o Francisco river basin, and Hopliasaimara, Arinos river (Amazon basin), were examined by conventional (C-banding, Ag-NOR, DAPI/CMA(3) double-staining) and fluorescent in situ hybridization (FISH) with 5S, 18S rDNA probes and cross-species Cot-1 DNA probing. Both species invariably had diploid chromosome number 2n = 50 and identical karyotypes composed of 10 pairs of metacentric and 15 pairs of submetacentric chromosomes. On the other hand, staining with base-specific fluorochromes (CMA(3), DAPI) and FISH mapping of repetitive DNA sequences showed extensive interspecific differences: while the genome of H. aimara had one submetacentric pair bearing CMA(3)-positive (DAPI-negative) sites, that of H. intermedius had 4 such pairs; while FISH with a 5S rDNA probe showed one (likely homologous) signal-bearing pair, that with 18S rDNA displayed one signal-bearing pair in H. intermedius and 2 such pairs in H. aimara. Cross-species FISH probing with Cot-1 DNA prepared from total DNA of both species showed no signals of Cot-1 DNA from H. aimara on chromosomes of H. intermedius but reciprocally (Cot-1 DNA from H. intermedius on chromosomes of H. aimara) displayed signals on at least 4 chromosome pairs. Present findings indicate (i) different composition of repetitive sequences around centromeres, (ii) different NOR phenotypes and (iii) distinct taxonomic status of both giant trahira species.     

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajsti?, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.     

Cytogenetic characterization of <Emphasis Type="Italic">Eurysternus caribaeus</Emphasis> (Coleoptera: Scarabaeidae): evidence of sex-autosome fusion and diploid number reduction prior to species dispersion

Mitotic and meiotic chromosomes of several populations of Eurysternus caribaeus (Coleoptera: Scarabaeidae) were analysed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining and fluorescent in situ hybridization (FISH). All specimens showed 2n = 8 in their karyotypes, with a neo-XY sex system (Y is a submetacentric and X a metacentric) and three pairs of submetacentric autosomes. The analysis of constitutive heterochromatin (CH) revealed small blocks located in the centromeric region of all chromosomes which do not present positive staining under the fluorochromes CMA3 and DAPI. Silver nitrate staining revealed that the nucleolar organizer region (NORs) is associated with the sex chromosomes. The FISH technique revealed that rDNA sites in the X and Y are different in size. Data from different populations indicate that the diploid number reduction (2n = 8) observed in E. caribaeus is established and presumably has preceded the dispersion of this species. Moreover, this reduction occasioned the translocation of rDNA sites to the sex chromosomes, X and Y, an uncommon pattern in Scarabaeidae that was observed for the first time by the FISH in this work.     

Cytogenetic analysis of three species of the genus <Emphasis Type="Italic">Haemulon</Emphasis> (Teleostei: Haemulinae) from Margarita Island,Venezuela

This paper describes the karyotype analysis of Haemulon aurolineatum, Haemulon bonariensis and Haemulon plumierii, by Giemsa staining, C-banding, Ag-staining and fluorescent in situ hybridization (FISH), to locate the 18S and 5S rRNA genes. Diploid modal count in the three species was 2n = 48 acrocentric elements. Except for pair 24, which exhibited an unmistakable secondary constriction in all three species, it was not possible to classify them as homologous to each other because differences in chromosome size were too slight between adjacent pairs within a size-graded series. Ag-NOR clusters were located in pair 24 in the three species with signal located on the secondary constriction of these chromosomes. C-banding demonstrated that the three species share the same distribution pattern of the constitutive heterochromatin with centromeric heterochromatic blocks in the 23 chromosome pairs and a pericentromeric block in pair 24 which is coincident with the NORs. FISH experiments showed that 18S rDNA sequences were located coincident with the Ag-NOR site in the three species; however, differences in both the number and chromosome distribution of 5S-rDNA cluster were detected among them. Our data suggest that chromosome evolution of Haemulon has been preserved from major changes in the karyotypic macrostructure, whereas microstructural changes have occurred.     

CPD staining: an effective technique for detection of NORs and other GC-rich chromosomal regions in plants.

Mitotic chromosome spreads of 16 plant species belonging to six families were analyzed using an improved combined PI and DAPI (CPD) staining procedure. Fluorescence in situ hybridization (FISH) with 45S rDNA probe was conducted sequentially on the same spreads to evaluate the efficiency and sensitivity of the technique. Fluorochrome staining with chromomycin A3 (CMA)-DAPI also was conducted to clarify the properties of the sequences involved in the CPD banded regions. Our results revealed that all of the NORs (rDNA sites) in the species tested were efficiently shown as red bands by CPD staining, and the number and position of the bands corresponded precisely to those of the 45S rDNA FISH signals, indicating that the detection sensitivity of CPD staining is similar to that of FISH. In 10 of the species tested including Aegilops squarrosa, Allium sativum, Oryza sativum ssp. indica, Oryza officinalis, Pisum sativum, Secale cereale, Setaria italica, Sorghum vulgare, Vicia faba and Zea mays, CPD bands were exhibited exclusively in their NORs, while in other six species including Hordeum vulgare, Allium cepa, Psophocarpus tetragonolobus, Arabidopsis thaliana, Brassica oleracea var. capitata and Lycopersicon esculentum, CPD bands appeared in chromosomal regions other than their NORs. The CPD bands were in accordance with the CMA bands in all species tested, indicating GC-rich sequences in the CPD bands and that the improved CPD staining procedure is specific for GC-rich regions in plant genomes. Our investigation not only elucidated the banding mechanisms of CPD, but also demonstrated that the CPD staining technique, which may be preferable to CMA staining, is an effective tool for detecting NORs and other GC-rich chromosomal regions in plants.     

Occurrence of ZZ/ZW sex chromosomes in <Emphasis Type="Italic">Thoracocharax stellatus</Emphasis> fish (Characiformes,Gasteropelecidae) from the Araguaia River,South America

The karyotypic and chromosomal characteristics of the hatchetfish Thoracocharax stellatus from the Araguaia River, Brazil (Araguaia-Tocantins basin) were analyzed using Giemsa, AgNO(3), and CMA(3) fluorescent staining, and C-banding. The diploid chromosome number was 54 and the karyotypes of females and males were composed of six metacentrics, six submetacentrics, six subtelocentrics and 36 acrocentrics. Two unpaired acrocentric chromosomes were detected in the female karyotype. C-banding showed heterochromatic blocks at several chromosomes and an entirely heterochromatic acrocentric chromosome in females that was lacking in the male karyotype. This discovery indicated a heteromorphic sex chromosome system of the ZZ/ZW type. Ag-staining and CMA(3) fluorescence revealed one major chromosome pair bearing the NORs with the presence of additional signals in some metaphases. Both heterochromatic segments associated with Ag-NORs and the W chromosome were positively stained by CMA(3). Considering the present data and previous findings it is hypothesized that the occurrence of ZW sex chromosome system is widespread in the genus Thoracocharax.     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

NOR regions of polychaete worms of the genus Ophryotrocha studied by chromosome banding techniques and FISH

This article reports the results of cytogenetic analyses carried out on 10 species of polychaete worms belonging to the genus Ophryotrocha (Dorvilleidae). Nucleolar organizer regions (NORs) were characterized by Ag staining, C-banding, CMA3 staining, and ribosomal fluorescent in situ hybridization (rDNA FISH). Extensive intraspecific variation in NOR number and distribution were observed in O. costlowi, O. sp. macrovifera, O. notoglandulata, O.l. labronica, O. l. pacifica (2n = 6), O. p. puerilis, O. diadema (2n = 8), O. hartmanni, O. gracilis (2n = 10). In O. sp. robusta (2n = 10), Ag-NORs were always located on a single chromosome pair. CMA3 staining suggests a possible trend toward a GC-rich rDNA compartmentalization. In O.l. labronica, O. p. puerilis, O. diadema, and O. sp. robusta rDNA FISH shows that Ag and FISH signals coincide. Results from C-banding seem to indicate that the increased genome size (GS) observed in O. sp. macrovifera (0.8 pg) and O. hartmanni (1.16 pg) compared to the base GS value of the genus (0.4 pg) cannot be attributed to variation in the heterochromatin content.