Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae).

Holocentric karyotypes are assumed to rapidly evolve through chromosome fusions and fissions due to the diffuse nature of their centromeres. Here, we took advantage of the recent availability of a chromosome-scale reference genome for Rhynchospora breviuscula, a model species of this holocentric genus, and developed the first set of oligo-based barcode probes for a holocentric plant. These probes were applied to 13 additional species of the genus, aiming to investigate the evolutionary dynamics driving the karyotype evolution in Rhynchospora. The two sets of probes were composed of 27,392 (green) and 23,968 (magenta) oligonucleotides (45-nt long), and generated 15 distinct FISH signals as a unique barcode pattern for the identification of all five chromosome pairs of the R. breviuscula karyotype. Oligo-FISH comparative analyzes revealed different types of rearrangements, such as fusions, fissions, putative inversions and translocations, as well as genomic duplications among the analyzed species. Two rounds of whole genome duplication (WGD) were demonstrated in R. pubera, but both analyzed accessions differed in the complex chain of events that gave rise to its large, structurally diploidized karyotypes with 2n = 10 or 12. Considering the phylogenetic relationships and divergence time of the species, the specificity and synteny of the probes were maintained up to species with a divergence time of ~25 My. However, karyotype divergence in more distant species hindered chromosome mapping and the inference of specific events. This barcoding system is a powerful tool to study chromosomal variations and genomic evolution in holocentric chromosomes of Rhynchospora species.

Macroevolutionary trends of the Neotropical genus Ameroglossum (Linderniaceae) in rocky outcrop environments.

Ameroglossum is a rare plant genus endemic to northeastern of Brazil, initially monospecific (A. pernambucense) and recently expanded by the description of eight new species and two related genera. The genus was initially placed in the family Scrophulariaceae, but this has never been phylogenetically tested. This group is ecologically restricted to rocky inselberg habitats that function as island-like systems (ILS) with spatial fragmentation, limited area, environmental heterogeneity, temporal isolation and low connectivity. Here we use a phylogenetic perspective to test the hypothesis that Ameroglossum diversification was related to island-like radiation in inselbergs. Our results support that Ameroglossum is monophyletic only with the inclusion of Catimbaua and Isabelcristinia (named here as Ameroglossum sensu lato) and this group was well-supported in the family Linderniaceae. Biogeographic analyses suggest that the ancestral of Ameroglossum and related genus arrived in South America c.a. 15 million years ago by long-distance dispersal, given the ancestral distribution of Linderniaceae in Africa. In rocky outcrop habitats, Ameroglossum s.l. developed floral morphological specialization associated with pollinating hummingbirds, compatible with an island-like model. However, no increase in speciation rate was detected, which may be related to high extinction rates and/or slow diversification rate in this ecologically restrictive environment. Altogether, in Ameroglossum key innovations involving flowers seem to have offered opportunities for evolution of greater phenotypic diversity and occupation of new niches in rocky outcrop environments.

Are chromosome number and genome size associated with habit and environmental niche variables? Insights from the Neotropical orchids.

BACKGROUND AND AIMS: The entangled relationship of chromosome number and genome size with species distribution has been the subject of study for almost a century, but remains an open question due to previous ecological and phylogenetic knowledge constraints. To better address this subject, we used the clade Maxillariinae, a widely distributed and karyotypically known orchid group, as a model system to infer such relationships in a robust methodological framework. METHODS: Based on the literature and new data, we gathered the chromosome number and genome size for 93 and 64 species, respectively. We built a phylogenetic hypothesis and assessed the best macroevolutionary model for both genomic traits. Additionally, we collected together ecological data (preferences for bioclimatic variables, elevation and habit) used as explanatory variables in multivariate phylogenetic models explaining genomic traits. Finally, the impact of polyploidy was estimated by running the analyses with and without polyploids in the sample. KEY RESULTS: The association between genomic and ecological data varied depending on whether polyploids were considered or not. Without polyploids, chromosome number failed to present consistent associations with ecological variables. With polyploids, there was a tendency to waive epiphytism and colonize new habitats outside humid forests. The genome size showed association with ecological variables: without polyploids, genome increase was associated with flexible habits, with higher elevation and with drier summers; with polyploids, genome size increase was associated with colonizing drier environments. CONCLUSIONS: The chromosome number and genome size variations, essential but neglected traits in the ecological niche, are shaped in the Maxillariinae by both neutral and adaptive evolution. Both genomic traits are partially correlated to bioclimatic variables and elevation, even when controlling for phylogenetic constraints. While polyploidy was associated with shifts in the environmental niche, the genome size emerges as a central trait in orchid evolution by the association between small genome size and epiphytism, a key innovation to Neotropical orchid diversification.

First complete NMR data and theoretical study of an antimicrobial formylated dihydrochalcone from Psidium guineense Sw.

Chalcones are a widespread group of natural compounds characterized by the basic structure 1,3-diaryl-2-propen-1-one. In this study, we focused on the uncommon formylated chalcone 3'-formyl-2',4',6'-trihydroxy-5'-methyldihydrochalcone that possess interesting antimicrobial and anticancer potential. Considering the pharmacological relevance of this compound, we report its isolation and the first complete and unequivocal NMR data for this substance, corroborated by in silico theoretical study. In addition to that, its antibacterial potential was evaluated and the compound presented strong activity against Pseudomonas aeruginosa.[Formula: see text].

Satellite DNA probes of Alstroemeria longistaminea (Alstroemeriaceae) paint the heterochromatin and the B chromosome, reveal a G-like banding pattern, and point to a strong structural karyotype conservation.

Alstroemeria species present a well-conserved and asymmetric karyotype. The genus is divided into a Chilean clade, rich in heterochromatin, and a Brazilian clade, poor in heterochromatin. We investigated the distribution of the main repetitive sequences in the chromosomes of the Brazilian species A. longistaminea (2n = 16 + 0-6B) aiming to evaluate the role played by these sequences on the structural organization of the karyotype. In situ hybridization of the three most abundant retrotransposons, corresponding to ~ 45% of the genome, was uniformly distributed. Three satellite DNA sequences, representing near half of the whole satellite fraction (1.93% of the genome), were mainly concentrated on the heterochromatin and one of them painted the whole B chromosome. Noteworthy, some satellites were located on euchromatin, either dispersed or concentrated in clusters along the chromosomes, revealing a G-band-like pattern. The two satellites that presented more C-band- and G-band-like labeling were also hybridized in situ in two other Alstroemeria species. They revealed astonishing similar patterns of distribution, indicating an unusually structural karyotype conservation among Brazilian species.

Origin and evolution of highly polymorphic rDNA sites in Alstroemeria longistaminea (Alstroemeriaceae) and related species.

Alstroemeria (Alstroemeriaceae) displays a conserved and highly asymmetric karyotype, where most rDNA sites can be properly recognized by the size and morphology of the chromosomes. We analyzed the intraspecific variation of rDNA sites in A. longistaminea and compared with their distribution in other species (A. caryophyllaea and A. piauhyensis) and a representative of a sister genus, Bomarea edulis. All three species of Alstroemeria presented 2n = 16, and one to six B chromosomes were found in some individuals of A. longistaminea. There was a set of 12 conserved rDNA sites (four 5S and eight 35S) and up to 11 variable sites. B chromosomes were almost entirely covered by 35S signals, coupled with tiny 5S sites. Noteworthy, most rDNA sites found in A. caryophyllaea and A. piauhyensis were localized in chromosome positions similar to those in A. longistaminea, suggesting the existence of conserved hotspots for rDNA accumulation. Some of these hotspots were absent in Chilean Alstromeria as well in B. edulis. We propose that insertions of rDNA sequences on chromosomes do not occur randomly but rather on preferential sites or hotspots for insertions. The maintenance of these arrays, however, may be favored/constrained by different factors, resulting in stable or polymorphic sites.

Heterochromatin and numeric chromosome evolution in Bignoniaceae, with emphasis on the Neotropical clade Tabebuia alliance.

Bignoniaceae is a diverse family composed of 840 species with Pantropical distribution. The chromosome number 2n = 40 is predominant in most species of the family, with n = 20 formerly being considered the haploid base number. We discuss here the haploid base number of Bignoniaceae and examine heterochromatin distributions revealed by CMA/DAPI fluorochromes in the Tabebuia alliance, as well as in some species of the Bignonieae, Tecomeae, and Jacarandeae tribes. When comparing the chromosome records and the phylogenies of Bignoniaceae it can be deduced that the base number of Bignoniaceae is probably n = 18, followed by an ascendant dysploidy (n = 18 → n = 20) in the most derived and diverse clades. The predominant heterochromatin banding patterns in the Tabebuia alliance were found to be two terminal CMA+ bands or two terminal and two proximal CMA+ bands. The banding pattern in the Tabebuia alliance clade was more variable than seen in Jacarandeae, but less variable than Bignonieae. Despite the intermediate level of variation observed, heterochromatin banding patterns offer a promising tool for distinguishing species, especially in the morphologically complex genus Handroanthus.

Chromosomal variability in Brazilian species of Anthurium Schott (Araceae): Heterochromatin, polyploidy, and B chromosomes.

The genus Anthurium has a Neotropical distribution, with karyotype predominance of x = 15, although some species show disploidy or polyploid variations. The karyotypes of seven species and different populations of Anthurium were analyzed using fluorochrome CMA and DAPI staining. The karyotypes were composed of meta- and submetacentric chromosomes, with numbers varying from 2n = 30 to 2n = 60. Supernumerary euchromatic chromosomes were observed in A. affine, and supernumerary heterochromatic chromosomes were observed in A. gladiifolium and A. petrophilum. Polyploidy was recurrent in the Anthurium species analyzed, with records of 2n = 30 and 60 in different A. pentaphyllum populations. Fluorochrome staining revealed different CMA+ banding distributions between diploid and polyploid cytotypes of A. pentaphyllum, suggesting structural alteration events. Anthurium petrophilum, on the other hand, showed a more consistent banding profile, with 10 to 12 proximal CMA bands in the three populations analyzed. DAPI+/CMA0 regions occurred exclusively in populations of A. gracile and A. pentaphyllum. The heterochromatic fraction in Anthurium was found to be quantitatively variable among species and populations, which may be related with adaptive aspects, different environmental conditions, or phylogenetic position.

Comparative cytogenetics of the ACPT clade (Anacampserotaceae, Cactaceae, Portulacaceae, and Talinaceae): a very diverse group of the suborder Cactineae, Caryophyllales.

The clade ACPT (Anacampserotaceae, Cactaceae, Portulacaceae, and Talinaceae) is the most diverse lineage of the subordem Cactineae. The relationships between these families are still uncertain, with different topologies suggested by phylogenetic analyses with several combinations of markers. Different basic numbers (x) have been suggested for each family and for the subord, often in a contestable way. Comparative cytogenetic has helped to understand the evolutionary relationships of phylogenetically poorly resolved groups, as well as their mechanisms of karyotype evolution. The karyotype evolution in representatives of Cactineae was analyzed, focusing on the ACPT clade, through the analysis of chromosome number in a phylogenetic bias. The phylogeny obtained showed a well-resolved topology with support for the monophyly of the five families. Although a chromosomal number is known for less than 30% of the Cactineae species, the analyses revealed a high karyotype variability, from 2n = 8 to 2n = 110. The analysis of character reconstruction of the ancestral haploid numbers (p) suggested p = 12 for Cactineae, with distinct basic numbers for the clade family ACPT: Cactaceae and Montiaceae (p = 11), Talinaceae (p = 12), and Anacampserotaceae and Portulacaceae (p = 9). Talinaceae, Anacampserotaceae, and Cactaceae were stable, while Portulaca and Montiaceae were karyotypically variable. The chromosome evolution of this group was mainly due to events of descending disploidy and poliploidy. Our data confirm that the low phylogenetic resolution among the families of the ACPT clade is due to a divergence of this clade in a short period of time. However, each of these families can be characterized by basic chromosome numbers and unique karyotype evolution events.

Leaf morphoanatomy of "mororó" (Bauhinia and Schnella, Fabaceae)

Abstract Bauhinia L. and Schnella (Raddi.) Wund. are popularly known in Brazil as "mororó". The leaves and stem bark are used in folk medicine for various purposes, especially against diabetes. Morphoanatomical studies of the leaves of Bauhinia cheilantha (Bong.) Steud., B. pentandra (Bong.) Steud., B. ungulata L. and Schnella outimouta (Aublet) Wund., tribe Cercidae, subtribe Bauhiniinae (Benth.) Walp., were carried out as subsidies to the quality control of their etnodrugs and their derivatives, as well as an additional support to their taxonomy. The morphological and anatomical studies employed traditional techniques of stereo- and light microscopy. All species showed bifoliate leaves, a dorsiventral mesophyll, epidermis with a papillose abaxial surface, anomocytic stomata at the level of the epidermis, and tector trichomes. Schnella outimouta showed leaf characters distinctive from the three species of Bauhinia: indument puberulous on the abaxial surface, leaves hypostomatic, midrib with two collateral bundles, and a cylindrical petiole. The species of Bauhinia have a sericeous-pubescent indument, amphistomatic leaves with boat-shaped glands, midrib with a single bundle, and a canaliculate petiole with lateral projections. Our results provide leaf morphological and anatomical parameters, useful to distinguish the four species studied, which support the quality control of its ethnodrugs.

Karyotype analysis in Bignonieae (Bignoniaceae): chromosome numbers and heterochromatin.

Chromosome numbers and heterochromatin banding pattern variability have been shown to be useful for taxonomic and evolutionary studies of different plant taxa. Bignonieae is the largest tribe of Bignoniaceae, composed mostly by woody climber species whose taxonomies are quite complicated. We reviewed and added new data concerning chromosome numbers in Bignonieae and performed the first analyses of heterochromatin banding patterns in that tribe based on the fluorochromes chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI). We confirmed the predominant diploid number 2n = 40, as well as variations reported in the literature (dysploidy in Mansoa [2n = 38] and polyploidy in Dolichandra ungis-cati [2n = 80] and Pyrostegia venusta [2n = 80]). We also found a new cytotype for the genus Anemopaegma (Anemopaegma citrinum, 2n = 60) and provide the first chromosome counts for five species (Adenocalymma divaricatum, Amphilophium scabriusculum, Fridericia limae, F. subverticillata, and Xylophragma myrianthum). Heterochromatin analyses revealed only GC-rich regions, with six different arrangements of those bands. The A-type (one large and distal telomeric band) were the most common, although the presence and combinations of the other types appear to be the most promising for taxonomic studies.

Karyotype analysis in Bignonieae (Bignoniaceae): chromosome numbers and heterochromatin

ABSTRACT Chromosome numbers and heterochromatin banding pattern variability have been shown to be useful for taxonomic and evolutionary studies of different plant taxa. Bignonieae is the largest tribe of Bignoniaceae, composed mostly by woody climber species whose taxonomies are quite complicated. We reviewed and added new data concerning chromosome numbers in Bignonieae and performed the first analyses of heterochromatin banding patterns in that tribe based on the fluorochromes chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI). We confirmed the predominant diploid number 2n = 40, as well as variations reported in the literature (dysploidy in Mansoa [2n = 38] and polyploidy in Dolichandra ungis-cati [2n = 80] and Pyrostegia venusta [2n = 80]). We also found a new cytotype for the genus Anemopaegma (Anemopaegma citrinum, 2n = 60) and provide the first chromosome counts for five species (Adenocalymma divaricatum, Amphilophium scabriusculum, Fridericia limae, F. subverticillata, and Xylophragma myrianthum). Heterochromatin analyses revealed only GC-rich regions, with six different arrangements of those bands. The A-type (one large and distal telomeric band) were the most common, although the presence and combinations of the other types appear to be the most promising for taxonomic studies.

Intrachromosomal karyotype asymmetry in Orchidaceae

Abstract The asymmetry indexes have helped cytotaxonomists to interpret and classify plant karyotypes for species delimitation efforts. However, there is no consensus about the best method to calculate the intrachromosomal asymmetry. The present study aimed to compare different intrachromosomal asymmetry indexes in order to indicate which are more efficient for the estimation of asymmetry in different groups of orchids. Besides, we aimed to compare our results with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971). Through a literature review, karyotypes were selected and analyzed comparatively with ideal karyotypes in a cluster analysis. All karyotypes showed some level of interchromosomal asymmetry, ranging from slightly asymmetric to moderately asymmetric. The five tested intrachromosomal asymmetry indexes indicated Sarcoglottis grandiflora as the species with the most symmetrical karyotype and Christensonella pachyphylla with the most asymmetrical karyotype. In the cluster analysis, the largest number of species were grouped with the intermediary ideal karyotypes B or C. Considering our results, we recommend the combined use of at least two indexes, especially Ask% or A1 with Syi, for cytotaxonomic analysis in groups of orchids. In an evolutionary perspective, our results support Stebbins' hypothesis that asymmetric karyotypes derive from a symmetric karyotypes.

Phytochemical study of Pilosocereus pachycladus and antibiotic-resistance modifying activity of syringaldehyde

ABSTRACT Pilosocereus pachycladus F. Ritter, Cactaceae, popularly known as "facheiro", is used as food and traditional medicine in Brazilian caatinga ecoregion. The plant is used to treat prostate inflammation and urinary infection. The present work reports the first secondary metabolites isolated from P. pachycladus. Therefore, the isolated compound 4-hydroxy-3,5-dimethoxy benzaldehyde (syringaldehyde) was evaluated as modulator of Staphylococcus aureus pump efflux-mediated antibiotic resistance. The isolation of compounds was performed using chromatographic techniques and the structural elucidation was carried out by spectroscopic methods. In order to evaluate syringaldehyde ability to modulate S. aureus antibiotic resistance, its minimum inhibitory concentrations (µg/ml) was first determinate, then, the tested antibiotics minimum inhibitory concentrations were determined in the presence of the syringaldehyde in a sub-inhibitory concentration. The chromatographic procedures led to isolation of twelve compounds from P. pachycladus including fatty acids, steroids, chlorophyll derivatives, phenolics and a lignan. The syringaldehyde did not show any antibacterial activity at 256 µg/ml against S. aureus. On the other hand the compound was able to reduce the antibiotic concentration (tetracycline, norfloxacin, ethidium bromide) required to inhibit the growth of drug-resistant bacteria, showing the ability of syringaldehyde of inhibiting the efflux pump on these bacteria.

Intrachromosomal karyotype asymmetry in Orchidaceae.

The asymmetry indexes have helped cytotaxonomists to interpret and classify plant karyotypes for species delimitation efforts. However, there is no consensus about the best method to calculate the intrachromosomal asymmetry. The present study aimed to compare different intrachromosomal asymmetry indexes in order to indicate which are more efficient for the estimation of asymmetry in different groups of orchids. Besides, we aimed to compare our results with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971). Through a literature review, karyotypes were selected and analyzed comparatively with ideal karyotypes in a cluster analysis. All karyotypes showed some level of interchromosomal asymmetry, ranging from slightly asymmetric to moderately asymmetric. The five tested intrachromosomal asymmetry indexes indicated Sarcoglottis grandiflora as the species with the most symmetrical karyotype and Christensonella pachyphylla with the most asymmetrical karyotype. In the cluster analysis, the largest number of species were grouped with the intermediary ideal karyotypes B or C. Considering our results, we recommend the combined use of at least two indexes, especially Ask% or A1 with Syi, for cytotaxonomic analysis in groups of orchids. In an evolutionary perspective, our results support Stebbins' hypothesis that asymmetric karyotypes derive from a symmetric karyotypes.

New Alcamide and Anti-oxidant Activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. ex Rowl. (Cactaceae).

The Cactaceae family is composed by 124 genera and about 1438 species. Pilosocereus gounellei, popularly known in Brazil as xique-xique, is used in folk medicine to treat prostate inflammation, gastrointestinal and urinary diseases. The pioneering phytochemical study of P. gounellei was performed using column chromatography and HPLC, resulting in the isolation of 10 substances: pinostrobin (1), ß-sitosterol (2), a mixture of sitosterol 3-O-ß-d-glucopyranoside/stigmasterol 3-O-ß-d-glucopyranoside (3a/3b), 13²-hydroxyphaeophytin a (4), phaeophytin a (5), a mixture of ß-sitosterol and stigmasterol (6a/6b), kaempferol (7), quercetin (8), 7'-ethoxy-trans-feruloyltyramine (mariannein, 9) and trans-feruloyl tyramine (10). Compound 9 is reported for the first time in the literature. The structural characterization of the compounds was performed by analyses of 1-D and 2-D NMR data. In addition, a phenolic and flavonol total content assay was carried out, and the anti-oxidant potential of P. gounellei was demonstrated.

Rock outcrop orchids reveal the genetic connectivity and diversity of inselbergs of northeastern Brazil.

BACKGROUND: Because of their fragmented nature, inselberg species are interesting biological models for studying the genetic consequences of disjoint populations. Inselbergs are commonly compared with oceanic islands, as most of them display a marked ecological isolation from the surrounding area. The isolation of these rock outcrops is reflected in the high number of recorded endemic species and the strong floristic differences between individual inselbergs and adjacent habitats. We examined the genetic connectivity of orchids Epidendrum cinnabarinum and E. secundum adapted to Neotropical inselbergs of northeastern Brazil. Our goals were to identify major genetic divergences or disjunctions across the range of the species and to investigate potential demographic and evolutionary mechanisms leading to lineage divergence in Neotropical mountain ecosystems. RESULTS: Based on plastid markers, high genetic differentiation was found for E. cinnabarinum (FST = 0.644) and E. secundum (FST = 0.636). Haplotypes were not geographically structured in either taxon, suggesting that restricted gene flow and genetic drift may be significant factors influencing the diversification of these inselberg populations. Moreover, strong differentiation was found between populations over short spatial scales, indicating substantial periods of isolation among populations. For E. secundum, nuclear markers indicated higher gene flow by pollen than by seeds. CONCLUSIONS: The comparative approach adopted in this study contributed to the elucidation of patterns in both species. Our results confirm the ancient and highly isolated nature of inselberg populations. Both species showed similar patterns of genetic diversity and structure, highlighting the importance of seed-restricted gene flow and genetic drift as drivers of plant diversification in terrestrial islands such as inselbergs.

Chromosome characterization and variability in some Iridaceae from Northeastern Brazil.

The chromosomes of 15 species of Iridaceae of the genera Alophia, Cipura, Eleutherine, Neomarica and Trimezia (subfamily Iridoideae) were examined after conventional Giemsa staining. The karyotypes of Alophia drummondii (2n = 14+1B, 28, 42 and 56), Cipura paludosa (2n = 14), C. xanthomelas (2n = 28) and Eleutherine bulbosa (2n = 12) were asymmetric; Neomarica candida, N. caerulea, N. humilis, N. glauca, N. gracilis, N. northiana and Neomarica sp. (2n = 18); N. cf. paradoxa (2n = 28), Trimezia fosteriana (2n = 52), T. martinicensis (2n = 54) and T. connata (2n = 82) were all generally symmetric. New diploid numbers of 2n = 56 for Alophia drummondii, 2n = 18 for N. candida, N. humilis, N. glauca, and N. gracilis, 2n = 28 for N. cf. paradoxa, and 2n = 82 for T. connata are reported. The karyotypic evolution of the studied species is discussed.

Chromosome characterization and variability in some Iridaceae from Northeastern Brazil

The chromosomes of 15 species of Iridaceae of the genera Alophia, Cipura, Eleutherine, Neomarica and Trimezia (subfamily Iridoideae) were examined after conventional Giemsa staining. The karyotypes of Alophia drummondii (2n = 14+1B, 28, 42 and 56), Cipura paludosa (2n = 14), C. xanthomelas (2n = 28) and Eleutherine bulbosa (2n = 12) were asymmetric; Neomarica candida, N. caerulea, N. humilis, N. glauca, N. gracilis, N. northiana and Neomarica sp. (2n = 18); N. cf. paradoxa (2n = 28), Trimezia fosteriana (2n = 52), T. martinicensis (2n = 54) and T. connata (2n = 82) were all generally symmetric. New diploid numbers of 2n = 56 for Alophia drummondii, 2n = 18 for N. candida, N. humilis, N. glauca, and N. gracilis, 2n = 28 for N. cf. paradoxa, and 2n = 82 for T. connata are reported. The karyotypic evolution of the studied species is discussed.

Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae)

We investigated four orchids of the genus Maxillaria (M. discolor, M. acicularis, M. notylioglossa and M. desvauxiana) in regard to the position of heterochromatin blocks as revealed using chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) fluorochrome staining and 5S and 45S rDNA sites using fluorescence in situ hybridization (FISH). The species showed differences in chromosome number and a diversified pattern of CMA+ and DAPI+ bands, including heteromorphism for CMA+ bands. The 5S and 45S rDNA sites also varied in number and most of them were co-localized with CMA+ bands. The relationship between 5S rDNA sites and CMA+ bands was more evident in M. notylioglossa, in which the brighter CMA+ bands were associated with large 5S rDNA sites. However, not all 5S and 45S rDNA sites were co-localized with CMA+ bands, probably due to technical constraints. We compare these results to banding data from other species and suggest that not all blocks of tandemly repetitive sequences, such as 5S rDNA sites, can be observed as heterochromatin blocks.