Cytogenetic studies on populations of Camponotus rufipes (Fabricius, 1775) and Camponotus renggeri Emery, 1894 (Formicidae: Formicinae).

Two valid ant species, Camponotus rufipes and Camponotus renggeri, have recently been the subject of a broad discussion with reference to taxa synonymization. Both species are quite common among the Neotropical myrmecofauna and share some unique traits, such as the shape of the scape and the pilosity patterns of the tibiae and scapes. A single morphological trait can help distinguish these species; however, only a combination of different approaches can enlighten our view of the complex phylogenetic relationships prevailing in the different populations of these two taxa. Therefore, focusing on the taxonomic issues concerning these two species, a cytogenetic survey including 10 populations of C. rufipes and two populations of C. renggeri was performed. In order to better understand the extent of the relationship between C. rufipes and C. renggeri, two common Neotropical Camponotus species, C. atriceps and C. cingulatus were taken as outgroups. All four species of Camponotus that were studied had 2n = 40 chromosomes (4sm+34st+2t); however, the abundance of chromosome rearrangements observed, combined with several chromosome markers, suggest that C. rufipes and C. renggeri are two good distinct species although closely related. The already reported chromosome translocation 2n = 39 (1m+4sm+32st+2t) for C. rufipes has been found in different populations as in the unprecedented chromosome inversions found both in C. rufipes and in C. renggeri populations. Within the C. renggeri chromosome inversions, both the heterozygous state 2n = 40 (1m+3sm+34st+2t) and the homozygous state, 2n = 40 (2m+2sm+34st+2t) were identified. However, only heterozygous specimens for chromosome inversions were found among C. rufipes, with karyotype configurations distinct from those found in C. renggeri, with 2n = 40 (1m+4sm+34st+2t). None of the populations studied showed signs of mosaic individuals. With respect to rDNA clusters, the 18S rDNA seemed to be more restricted inside the genome, as C. renggeri showed four 18S rDNA clusters, whereas, C. rufipes, C. atriceps, and C. cingulatus showed only two clusters. The chromosome locations of the 5S rDNA clusters were pointed for the first time in Formicidae, and showed itself to be more widely spread over the genome. By combining different chromosome banding approaches it was possible to demonstrate the crucial importance that chromosome inversions played on the karyotype evolution within these ants. The results also showed that chromosome translocations might be a consequence of the chromatin dynamic condition observed among Camponotus species. The homozygosis condition found in a C. renggeri from a Brazilian savanna population for chromosome inversions and the contrasting heterozygous condition for a different kind of chromosome inversion in C. rufipes from the Brazilian coastal rainforest, opens the window for a chromosome race hypothesis within the group C. renggeri and C. rufipes. The wide distribution, rich ecological interactions, genetic diversity, and morphological variability among C. renggeri and C. rufipes justify questioning of the actual taxonomic status of these species. The answer of this puzzle is clear when observing the number of 18S rDNA clusters of these ants, as C. rufipes has only two clusters whereas C. renggeri has four.

Cytogenetic data on six leafcutter ants of the genus Acromyrmex Mayr, 1865 (Hymenoptera, Formicidae, Myrmicinae): insights into chromosome evolution and taxonomic implications.

Cytogenetic data for the genus Acromyrmex Mayr, 1865 are available, to date, for a few species from Brazil and Uruguay, which have uniform chromosome numbers (2n = 38). The recent cytogenetic data of Acromyrmex striatus (Roger, 1863), including its banding patterns, showed a distinct karyotype (2n = 22), similar to earlier studied Atta Fabricius, 1804 species. Karyological data are still scarce for the leafcutter ants and many gaps are still present for a proper understanding of this group. Therefore, this study aimed at increasing cytogenetic knowledge of the genus through the characterization of other six species: Acromyrmex balzani (Emery, 1890), Acromyrmex coronatus Fabricius, 1804, Acromyrmex disciger (Mayr, 1887), Acromyrmex echinatior (Forel, 1899), Acromyrmex niger (Smith, 1858) and Acromyrmex rugosus (Smith, 1858), all of which were collected in Minas Gerais - Brazil, except for Acromyrmex echinatior which was collected in Barro Colorado - Panama. The number and morphology of the chromosomes were studied and the following banding techniques were applied: C-banding, fluorochromes CMA3 and DAPI, as well as the detection of 45S rDNA using FISH technique. All the six species had the same chromosome number observed for already studied species, i.e. 2n = 38. Acromyrmex balzani had a different karyotype compared with other species mainly due to the first metacentric pair. The heterochromatin distribution also showed interspecific variation. Nevertheless, all the studied species had a pair of bands in the short arm of the first subtelocentric pair. The fluorochrome CMA3 visualized bands in the short arm of the first subtelocentric pair for all the six species, while Acromyrmex rugosus and Acromyrmex niger also demonstrated in the other chromosomes. The AT-rich regions with differential staining using DAPI were not observed. 45S ribosomal genes were identified by FISH in the short arm of the first subtelocentric pair in Acromyrmex coronatus, Acromyrmex disciger and Acromyrmex niger. The uniform chromosome number in the genus Acromyrmex (2n = 38) suggests that Acromyrmex striatus (2n = 22) should be transferred to a new genus. Other aspects of the chromosome evolution in ants are also discussed.

New sex-determination system in the genus Panstrongylus (Hemiptera: Reduviidae) revealed by chromosomal analysis of Panstrongylus lutzi.

BACKGROUND: Panstrongylus lutzi (Neiva & Pinto, 1923) is a triatomine species native to Caatinga habitats in north-eastern Brazil. It is considered an important vector of Chagas disease in this region, presenting high rates of natural infection with Trypanosoma cruzi Chagas, 1909, and readily invading houses by flight. This study describes a previously unknown chromosomal sex system in the genus Panstrongylus based on P. lutzi. METHODS: Fifth-instar and male adults of P. lutzi originating from municipality of Várzea Alegre, Ceará (Brazil) were analysed. Chromosomal analyses of male meiotic process were done by Giemsa staining. RESULTS: Chromosomal analyses of male meiosis reveal a diploid chromosome number of 24 chromosomes (20 autosomes plus X1X2X3Y). During meiotic prophase I, the sex chromosomes remained close together, forming four heteropycnotic chromocenters in zygotene, and a single chromocenter in pachytene and diplotene. Still at the diplotene stage, each one of the ten autosomal bivalents showed an evident chiasma. In metaphase I, the four sex chromosomes appeared clearly separated. The three X chromosomes were the smallest of the complement and isopycnotic with respect to the Y chromosome. Two bivalents appear larger, whereas the other eight showed no significant difference in size. CONCLUSION: Karyotype analysis of P. lutzi revealed a new sex system in the genus Panstrongylus. This result is of utmost importance to karyosystematics of P. lutzi, and demonstrates the need for further studies of this type in the subfamily Triatominae.

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 first cytogenetic data on Strumigenys louisianae Roger, 1863 (Formicidae: Myrmicinae: Dacetini): the lowest chromosome number in the Hymenoptera of the neotropical region.

In the present study, the first cytogenetic data was obtained for the ant species Strumigenys louisianae, from a genus possessing no previous cytogenetic data for the Neotropical region. The chromosome number observed was 2n = 4, all possessing metacentric morphology. Blocks rich in GC base pairs were observed in the interstitial region of the short arm of the largest chromosome pair, which may indicate that this region corresponds to the NORs. The referred species presented the lowest chromosome number observed for the subfamily Myrmicinae and for the Hymenoptera found in the Neotropical region. Observation of a low chromosome number karyotype has been described in Myrmecia croslandi, in which the occurrence of tandem fusions accounts for the most probable rearrangement for its formation. The accumulation of cytogenetic data may carry crucial information to ensure deeper understanding of the systematics of the tribe Dacetini.

Cytogenetics of Melitoma segmentaria (Fabricius, 1804) (Hymenoptera, Apidae) reveals differences in the characteristics of heterochromatin in bees.

To date, more than 65 species of Brazilian bees (of the superfamily Apoidea) have been cytogenetically studied, but only a few solitary species have been analyzed. One example is the genus Melitoma Lepeletier & Serville, 1828, for which there is no report in the literature with regard to cytogenetic studies. The objective of the present study is to analyze the chromosome number and morphology of the species Melitoma segmentaria (Fabricius, 1804), as well as to determine the pattern of heterochromatin distribution and identify the adenine-thymine (AT)- and guanine-cytosine (GC)-rich regions. Melitoma segmentaria presents chromosome numbers of 2n=30 (females) and n=15 (males). With C-banding, it is possible to classify the chromosomes into seven pseudo-acrocentric pairs (A(M)), seven pseudo-acrocentric pairs with interstitial heterochromatin (A(Mi)), and one totally heterochromatic metacentric pair (M(h)). Fluorochrome staining has revealed that heterochromatin present in the chromosomal arms is rich in GC base pairs (CMA3 (+)) and the centromeric region is rich in AT base pairs (DAPI(+)). The composition found for Melitoma diverges from the pattern observed in other bees, in which the heterochromatin is usually rich in AT. In bees, few heterochromatic regions are rich in GC and these are usually associated with or localized close to the nucleolus organizer regions (NORs). Silver nitrate impregnation marks the heterochromatin present in the chromosome arms, which makes identification of the NOR in the chromosomes impossible. As this technique reveals proteins in the NOR, the observation that is made in the present study suggests that the proteins found in the heterochromatin are qualitatively similar to those in the NOR.

Occurrence of pre-nucleolar bodies and 45S rDNA location on the chromosomes of the ant Mycocepurus goeldii (Forel) (Formicidae, Myrmicinae, Attini).

The ant Mycocepurus goeldii (Forel) is known for having a relict karyotype with low chromosome number and the present study help the understanding of this ant cytogenetics by describing the occurrence of pre-nucleolar bodies in their chromosomes using impregnation with silver nitrate (Ag-NOR) and the location of 45S rDNA sites by means of the FISH (fluorescent in situ hybridization) technique. Several spots were observed surrounding all chromosomes when submitted to the Ag-NOR technique. These unusual markings were observed in both chromatids of metaphase and early anaphase chromosomes, and are associated to the presence of pre-nucleolar bodies, allowing the observation of the phenomenon of nucleologenesis. Although recent studies have shown that all chromosomes of M. goeldii exhibit centromeric or pericentromeric markings for the CMA(3) fluorochrome, the FISH technique indicated the presence of 45S rDNA in only one pair of chromosomes that differed in the number of CMA(3) markings observed for this species, pointing that the other markings observed with this fluorochrome do not match the sequences in ribosomal genes.

First cytogenetic characterization of a species of the arboreal ant genus Azteca Forel, 1978 (Dolichoderinae, Formicidae).

In this paper we present, for the first time, a detailed karyotype characterization of a species of the genus Azteca (Dolichoderinae, Formicidae). Cerebral ganglia from Azteca trigona Emery, 1893 were excised and submitted to colchicine hypotonic solution and chromosomal preparations were analyzed through conventional staining with Giemsa, C-banding, silver nitrate staining (AgNO3) and sequential base-specific fluorochromes. The analysis shows that Azteca trigona has a diploid number of 28 chromosomes. The karyotype consists of five metacentric pairs, seven acrocentric pairs and two pseudo-acrocentric pairs, which represents a karyotype formula 2K= 10M + 14A + 4A(M) and a diploid number of the arms 2AN = 38. The analysis of heterochromatin distribution revealed a positive block on distal region of the short arm of fourth metacentric pair, which was coincident with Ag-NOR band and CMA3 fluorochrome staining, meaning that rDNA sequences are interspaced by GC-rich base pairs sequences. The C-banding also marked short arms of other chromosomes, indicating centric fissions followed by heterochromatin growth. The karyotype analysis of Azteca trigona allowed the identification of cytogenetic markers that will be helpful in a difficult taxonomic group as Azteca and discussion about evolutionary aspects of the genome organization.

Occurrence of B chromosomes in Tetragonisca Latreille, 1811 (Hymenoptera, Apidae, Meliponini): A new contribution to the cytotaxonomy of the genus.

Tetragonisca angustula and Tetragonisca fiebrigi have recently been listed as valid species. This study aimed to cytogenetically investigate both species, emphasizing the new registry of B chromosomes in the tribe Meliponini. We analyzed colonies of T. angustula and T. fiebrigi collected at Tangará da Serra, Mato Grosso, Brazil, through conventional Giemsa staining, C-banding, and base-specific fluorochrome staining (CMA(3)/DAPI). T. angustula showed 2n = 34 chromosomes in females and n = 17 in males, with karyotype formula 2K = 34A(M). T. fiebrigi showed numeric variation, with chromosome number varying from 2n = 34 to 2n = 36 in females and from n = 17 to n = 18 in males, with karyotype formula 2K = 32A(M)+2A(Mc) and 2K = 32A(M)+2A(Mc) + 1 or 2 B-chromosomes. The B chromosomes are heterochromatic. In T. fiebrigi, the CMA(3)/DAPI staining revealed four chromosomes with a CMA(3) positive band. All individuals from the same colony showed the same number of B chromosomes. T. angustula and T. fiebrigi showed karyotype divergence, principally due to the presence of B chromosomes, which are found only in T. fiebrigi. Our data corroborate the status of valid species for both T. angustula and T. fiebrigi, as recently proposed.

Occurrence of B chromosomes in Tetragonisca Latreille, 1811 (Hymenoptera, Apidae, Meliponini): a new contribution to the cytotaxonomy of the genus

Tetragonisca angustula and Tetragonisca fiebrigi have recently been listed as valid species. This study aimed to cytogenetically investigate both species, emphasizing the new registry of B chromosomes in the tribe Meliponini. We analyzed colonies of T. angustula and T. fiebrigi collected at Tangará da Serra, Mato Grosso, Brazil, through conventional Giemsa staining, C-banding, and base-specific fluorochrome staining (CMA3/DAPI). T. angustula showed 2n = 34 chromosomes in females and n = 17 in males, with karyotype formula 2K = 34A M. T. fiebrigi showed numeric variation, with chromosome number varying from 2n = 34 to 2n = 36 in females and from n = 17 to n=18in males, with karyotype formula 2K = 32A M+2A Mc and 2K = 32A M+2A Mc + 1 or 2 B-chromosomes. The B chromosomes are heterochromatic. In T. fiebrigi, the CMA3/DAPI staining revealed four chromosomes with a CMA3 positive band. All individuals from the same colony showed the same number of B chromosomes. T. angustula and T. fiebrigi showed karyotype divergence, principally due to the presence of B chromosomes, which are found only in T. fiebrigi. Our data corroborate the status of valid species for both T. angustula and T. fiebrigi, as recently proposed.

Karyotypic description of the stingless bee Oxytrigona cf. flaveola (Hymenoptera, Apidae, Meliponina) of a colony from Tangará da Serra, Mato Grosso State, Brazil.

The aim was to broaden knowledge on the cytogenetics of the subtribe Meliponina, by furnishing cytogenetic data as a contribution to the characterization of bees from the genus Oxytrigona. Individuals of the species Oxytrigona cf. flaveola, members of a colony from Tangará da Serra, Mato Grosso State, Brazil, were studied. The chromosome number was 2n = 34, distributed among four chromosomal morphologies, with the karyotype formula 8m+8sm+16st+2t. Size heteromorphism in the first metacentric pair, subsequently confirmed by sequential staining with fluorochrome (DA/DAPI/CMA(3) ), was apparent in all the examined individuals The nucleolar organizing regions (NORs) are possibly located in this metacentric chromosome pair. These data will contribute towards a better understanding of the genus Oxytrigona. Given that species in this group are threatened, the importance of their preservation and conservation can be shown in a sensible, concise fashion through studies such as this.

Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae) by fluorochromes.

Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae) were cytogenetically analyzed using conventional staining and the fluorochromes CMA(3) e DAPI. The females have 2n = 34 chromosomes (2K = 32 M¯+2 A¯). Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA (3) (+) bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.

Karyotypic description of the stingless bee Oxytrigona cf. flaveola (Hymenoptera, Apidae, Meliponina) of a colony from Tangará da Serra, Mato Grosso State, Brazil

The aim was to broaden knowledge on the cytogenetics of the subtribe Meliponina, by furnishing cytogenetic data as a contribution to the characterization of bees from the genus Oxytrigona. Individuals of the species Oxytrigona cf. flaveola, members of a colony from Tangará da Serra, Mato Grosso State, Brazil, were studied. The chromosome number was 2n = 34, distributed among four chromosomal morphologies, with the karyotype formula 8m+8sm+16st+2t. Size heteromorphism in the first metacentric pair, subsequently confirmed by sequential staining with fluorochrome (DA/DAPI/CMA3), was apparent in all the examined individuals The nucleolar organizing regions (NORs) are possibly located in this metacentric chromosome pair. These data will contribute towards a better understanding of the genus Oxytrigona. Given that species in this group are threatened, the importance of their preservation and conservation can be shown in a sensible, concise fashion through studies such as this.

Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae) by fluorochromes

Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae) were cytogenetically analyzed using conventional staining and the fluorochromes CMA3 e DAPI. The females have 2n = 34 chromosomes (2K=32+2). Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA3+ bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.

Cytogenetic characterization of Melipona rufiventris Lepeletier 1836 and Melipona mondury Smith 1863 (Hymenoptera, Apidae) by C banding and fluorochromes staining

The stingless bees Melipona rufiventris and M. mondury were analyzed cytogenetically by conventional staining with Giemsa, C-banding and sequential staining with the fluorochromes CMA3/DA/DAPI. Both species presented 2n = 18 and n = 9, except for one colony of M. rufiventris, in which some individuals had 2n = 19 due to the presence of a B chromosome. After Giemsa staining and C-banding the chromosomes appeared very condensed and presented a high heterochromatic content, making it difficult to localize the centromere and therefore to visualize the chromosomes morphology. The constitutive heterochromatin was located in interstitial chromosome regions covering most of the chromosomes extension and consisted mainly of AT, as shown by DAPI staining. The euchromatin was restricted to the chromosome extremities and was GC-rich, as evidenced by CMA3 staining. The B chromosome was CMA3-negative and DAPI-positive, a heterochromatic constitution similar to that of the A genome chromosomes.

Melípona: seis décadas de citogenética / Melípona: six decade of cytogenetic

São 59 anos de estudos citogenéticos no gênero Melipona e esse artigo é uma revisão dessa história, que vai desde trabalhos com apenas a determinação do número cromossômico até os trabalhos de citogenética molecular. Os principais focos do artigo são: o número e morfologia dos cromossomos, conteúdo heterocromático e a natureza da cromatina. Fundamentadas nesses dados são feitas inferências sobre a evolução cariotípica do gênero.

They are 59 years of cytogenetics study in Melipona genus and this paper has a review about this history, going to the works only with the chromosome number determination, up to molecular cytogenetic. The mainly focuses of this paper are: chromosome number and morphology, heterochromatin content and the chromatin nature. With base of this data they are realized inferences about the karyotype evolution of this genus.

Exploratory studies on the karyotypes of seven species of the ant neotropical genus pseudomyrmex (HYMENOPTERA: FORMICIDAE: PSEUDOMYRMECINAE)

The karyotypes of Neotropical Pseudomyrmecinae were analyzed for the first time. Seven species belongingto the Pseudomyrmex genus from four Brazilian localities had their chromosome number and morphologystudied. Six of the nine species groups of Pseudomyrmex were sampled. Chromosome numbers rangedfrom 2n=24 to 2n=70, characterized in acrocentrics and metacentrics. Our cytogenetic studies indicatethat, as in other ants, karyoptype evolution in Pseudomyrmex may have evolved increasing chromosomenumber and diversifying chromosome morphology, minimizing genetics risks for deleterious mutationsthrough centric fission and pericentric inversion. Such a karyotypic diversity is also recognized in otherants’ subfamilies, especially Myrmeciinae, Ponerinae and Myrmicinae. These first cytogenetic studies in thegenus Pseudomyrmex should contribute with other data to the evolutionary history of the Pseudomyrmecinaesubfamily.

Cytogenetic data of Partamona peckolti (Hymenoptera, Apidae, Meliponini) by C banding and fluorochrome staining with DA/CMA(3) and DA/DAPI

The stingless bees of the Partamona genus have been studied taxonomically, ecologically and behaviourally, but cytogenetic studies are still rare. The objective of this study was to obtain cytogenetic data to contribute to Partamona peckolti species characterization. Heterochromatin was localized in all chromosome pericentromeric regions but some blocks could be visualized on some large chromosomes arms. A large heterozygous DA-CMA(3)-positive band was observed on one large chromosome arm, but was completely absent when C banding was applied before fluorochrome staining, with only one small positive band being visualized. Sequential DA-CMA(3)-NOR staining of interphase nuclei provided coincident positive responses. This suggests that DA-CMA(3)-positive bands of P. peckolti correspond to nucleolar organizer regions, as previously confirmed for another Partamona species by FISH

Evolutionary dynamics of the karyotype of the wasp Trypoxylon (Trypargilum) nitidum (Hymenoptera, Sphecidae) from the Rio Doce State Park, Minas Gerais, Brazil

Cytogenetic analysis based on the distribution of C-bands showed two groups of karyotypes in a Trypoxylon nitidum population from the Rio Doce Park, State of Minas Gerais, Brazil. One of these groups, that was identical to a previously described karyotype (n = 15; 2n = 30), had a stable chromosome number and was rich in acrocentric chromosomes, whereas the other had a variable chromosome number (n = 12 to 14; 2n = 25 to 28) and was rich in pseudo-acrocentric chromosomes. We propose a hypothesis explaining the dynamics of the modifications which occurred in the karyotype of this species, based on the minimum interaction theory of Imai et al. (1986, 1988, 1994) and on the chromosome rearrangements and heteromorphisms observed by us

DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera, Meliponini).

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.