Cytogenetic Characterization of Two Partamona Species (Hymenoptera, Apinae, Meliponini) by Fluorochrome Stain- Ing and Localization of 18S Rdna Clusters by FISH

Home , Partamona

Cytogenetic Characterization of Two Partamona Species (Hymenoptera, Apinae, Meliponini) by Fluorochrome Stain- ing and Localization of 18S rDNA Clusters by FISH

Rute Magalhães Brito1, Silvia das Graças Pompolo2,*, Marta Fonseca Martins Magalhães2, Everaldo Gonçalves de Barros2 and Elza Tieme Sakamoto-Hojo3

1 Departamento de Biologia/Genética-IB/USP-R. do Matão, 277-Butantã-São Paulo, SP. 05508–090 2 Departamento de Biologia Geral-UFV-. Viçosa MG, 36570–000 Brazil. 3 Faculdade de Filosoﬁa Cieˆncias e Letras de Ribeirão Preto-USP. Av. Bandeirantes, 3900, Vila Monte Alegre-Ribeirão Preto-SP, Brazil. 14040–901

Received July 28, 2005; accepted August 23, 2005

Summary The tribe Meliponini comprises several hundred species of stingless bees which are major pollinators of many tree species. In the present work we studied two Partamona species: Par- tamona helleri and Partamona seridoensis. Both species presented similar karyotypes with 2n34 chromosomes for females, pericentromeric heterochromatic blocks and terminal GC rich heterochro-

matic segments in chromosome pairs 2, 9, 10 and 15, as shown by sequential C banding-DA/CMA3 staining. The heterochromatin is heterogeneous: pericentromeric and the terminal blocks behave quite differently. FISH with 18S rDNA probe showed that NORs were in terminal blocks. In addition, B chromosomes were observed in P. helleri. The results of the different treatments employed such as C banding and ﬂuorochrome staining led us to believe that their origin was not due to non- disjunction of A genome chromosomes. Supernumerary pericentromeric heterochromatin and the non-homologous portion of a heteromorphic pair behaved in a similar fashion, indicating that may be originated by ﬁssion. The FISH assay showed, however that there was no sequence correspondence between these segments.

Key words Partamona, Meliponini, 18S rDNA, B chromosomes.

The Meliponini are known as stingless bees and are spread over the Pantropical region. This tribe presents a great diversity comprising several hundred species. However, the precise number is not known because of the abundance of cryptic species (Michener 2000). Meliponini are believed to pollinate from 40 to 90% of tree species depending on the ecosystem (Kerr et al. 1996). Accord- ing to Kerr et al. (1996) almost 100 species of this tribe are already endangered, and the destruction of native vegetation in Brazil endangers bee species that nest in tree holes. As a consequence, many Meliponini are frequently observed nesting in wall crevices and even in plant vases in urban areas. Kerr and Silveira (1972) hypothesized that the different bee chromosome numbers resulted from polyploidization of a basic number, n8 of bee species. Alternatively, the Minimum Interac- tion Theory (Imai et al. 1986, 1988, 1994), based on ants, has also been proposed to explain the karyotype evolution of Meliponini. According to this theory, primitive karyotypes had a small number of large chromosomes and, with time the chromosomes got smaller and increased in number by centric ﬁssion. This process would prevent deleterious interactions such as translocations within the interphase nucleus. For Partamona, seven species have been studied. All presented 2n34 chromosomes for females: P. pearsoni and P. helleri (cited as cupira) (Tarelho 1973), P. mulata, P. aiylae, P. vicina and P. sp. (Brito-Ribon et al. 1999), P. peckolti (Brito et al. 2003). In P. helleri a system of B (or super-

* Corresponding author, e-mail: [email protected] 374 Rute Magalhaes Brito et al. Cytologia 70(4) numerary) chromosomes was observed, in addition to the normal complement, varying from 0 to 4 B chromosome inside and among colonies (Costa et al. 1992, Brito et al. 1997, Tosta et al. 2004). B chromosomes are quite rare in Hymenoptera. They have been observed in eight ant species (Crozier 1975, Imai 1974, Imai et al. 1977, 1984, Mariano et al. 2001, Lorite et al. 2000, Palomeque et al. 1990); in wasps Nasonia vitripennis wasps (Nur et al. 1988); Trichograma kaykai (Stouthamer et al. 2001) Trypoxylon albitarse (Araújo et al. 2000); and in another Meliponini bee Melipona quinquefasciata, with a system varying from 0 to 4 (Pompolo et al. 2004). According to Zurita et al. (1998), B chromosomes may behave as selfish genetic elements, which would increase their transmission beyond the Mendelian rate of 0.5. Over time, these chromosomes would loose the sequences responsible for their selfish behavior, being neutralized by the A genome. The cycle would then be closed with the loss of neutral or near-neutral Bs over the gen- erations, and the appearance of another selfish B by mutation. Previously we have detected a heteromorphism in the second pair of the karyotype of some colonies of P. helleri that was hypothesized to be related to the origin of B chromosomes (Brito et al. 1997). Nevertheless, a thorough chromosomal characterization of the A genome of this species is needed to reinforce or refute this possibility. In this work, we described the karyotype of Partamona seridoensis and P. helleri, from the At- lantic Forest bioma. We have also investigated the structure of the B chromosomes and their rela- tion with the A genome in P. helleri, using several techniques such as C banding, fluorochrome staining and fluorescent in situ hybridization.

Materials and methods We have analyzed 11 colonies of Partamona helleri: six from the town of Viçosa (20°45LS, 42°52LW), one from São Miguel do Anta (20°42LS, 42°43LW), one from Argirita (21°36LS, 46°08LW), and three from Porto Firme (20°40LS, 43°05LW), all in Minas Gerais state, Brazil. Three Partamona seridoensis nests were collected at Sant’Ana do Seridó city (6°35LS, 36°46LW) in the Rio Grande do Norte state, Brazil; two of them were orphans. The species were identified by Dr. Silvia Regina de Menezes Pedro, University of São Paulo, Ribeirão Preto, SP (Brazil). Cerebral ganglion metaphase slides were obtained of post-defecating larvae as described by Imai et al. (1988). The following chromosome banding techniques were applied: C banding according to Sumner (1972), GTG banding (Seabright 1971), fluorochrome staining with DA/Chro- momycin A3 (Schweizer, 1980) and Q banding with mustard quinacrine (Schmid 1980). Sequential treatments, BS-DA/CMA3, with C banding followed by DA/CMA3 and Giemsa staining were performed using the same procedures used in the individual treatments. Fluorescent in situ hybridization (FISH) was performed by the method of Pinkel et al. (1986) using an 18S rDNA probe from maize, Zea mays, kindly provided by Dr. Newton Carneiro, EM- BRAPA, Sete Lagoas, MG (Brazil). This probe was labelled with fluorescein (fd-12-dUTP) by ran- dom priming with a STRATAGENE labeling kit as recommended by manufacturer. The probe was denatured at 70°C for 5 min and the chromosomal DNA was denatured in a solution of 70% formamide 2SSC, at 80°C for 5 min. Hybridization was performed overnight in a moist chamber at 37°C in the presence of 60% formamide. After hybridization, three 0.1SSC washes were performed at 37°C. Chromosomes were classified according to Imai (1991) and mounted by pairing the chromosomes in decreasing size order.

Results and discussion General features of P. helleri and P. seridoensis karyotypes From eleven analyzed colonies of P. helleri, ﬁve had B chromosomes (2K 2005 Cytogenetic characterization of two Partamona species 375

20M¯ cc4M¯ c8M¯ ct2M¯ cci) of B1 type. This B type was described by Brito et al. (1997). P. seridoensis had 2n34 chromosomes (2K22M¯ c4M¯ cc4M¯ ct4M¯ cct) in females and n17 for males. The orphan colonies of P. seridoensis had only haploid larvae, probably sons of workers. Workers may develop ovaries when the queen is absent; a fact observed by Sakagami et al. (1963) for the Partamona. The C banding procedure revealed that the heterochromatin localization is similar in both species, with most of chromosomes with metacentric morphology (M¯ cc and M¯ c) i.e. heterochromatic blocks at the pericentromeric region (Figs. 1A, 2A). Chromosome pairs 2, 4, 9, 10 and 15, in both species presented terminal heterochromatic blocks, (Figs. 1A, 2A). The presence of this chromatin, around the centromere had been previously observed in P. sp. (Brito-Ribon et al. 1999) and in P. peckolti (Brito et al. 2003). The heteromorphism observed by Brito et al. (1997) in P. helleri has been detected in some of the colonies analyzed. This fact could not be associated with the presence of B chromosomes in the karyotype as it was also been observed in regular 2n34 cells. As the heteromorphism was easily visible in colony G15, we subjected this colony to other cytogenetic techniques to verify the possibility of this structural feature was directly related with the B chromosome origin. According to the Minimum Interaction Theory of Imai et al. (1986, 1988, 1994) metacentric chromosomes (M¯) such as those of P. helleri and P. seridoensis would have evolved by centric ﬁs- sions from large euchromatic metacentric chromosomes of an ancestral karyotype, followed by in tandem heterochromatin increase, and posterior chromosomic rearrangements such as pericentric inversions and fusions with inactivation of one of the centromeres. This heterochromatin growth would be the result of telomerase action, unequal crossing-over, genic ampliﬁcation or telomere- telomere recombination (revised by Imai 1991). Hoshiba and Imai (1993) have proposed the Minimum Interaction Theory to explain the karyotype evolution of hymenopterans as a whole. It has been supported by data obtained from wasps of the genus Microstigmus (Costa et al. 1993) and Trypoxylon (Gomes et al. 1995, Scher and Pompolo

A A

B B

C C

D D

Fig. 1. Karyotype of Partamona helleri submitted to: A) Fig. 2. Karyotype of P. seridoensis submitted to: A) C C banding; B) GTG banding; C) Mustard banding; B) GTG banding; C) Mustard

quinacrine staining; D) DA/CMA3 staining. quinacrine staining; D) DA/CMA3 staining. Bar5 mm. Bar5 mm. 376 Rute Magalhaes Brito et al. Cytologia 70(4)

2003). We wish to gain a better understanding of the nature of heterochromatin in the tribe Meliponini to verify if the Minimum Interaction Theory can be applied to stingless bees. Pertinent studies on Meliponini bees include those of Pompolo and Campos (1995) in Leurotrigona, Rocha et al. (2003) in Friesomiellita and Tetragonisca angustula, Caixeiro (1999) in Plebeia, and Costa et al. (2004) in Trigona. However, rather few species (about 80 out of 400 species) of stingless bees have been studied.

Localization of GC rich regions To characterize the karyotypes of P. helleri and P. seridoensis we have applied GTG banding according to Seabright (1971). Trypsin digested proteins that allowed us to recognize easily the chromosome pairs: 2, 9, 10 and 15 in both species. Even though as good as G band pattern cannot be achieved in insects as in mammals, the digestion of the terminal portions of those chromosome pairs matched to the positive GC bands highlighted by the DA/CMA3 staining and also to the faint bands of mustard quinacrine coloration (Figs. 1B, C, D and 2B, C, D). We conclude that the proteins digested by the trypsin have afﬁnity for AT rich regions of Partamona. The sequential treatment BS-DA/CMA3 revealed that not all positive DA/CMA3 bands were heterochromatic. Previous C banding treatment prevented the appearance of the bright terminal bands of chromosome pair number 4, which were observed when the ﬂuorochrome was applied separately in P. helleri. These results showed that heterochromatin is not homogeneous in the karyotype of the two species. This has also been previously demonstrated in Eufriesea violacea (Gomes et al. 1998), an orchid bee species which possesses large and predominantly heterochromatic chromosomes and has three to four GTG bands per chromosome when treated with trypsin. The composition difference between the pericentromeric heterochromatin and the ones located on distal portions of chromosome pairs 2, 9, 10 and 15, in P. helleri and P. seridoensis (Table 1), led us to conclude that it is im- probable that these chromatin regions have the same origin.

Number and localization of NOR sites by FISH

With the objective to further study the DNA composition of the CMA3 positive heterochromatic regions we used an in situ hybridization assay, since there are many examples on the literature of coincidental C /CMA3 /NOR marks. The FISH results with rDNA 18S probe conﬁrmed that pairs number 2, 9, 10 and 15 carry cistrons for the ribosomal RNA. The same correspondence of CMA3 /NOR has been observed in the coleopteran Olla v-nigrum (Maffei et al. 2001a); and in the wasp Trypoxylon albitarse (Araújo et al. 2000); the stingless bees Plebeia and Melipona (Maffei et al. 2001b); Friesella schrottkyi (Mampumbu 2002) and Partamona peckolti (Brito et al. 2003). However, we have noticed a difference in the number of rDNA carrier chromosomes among colonies of the same species and between species. In P. helleri, individuals of colony PK had six

Table 1. Comparision of the techniques applied to chromosomes of Partamona helleri and P. seridoensis. (andmean positive and negative responses to the treatments employed, respectively)

Chromosome pairs B chromosomes of (Terminal portion of the Technique large arm) Pericentromeric P. helleri heterochromatin Pericetromeric Euchromatin 2910 15 heterochromatin

C banding Q banding DA/CMA3 FISH (18S rDNA) 2005 Cytogenetic characterization of two Partamona species 377 carrier chromosomes and the workers of colony G15 had eight carriers chromosomes, while in the P. seridoensis non-orphan colonies individuals, seven chromosomes showed hybridization with the 18S rDNA probe (Fig. 3). The odd numbers of NORs can be explained by heterozygosity due to NOR presence in two chromosome pairs of P. helleri in colony PK and in one pair of colony 153 of P. seridoensis. Hirai et al. (1994, 1996) have reported interspecific numerical variation of NORs among Myrmecia ants. Their results showed that species presenting higher chromosome numbers also presented higher numbers of 28S rDNA positive hybridization regions. These results allowed the authors to infer that the karyotype evolution theory proposed by Imai et al. (1986, 1988, 1994) could explain the pattern observed and, also to propose that the fission- fusion cycle would also be responsible for the rDNA dispersion observed in Myrmecia. The difference in rDNA chromosome carrier number observed between P. helleri and P. seridoensis may be related to the chromosome evolutionary process that the species have under- gone. However, we believe that more species must be assayed before an analogy to the Myrmecia case may be proposed. The largest chromosome of the hetero- Fig. 3. Metaphases submitted to fluorescent in situ hy- morphic pair of P. helleri of colony G15 indi- bridization with an 18S rDNA probe. Partamona viduals showed an intense hybridization with helleri: a) colony G15; b) colony PK. Partamona seridoensis: c) colony. 153 Arrows: positive hy- the probe on the heterochromatic segment of bridization signals in the normal complement. the long arm. That may represent a larger Arrowhead: B chromosome with no hybridiza- rDNA gene copy number, if one considers the tion signals. Bar5 mm. intensity of the signals in the other two colonies (Fig. 3A). NOR heteromorphisms have been reported in the literature for many species such as Glossina flies (Willhoeft 1997); in two Myzus aphids (Bizzaro et al. 1999) and in the fish Pinirampus (Swarça et al. 2001). Unequal crossing-over between the homologous chromosomes and in tandem gene amplification of rDNA sequences may be a possible explanation for such heteromorphisms. This idea has gained support by the results of Mandriolli et al. (1999) with the analysis of the rDNA intergenic spacers of the aphid Acyrthosiphon pisum. Another fact that could increase the rearrangement frequency in P. helleri cells would be the presence of B chromosomes as seen in the grasshopper Eyprepocnemis plorans (Henriques-Gil et al. 1984) and in rye, Secale cereale (Jones 1991). There were modifications not only in frequency but also in chiasma distribution at the normal complement homologous chromosomes. In the case of Partamona helleri, this event must have happened in the queen gametic cells, since we have noticed such heteromorphism in all cells of the individual of colony G15. A population study com- bined with molecular cytogenetics should be applied to show if the structural abnormality on the P. 378 Rute Magalhaes Brito et al. Cytologia 70(4) helleri karyotype is not a polymorphism. The 18S rDNA nuclear genes were the first to be mapped in the Partamona genus. The analysis of this chromosome marker can aid the construction of a particular model for the karyotype evolution of the Meliponini bees. However, analyses of the 18S rDNA region in Meliponini are still scarce. Only Melipona (Rocha et al. 2002) and one Friesella species (Mampumbu 2002) have been analyzed so far.

B chromosomes and the structure of the A genome of P. helleri Even though B chromosomes have been studied since 1907, when they were first described (for revision see Camacho et al. 2000). Knowledge about their origin is still quite controversial. The various treatments applied in this work allowed us to conclude that the pericentromeric heterochromatin of the normal complement and that of B chromosomes of P. helleri are not similar. It suggests that the B chromosome have not originated from the A genome by non-disjunction followed by accumulation. However, the B chromosomes heterochromatin showed the same responses to the treatments of C, G, CMA3 and mustard quinacrine banding when compared to the terminal portions of the chromosome pairs 2, 9, 10 and 15 (Fig. 1 and Table 1). Such numerical alterations could have appeared from the A genome of P. helleri through fission, in particular at the heteromorphic pair. It was supported by the similar responses of the heterochromatin of the non-homologous portion of heteromorphic pair and of the supernumerary chromosome to the treatments used. However, the FISH data showed that the terminal portions of chromosome pair number two and the B chromosome have different DNA compositions (Fig. 3A). A RAPD marker band associated with the B chromosome has already been found in individuals carrying only one B chromosome (Tosta et al. 2004). The hybridization of this sequence as a probe on P. helleri chomosomes may show homologies between A and B chromosomes, if the su- pernumeraries were originated by fissions of the normal complement. However, if homologies would be observed between chromosomes of the different Partamona species, it will suggest an origin by interspecific hybridization for the Bs, as noticed for the extra PSR chromosome of Nasonia vitripennis (McAllister and Werren 1997, Perfeccti and Werren 2001). The genus Partamona has been morphologically characterized as a monophyletic group but there are many unsolved questions concerning to the phylogeny of this genus (Camargo and Pedro 2003, Pedro and Camargo 2003). Cytogenetics and molecular approaches of nuclear and mitochon- drial genomes can clarify these relations within the genus. We believe that cytogenetic data are strong phylogenetical tools, and are indispensable in the study of evolutionary pathways. Such data include: chromosome number and length; arm number and length; heterochromatin position; NORs number, and GC and AT rich “islands” position, among others. A good begining has been done with Frieseomelitta (Rocha et al. 2003); Partamona (Brito-Ribon et al. 1999, Brito et al. 2003); Plebeia (Caixeiro, 1999); and Melipona (Rocha 2002, Rocha and Pompolo 1998, Rocha et al. 2002). Our expectations are that our results will contribute for the evolutionary study of the Parta- mona genus.

Acknowledgements Research supported by the “Fundação de Amparo à Pesquisa do Estado de Minas Gerais” (FAPEMIG), “Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES) and “Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico” (CNPq). We are grateful to Dr Newton Carneiro, EMBRAPA, Sete Lagoas, MG, Brazil for providing the probe; to Dr Giselle Garcia Azevedo for the assistance with Partamona colonies; to Dr Lucio A. O. Campos for the ad- visory and encouragement. 2005 Cytogenetic characterization of two Partamona species 379

References

Araujo, S. M. R., Pompolo, S. G., Dergam, J. A. S. and Campos, L. A. O. 2000. The B chromosome system of Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae). 1. Banding analysis. Cytobios 101: 7–13. Bizzaro, D., Barbolini, E., Mandrioli, M., Mazzoni, E. and Manicardi, G. C. 1999. Cytogenetic characterization of the holo- centric chromosomes in the aphids Myzus varians and Myzus cerasi. Caryologia 52: 81–85. Brito, R. M., Costa, M. A. and Pompolo, S. G. 1997. Characterization and distribution of supernumerary chromosomes in 23 colonies of Partamona helleri (Hymenoptera, Apidae, Meliponinae). Brazil. J. Genet. 20: 185–188. —, Caixeiro, A. P. A., Pompolo, S. G. and Azevedo, G. G. 2003. Cytogenetic data of Partamona peckolti (Hymenoptera,

Apidae, Meliponini) by C banding and ﬂuorochrome staining with DA/CMA3 And Da/Dapi. Genet. Mol. Biol. 26: 53—57. Brito-Ribon, R. M., Miyazawa, C. S. and Pompolo, S. G. 1999. First karyotype characterization of four species of Parta- mona (Friese, 1980) (Hymenoptera, Apidae, Meliponini) in Mato Grosso state-Brazil. Cytobios 100: 19–26. Caixeiro, A. P. A. 1999. Caracterização citogenética da heterocromatina constitutiva e sua implicação na evolução do cariótipo de espécies do gênero Plebeia (Hymenoptera: Apinae, Meliponini). Magister Scientiae thesis, Universi- dade Federal de Viçosa. Viçosa. Brazil. Camacho, J. P. M., Sharbel, T. F. and Beukeboom, L. W. 2000. B-chromosome evolution. Phil. Trans. R. Soc. Lond. 355: 163–178. Camargo, J. M. F. and Pedro, S. R. M. 2003. Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae, Apinae)—bionomia e biogeograﬁa. Rev. Brasil. Entomol. 47: 311–372. Costa M. A., Pompolo S. G. and Campos L. A. O. 1992. Supernumerary chromosomes in Partamona cupira (Hymenoptera, Apidae, Meliponinae). Rev. Brasil. Genet. 15: 801–806. —, Melo, G. A. R., Pompolo, S. G. and Campos, L. A. O. 1993. Karyotypes and heterochromatin distribuition (C-band patterns) in three species of Microstigmus wasps (Hymenoptera, Sphecidae, Pemphredoninae). Rev. Brasil. Genet. 15: 923–926. Costa, K. F., Brito, R. M. and Miyazawa, C. S. 2004. Karyotypic description of four species of Trigona (Jurine, 1807) (Hy- menoptera, Apidae, Meliponini) from the Mato Grosso, Brazil. Genet. and Mol. Biol. 27: 187–190. Cyozier, R. H. 1975. Hymenoptera. In John B. ed. Animal Cytogenetics, Insecta 7.Gebruder. Gebruder Borntraeger, Berlrin. Crozier, R. H. 1977. Evolutionary genetics of Hymenoptera. Ann. Rev. Entomol. 22: 236–288. Gomes, L. F., Pompolo, S. G. and Campos, L. A. O. 1995. Cytogenetic analysis of three species of Trypoxylon (Trypoxylon) (Hymenoptera, Sphecidae, Larrinae). Rev. Brasil. Genet. 18: 173–176. —, Brito, R. M. and Pompolo, S. G. 1998. Karyotype and C- and G-banding patterns of Eufriesea violacea (Hymenoptera, Apidae, Euglossinae). Hereditas 128: 73–76. Henriques-Gil, N., Santos, J. L. and Arana, P. 1984. Evolution of a complex B-chromosome polymorphism in the grasshopper Eyprepocnemis plorans. Chromosoma 89: 290–293. Hirai, H., Yamamoto, M. and Ogura, K. 1994. Multiplication of 28S and NOR activity in chromosome evolution among ants of the Myrmecia pilosula species complex. Chromosoma 103: 171–178. —, — and Taylor, R. W. 1996. Genomic dispersion of 28S rDNA during karyotypic evolution in the genus Myrmecia (Formicidae). Chromosoma 105: 190–196. Hoshiba, H. and Imai, H. T. 1993. Chromosome evolution of bees and wasps (Hymenoptera, Apocrita) on the basis of C- banding patterns analyses. Jpn. J. Genet. 61: 465–492. Imai, H. T. 1974. B-chromosomes in the mymercine ant Lepthotorax spinosior. Chromosoma 36: 431–444. — 1991. Mutability of constitutive heterochromatin (C-bands) during eukaryotic chromosomal evolution and their cytologi- cal meaning. Jpn. J. Genet. 66: 635–661. —, Crozier, R. H. and Taylor, R. W. 1977. Karyotype evolution in Australian ants. Chromosoma 59: 341–393. —, Brown Jr, W. L., Kubota, M., Young, H. S. and Tho, Y. P. 1984. Chromosomes observations of tropical ants in western Malasya. Ann. Rep. Natl. Inst. Genet. 34: 66–69. —, Maruyama, T., Gojobori, T., Inoue, Y. and Crozier, R. H. 1986. Theoretical bases for karyotype evolution. The minimum-interaction hypothesis. Am. Nat. 128: 900–920. —, Taylor, R. W. and Crozier, R. H. 1988. Modes of spontaneous chromosomal mutation and karyotype evolution in ants with reference to the minimum interaction hypothesis. Jap. J. Genet. 63: 159–185. —, — and — 1994. Experimental bases for the minimum interaction theory. I. Chromosome Evolution in ants of the Myrmecia pilosula species complex (Hymenoptera: Formiciinae: Myrmecinae). Jpn. J. Genet. 69: 137–182. Jones, R. N. 1991. Cytogenetics of B-chromosomes in crops. In: Gupta P. K. and Tsuchiya T. (eds.) Chromosome engineer- ing in Plants: Genetics, Breeding, Evolution. Elsevier, Amsterdam. pp. 141–158. Kerr, W. E., Carvalho, G. A. and Nascimento. V. A. 1996. Abelha urucu. Biologia, manejo e conservação. Acangaú, Belo Horizonte. — and Silveira, Z. V. 1972. Karyotypic evolution and corresponding taxonomic implications. Evolution 26: 197–202. Lorite, P., Palomeque, T., Garneria, I. and Petitpierre, E. 2000. Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae). Genetica 110: 143–150. Maffei, E. M. D., Pompolo, S. G., Campos, L. A. O. and Petitpierre, E. 2001a. Sequential FISH analysis with rDNA genes and Ag-NOR banding in the lady beetle Olla v-nigrum (Coleoptera: Coccinellidae). Hereditas 135: 13–18. 380 Rute Magalhaes Brito et al. Cytologia 70(4)

—, —, Silva-Jr, J. C., Caixeiro, A. P. A., Rocha, M. P. and Dergam, J. A. 2001b. Silver staining of nucleolar organizer regions (NORs) in some species of Hymenoptera (bees and parasitic wasps) and Coleoptera (lady beetle). Cytobios 104: 119–125. Mampumbu, A. R. 2002. Análise citogenética da heterocromatina e da NOR em populações de abelhas elm ferrão Friesella schrottkyi (FRIESE, 1900) (Hymenoptera: Apidae: Meliponini). Magister Scientiae dissertation, Unicamp. Camp- inas. Brazil. Mandrioli, M., Manicardi, G. C., Bizarro, D. and Bianchi, U. 1999. NOR heteromorphism within a parthenogenetic lineage of aphid Megoura viciae. Chrom. Res. 7: 157–162. Mariano, C. S. F., Pompolo, S. G., Delabie, J. H. C. and Campos, L. A. O. 2001. Estudos cariotípicos de algumas espécies neotropicais de Camponotus Mayr (Hymenoptera, Formicidae). Rev. Brasil. Entomol. 45: 267–274. McAllister, B. F. and Werren, J. H. 1997. Hybrid origin of a B chromosome (PSR) in the parasitic wasp Nasonia vitripennis. Chromosoma 106: 243–253. Michener, C. D. 2000. The Bees of the World. The Johns Hopkins University Press, Baltimore and London. Nur, U., Werren, J. H., Eickbush, D. G., Burke, W. D. and Eickbush, T. H. 1988. A “selfish” B chromosome that enhances its transmission by eliminating the paternal genome. Science 240: 512–514. Palomeque, T., Chica, E. and Guardia, R. D. 1990. Karyotypes, C-banding, chromosomal location of active nucleolar orga- nizing regions, and B-chromosomes in Lasius niger (Hymenoptera, Formicidae). Genome 33: 277–280. Pedro, S. R. M. and Camargo, J. M. F. 2003. Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae). Rev. Bras. Entomol. 47: 1–117. Perfectti, F. and Werren, J. H. 2001. The interspecific origin of B chromosomes: experimental evidence. Evolution 55: 1069–1073. Pinkel, D., Straume, T. and Gray, J. W. 1986. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. 83: 2934–2938. Pompolo, S. G. and Campos, L. A. O. 1995. Karyotypes of two species of stingless bees, Leurotrigona muelleri and Leu- rotrigona pusilla (Hymenoptera, Meliponinae). Rev. Brasil. Genet. 18: 181–184. Rocha, M. P. 2002. Análises citogenéticas em abelhas do gênero Melipona (Hymenoptera Meliponinae). Doctoral Scientiae thesis, Unicamp, Campinas, Brazil. —, Rocha, M. P., Fernandes, A. and Aguiar, H. 2004. The B chromosome in the stingless bee Melipona quinquefasciata (Meliponina, Hymenoptera). Second B Chromosome Conference, Bubión , ES: 13. Rocha, M. P. and Pompolo, S. G. 1998. Karyotypes and heterochromatin variation (C-bands) in Melipona species (Hy- menoptera, Apidae, Meliponinae). Genet. Mol. Biol. 21: 41–45. —, —, Dergam, J. A., Fernandes, A., and Campos, L. A. O. 2002. DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera, Meliponini). Hereditas 136: 19–27. —, —, Campos, L. A. O. 2003. Citogenética da tribo Meliponini (Hymenoptera, Meliponini). In: Melo, G. A. R. & Santos, I. A. (eds). Apoidea Neotropica: Homenagem aos 90 anos de Jesus Santiago Moure. Editora UNESC, Criciuma, SC. pp: 311–320. Sakagami, S. F., Beig, D., Zucchi, R. and Akahira, Y. 1963. Occurence of ovary-developed workers in queenright colonies of stingless bees. Rev. Bras. Biol. 23: 115–129. Scher, R. and Pompolo, S. G. 2003. Evolutionary dynamics of the karyotype of the wasp Trypoxylon (Trypargilum) nitidum (Hymenoptera, Sphecidae) from the Rio Doce State Park, Minas Gerais, Brazil. Genet.Mol. Biol. 26: 307–311. Schweizer, D. 1980. Simultaneous fluorescent staing of R bands and specific heterochromatic regions (DA DAPI-bands) in human chromosomes. Cytogenet. Cell Genet. 27: 190–193. Schmid, M. 1980. Chromosome banding in Amphibia. IV—Differentiation of GC and AT-rich regions in Anura. Chromo- soma 77: 83–103. Seabright, M. 1971. A rapid banding technique for human chromosomes. Lancet. 2: 971–972. Stouthamer, R., Van Tilborg, M., De Jong, J. H., Nunney, L. and Luck, R. F. 2001. Selfish element maintains sex in natural populations of a parasitoid wasp. Proc. R. Soc. London 268: 617–622. Sumner, A. T. 1972. A simple technique for demonstrating centromeric heterochromatin. Exp. Cell. Res. 75: 304–306. Swarça, A., Giuliano-Caetano, L., Vanzela, A. L. L. and Dias, A. L. 2001. Heteromorphism of rDNA size in Pinirampus pirinampu (Pisces: Pimelodidae) detected by in situ hybridization. Cytologia 66: 275–278. Tarelho, Z. V. S. 1973. Contribuição ao estudo citogenético dos Apoidea. Magister Scientiae thesis, Universidade de São Paulo. Ribeirão Preto. Brazil. Tosta, V. C., Fernandes-Salomão, T. M., Tavares, M. G., Pompolo, S. G., Barros, E. G. and Campos, L. A. O. 2004. A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae). Cytogenet Genome Res. 106: 279–283. Willhoeft, U. 1997. Fluorescence in situ hybridization of ribosomal DNA to mitotic chromosomes of tsetse flies (Diptera : Glossonidae : Glossina). Chromos. Res. 5: 262–267. Zurita, S., Cabrero, J., López-León, M. D. and Camacho, J. P. M. 1998. Polymorphism regeneration for a neutralized selfish B chromosome. Evolution 52: 274–277.