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-CMA3-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-CMA3-NOR staining of interphase nuclei provided coincident positive responses. This suggests that DA-CMA3-positive bands of P. peckolti correspond to nucleolar organizer regions, as previously confirmed for another Partamona species by FISH.


Introduction
The first karyotype evolution hypothesis elaborated for Meliponini bees postulated that the different chromosome numbers observed today are the product of polyploidy events from a basic number, n = 8 of Melipona species (Kerr et al., 1952).Several Meliponini genus have been studied in detail at the cytogenetic level aiming at the comprehension of the karyotype evolution of stingless bees such as: Leurotrigona (Pompolo and Campos, 1995); Frieseomelitta (Moreira, 1997), Plebeia (Caixeiro and Pompolo, 1998) and Melipona (Rocha and Pompolo, 1998;Rocha et al., 2002).These modern cytogenetic data have shown interespecific differences that cannot sustain the Polyploidy Hypothesis mentioned.Alternatively another karyotype evolution theory, the Minimum Interaction Theory (Imai et al. 1986(Imai et al. , 1988(Imai et al. , 1994)), postulated for Australian ants, has also been accepted as a possible model for the karyotype evolution of Meliponini bees (Pompolo, 1992).This theory states that primitive karyotypes had small numbers of large chromosomes and, as time went by, theses chromosomes got smaller and increased in number by centric fission that would prevent deleterious interactions within the interphase nucleus, such as translocations.
Detailed study of each Meliponini genus at the cytogenetic level will provide the necessary data to verify if this Theory could also be applied to the stingless bees or whether it would be necessary to postulate another hypothesis to understand the karyotype evolution of this important group which is responsible for the pollination of 40 to 90% of wild plant species, depending on the ecosystem (Kerr, et al. 1996).
In this short communication, we describe the karyotype of Partamona peckolti, a species that occurs in the tropical rainforests of central America, the Caribbean

Material and Methods
The biological material was collected from a nest associated with a Bromeliacea plant, in San Francisco de Las Pampas village (00°25'182" S and 79°00'183" W), Cotopaxi province, Ecuador.Slides with metaphases were prepared from 30 post-defecant larvae cerebral ganglion according to the technique described by Imai et al. (1988).Chromosome banding techniques were applied as C banding according to Sumner (1972) and fluorochrome sequential staining DA -DAPI -Chromomycin A 3 (Schweizer, 1980).Sequential treatment, C banding followed by DA -DAPI -CMA 3 staining was done using the techniques in the same way as done separately.Another sequential staining with DA -CMA 3 followed by AgNOR treatment was also applied using the Schweizer (1980) protocol and silver impregnation as described by Howell and Black (1980).The metaphases were photographed in photomicroscope Olympus BX60, with FUJI HR II ISO 40 and KODAK GOLD ULTRA ISO 100 films.The karyotypes were mounted in increasing order of the chromosome length.

Results and Discussion
Partamona peckolti presented 2n = 34 chromosomes as observed in other species of the genus (Tarelho, 1973;Brito 1998;Brito-Ribon et al., 1999) and conventional staining revealed a secondary constriction in the first pair of the karyotype not observed in any other Partamona species studied cytogenetically (Brito 1998, Brito-Ribon et al., 1999) (Figure 1A).
The C-banding pattern showed heterochromatin in the pericentromeric region of all chromosomes, as already observed in P. helleri (Brito, 1998) and Partamona sp.n.(Brito-Ribon et al., 1999) as well as a large hetercromatic block on the first karyotype pair, similarly to P. aylae, P. mulata and P. vicina (Brito-Ribon et al., 1999) (Table 1 and Figure 1B).
Sequential staining with DA-DAPI-CMA 3 revealed a wide positive band in one large chromosome with both fluorochromes (Figure 2A and B, arrows).A similar wide CMA 3 -positive band has already been observed in other Partamona species (Table 1).However, this large band disappeared when C banding was applied before sequential staining with DA-DAPI-CMA 3 (Figure 3A, B and C).Another small DA/CMA 3 + band seen with or without the C banding treatment which presented the shape and richness in GC base pairs led us to believe that we were evidencing a nucleolar organizer region (NOR) (Figure 3 arrowhead).This correlation between CMA 3 + bands and NORs is quite common among animals and in insects has been observed in: the grasshoppers Eyprepocnemis plorans and Locusta migratoria (Camacho, et al., 1991); the wasp Trypoxylon albitarse (Araújo et al., 2000;2002); the lady beetle Olla v-nigrum (Maffei et al., 2001a); and in other Meliponini bees such as Melipona (Rocha and Pompolo, 1998;Rocha et al., 2002); Plebeia (Caixeiro, 1999) and Friesella  schrottkyi (Mampumbu, 2002).The location of these regions by the classical technique of Howell and Black (1980) is difficult on Meliponinae chromosomes that was effectively achieved only in Tetragonisca angustula (Menezes, 1997), Melipona (Maffei et al., 2001b;Rocha et al., 2002); Plebeia (Maffei et al., 2001b) and Friesella schrottkyi (Mampumbu, 2002).Conversely, correspondence of DA-CMA 3 and AgNOR staining could be observed at interphase nuclei (Figure 4A and B).As the correlation among positive staining with DA-CMA 3 ; AgNOR and fluorescent in situ hybridization with 18S rDNA probe has been observed for two another Partamona species, P. helleri and P. aff.nigrior (Brito, 1988), we indeed accepted that structure (Figure 3C) as a NOR.Many studies have been carried out for a better understanding of the evolution of the Partamona genus: taxonomy revision (Pedro, 1998), provisioning and oviposition process behavior of nine species (Azevedo, 2001); molecular study of B chromosomes of P. helleri (Tosta, 2001); mi-tochondrial genome characterization of P. mulata and P. helleri (Brito and Arias, 2001).In this context, Cytogenetics can still contribute greatly to the knowledge of the relations among Partamona species to further cytotaxonomy treatment of the data.Nevertheless, to achieve this purpose, more karyotype characterizations are needed as many Partamona species persist even without their chromosome number known.Furthermore, the better comprehension of the relations among Partamona species, especially at the cytogenetic level, will contribute to the comprehension of the karyotype evolution of the Meliponinae bees as a whole.