Hereditas 128: 73-76 (1998)

Karyotype and C- and G-banding patterns of Eufriesea violacea (Hymenoptera, Apidae, Euglossinae) LUIZ FERNANDO GOMES, RUTE MAGALHAES BRITO, SILVIA DAS GRACAS POMPOLO, LUCIO ANTONIO DE OLIVEIRA CAMPOS and RUI CARLOS PERUQUETTI Departamento de Biologia Geral, Universidade Federal de Vigosu, ViGosu, MG, Brasil

Gomes, L. F., Brito, R. M., Pompolo, S. G., Campos, L. A. 0. and Peruquetti, R. C. 1998. Karyotype and C- and G-banding patterns of Eufriesea uiolacea (Hymenoptera, Apidae, Euglossinae). - Hereditas 128: 73-76. Lund, Sweden. ISSN 0018-0661. Received June 1 I , 1997. Accepted December 10, 1997

Euglossinae represent an exclusively neotropical bee group, the cytogenetics of which is poorly known. We have studied ten specimens of Eujriesea uiolacea, nine males (K = 15 M) and one female (2K = 30 M). C-banding revealed constitutive heterochromatin throughout the extension of the long arm of 13 chromosome pairs. The end of the long arm had a lighter region in two chromosome pairs. The proximal region of the centromere of the short arm of all chromosomes was also heterochromatic. G-banding revealed three to four positive bands in both the heterochromatic and euchromatic regions of most chromosomes. The small number of G bands per chromosome can perhaps be explained by the structural arrangement of chromatin and they do not need to correspond to chromomeric regions.

Luiz Fernando Gornes, Departamento de Biologiu Gerai, Uniuersidade Federal de Vicosa, 36571 -000 ViCosa, MG, Brasil. e-mail: Ifgomes@alunos.ufv.br

Euglossinae are an exclusively neotropical bee group occurring from Argentina to northern Mexico (PEAR- SON and DRESSLER 1985). Three genera in this group (Euglossa, Eulaema, and Eufriesea) are orchid polli- nators and two, Exaeretr and Aglae, are parasites.

Few cytogenetic data are available for the genus Eufriesea. According to TARELHO (1973), the kary- otype of Eujyiesea violaceu has n = 15 chromosomes with a larger number of secondary constrictions than observed in other bees. In that study it was not possible to determine the position of the centromere because the chromosomes were highly condensed.

Evidently, further cytogenetic data are needed for this genus, Among the techniques used, chromosome banding is of great importance for the understanding of karyotype evolution in a species.

G-banding patterns have been observed in Hy- menoptera such as Encarsiu berlesei (Aphelinidae), a parasitoid species (ODTERNA et al. 1993), species of the family Vespidae, Vespa simillimu Smith and Vespa simillima xanthoptera Cameron (HOSHIBA 1985a) and Polistes and Purapolybiu indica (HOSHIBA 1985b), the ant Tupinomu nigerrimum (LORITE et al. 1996), and Apis mellijkru (HOSHIBA 1984).

Here we characterize the karyotype of Eujriesea violacea with more refined cytogenetic techniques than the ones employed earlier for this species.

MATERIAL AND METHODS

Nests of Eufrieseu violaceu were collected from cracks in a wood that supported a roof on the Campus of the Federal University of Vigosa, Vigosa, MG

(20"45'S, 42'52'W). Nests were also collected at the same site, using trap nests as GAROFALO et al. (1993) used to collect some species of Euglossinii.

Metaphase cells were obtained by the technique of IMAI et al. (1988). C-banding was carried out by the method of SUMNER (1972, 1990) as modified by POMPOLO and TAKAHASHI (1990).

G-banding was done using the technique of SEABRIGHT (1971). Chromosome preparations aged for a maximum of four days were hydrolysed in 2 x SSC for 10 min in a water bath at 60°C and quickly washed in distilled water. The preparations were then digested with a 0.03 YO trypsin solution (Merck 40 U/mg) in a water bath at 37°C for 45 to 60 s, quickly washed with distilled water, stained with a Giemsa solution (Merck) in 0.06 M phosphate buffer (1 :30), pH 6.8, for 10 min and carefully rinsed in running water.

The metaphases were photographed with a Zeiss photomicroscope using Agfa Copex Pan A.H.U. film.

RESULTS

Ten Eufriesea violucea individuals, nine males and one female, were analyzed. About ten metaphase cells per slide were analyzed in three slides per individual.

The female had 2n = 30 submetacentric chromo- somes and the male n = 15, with karyotype formulae of 2K = 30 M and K = 15 M, respectively (Fig. 1). A weakly staining region was observed in the terminal portion of the long arm of two chromosomes, possi- bly corresponding to the region of the secondary constriction (Fig. 1 C, arrowheads).

74 L. F. Gomes et al. Hereditas I28 (1 998)

A

B

C

D

Fig. 1A-D. Karyotypes of Eufrieseu uiolucea. A female, standard staining. B male, standard staining. C male, C-banding patterns. D male, G-banding patterns. Bar = 5 pm.

Hereditas 128 (1998) Cytogenetics of an Eunlossinae bee suecies 75

C-banding showed heterochromatin blocks along the chromosomes, with euchromatin restricted to the distal regions of the short arm (Fig. 1C).

G-banding showed differential trypsin digestion along the chromosomes, with three to four positive bands differentiated by C-banding within the hete- rochromatic and euchromatic regions (Fig. 1D). These bands appear to coincide with light bands observed in some metaphases submitted to standard staining (Fig. 1B).

DISCUSSION

We may now precisely characterize the karyotype of Eufriesea violacea. It consists of 15 chromosomes in males, as observed by TARELHO (1973). The chromo- somes are submetacentric, what the author did not observe because of the high degree of condensation of her preparations. The number of n = 16 obtained by KERR (1952) probably is not representative of the species.

These data are important to phylogenetic studies because, according to the Minimal Interaction Theory of IMAI et al. (1986, 1988, 1994), the karyotype evolution proceeds towards the enlarging of the chro- mosome number, with fissions and pericentric inver- sions prevailing. Thus, primitive species would have a lower number of longer chromosomes while less prim- itive species would have a larger number of smaller ones. Indeed, another species of the Euglossine tribe has shown a haploid number of n = 15 as Eufriesea surinamensis and Eulaema nigrita (unpublished data).

The C band results showed that the chromosomes of Eufriesea violacea have euchromatin restricted to the extremity of the short arms. According to IMAI (1991), this can be a result of a rearrangement such as fission, heterochromatin addition, and posterior func- tional centromere change. However, this C-banding pattern is not maintained within the genus because Eufriesea surinamensis has euchromatin in the pericen- tromeric region and some interstitial bands (unpub- lished data). The difference of the C-banding patterns between these two species is in agreement with what has been observed in relation to other Hymenoptera species like wasps of the genus Trypoxylon (GOMES et al. 1995) and some bee species of the genus Melipona (Rocha and Pompolo, personal communication).

We used G-banding to observe the constitution of heterochromatic blocks seen in the karyotype of this species. However, positive bands were found in both euchromatic and heterochromatic regions. Many au- thors have been trying to explain the nature of this kind of chromosome banding. According to BICK- MORE and SUMNER (1989) and HOLMQUIST (1989), the broadest explanation of G bands is that the

pattern reflects euchromatin packing (which, however, can be visualized as chromomeres in meiotic chromo- somes), and corresponds to the replication pattern. We believe that the positive G bands observed in the karyotype of Eufriesea violacea do not correspond to chromomeric regions, as is the case in mammals, because of their small number per chromosome.

The correlation found in mammals, between spe- cific fluorochromes to A-T-rich regions and G bands, does not seem to occur in Hymenoptera. According to ODIERNA et al. (1993), the chromosomes of the para- sitoid Encarsia berlesei stain uniformly with specific DAPI dA-dT staining. Also, LORITE et al. (1996) observed that only one G band stained positively by the DAPI method in Tapinoma nigerrimum (an ant species), and that the banding pattern obtained for this species was probably the consequence of the local arrangement of chromatin and/or altered proteins. In this study, this type of correlation with the base composition of positive bands was not possible since we did not use techniques for this purpose. However, when comparing C-banding and G-banding results, we noted that the constitution of heterochromatin blocks was not homogeneous and that subtypes of constitutive heterochromatin that were not differenti- ated by C-banding, may have been present. Further utilization of fluorochromes can give us more infor- mation about the nature of the heterochromatic blocks.

In contrast to the data reported by TARELHO (1973), possible secondary constriction regions were observed in only two chromosomes (Fig. lC, arrow- heads). The light bands observed in chromosomes of some metaphases submitted to standard staining, sim- ilar to those observed with G-banding (Fig. ID), were probably considered to be secondary constrictions by the author.

According to ODIERNA et al. (1993), this type of staining is important for an accurate identification of chromosome pairs and their linkage groups, a fact that is essential in advanced genetic studies, especially in insects that do not have polytene chromosomes. Applying this technique to bees is of great impor- tance. It would allow a more precise homologue pairing in species such as Melipona erbunea fuscopi- losa, Melipona captiosa, Melipona capixaba, and Melipona scutellaris, which according to ROCHA and POMPOLO (personal communication), have a large amount of heterochromatin which does not permit centromere disclosure.

ACKNOWLEDGEMENTS

Research supported by “Fundaqiio de Amparo a Pesquisa do Estado de Minas Gerais” (FAPEMIG), “Coordenaqgo

Hereditas 128 (1998) 76 L. F. Gornes et al.

de Aperfeiqoamento de Pessoal de Nivel Superior” (CAPES) “Conselho Nacional de Pesquisa” (CNPq). We are grateful to Dr. Eldo A. M. da Silva and Dr. Aristeia A. de Azevedo for supplying the photographic equipment, and to Dr. Catarina Satie Takahashi for manuscript review and suggestions.

REFERENCES Bickmore WA and Sumner AT, (1989). Mammalian chro-

mosome banding an expression of genome organization. Trends Genet. 5: 144-148.

Garofalo CA, Camillo E, Serrano JC and Rebelo JM, (1993). Utilization of trap nests by Euglossinii species (Hymenoptera: Apidae). Rev. Brasil. Biol. 53: 177- 187.

Gomes LF, Pompolo SG and Campos LAO, (1995). Cyto- genetic analysis of three species of Trypoxylon (Trypoxy- lon) (Hymenoptera, Sphecidae, Laminae). Brazil. J. Genet. 15: 173-176.

Holmquist GP, (1989). Evolution of chromosome bands: molecular ecology of non-coding DNA. J. Mol. Evol. 28: 469-486.

Hoshiba H, (1984). Karyotype and banding analysis on haploid males of the honey bee (Apis mellifera). Proc. Jpn. Acad. 60(B): 122-124.

Hoshiba H, (198%). G-banding analysis of two species of the hornets, Vespa mandarinia Smith and Vespa simil- lima xanthoptera Cameron (Vespidae, Hymenoptera). Proc. Jpn. Acad. 61(B): 116-118.

Hoshiba H, (1985b). The karyological and the G-banding analyses of a Polistes male wasp, Parapolybia indica Saussure (Vespidae, Hymenoptera). Proc. Jpn. Acad. 61(B): 119-120.

Imai HT, (1991). Mutability of constitutive heterochro- matin (C-bands) during eukaryotic chromosomal evolu- tion and their cytological meaning. Jpn. J. Genet. 66: 635-661.

Imai HT, Maruyama T, Gojobori YI and Crozier RS,

(1986). Theoretical bases for karyotype evolution. I . The minimum-interaction hypothesis. Am. Nat. 128: 900- 920.

Imai HT, Taylor RW and Crozier RH, (1988). Modes of spontaneous chromosomal mutation and karyotype evo- lution in ants with reference to the minimum interaction hypothesis. Jpn. J. Genet. 63: 159-185.

Imai HT, Taylor RW and Crozier RH, (1994). Experimen- tal bases for the minimum interaction theory. I Chromo- some evolution in ants of Myrmecia pilosula species complex. (Hymenoptera: Formicidae: Myrmeciinae). Jpn. J. Genet. 69: 137-181.

Kerr WE, (1952). A variaqiio do numero de cromossomos na evoluqiio dos Hymenoptera. Sci. Genet. 4: 182-190.

Lorite P, Chica E and Palomeque T, (1996). G-banding and chromosome condensation in the ant, Tapinoma nigerri- mum. Chromosome Res. 4: 77-79.

Odierna G, Baldanzana F, Aprea G and Olmo E, (1993). Occurrence of G-Banding in metaphase chromosomes of Encarsia berlesei (Hymenoptera, Aphelinidae). Genome 36: 662-667.

Pearson DL and Dressler RL, (1985). Two-year study of male orchid bee (Hymenoptera, Apidae, Euglossinae) attraction to chemical baits in lowland south-eastern Peru. J. Trop. Ecol. 1: 37-54.

Pompolo SG and Takahashi CS, (1990). Chromosome numbers and C-banding in two wasp species of the genus Polistes (Hymenoptera, Polistinae, Polistini). Insectes So- ciaux 37: 251-257.

Seabright M, (1971). A rapid banding technique for human chromosomes. Lancet (2): 971 -972.

Sumner AT, (1972). A simple technique for demonstrating centromeric heterochromatin. Exp. Cell. Res. 75: 304- 306.

Sumner AT, (1990). Chromosome Banding. Unwin Hyman Ltd, London, UK.

Tarelho ZVS, (1973). Contribuiqiio ao estudo citogenktico dos Apoidea. Master’s Thesis, Faculdade de Medicina de Ribeiriio Preto, USP, Ribeiriio Preto, SP, Brasil.