Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flank- ing regions from three Melipona species were PCR-ampli- fied, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approxi- mately 80 bp. The low variation level between M. quadrifas- ciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological charac- ters. Key words: Melipona, molecular phylogeny, ITS-1 spacer. Introduction The neotropical bee genus Melipona (Illiger, 1806) belongs to the tribe Meliponini and represents a group of stingless bees of about 40 described species (Michener, 2000). Moure (1992) suggested four subgenera for Melipona (Eomelipona, Melikerria, Melipona, and Michemelia), based on morpho- logical characters. Michener (2000) however, considered the Melipona species morphologically similar and did not acknowledge the subgenera. Silveira et al. (2002) also acknowledge these subgenera, suggesting some modifica- tions in the classification proposed by Moure. Despite the fact that the biology of some Melipona species is well documented, many systematic aspects of the genus still remain a mystery. We believe that a robust phy- logeny based on both morphological and molecular data should be useful for a taxonomic review of Melipona. Recent work has shown that single-copy nuclear gene sequences can provide a good data source for higher level studies of bee phylogeny (Mardulyn and Cameron, 1999; Danforth et al., 1999, 2003; Danforth, 2002; Leys et al., 2002; Bull et al., 2003; Schuarz et al., 2003). On the other hand, comparisons among ribosomal DNA internal tran- scribed spacer (rDNA ITS) sequences have been helpful to ascertain evolutionary relatedness of closely related species in diverse insect groups, owing to their high evolutionary rates (Vogler and DeSalle, 1994; Schlötterer et al., 1994: Di Muccio et al., 2000; Weekers et al., 2001, Mukha et al., 2002). Recently, studies of Coleoptera and Hemiptera demonstrated the usefulness of ITS-1 and ITS-2 markers in discovering phylogenetic relationships at higher levels such as tribe and subfamily (Marcilla et al., 2001; Von der Schu- lenburg, 2001). In spite of the importance of the ITS-1 region in phyloge- netic studies, to date only one study of this region in bees has been carried out by Sheppard and McPheron (1991). These authors amplified partial ITS-1 sequences of Anthophora abrupta, Apis mellifera intermissa, Apis mellifera ligustica, and Apis mellifera from Argentina. Sequences were only obtained for Anthophora abrupta (83 pb) and the Argentinian Apis mellifera (132 pb), so the usefulness of the ITS region for phylogenetic studies of the group in question could not be determined. Insect. Soc. 52 (2005) 11 – 18 0020-1812/05/010011-08 DOI 10.1007/s00040-004-0767-8 © Birkhäuser Verlag, Basel, 2005 Insectes Sociaux Research article The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera,Apidae, Meliponini): characterization and phylogenetic analysis T. M. Fernandes-Salomão 1,2 ; R. B. Rocha 3 ; L. A. O. Campos 1 and E. F.Araújo 3, * 1 Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil 2 Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil 3 Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil, e-mail: ezfa@ufv.br Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004. * Author for correspondence.