Sequence and RFLP analysis of the ITS2 ribosomal DNA in two Neotropical social bees, Melipona beecheii and Melipona yucatanica (Apidae, Meliponini)

Abstract

Two stingless bees species of the genus Melipona, M. beecheii and M. yucatanica, are the only ones reported for the Yucatan Peninsula. The natural distribution of M. beecheii ranges from southern Mexico to Costa Rica, that of M. yucatanica from south Mexico to Guatemala. Colonies of both species occur in a variety of habitats and show adaptations to local conditions denoting the occurrence of ecotypes. The ITS2 of ribosomal DNA has been characterized in both species and its utility to discriminate among colonies has been investigated through RFLP experiments. The ITS2 region is unusually long, 1788 bp in M. beecheii and 1845 bp in M. yucatanica (including the 3' end of the 5.8S gene and partial 5' of the 28S gene). Mean nucleotide divergence between both ITS2 sequences is 16 % (excluding sites with insertions/ deletions) and 20 % when the insertions/deletions are taken into account. The G+C content in both sequences is close to 53 %. The PCR-RFLP assay was performed with 12 restriction enzymes on colonies of M. beecheii from Mexico (Yucatan, Campeche and Chiapas) Costa Rica, El Salvador and Guatemala, and of M. yucatanica from Mexico (Yucatan) and Guatemala. The restriction patterns obtained allow to discriminating colonies of both species with different origins. Both kinds of data are thus useful for assessing intra and interspecific genetic variability and for developing appropriate conservation strategies for these species.

Geometric morphometrics of the wing as a tool for assigning genetic lineages and geographic origin to Melipona beecheii (Hymenoptera: Meliponini)

“…The short swarming ranges known for stingless bee species (Nogueira-Neto 1997) can help us to explain the local variation among the populations; this limited dispersal mechanism may contribute to the geographical isolation of the populations. The variability that we found indicates the existence of locally adapted ecotypes; it also corroborates conclusions made with various types of markers including standard morphometrics and microsatellites (QuezadaEuán et al 2007) RFLP and nuclear gene sequences, such as ITS2 (De la Rúa et al 2007) and ITS1 (May-Itzá et al 2009). …”

“…Quezada-Euán et al (2007) used morphometrics and microsatellite DNA to compare M. beecheii from Mexico and Costa Rica and detected substantial differences between these populations. These results were later confirmed and expanded to other populations based on variation in the ITS1 and ITS2 ribosomal and mitochondrial CO-1 genes among bees from Mexico, Guatemala, El Salvador, and Costa Rica (De la Rúa et al 2007;May-Itzá et al, unpublished data). Thus, evidence from various studies has demonstrated considerable genetic differentiation between populations of M. beecheii, suggesting various genetic lineages and that M. beecheii could be in fact a complex of species (May-Itzá et al, unpublished data).…”

Utility of the ITS1 Region for Phylogenetic Analysis in Stingless Bees: a Case Study of the Endangered Melipona yucatanica Camargo, Moure and Roubik (Hymenoptera: Meliponini).

“…The ITS1 clades separate between Mexican and Guatemalan populations showing the same phylogeographic pattern found in previous markers (ITS2, De la Rúa et al, 2007;cox1 and microsatellites May-Itzá et al, 2010). These results are congruent with the fact that both populations are located in opposite extremes of the species distribution range (Ayala, 1999).…”

“…This species is associated with primary forest, and based on its patched distribution, various authors have suggested a recent fragmentation of M. yucatanica populations due to massive deforestation (Camargo et al 1988;Ayala, 1999). Preliminary molecular studies in Mexican and Guatemalan populations of M. yucatanica yielded different RFLP patterns in the ITS2 region (De la Rúa et al, 2007), suggesting allopatric speciation in populations geographically separated. Furthermore, morphometric and Bayesian analyses of the mitochondrial cox1 region and microsatellite loci revealed geographic differences between Guatemalan and Mexican populations, suggesting that M. yucatanica from México and Guatemala could represent two distinct species (May-Itzá et al, 2010).…”

“…Herein, the entire ITS1 region of M. yucatanica has been sequenced and compared with available ITS1 (complete or 3' end) from other Melipona species as well as with previous results on M. yucatanica based on morphometry and molecular markers (De la Rúa et al, 2007;May-Itzá et al, 2010). We aimed to i) analyze the molecular diversity underlying this marker, and ii) test its phylogenetic signal to verify whether this species is included within the morphologically described subgenus Melipona s. str.…”