Denilce Meneses Lopes

Genetic divergence between populations of the stingless bee uruçu amarela (Melipona rufiventris group, Hymenoptera, Meliponini): is there a new Melipona species in the Brazilian state of Minas Gerais?

Allozyme, microsatellite and random amplified polymorphic DNA (RAPD) molecular markers were used to investigate the within and between population genetic variability and between population genetic differentiation of the Brazilian stingless bee urucu amarela (nominally Melipona rufiventris Lepeletier, 1836) present in savanna and Atlantic forest habitats of the Brazilian state of Minas Gerais (MG). We found low levels of within population variability, although there were a large number of private alleles that specifically characterized these populations. The FST values indicated a high level of genetic diversity between populations. Analysis of molecular variance (AMOVA) showed a high degree of population differentiation between the savanna and Atlantic forest habitats, confirmed by population pairwise FST data. Principal coordinates analysis and unweighted pair-group method using an arithmetic average (UPGMA) dendrograms also confirmed that in Minas Gerais the savanna populations (M. rufiventris) were genetically distinct from those present in the Atlantic forest (M. mondury). In addition, populations from locations near the towns of Dom Bosco and Brasilândia de Minas were genetically different from those collected in other localities in the savanna. Our data indicate that populations of urucu amarela found in the savanna and Atlantic forest habitats of Minas Gerais state should be treated separately for conservation purposes and that special attention should be given to the populations found in the region of Dom Bosco and Brasilândia de Minas until their taxonomic status is clarified.

Cytogenetic characterization of Melipona rufiventris Lepeletier 1836 and Melipona mondury Smith 1863 (Hymenoptera, Apidae) by C banding and fluorochromes staining

The stingless bees Melipona rufiventris and M. mondury were analyzed cytogenetically by conventional staining with Giemsa, C-banding and sequential staining with the fluorochromes CMA3/DA/DAPI. Both species presented 2n = 18 and n = 9, except for one colony of M. rufiventris, in which some individuals had 2n = 19 due to the presence of a B chromosome. After Giemsa staining and C-banding the chromosomes appeared very condensed and presented a high heterochromatic content, making it difficult to localize the centromere and therefore to visualize the chromosomes morphology. The constitutive heterochromatin was located in interstitial chromosome regions covering most of the chromosomes extension and consisted mainly of AT, as shown by DAPI staining. The euchromatin was restricted to the chromosome extremities and was GC-rich, as evidenced by CMA3 staining. The B chromosome was CMA3-negative and DAPI-positive, a heterochromatic constitution similar to that of the A genome chromosomes.

Microsatellite loci for the stingless bee Melipona rufiventris (Hymenoptera: Apidae).

Eight microsatellite primers were developed from ISSR (intersimple sequence repeats) markers for the stingless bee Melipona rufiventris. These primers were tested in 20 M. rufiventris workers, representing a single population from Minas Gerais state. The number of alleles per locus ranged from 2 to 5 (mean = 2.63) and the observed and expected heterozygosity values ranged from 0.00 to 0.44 (mean = 0.20) and from 0.05 to 0.68 (mean = 0.31), respectively. Several loci were also polymorphic in M. quadrifasciata, M. bicolor, M. mandacaia and Partamona helleri and should prove useful in population studies of other stingless bees.

A scientific note on the characterization of microsatellite loci for Melipona mondury (Hymenoptera: Apidae)

Microsatellites are a class of markers much usedin population, phylogenetic and parentage studiesbecause of their high degree of polymorphism, co-dominance and high reproducibility (De la Ruaet al., 2001; Oliveira et al., 2006). Microsatellite se-quences are amplified by PCR, using primers thatflank the repeat sequences of nuclear DNA. The de-velopment of these primers, however, is the lim-iting factor for the use of these markers. Becauseof this difficulty, there are microsatellite primersdeveloped for only three species of stingless bee,

Genome size estimation of three stingless bee species (Hymenoptera, Meliponinae) by flow cytometry

The present study standardizes a reproducible flow cytometry (FCM) protocol for DNA content measurement of bee species and applied it to Scaptotrigona and Melipona species. The mean nuclear DNA content value of male and female S. xantotricha was 0.42 pg (410.8 Mbp) and 0.44 pg (430.3 Mbp), respectively, while the mean haploid genome size was determined to be 0.93 pg (909.5 Mbp) for M. rufiventris and 0.95 pg (929.1 Mbp) for M. mondury. The variation observed in this study, albeit in a preliminary way, may be related with the variation in the heterochromatin content in the chromosomes of Scaptotrigona and Melipona species. The results provide a starting point for comparative analysis on the patterns of genome size variation in the stingless bees.ZusammenfassungKenntnisse über Genomgrössen sind von Bedeutung für den Entwurf von Sequenzierungesprojekten und für vergleichende Studien unterschiedlicher taxonomischer Gruppen. Nichtsdestoweniger ist die Genomgröße verschiedener Insektengruppen, einschliesslich der Stachellosen Biene nahe unbekannt, trotz ihrer ökologischen Bedeutung. Ziele dieser Untersuchung waren: (1) die Standardisierung eines Protokolls zur Gewinnung von Zellkernen für Durchflusszytometrieanalysen (FCM) bei Bienen, und (2) die Bestimmung des DNA-Gehalts für Scaptotrigona xantotricha, Melipona rufiventris und M. mondury, als Startpunkt für vergleichende Analysen der Variation der Genomgrössen Stachelloser Bienen. Das entwickelte Protokoll (Tab. I) erlaubte es, adequate Kernsuspensionen mit niedrigem Fluoreszenzhintergrund bedingt durch Kernfragmente und andere Organelle zu gewinnen, und es lieferte Histogramme mit Variationskoeffizienten zwischen 2,87 und 4,14 % (Abb. 1). Der mittlere DNA-Gehalt für Männchen von S. xantotricha lag bei 0,42 pg (410,8 Mbp), der von Weibchen betrug 0,44 pg (430,3 Mbp). Für M. rufiventris betrug die Größe des haploiden Genoms 0,93 pg (909,5 Mbp) und für M. mondury lag der Wert bei 0,95 pg (929,1 Mbp) (Tab. II). Diese Unterschiede im DNA-Gehalt könnten ihre Ursache in unterschiedlichen Heterochromatingehalten der Chromosomen von Scaptotrigona und Melipona-Arten haben. Die Ergebnisse stellen einenAusgangspunkt für vergleichende Untersuchungen zur Genomgröße und der entsprechenden Variationen bei Stachellosen Bienen dar.

Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae)

Due to their high degree of polymorphism, microsatellites are considered useful tools for studying population genetics. Nevertheless, studies of genetic diversity in stingless bees by means of these primers have revealed a low level of polymorphism, possibly the consequence of the heterologous primers used, since in most cases these were not specifically designed for the species under consideration. Herein we compared the number of polymorphic loci and alleles per locus, as well as observed heterozygosity in Melipona rufiventris and M. mondury populations, using specific and heterologous primers. The use of specific primers placed in evidence the greater frequency of polymorphic loci and alleles per locus, besides an expressive increase in observed heterozygosity in M. rufiventris and M. mondury, thereby reinforcing the idea that populational studies should be undertaken by preferably using species-specific microsatellite primers.

Sperm utilisation by Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) queens subjected to multiple mating

SummaryIn most Hymenoptera species the queen mates once but in a small number of species, multiple matings can occur normally. So, in this study, physogastric M. quadrifasciata queens were mated with a second male to investigate how these queens, naturally inseminated and laying eggs, use spermatozoa stored in their spermatheca, when they are mated with a second male. Results demonstrate that spermatozoa of different males mix in the spermatheca of M. quadrifasciata queens and that there is a gradual increase in the utilisation of spermatozoa of the second male, which could be explained by a competition among spermatozoa of different drones over the way in which spermatozoa are stored in the spermatheca.

An overview of cytogenetics of the tribe Meliponini (Hymenoptera: Apidae)

The present study provides a comprehensive review of cytogenetic data on Meliponini and their chromosomal evolution. The compiled data show that only 104 species of stingless bees, representing 32 of the 54 living genera have been studied cytogenetically and that among these species, it is possible to recognize three main groups with n = 9, 15 and 17, respectively. The first group comprises the species of the genus Melipona, whereas karyotypes with n = 15 and n = 17 have been detected in species from different genera. Karyotypes with n = 17 are the most common among the Meliponini studied to date. Cytogenetic information on Meliponini also shows that although chromosome number, in general, is conserved among species of a certain genus, other aspects, such as chromosome morphology, quantity, distribution and composition of heterochromatin, may vary between them. This reinforces the fact that the variations observed in the karyotypes of different Meliponini groups cannot be explained by a single theory or a single type of structural change. In addition, we present a discussion about how these karyotype variations are related to the phylogenetic relationships among the different genera of this tribe.

Cytogenetics of Melitoma segmentaria (Fabricius, 1804) (Hymenoptera, Apidae) reveals differences in the characteristics of heterochromatin in bees

Abstract To date, more than 65 species of Brazilian bees (of the superfamily Apoidea) have been cytogenetically studied, but only a few solitary species have been analyzed. One example is the genus Melitoma Lepeletier & Serville, 1828, for which there is no report in the literature with regard to cytogenetic studies. The objective of the present study is to analyze the chromosome number and morphology of the species Melitoma segmentaria (Fabricius, 1804), as well as to determine the pattern of heterochromatin distribution and identify the adenine–thymine (AT)- and guanine–cytosine (GC)-rich regions. Melitoma segmentaria presents chromosome numbers of 2n=30 (females) and n=15 (males). With C-banding, it is possible to classify the chromosomes into seven pseudo-acrocentric pairs (AM), seven pseudo-acrocentric pairs with interstitial heterochromatin (AMi), and one totally heterochromatic metacentric pair (Mh). Fluorochrome staining has revealed that heterochromatin present in the chromosomal arms is rich in GC base pairs (CMA3+) and the centromeric region is rich in AT base pairs (DAPI+). The composition found for Melitoma diverges from the pattern observed in other bees, in which the heterochromatin is usually rich in AT. In bees, few heterochromatic regions are rich in GC and these are usually associated with or localized close to the nucleolus organizer regions (NORs). Silver nitrate impregnation marks the heterochromatin present in the chromosome arms, which makes identification of the NOR in the chromosomes impossible. As this technique reveals proteins in the NOR, the observation that is made in the present study suggests that the proteins found in the heterochromatin are qualitatively similar to those in the NOR.

Similarity of heterochromatic regions in the stingless bees (Hymenoptera: Meliponini) revealed by chromosome painting

Most Meliponini share a distinctive pattern of heterochromatin distribution in relation to other bees. In general, they present one euchromatic and one heterochromatic chromosome arm, a feature explained by minimum interaction theory, which involved centric fissions followed by heterochromatin addition. In this work, two Meliponini with a distinct pattern of heterochromatin distribution, Tetragonisca fiebrigi and Melipona rufiventris, were analyzed using chromosomal microdissection of the heterochromatin region followed by FISH (fluorescent in situ hybridization). Hybridization revealed FISH signals equivalent to location of the isolated fragment that were widespread over heterochromatic portions of other chromosomes. This result showed that the heterochromatic sequences were very similar among chromosomes in the same species. Cross-hybridization of each probe in M. rufiventris and T. fiebrigi yielded no signals, revealing that both species presented differentiated and non-homologous heterochromatin sequences.