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Stingless bees of the genus Melipona are subdivided into 4 subgenera called Eomelipona, Melikerria, Melipona sensu stricto, and Michmelia according to species morphology. Cytogenetically, the species of the genus Melipona show variation in the amount and distribution of heterochromatin along their chromosomes and can be separated into 2 groups: the first with low content of heterochromatin and the second with high content of heterochromatin. These heterochromatin patterns and the number of chromosomes are characteristics exclusive to Melipona karyotypes that distinguish them from the other genera of the Meliponini. To better understand the karyotype organization in Melipona and the relationship among the subgenera, we mapped repetitive sequences and analyzed previously reported cytogenetic data with the aim to identify cytogenetic markers to be used for investigating the phylogenetic relationships and chromosome evolution in the genus. In general, Melipona species have 2n = 18 chromosomes, and the species of each subgenus share the same characteristics in relation to heterochromatin regions, DAPI/CMA3 fluorophores, and the number and distribution of 18S rDNA sites. Microsatellites were observed only in euchromatin regions, whereas the (TTAGG)6 repeats were found at telomeric sites in both groups. Our data indicate that in addition to the chromosome number, the karyotypes in Melipona could be separated into 2 groups that are characterized by conserved cytogenetic features and patterns that generally are shared by species within each subgenus, which may reflect evolutionary constraints. Our results agree with the morphological separation of the Melipona into 4 subgenera, suggesting that they must be independent evolutionary lineages.

The species Melipona rufiventris Lepeletier, 1836 is a Brazilian native stingless bee that is part of a species complex known as the &lsquo, rufiventris group&rsquo, making it difficult to distinguish between the different species. Populations in this group are facing a severe decline, leading to the risk of local extinction, and therefore, their conservation should be treated as a major concern. This study describes the first set of tri- and tetranucleotide microsatellite markers, using next-generation sequencing technology for use in the identification of genetic diversity and population structure in the &lsquo, A total of 16 microsatellite loci displayed polymorphism. Analysis of the whole data set (n = 50) detected 63 alleles in all loci, ranging from 2 to 7 with a mean of 3.9 alleles/locus. A genetic diversity analysis revealed high values for population differentiation estimates (FST = 0.252, RST = 0.317, and DEST = 0.284) between the Atlantic Forest, Cerrado, and Caatinga biomes. An additional evidence for genetic divergence among populations was also found in the &rsquo, these should be treated as separate conservation units or even as separate species. These microsatellite markers have demonstrated a strong potential for assessing population discrimination in this threatened stingless bee group.

It is thought that two evolutionary mechanisms gave rise to chromosomal variation in bees: the first one points to polyploidy as the main cause of chromosomal evolution, while the second, Minimum Interaction Theory (MIT), is more frequently used to explain chromosomal changes in Meliponini and suggests that centric fission is responsible for variations in karyotype. However, differences in chromosome number between Meliponini and its sister taxa and in the karyotype patterns of the Melipona genus cannot be explained by MIT, suggesting that other events were involved in chromosomal evolution. Thus, we assembled cytogenetical and molecular information to reconstruct an ancestral chromosome number for Meliponini and its sister group, Bombini, and propose a hypothesis to explain the evolutionary pathways underpinning chromosomal changes in Meliponini. We hypothesize that the common ancestor shared by the Meliponini and Bombini tribes possessed a chromosome number of n = 18. The karyotype with n = 17 chromosomes was maintained in Meliponini, and variations of haploid numbers possibly originated through additional Robertsonian fissions and fusions. Thus, the low chromosome number would not be an ancestral condition, as predicted by MIT. We then conclude that Robertsonian fission and fusions are unlikely to be the cause of chromosomal rearrangements that originated the current karyotypes in Meliponini.

Stingless bees of the genus Melipona are subdivided into 4 subgenera called Eomelipona, Melikerria, Melipona sensu stricto, and Michmelia according to species morphology. Cytogenetically, the species of the genus Melipona show variation in the amount and distribution of heterochromatin along their chromosomes and can be separated into 2 groups: the first with low content of heterochromatin and the second with high content of heterochromatin. These heterochromatin patterns and the number of chromosomes are characteristics exclusive to Melipona karyotypes that distinguish them from the other genera of the Meliponini. To better understand the karyotype organization in Melipona and the relationship among the subgenera, we mapped repetitive sequences and analyzed previously reported cytogenetic data with the aim to identify cytogenetic markers to be used for investigating the phylogenetic relationships and chromosome evolution in the genus. In general, Melipona species have 2n = 18 chromosomes, and the species of each subgenus share the same characteristics in relation to heterochromatin regions, DAPI/CMA3 fluorophores, and the number and distribution of 18S rDNA sites. Microsatellites were observed only in euchromatin regions, whereas the (TTAGG)6 repeats were found at telomeric sites in both groups. Our data indicate that in addition to the chromosome number, the karyotypes in Melipona could be separated into 2 groups that are characterized by conserved cytogenetic features and patterns that generally are shared by species within each subgenus, which may reflect evolutionary constraints. Our results agree with the morphological separation of the Melipona into 4 subgenera, suggesting that they must be independent evolutionary lineages.

Melipona capixaba, popularly known as “uruçu preta”, is a stingless bee restricted to the mountainous Atlantic Rainforest areas of Espírito Santo State, Brazil. Due to the endemism and small population size, this species discovered in 1994 is now considered “vulnerable to extinction”. Using ISSR, PCR–RFLP and microsatellites markers, we studied the genetic variability and structure of M. capixaba from 88 colonies collected throughout the distribution area of the species within Espírito Santo State. The microsatellite, ISSR and mitochondrial haplotype analyses showed that M. capixaba has low genetic variability compared to other insect species. The molecular analyses also indicated a high genetic similarity among the M. capixaba samples, with no clear pattern of structuring. The analyses of molecular variance results indicated that most of the total genetic variation in M. capixaba was explained by the genetic diversity within local populations. Results suggest that the analyzed samples could be treated as a single population for preservation purposes. Thus, given its endemism, local adaptation and low number of natural colonies, efforts for the conservation of M. capixaba should focus on preservation and increasing the number of colonies in the wild, so that M. capixaba can support constant captures and the effects of habitat deforestation in Espírito Santo State.

The incorporation of fragments of mitochondrial DNA (mtDNA) in the nuclear genome, known as numts (nuclear mitochondrial pseudogenes), undermines general assumptions concerning the use of mtDNA in phylogenetic and phylogeographic studies. Accidental amplifications of these nuclear copies instead of the mitochondrial target can lead to crucial misinterpretations, thus the correct identification of numts and their differentiation from true mitochondrial sequences are important in preventing this kind of error. Our goal was to describe the existence of cytochrome b (cytb) numts in the leafcutter ant Acromyrmex striatus (Roger, 1863). PCR products were directly sequenced using a pair of universal primers designed to amplify the cytb gene of these insects. Other species of leafcutter ants were also sequenced. The sequences were analyzed and the numts were identified by the presence of double peaks, indels and premature stop codons. Only A. striatus clearly showed the presence of numts, while the other species displayed the expected amplification of the mtDNA cytb gene target using the same primer pair. We hope that our report will highlight the benefits and challenges of using mtDNA in the molecular phylogenetic reconstruction and phylogeographic studies of ants, while establishing the importance of numts reports for future studies.

The origin of supernumerary (B) chromosomes is still a debated topic, with intra- and interspecific origins being the most plausible options. In the bee Partamona helleri, a sequence-characterized amplified region (SCAR) marker being specific to B chromosomes suggested the possibility of interspecific origin. Here, we search for this marker in 3 close relative species and perform DNA sequence comparison between species. The SCAR sequence does not show homology with other sequences in the databases, but does contain an open reading frame with sequence homology with a reverse transcriptase. Dot-blot hybridization using the SCAR marker as a probe confirmed that it is present in B-carrying, but not B-lacking larvae of P. helleri, and indicated its presence in adult individuals of P. cupira and P. criptica. Additionally, PCR amplification of the SCAR marker was successful on genomic DNA obtained from P. helleri and P. rustica larvae carrying B chromosomes, and on genomic DNA obtained from adult individuals of P. cupira, P. criptica and P. rustica. Finally, a comparison of the DNA sequence of the SCAR markers amplified from these 4 species showed very few nucleotide differences between the species. The complete association between B chromosome and SCAR presence and the scarce divergence observed for this DNA sequence between the 4 species analyzed suggest the possibility that this B chromosome has recently been transferred between species through several episodes of interspecific hybridization.

Physicochemical analyses were carried out to evaluate 27 samples of honeys from three species of the Brazilian genus Melipona (M. capixaba, M. rufiventris and M. mondury) from Espírito Santo and Minas Gerais States. The parameters water activity (Aw), percentage of soluble solids (Brix %), pH, acidity (meq/Kg) and moisture (%) were evaluated. The honey characteristics obtained from these samples were very similar to the ones from other Melipona species. However, regarding the honey from Apis (honey bee), only the pH values were similar. The low pH value and the high acidity detected in Melipona honey are potential factors for increasing the honey shelf life because they do not provide favorable conditions for the microbial development. On the other hand, the high level of water activity favors the growth of microorganisms, especially yeast, which demands a more careful handled and storage. The observed differences between Melipona and Apis honey reinforce the need for specific quality settings for stingless bee honey.Análises fisico-quimicas foram realizadas para avaliar vinte e sete amostras de mel de três espécies do gênero Melipona (M. capixaba, M. rufiventris e M. mondury) coletadas nos Estados do Espírito Santo e de Minas Gerais. Os parâmetros atividade de água (aW), porcentagem de sólidos solúveis (Brix %), pH, acidez (meq.Kg-1) e umidade (%) foram avaliados. As características do mel dessas amostras foram muito similares às do mel de outras espécies de Melipona. Entretanto, para o mel de Apis apenas os valores de pH foram similares. O baixo valor de pH e a elevada acidez detectados no mel de Melipona são fatores potenciais para uma maior vida útil do mesmo por não oferecerem condições favoráveis ao desenvolvimento microbiano. Por outro lado, o alto teor de atividade de água favorece o crescimento de microrganismos, especialmente de leveduras o que requer maior cuidado no manuseio e armazenamento do mel. As diferenças observadas entre o mel de Melipona e o de Apis reforçam a necessidade de estabelecimento de padrões de qualidade específicos para o mel de abelhas sem ferrão.

Pollen samples were collected in three different periods from 11Melipona capixabaMoure & Camargo hives and analyzed with melissopalynological methodology. A total of 33 pollen types were identified, of which 23 genera and 15 families were identified. The following families showed the highest pollen richness: Fabaceae (7), Myrtaceae (3), Solanaceae (3), Arecaceae (2), Asteraceae (2), Euphorbiaceae (2), Melastomataceae/Combretaceae (2), Rubiaceae (2), and Sapindaceae (2). The most frequent pollen types (>45%) wereEucalyptus, which generated great similarities between the samples, except one in which theTibouchinawas predominant. Although the majority of the pollen types showed low percentage values, the results demonstrated thatM. capixabahas taken advantage of the polliniferous sources available in the Atlantic Rainforest as well as in the “Capoeira” (brushwood, secondary forest) and “ruderal” (field) plants, probably implying its importance as a pollinator of the native flora and of the exotic species.

Dinoponera lucida (Formicidae; Ponerinae) is an extinction-threatened species of ant which is endemic in the central corridor of the Atlantic Forest. We used mitochondrial sequences of the Cox1, Cox2 and Cytb genes in order to infer some aspects of the evolutionary history and phylogeography of this ant. High genetic divergence and population structure were observed for the whole species. The current pattern of D. lucida diversity seems to be shaped during different geological times: middle Pliocene, early Pleistocene and mainly late Pleistocene, when the reduction of populations generated a structure pattern of the genetic variation of this species. Our data show that this structure results from the maintenance of populations of D. lucida within very small putative refuges to the south of the central Bahia refugium. We thus argue that, for some Atlantic forest endemic species, especially those resistant to very small fragments of forest, such as D. lucida, the small putative refuges were as important as, or even more important than, larger and stable refuges for the creation and maintenance of diversity, adding another piece to the puzzle of the mechanisms underlying local endemism. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101, 949–960.

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

The stingless bee Melipona capixaba Moure and Camargo, 1994 is endemic to the Brazilian Atlantic Forest. Its occurrence is restricted to highlands in the Espirito Santo State, and it has possibly the smallest known geographic distribution among the cataloged stingless bees. It is therefore considered to be an endangered species. Perhaps because of its small area of occurrence, or because it was only identified two decades ago, little is known about the biology of this species, its current geographic distribution, or its actual preservation status. Here, we present the results from the largest sampling of M. capixaba conducted in its natural habitat. We developed a distribution map by using a geographic information system. Our data indicate that M. capixaba is found in the municipalities of Espirito Santo State at altitudes between 800 m and 1,200 m; with annual average temperatures around 18–23°C; precipitation more than 1,200 mm per year; and vegetation cover-type Mountain Dense Ombrophylous Forest, restricted to an area of approximately 3,450 km 2 . We observed colonies both in their natural habitat and under conditions of ex situ maintenance, and identified the key challenges and prospects for the conservation of this endangered bee.

The origin of supernumerary (B) chromosomes is still a debated topic, with intra- and interspecific origins being the most plausible options. In the bee Partamona helleri, a sequence-characterized amplified region (SCAR) marker being specific to B chromosomes suggested the possibility of interspecific origin. Here, we search for this marker in 3 close relative species and perform DNA sequence comparison between species. The SCAR sequence does not show homology with other sequences in the databases, but does contain an open reading frame with sequence homology with a reverse transcriptase. Dot-blot hybridization using the SCAR marker as a probe confirmed that it is present in B-carrying, but not B-lacking larvae of P. helleri, and indicated its presence in adult individuals of P. cupira and P. criptica. Additionally, PCR amplification of the SCAR marker was successful on genomic DNA obtained from P. helleri and P. rustica larvae carrying B chromosomes, and on genomic DNA obtained from adult individuals of P. cupira, P. criptica and P. rustica. Finally, a comparison of the DNA sequence of the SCAR markers amplified from these 4 species showed very few nucleotide differences between the species. The complete association between B chromosome and SCAR presence and the scarce divergence observed for this DNA sequence between the 4 species analyzed suggest the possibility that this B chromosome has recently been transferred between species through several episodes of interspecific hybridization.

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

Chromosomes in hymenopteran insects cannot currently be analysed in adult individuals. The only available cytogenetic techniques need to be performed in larvae. Here we develop and implement a SCAR (Sequence Characterized Amplified Region) marker, associated with B chromosomes in the bee Partamona helleri, which has proven to be very useful to reveal B chromosome presence in adults from natural populations. The marker was tested in ten different colonies simultaneously analysed by both molecular (ten adults per colony) and cytogenetic (20 larvae per colony) techniques. The presence of the SCAR marker always showed the same pattern as B chromosome presence: both were present or absent in all individuals from a same colony, or both were present in only part of the individuals from a same colony. This molecular marker is thus a useful tool for analysing new aspects of this B chromosome system such as B frequency and geographical distribution, B transmission, or B effects in adult individuals.

The leafcutter ants, which consist of Acromyrmex and Atta genera, are restricted to the New World and they are considered the main herbivores in the neotropics. Cytogenetic studies of leafcutter ants are available for five species of Atta and 14 species of Acromyrmex, both including subspecies. These two ant genera have a constant karyotype with a diploid number of 22 and 38 chromosomes, respectively. The most distinct Acromyrmex species from Brazil is A. striatus, which is restricted to the southern states of Santa Catarina and Rio Grande do Sul. Several cytogenetic and phylogenetic studies have been conducted with ants, but the karyotypic characterization and phylogenetic position of this species relative to leafcutter ants remains unknown. In this study, we report a diploid number of 22 chromosomes for A. striatus. The phylogenetic relationship between A. striatus and other leafcutter ants was estimated based on the four nuclear genes. A. striatus shared the same chromosome number as Atta species and the majority of metacentric chromosomes. Nuclear data generated a phylogenetic tree with a well-supported cluster, where A. striatus formed a different clade from other Acromyrmex spp. This combination of cytogenetic and molecular approaches provided interesting insights into the phylogenetic position of A. striatus among the leafcutter ants and the tribe Attini.

International audience; The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.

Melipona quadrifasciata Lepeletier, 1836, regionally known as "mandaçaia", has been traditionally divided in two distinct subspecies: M. quadrifasciata anthidioides and M. quadrifasciata quadrifasciata. The main difference between the subspecies refers to the yellow metasomal stripes which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. This study investigated the geographic differentiation in the metasomal stripes and characterized the restriction sites in the mtDNA of both chromatic types. Specimens from 198 localities were examined, and the variation observed in the pattern of stripes was grouped into distinct classes. The distribution pattern found in the present work agrees with the previously reported pattern: M. q. quadrifasciata inhabits the southern portion of the distribution, from Misiones, Argentina, southeastern Paraguay and Rio Grande do Sul to southern São Paulo, and M. q. anthidioides ranges from northeastern São Paulo to the northern Diamantina Plateau, Bahia, and westwards to the central portion of the Goiás state. It is documented for the first time the occurrence of two populations with continuous stripes inhabiting disjunct areas in relation to M. q. quadrifasciata - one in northern Minas Gerais and another in northeastern Bahia and Sergipe. The data of RFLP showed two restriction patterns, one present in M. q. quadrifasciata, and another in M. q. anthidioides and in populations with continuous metasomal stripes from northern Minas Gerais and northeastern Bahia and Sergipe. The observed patterns of geographic differentiation of M. quadrifasciata suggests the occurrence of repeated events of geographical isolation, followed by range expansion, that occurred probably during the cycles of climatic changes in the Pleistocene.

A abelha sem ferrão Melipona capixaba Moure & Camargo é uma espécie restrita à Mata Atlântica nos municípios de Domingos Martins, Conceição do Castelo, Venda Nova do Imigrante e Afonso Cláudio, ES. Apesar de sua importância ecológica como polinizador, poucos estudos têm investigado a biologia e ecologia dessa abelha, popularmente conhecida como uruçu-negra, pé-de-pau, ou uruçu-capixaba. Esta nota relata uma observação de operárias de M. capixaba carregando polinário preso ao escutelo. Os polinários foram identificados como pertencentes a uma espécie da subtribo Maxillariinae e, possivelmente, a um dos gêneros Maxillaria sensu lato ou Xylobium. The stingless bee Melipona capixaba Moure & Camargo is a species restricted to the Atlantic forest in the Domingos Martins, Conceição do Castelo, Venda Nova do Imigrante and Afonso Cláudio County, in the Espírito Santo State, Brazil. Despite its cological importance as pollinator few studies have examined the ecology and biology of this bee. This note relates a case of the M. capixaba workers carrying pollinarium attached to the scuttellum. The pollinaria were identified as belonging to the orchid subtribe Maxillariinae species possibly of the genus Maxillaria sensu lato or Xylobium.

The stingless bee Melipona capixaba MoureCamargo is a species restricted to the Atlantic forest in the Domingos Martins, Conceição do Castelo, Venda Nova do Imigrante and Afonso Cláudio County, in the Espírito Santo State, Brazil. Despite its cological importance as pollinator few studies have examined the ecology and biology of this bee. This note relates a case of the M. capixaba workers carrying pollinarium attached to the scuttellum. The pollinaria were identified as belonging to the orchid subtribe Maxillariinae species possibly of the genus Maxillaria sensu lato or Xylobium.