Isolation and characterization of ten microsatellite loci in\ud stingless bee Trigona spinipes (Apidae: Meliponini)

Stingless bees are the most abundant pollinators of Brazilian tropical flora. Trigona spinipes has some of the\ud largest colonies of any stingless bee species found in several types of environment. This work describes the\ud isolation and characterization of microsatellite loci for this species. A microsatellite-enriched genomic library\ud was constructed and ten primer pairs were designed for T. spinipes. The primers were tested in 20 unrelated\ud individuals. The mean number of alleles was 8.10 and mean observed and expected heterozygosity were\ud 0.655 and 0.680, respectively. Primers were also tested in cross-species amplification and five loci were\ud successfully amplified inTrigona chanchamayoensis,Trigona hyalinata,Tetragonisca angustula, Partamona\ud mulata and Frieseomelitta varia. The microsatellite primers described herein will be useful for evaluating\ud genetic variability and gaining a better understanding of the population structure of T. spinipes as well as\ud other species of stingless bees.Fundação de Amparo à Pesquisa do Estado de São Paulo (2004/15801-0, 2010/50597-5

Molecular and population analysis of Partamona mulata (Moure In Camargo, 1980)and Partamona helleri (Friese, 1900) (Hymenoptera, Apidae, Meliponini)

O gênero Partamona compreende 33 espécies, distribuídas do sul do México ao sul do Brasil. O gênero tem sido amplamente estudado em diferentes níveis: citogenético, etológico e morfológico. O presente trabalho teve como objetivo contribuir com dados moleculares para o conhecimento do grupo, realizando estudos populacionais por meio da caracterização do DNA mitocondrial por PCR+RFLP e da análise de regiões de microssatélites do DNA genômico de duas espécies: P. mulata de distribuição restrita ao sul de Mato Grosso e norte do Mato Grosso do Sul, e P. helleri de distribuição mais ampla, do sul da Bahia até Santa Catarina. Foram detectados apenas dois haplótipos em P. mulata, os quais diferiram entre si por apenas um sítio de restrição. As análises estatísticas demonstraram não haver estruturação entre as populações sugerindo que esta espécie possa ter passado por recente afunilamento populacional. Em P. helleri foram observados dez haplótipos sendo alguns exclusivos e outros compartilhados. Análises estatísticas apontaram alta estruturação entre as populações e a distribuição filogeográfica observada sugere um possível isolamento por fragmentação da Mata Atlântica durante o Pleistoceno. A análise dos locos microssatélites mostrou baixa variabilidade genética em ambas espécies e discreta estruturação entre as populações, não relacionada com a distribuição geográfica das mesmas. Isto pode ser conseqüência de migração de machos entre populações visto que as rainhas são filopátricas ou, fragmentação dos habitats pela rápida degradação do cerrado e da Mata Atlântica, ou por alelos nulos causados pelo uso de primers heteroespecíficos. A análise de parentesco entre abelhas de um mesmo ninho apontou a existência de apenas uma patrilínea em P. mulata sugerindo monoandria para esta espécie. Foram encontradas duas patrilíneas em algumas colônias de P. helleri, o que pode ser resultante de fecundação por mais de um macho ou substituição recente da rainha. A caracterização parcial do DNAmt de duas espécies de Partamona poderá contribuir em estudos filogenéticos tanto do gênero quanto de outras espécies de Meliponini. A análise populacional mostrou o status da variabilidade genética das espécies, suas possíveis histórias evolutivas e a possível relação desta com degradação dos ambientes onde estas estão distribuídas.The Partamona genus comprises 33 species distributed from south Mexico to south Brazil. This genus has been studied at different levels: cytogenetical, ethological and morphological. This work aimed at to contribute with molecular data for the knowledge about the group performing a population study employing the PCR+RFLP of mtDNA, and analysis of microsatellite loci from nuclear DNA of two species, P. mulata which is distributed in south Mato Grosso and north Mato Grosso do Sul, and P. helleri which geographic distribution is wider, from Santa Catarina to southern Bahia. It was detected two haplotypes in 58 colonies of P. mulata, each one differing by one single restriction site. The statistical analyses indicated no differentiation among populations suggesting that the species could have passed through a recent populational bottleneck. It was observed ten haplotypes in 47 colonies of P. helleri, some exclusive and others shared among populations. Statistical analysis pointed high population differentiation and the observed phylogeography distribution suggested a possible recent isolation probably by Atlantic Forest fragmentation during the Pleistocene. The microsatellite analysis showed low genetic variability in both species and discrete population structuring, not related to the geographic distribution. This might be consequence of migration of males, since the queens are highly phylopatric, or habitat fragmentation by degradation of savanna and Atlantic forest areas, or null alleles caused by the use of heterospecific primers. The relatedness investigation revealed only one patriline in nest mates of P. mulata that suggests monoandry for this species. It was found two patrilines in P. helleri that can be resulted from more than one mating or recent queen replacement. The partial characterization of the mtDNA of two Partamona species can contribute to further phylogenetic studies among bees of this genus or among other Meliponini species. The populational analysis showed the genetic variability status of the species, their putative evolutionary histories and the possible relation between the results and the environmental degradation in their distribution areas

Mitochondrial DNA characterization of two Partamona species (Hymenoptera, Apidae, Meliponini) by PCR+RFLP and sequencing

We characterized the mitochondrial DNA of two stingless bee species of the genus Partamona. Partial restriction maps were obtained based on digestion of PCR amplified fragments with 8 restriction enzymes. Using Melipona bicolor mtDNA sequence as a model, we were able to amplify 12120 bp of P. mulata and 10300 bp of P. helleri, about 65.5% and 55.7% of their mitochondrial genome, respectively. The digestion assays showed 16 restriction sites for P. mulata and 20 for P. helleri, some of which were exclusive to the genus and others shared with other Meliponini species. The main mitochondrial genes could be mapped and through sequencing analysis we verified that the intergenic region that occurs between the genes CO I and CO II in Apis is absent in Partamona

Karyotypic description of four species of Trigona (Jurine, 1807) (Hymenoptera, Apidae, Meliponini) from the State of Mato Grosso, Brazil

The genus Trigona contains at least 31 species, but there have been few cytogenetic studies of this group. In this work, four species of Trigona (T. branneri, T. chanchamayoensis, T. hyalinata, and T. recursa) from the municipality of Cuiabá, in the State of Mato Grosso, Brazil, were studied. In all of the species, the females had 2n = 34 chromosomes and the males had n = 17. The C-banding patterns showed that the karyotypes of these species consisted mainly of acrocentric and pseudoacrocentric chromosomes. These cytogenetic findings should useful in future phylogenetic studies of this group

Genetic architecture of the Tetragonula carbonaria species complex of Australian stingless bees (Hymenoptera, Apidae, Meliponini)

A species complex is a group of closely related species whose ecological or morphological boundaries are sufficiently vague that delimiting one species from another is difficult. In Australia, a group of four stingless bee species Tetragonula carbonaria Smith, T. hockingsi Cockerell, T. mellipes Friese and T. davenporti Franck form a species complex in which gross morphology is clinal and overlapping. The species are most readily distinguished by the morphology of their brood combs. Here we genetically characterize bees sampled in areas in which the species do and do not have contact. Our data corroborate previous evidence that T. hockingsi and T. carbonaria are genetically distinct and that there are two genetically distinct groups of T. hockingsi, one in the north and the other in the south-east of Queensland. Curiously, northern populations of T. hockingsi, which are allopatric to T. carbonaria, are genetically closer to T. carbonaria than are southern populations of T. hockingsi, which are in sympatry with T. carbonaria. We detected three hybrid colonies that appear to have arisen because of anthropogenic movement of T. hockingsi colonies from north to south-east Queensland where males mated with local T. carbonaria queens. We discuss the status of T. davenporti, a recently-described species cryptically similar to T. hockingsi from south-east Queensland

Cytogenetic data of Partamona peckolti (Hymenoptera, Apidae, Meliponini) by C banding and fluorochrome staining with DA/CMA3 and DA/DAPI

The stingless bees of the Partamona genus have been studied taxonomically, ecologically and behaviourally, but cytogenetic studies are still rare. The objective of this study was to obtain cytogenetic data to contribute to Partamona peckolti species characterization. Heterochromatin was localized in all chromosome pericentromeric regions but some blocks could be visualized on some large chromosomes arms. A large heterozygous DA-CMA3-positive band was observed on one large chromosome arm, but was completely absent when C banding was applied before fluorochrome staining, with only one small positive band being visualized. Sequential DA-CMA3-NOR staining of interphase nuclei provided coincident positive responses. This suggests that DA-CMA3-positive bands of P. peckolti correspond to nucleolar organizer regions, as previously confirmed for another Partamona species by FISH

Cytogenetic data of Partamona peckolti (Hymenoptera, Apidae, Meliponini) by C banding and fluorochrome staining with DA/CMA3 and DA/DAPI

The stingless bees of the Partamona genus have been studied taxonomically, ecologically and behaviourally, but cytogenetic studies are still rare. The objective of this study was to obtain cytogenetic data to contribute to Partamona peckolti species characterization. Heterochromatin was localized in all chromosome pericentromeric regions but some blocks could be visualized on some large chromosomes arms. A large heterozygous DA-CMA3-positive band was observed on one large chromosome arm, but was completely absent when C banding was applied before fluorochrome staining, with only one small positive band being visualized. Sequential DA-CMA3-NOR staining of interphase nuclei provided coincident positive responses. This suggests that DA-CMA3-positive bands of P. peckolti correspond to nucleolar organizer regions, as previously confirmed for another Partamona species by FISH

Very low mitochondrial variability in a stingless bee endemic to cerrado

Partamona mulata is a stingless bee species endemic to cerrado, a severely threatened phytogeographical domain. Clearing for pasture without proper soil treatment in the cerrado facilitates the proliferation of termite ground nests, which are the nesting sites for P. mulata. The genetic consequences of these changes in the cerrado environment for bee populations are still understudied. In this work, we analyzed the genetic diversity of 48 colonies of P. mulata collected throughout the species' distribution range by sequencing two mitochondrial genes, cytochrome oxidase I and cytochrome B. A very low polymorphism rate was observed when compared to another Partamona species from the Atlantic forest. Exclusive haplotypes were observed in two of the five areas sampled. The sharing of two haplotypes between collection sites separated by a distance greater than the flight range of queens indicates an ancient distribution for these haplotypes. The low haplotype and nucleotide diversity observed here suggests that P. mulata is either a young species or one that has been through population bottlenecks. Locally predominant and exclusive haplotypes (H2 and H4) may have been derived from local remnants through cerrado deforestation and the expansion of a few colonies with abundant nesting sites

Molecular markers as a tool for population and evolutionary studies of stingless bees

Molecular markers are widely used in biology to address questions related to ecology, genetics and evolution. In bees, molecular studies addressing those issues have focused on Apis and Apis mellifera. Here we describe examples where molecular markers from mtDNA and microsatellite analyses were applied to stingless bees species. The data obtained, although in some cases preliminary, have already proven useful to infer hypotheses about phylogeny, population dynamics, species validity and the evolution of this group of bees