Flávio de Oliveira Francisco

Alelle number and heterozigosity for microsatellite loci in different stingless bee species (Hymenoptera: Apidae, Meliponini)

In the present study we compare genetic characteristics (allele diversity and observed heterozygosity) of microsatellite loci, from three stingless bee species (Plebeia remota Holmberg, Partamona mulata Moure In Camargo and Partamona helleri Friese), amplified by using heterospecific primers originally designed for Melipona bicolor Lepeletier and Scaptotrigona postica Latreille. We analyzed 360 individuals of P. remota from 72 nests, 58 individuals of R. mulata from 58 nests, and 47 individuals of P. helleri from 47 nests. The three species studied showed low level of polymorphism for the loci amplified with primers derived from M. bicolor. However, for the loci amplified with primers derived from S. postica, only P. remota presented low level of polymorphism.

Characterization of microsatellite loci of Tetragonisca angustula (Hymenoptera, Apidae, Meliponini)

An enriched genomic library was constructed from Tetragonisca angustula, a stingless bee species widely distributed in Brazil. The library was screened using two simple-repeat oligonucleotide probes and 21 microsatellite primer pairs were designed flanking a selection of repeat sequences within positive clones. The polymorphism of the microsatellite loci was analyzed by screening a sample of 19 unrelated T. angustula workers. Fifteen out of 21 loci were shown to be polymorphic, with observed heterozygosity estimates ranging from 0.00 to 0.89. The primers were also successfully used to amplify microsatellite loci from other stingless bee species, Tetragonisca fiebrigi, Tetragonisca weyrauchi, Lestrimelitta maracaia and Schwarziana quadripunctata. The results from variability analyses suggest that the microsatellite loci isolated from T. angustula will be useful in further population studies for the species and also for other Meliponini.

Isolation and characterization of 15 microsatellite loci in the stingless bee Plebeia remota (Apidae: Meliponini)

The destruction of Brazilian natural habitats has reduced bee populations and negative impacts of native flora pollination have been noticed. This work describes the isolation and characterization of microsatellite loci and evaluates them as molecular markers to study genetic variability of the stingless bee Plebeia remota. A microsatellite enriched genomic library was constructed and 15 primer pairs were designed for this species. The survey was conducted by analyzing 21 unrelated individuals. Genetic diversity indexes were calculated. The mean allelic richness was 6.3, the observed heterozygosity was 0.568, and the percentage of polymorphic loci was 93.33%. Also the primers were tested in cross-species amplification and showed promising results for P. droryana, P. emerina, P. lucii, P. meridionalis, P. pugnax, and P. saiqui. The microsatellite loci described here will be useful to evaluate genetic variability of stingless bees, and certainly will improve our knowledge about population dynamics especially in threatened environments.

Inferences of evolutionary and ecological events that influenced the population structure of Plebeia remota, a stingless bee from Brazil.

The present study characterised the population genetic structure of Plebeia remota through mitochondrial DNA (mtDNA) analysis and evaluated evolutionary and ecological processes that may have contributed to the species current genetic scenario. Seventy feral nests were sampled representing four geographic regions (Cunha, Curitiba, Prudentópolis, and Blumenau). Fifteen composite mtDNA haplotypes were determined and a high genetic structure was detected among all populations. The current population structure may be a result of queen philopatry and vegetation shifts caused by palaeoclimatic changes and uplift of Brazilian coastal ranges. Finally, this study strongly suggests a revision of the taxonomic status of P. remota from the Prudentópolis region.ZusammenfassungNeuere Studien haben gezeigt, dass innerhalb der Art Plebeia remota in ihrem Verbreitungsgebiet ausgeprägte Unterschiede in Verhalten, Morphometrie, chemischen Komponenten und genetischen Besonderheiten existieren. Die genetische Struktur von P. remota wurde mit Hilfe von mitochondrialer DNA (mtDNA) untersucht; evolutionäre und ökologische Prozesse, die zu diesem Szenarium geführt haben könnten wurden abgeleitet. Die Proben wurden in Cunha (São Paulo), Blumenau (Santa Catarina), sowie in Curitiba und Prudentópolis (Paraná) gesammelt (Abb. 1). Die Proben stammen von siebzig Nestern und wurden mittels RFLP Analyse untersucht, wobei 15 Restriktionsenzyme eingesetzt wurden. Die Länge des mtDNA-Moleküls betrug in allen Proben 18 500 bp. Mit zwei der Restriktionsenzyme konnten in keiner der Proben eine Schnittstelle nachgewiesen werden. Mit den restlichen 13 Enzymen wurden insgesamt 27 verschiedene Schnittstellen gefunden; daraus ergaben sich 15 Haplotypen, deren Verteilung gut mit der geographischen Herkunft der Proben korrelierte (Abb. 2). Dabei trat der gleiche Haplotyp nur selten in mehreren Populationen auf (Tab. I). Die Diversität der Haplotypen (h) und der Nukleotide (π) innerhalb jeder einzelnen Population waren vergleichsweise hoch, mit der Ausnahme von Prudentópolis (Tab. II). Die Werte für FSTund die Nukleotid-Divergenz zwischen den Populationen sind in Tabelle III dargestellt. Die Analyse der mtDNA zeigte große genetische Variabilität und Isolation zwischen den Populationen, was sowohl durch die Philopatrie der Königinnen als auch mit Habitatfragmentation in früheren Zeiten durch Klimaänderungen oder die Auffaltung des Küstengebirges erklärt werden kann. Zusammen mit bereits veröffentlichten Daten unterstützen die Ergebnisse die Vermutung, dass diese spezifische Population als eine eigene Art angesehen werden sollte. Die Untersuchung zusätzlicher Bienenarten und anderer Organismen aus denselben geographischen Gebieten wird unser Wissen über die Verbreitungsmuster der Arten und die möglichen Zusammenhänge mit historischen und evolutionären Ereignissen in dieser Region vermehren.

Molecular genetic diversity in populations of the stingless bee Plebeia remota: A case study

Genetic diversity is a major component of the biological diversity of an ecosystem. The survival of a population may be seriously threatened if its genetic diversity values are low. In this work, we measured the genetic diversity of the stingless bee Plebeia remota based on molecular data obtained by analyzing 15 microsatellite loci and sequencing two mitochondrial genes. Population structure and genetic diversity differed depending on the molecular marker analyzed: microsatellites showed low population structure and moderate to high genetic diversity, while mitochondrial DNA (mtDNA) showed high population structure and low diversity in three populations. Queen philopatry and male dispersal behavior are discussed as the main reasons for these findings.

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.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.ZusammenfassungDer Informationsgehalt des mitochondrialen Genoms (mtDNA) ist in weitem Mass von Bedeutung für populationsgenetische und evolutive Studien. Bei der Honigbiene, Apis mellifera, trug die Aufdeckung von Polymorphismen in diesem Molekül zu wichtigen Einsichten in die Populationsstruktur und zur Phylogenie bei. Obwohl A. mellifera die Hymenopterenart ist, für die als erste das mitochondriale Genom komplett sequenziert wurde, beschränken sich selbst bei dieser Art die meisten Populations- und Evolutionsstudien auf nur einige wenige mitochondriale Regionen. Für die Nutzung weiterer mtDNA Regionen ist die Entwicklung geeigneter Primer von zentraler Bedeutung für erfolgreiche PCR-Amplifikationen. Die Verwendung genereller Oligonukleotidprimer (universelle Primer) für das mitochondriale Genom von Insekten führte bei der Honigbiene oft zu Fehlschlägen oder lieferte keine hochwertigen PCR-Produkte. Wir beschreiben in der vorliegenden Arbeit einen Satz bestehend aus 24 Oligonukleotiden, mit denen es möglich war, das gesamte mitochondriale Genom der Honigbiene A. mellifera in Form von 12 Fragmenten zu amplifizieren (Abb. 1, Tab. I). Die Amplifikationsbedingungen für diese Primerkombinationen wurden etabliert und sind in Tabelle II zusammengestellt. Diese Fragmente können von Wichtigkeit sein für die Untersuchung mitochondrialer Gene bei verschiedenen Unterarten von A. mellifera und ebenso als heterospezifische Sonden zur Charakterisierung des mitochondrialen Genoms anderer Bienenarten dienen. Ausserdem kann die Verwendung eines Teilsatzes dieser Primer für die Amplifizierung homologer Regionen bei anderen Bienen genutzt werden (Tab. III) und damit künftige Populationsuntersuchungen und evolutive Studien innerhalb der Hymenopteren vorantreiben.

Genetic variability in captive populations of the stingless bee Tetragonisca angustula

Low genetic variability has normally been considered a consequence of animal husbandry and a major contributing factor to declining bee populations. Here, we performed a molecular analysis of captive and wild populations of the stingless bee Tetragonisca angustula, one of the most commonly kept species across South America. Microsatellite analyses showed similar genetic variability between wild and captive populations However, captive populations showed lower mitochondrial genetic variability. Male-mediated gene flow, transport and division of nests are suggested as the most probable explanations for the observed patterns of genetic structure. We conclude that increasing the number of colonies kept through nest divisions does not negatively affect nuclear genetic variability, which seems to be maintained by small-scale male dispersal and human-mediated nest transport. However, the transport of nests from distant localities should be practiced with caution given the high genetic differentiation observed between samples from western and eastern areas. The high genetic structure verified is the result of a long-term evolutionary process, and bees from distant localities may represent unique evolutionary lineages.

Eusocial bee male aggregations: spatially and temporally separated but genetically homogenous

Male insects mostly aggregate near sites where sexually reproductive virgin females are found and where mating occur. This reproductive strategy is quite common in Hymenoptera and appears to decrease the chances of inbreeding. In Hymenoptera, inbred mating frequently result in sterile diploid males. Production of diploid males may reach high proportions in small bee populations, and it usually lead to population extinction within a small number of generations. Aggregation of males during a short period of time allow the mixing of local genes. In this paper, we analyzed male aggregations of Tetragonisca angustula (Latreille) (Hymenoptera: Apidae), a eusocial stingless bee, using microsatellite molecular markers. We used population genetic statistics for haplodiploid organisms to address genetic structuring among male aggregations. Our findings indicate that, in general, male aggregations of T. angustula occurring over short time scales and in close proximity are genetically homogeneous as if a single aggregation. We conclude that T. angustula males randomly disperse within a population looking for mating chances rather than settle on a distinct male aggregation. This behavior seems to contribute to the reduction of inbreeding in this species.

A fauna apícola do Parque Municipal da Cachoeirinha (Iporá, GO)

Bees are dependent on flower nectar and pollen (main sources of protein and energy, respectively) and have an important economic and ecological role as pollinators, representing about 40 to 90% of phanerogams pollinators in several ecosystems. This work presents a description of bee species from Parque Municipal da Cachoeirinha in Ipora, Goias state, Brazil. A total of 58 hours of capturing were performed on November and December 2004 and on March and May 2005. A total of 933 individuals were collected, distributed in one family, 16 tribes, 33 genera, and 62 species. Meliponini tribe presented the highest number of individuals and species. Most abundant species were Trigona chanchamayoensis (16.51%), Tetragona clavipes (14.79%) and Apis mellifera and Trigona recursa (12.33%). This work presents the first occurrence in Goias state for six genera and 12 species.