Maria Cristina Arias

Genetic structure of Africanized honeybee populations (Apis mellifera L.) from Brazil and Uruguay viewed through mitochondrial DNA COI-COII patterns

Mitochondrial genotypes of Africanized honeybees from Brazil and Uruguay were surveyed by DraI restriction of the COI–COII region. Eleven mitotypes were found, three of which had not previously been described (A28–A30). Out of 775 samples (725 from Brazil, 50 from Uruguay), 197 were A1 and 520 were A4. A1 frequency increases toward the north of Brazil, whereas A4 frequency increases toward the south, a pattern echoing the African distribution. The origin of the A4 and most of the A1 African patterns can be attributed to the introduction of Apis mellifera scutellata into Brazil in 1956. The A29 and A30 patterns have the P1 sequence observed in many Iberian Peninsula samples, which represent the traces of the introductions into Brazil and Uruguay by settlers.

The mitochondrial genome of the stingless bee Melipona bicolor (Hymenoptera, Apidae, Meliponini): sequence, gene organization and a unique tRNA translocation event conserved across the tribe Meliponini

At present a complete mtDNA sequence has been reported for only two hymenopterans, the Old World honey bee, Apis mellifera and the sawfly Perga condei. Among the bee group, the tribe Meliponini (stingless bees) has some distinction due to its Pantropical distribution, great number of species and large importance as main pollinators in several ecosystems, including the Brazilian rain forest. However few molecular studies have been conducted on this group of bees and few sequence data from mitochondrial genomes have been described. In this project, we PCR amplified and sequenced 78% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was partially sequenced). We also report the genome organization (gene content and order), gene translation, genetic code, and other molecular features, such as base frequencies, codon usage, gene initiation and termination. We compare these characteristics of M. bicolor to those of the mitochondrial genome of A. mellifera and other insects. A highly biased A+T content is a typical characteristic of the A. mellifera mitochondrial genome and it was even more extreme in that of M. bicolor. Length and compositional differences between M. bicolor and A. mellifera genes were detected and the gene order was compared. Eleven tRNA gene translocations were observed between these two species. This latter finding was surprising, considering the taxonomic proximity of these two bee tribes. The tRNA Lys

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.

Phylogeographical autocorrelation of phenotypic evolution in honey bees (Apis mellifera L.)

The analysis of phenotypic divergence among local populations within a species has been traditionally performed in a spatial context, although advances in genetic analysis using mtDNA have permitted a simultaneous evaluation of geographical and historical patterns of variation, so-called phylogeographical analysis. In this paper, we combine these two dimensions of variation (geographical space and phylogenetic history) to evaluate patterns of phenotypic evolution in honey bees (Apis mellifera L.). Data on 39 phenotypic traits, derived from 417 colonies grouped into 14 subspecies, were analysed using autocorrelation methods. Mantel tests indicated that the relationship between phenotypic divergence, estimated by Euclidean distances among subspecies’ morphological centroids, was significant both when compared to geographical distance (r=0.371; P< 0.01) and to genetic distance (estimated as sequence divergence (%) in a mtDNA region encompassing part of the NADH dehydrogenase subunit 2 and isoleucine transfer RNA (r=0.329; P< 0.01)). For the analysis of each trait, the effects of the geographical co-ordinates (latitude and longitude of subspecies geographical range) and of the phylogenetic patterns (defined by eigenvectors of the genetic distance matrix) on phenotypic variation were simultaneously analysed using an extension of a recently developed model, called Phylogenetic Eigenvector Regression (PVR). In general terms, the partial regression slopes indicated that the variation in the characters traditionally associated with adaptive processes, such as body and wing size, were better explained by geographical position. However, characters usually thought to be neutral, such as wing venation angle, were more associated with phylogeny. This is expected because PVR can be interpreted as a partition model, in which adaptive variation tends to be independent of phylogeny (and, in this case, associated with geography). In addition, the first principal component derived from the expected values of the model for each trait, which can be interpreted as the phenotypic variation predicted by phylogeny, is more structured in a north–south cline than are the original data, supporting an adaptive interpretation. The phylogeographical autocorrelation analyses performed in this study show that different traits are more related to one of the two dimensions of variation (geography and phylogeny), and these patterns can furnish insights into the nature of phenotypic evolution in these organisms.

Monogamy in large bee societies: a stingless paradox

High genetic diversity is important for the functioning of large insect societies. Across the social Hymenoptera (ants, bees, and wasps), species with the largest colonies tend to have a high colony-level genetic diversity resulting from multiple queens (polygyny) or queens that mate with multiple males (polyandry). Here we studied the genetic structure of Trigona spinipes, a stingless bee species with colonies an order of magnitude larger than those of polyandrous honeybees. Genotypes of adult workers and pupae from 43 nests distributed across three Brazilian biomes showed that T. spinipes colonies are usually headed by one singly mated queen. Apart from revealing a notable exception from the general incidence of high genetic diversity in large insect societies, our results reinforce previous findings suggesting the absence of polyandry in stingless bees and provide evidence against the sperm limitation hypothesis for the evolution of polyandry. Stingless bee species with large colonies, such as T. spinipes, thus seem promising study models to unravel alternative mechanisms to increase genetic diversity within colonies or understand the adaptive value of low genetic diversity in large insect societies.

Hybridization and asymmetric introgression between Tetragonisca angustula and Tetragonisca fiebrigi

The broadly distributed Neotropical stingless bee Tetragonisca angustula was once regarded as having two subspecies, T. angustula angustula and T. angustula fiebrigi. In a recent taxonomic revision, these subspecies were elevated to species status (T. angustula and Tetragonisca fiebrigi) based on morphology and distribution. While molecular studies show two well-delineated subgroups within the Tetragonisca, they are inconclusive as to whether there is gene flow between T. angustula and T. fiebrigi. We characterize 1,003 specimens from southern and southeastern Brazil using mitochondrial DNA and microsatellite analysis and demonstrate that there is ongoing hybridization and introgression between T. angustula and T. fiebrigi and suggest that they may be better regarded as subspecies.

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.

Detection of mitochondrial DNA restriction site differences between the subspecies of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae: Meliponini)

A distribuicao geografica da abelha sem ferrao Melipona quadrifasciata Lepeletier, no Brasil, atinge do Rio Grande do Sul ate a Paraiba, habitando originalmente regioes de Mata Atlântica. A especie compreende duas subespecies, M. q. quadrifasciata e M. q. anthidioides, que sao reconhecidas morfologicamente pelo padrao das bandas tergais (listras amarelas no abdome). O presente trabalho teve por objetivo detectar diferencas no padrao de restricao do DNA mitocondrial entre as duas subespecies. Foram coletadas amostras de quatro colonias de cada subespecie e uma operaria por colonia foi utilizada nas analises moleculares. Nove regioes do genoma mitocondrial foram amplificadas via PCR e posteriormente digeridas com 13 enzimas de restricao (PCR+RFLP). Tres enzimas apresentaram padrao de restricao diferente entre as duas subespecies. Diferencas no padrao de restricao do genoma mitocondrial poderao contribuir na identificacao da origem materna de individuos hibridos de M. q. quadrifasciata e M. q. anthidioides, pois estes ocorrem naturalmente ou por influencia humana.

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.