Transferability of microsatellite markers in Syagrus coronata (Mart.) Becc. (Arecaceae), an iconic palm tree from the Brazilian semiarid region.

The licuri palm Syagrus coronata plays a key role in the ecology and economy of Brazilian semiarid region. Nonetheless, genetic data about populations of this species are absent even though the intensive and uncontrolled exploitation since colonial periods has threatened the sustainability and viability of licuri populations. Therefore, we attempted to test the efficacy of transferability of microsatellite loci isolated from three palm tree species to S. coronata to analyze the population of this species throughout their range. A set of 19 heterologous microsatellite loci was tested in three native populations of S. coronata from the State of Bahia, northeastern Brazil, which amplified using distinct annealing temperatures (50°-60°C). Based on the 10 most polymorphic loci, the selected populations exhibited a mean number of alleles per locus of 9.8, and high genetic diversity values since the expected heterozygosity ranged from 0.573 to 0.754, while the observed heterozygosity varied from 0.785 to 1.000. In conclusion, the tested loci are transferrable and highly efficient to population studies in S. coronata, thus minimizing the lack of species-specific loci to the genetic monitoring of licuri populations.

An optimized protocol for DNA extraction in plants with a high content of secondary metabolites, based on leaves of Mimosa tenuiflora (Willd.) Poir. (Leguminosae).

Some species are characterized by a high content of tannins, alkaloids, and phenols in their leaves. These secondary metabolites are released during DNA extraction and might hinder molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa tenuiflora, an important leguminous plant from Brazilian semiarid region used in popular medicine and as a source of fuelwood or forage, was used. Eight procedures previously reported for plants were tested and adapted from leaf tissues of M. tenuiflora stored at -20°C. The optimized procedure in this study encompassed the utilization of phenol during deproteinization, increased concentrations of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and lower temperature of incubation concerning other methods. The extracted DNA did not present degradation, and amplification via PCR was successful using ISSR, trnL, ITS, and ETS primers. Besides M. tenuiflora, this procedure was also tested and proved to be efficient in genetic studies of other plant species.

Population structure and genetic diversity in Gynaikothrips uzeli (Thysanoptera: Phlaeothripidae): is there a correlation between genetic and geographic proximity?

Gynaikothrips uzeli (Thysanoptera: Phlaeothripidae) is a minuscule insect species, which forms galls, is subsocial, and parthenogenetic. It is associated with Ficus benjamina L. (Moraceae) and has a pantropical occurrence. The paucity of genetic studies on the order Thysanoptera led us to use inter-simple sequence repeat molecular marker to assess intra- and inter-gall, as well as intra- and inter-site, genetic variability and population structure of G. uzeli. Analyses indicated low genetic variability, probably related to haplodiploidy, genetic drift, the galling habit, and the low dispersal ability of G. uzeli. Populations were highly structured, with higher variation within populations than among them. Geographic distance does not appear to affect structure and genetic diversity, the latter being influenced by G. uzeli's bioecological traits, by numerous introductions during a short period, and by a possible recent, common ancestry.

Genetic diversity and structure in natural populations of Maytenus truncata Reiss, 1861, a medicinal plant vulnerable to extractivism in Bahia State, Brazil.

Maytenus truncata (Celastraceae) is a plant species widely used in the treatment of ulcers and tumors. Despite the intensive harvest of native specimens in the State of Bahia, northeastern Brazil, there is no information about the genetic variability or structure of this species. Therefore, the goal of this study was to estimate the genetic diversity and population structure of M. truncata based on inter simple sequence repeat (ISSR) molecular markers. The samples comprised specimens from Jequié, Contendas do Sincorá, Boa Nova, and Boa Vista do Tupim in the State of Bahia. After selection of eight ISSR primers, the percentage of polymorphic loci was equal to 96.2% and genetic diversity was 0.3581. The Mantel test revealed positive correlation among genetic and geographic distances (r = 0.5462), but it was not significant (r ≥ 0, P = 0.8365). Even though AMOVA revealed that most variation was found within populations (68%), a high structuring was detected among them (ΦST = 0.31, P < 0.001). Both UPGMA and Bayesian analyses indicated that gene flow was higher between Jequié and Contendas do Sincorá, whereas samples from Boa Nova and Boa Vista do Tupim were more isolated. This result is likely because of the population decrease and restriction to gene flow associated with intensive extractivism of populations of this species.

Genetic variability in Melipona scutellaris from Recôncavo, Bahia, Brazil.

Bees play a key role in pollination and thereby help maintain plant diversity. The stingless bee Melipona scutellaris is an important pollinator in northeastern Brazil because it is endemic to this region. Both deforestation and timber harvesting have reduced the nesting sites for this species, thus reducing its population and range. Genetic studies may help reverse this process by providing important tools for their proper management with a view to conservation of this species. Microsatellite markers have proven to be ideal for mapping genes and population genetic studies. Our aim was to study, using microsatellite markers, the interpopulation genetic variability of M. scutellaris in different parts of the Recôncavo region in Bahia State, Brazil. In all, 95 adult workers from 11 localities in Recôncavo Baiano (Amargosa, Cabaceiras do Paraguaçu, Conceição da Feira, Conceição do Almeida, Domingos Macedo Costa, Governador Mangabeira, Jaguaripe, Jiquiriça, Maragojipe, São Felipe, and Vera Cruz) were analyzed using 10 pairs of microsatellite primers developed for different Meliponini species. The total number of alleles, allele richness, and genetic diversity ranged from 2 to 7 per locus (average = 4.4), 1.00 to 4.88, and 0.0 to 0.850, respectively. The expected and observed heterozygosities varied from 0.0 to 0.76 and 0.0 to 0.84, respectively. No locus showed deviation from the expected frequencies in the chi-square test or linkage disequilibrium. The fixation index, analysis of molecular variance, and unweighted pair-group method using the arithmetic average revealed the effects of human activities on the populations of M. scutellaris, as little genetic structure was detected.

Optimization of DNA extraction from fresh leaf tissues of Melanoxylon brauna (Fabaceae).

Melanoxylon brauna (Fabaceae - Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/µL). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/µL) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/µL DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies.

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona.

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.

Occurrence of multiple nucleolus organizer regions and intraspecific karyotype variation in Scaptotrigona xanthotricha Moure (Hymenoptera, Meliponini).

Scaptotrigona xanthotricha has a wide geographic distribution in the Brazilian Atlantic rainforest. One population from southeast and two from northeast Brazil were analyzed and were found to have chromosome polymorphisms. Although the chromosome number 2n = 34 is conserved in this species, karyotypic analysis revealed clear differences between the three populations. Congruent and ubiquitous multiple nucleolus organizer regions, heterochromatin and CMA(3)-positive blocks were found. The variations suggest that this species is in a process of genetic differentiation. This differentiation process might have been enhanced by restricted nesting preferences, combined with recent extensive fragmentation of the Atlantic rainforest, which limits gene flow between populations.

Genetics of Euglossini bees (Hymenoptera) in fragments of the Atlantic Forest in the region of Viçosa, MG.

With uncontrolled deforestation, forest fragments remain, which in most cases are in different stages of regeneration and present isolated populations. In the present study we analyzed the genetic patterns of Eulaema nigrita populations in seven Atlantic Forest fragments of different sizes and successional stages in the region of Viçosa, MG. This was done by RAPD molecular markers. We observed that the area of the fragments had no effect on the genetic variability of E. nigrita in the direction predicted by meta-population models. Medium-sized well-preserved woods presented the lowest variability, whereas large and small woods were statistically identical. The evidence supports the notion that rural areas present greater dispersal among fragments, implying greater similarity between the populations of fragments located in rural areas when compared to fragments in urban areas.

Genetics of Euglossini bees (Hymenoptera) in fragments of the Atlantic Forest in the region of Viçosa, MG

Com o desmatamento descontrolado das florestas há a formação de fragmentos de mata que, na maioria das vezes, se encontram em distintos estágios de regeneração, mantendo populações isoladas. Neste trabalho foi feita a análise dos padrões genéticos de populações de Eulaema nigrita de fragmentos de mata Atlântica de diferentes tamanhos e estágios sucessionais por meio de marcadores moleculares RAPD da região de Viçosa, MG. Pode-se verificar que a área dos fragmentos não apresentou efeito sobre a variabilidade genética em E. nigrita na direção predita pelos modelos de metapopulação. Uma mata de tamanho médio e bem preservada apresentou a menor variabilidade, enquanto matas grandes e pequenas foram estatisticamente iguais. As evidências sustentam que áreas rurais apresentam maior dispersão entre fragmentos, implicando maior similaridade entre as populações de fragmentos localizados em áreas rurais se comparados com fragmentos nas áreas urbanizadas.

Longitudinal differentiation in Melipona mandacaia (Hymenoptera, Meliponini) chromosomes.

Melipona mandacaia is a stingless bee endemic to northeast Brasil. We describe the M. mandacaia karyotype using C-banding technique. fluorochrome staining and treatment with restriction enzymes and discuss the position of this species in the context of the phylogeny of the genus. Melipona mandacaia has 2n = 18 (14 SM + 2 M + 2 A). Heterochromatin was detected in the pericentromeric region of pairs 1, 2 and 8 and in the form of small blocks in the remaining pairs. Staining with base-specific fluorochromes showed that this heterochromatin was rich AT (QM and DAPI), except in the region corresponding to the NOR which was rich GC (CMA3) and was cleaved by the HaeIII enzyme. Melipona mandacaia is a member of Group I Melipona. Treatment with DraI/Giemsa discloses a larger number of bands than treatment with DraI/QM. Pre-cleavage with DraI gave rise to a larger number of bands following QM staining; a circumstance evidently due to a removal of the DNA-protein complex that prevented the association of the fluorochrome with AT-rich DNA. The results highlight the complex nature of heterochromatin.

Genetic analysis of Melipona quadrifasciata Lep. (Hymenoptera: Apidae, Meliponinae) with RAPD markers

Melipona quadrifasciata ("mandaçaia") can be subdivided into two subspecies: M. q. anthidioides and M. q. quadrifasciata. In the present study we used RAPD markers to estimate intercolonial genetic variation among 69 colonies of Melipona quadrifasciata. Ten workers per colony were analyzed. The intercolony genetic distances based on RAPD markers ranged from 29.5 percent (colonies collected in the State of Säo Paulo vs colonies from the State of Minas Gerais) to 34.2 percent (Säo Paulo vs Santa Catarina). These results indicate a high genetic similarity among the colonies analyzed.According to the genetic distances two different groups could be distinguished. The first containing the samples from Santa Catarina region and the second, samples from Paraná, Säo Paulo, Minas Gerais, and Espírito Santo. Based on the molecular analysis, bees belonging to the different subspecies M. q. quadrifasciata (from Santa Catarina) and M. q. anthidioides (from the other regions) were distinguished

Genetic analysis of Melipona quadrifasciata LEP. (Hymenoptera: Apidae, Meliponinae) with RAPD markers.

Melipona quadrifasciata ("mandaçaia") can be subdivided into two subspecies: M. q. anthidioides and M. q. quadrifasciata. In the present study we used RAPD markers to estimate intercolonial genetic variation among 69 colonies of Melipona quadrifasciata. Ten workers per colony were analyzed. The intercolony genetic distances based on RAPD markers ranged from 29.5% (colonies collected in the State of São Paulo vs colonies from the State of Minas Gerais) to 34.2% (São Paulo vs Santa Catarina). These results indicate a high genetic similarity among the colonies analyzed. According to the genetic distances two different groups could be distinguished. The first containing the samples from Santa Catarina region and the second, samples from Paraná, São Paulo, Minas Gerais, and Espírito Santo. Based on the molecular analysis, bees belonging to the different subspecies M. q. quadrifasciata (from Santa Catarina) and M. q. anthidiodes (from the other regions) were distinguished.