Ana Maria Waldschmidt

Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)

O objetivo deste trabalho foi o de testar tres protocolos de extracao de DNA utilizados para Apis, plantas e fungos, visando determinar um que seja eficiente para extracao de DNA de Melipona quadrifasciata. Esses metodos diferem nas concentracoes de componentes especificos do tampao de extracao. Os resultados obtidos mostraram que a metodologia recomendada para extracao de DNA de Apis nao e adequada para a extracao de DNA de M. quadrifasciata. Entretanto, com pequenas modificacoes, este metodo, bem como aquele utilizado para a extracao de DNA de plantas e fungos, mostrou-se eficiente para a extracao de DNA desta abelha sem ferrao, tanto de individuos adultos como de larvas

Phylogeography and historical demography of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera, Apidae): incongruence between morphology and mitochondrial DNA

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.ZusammenfassungStachellose Bienen gehören zu den häufigsten und ökologisch bedeutsamsten wirbellosen Tieren in tropischen Lebensgemeinschaften. Die neotropische stachellose Biene Melipona quadrifasciata hat zwei Unterarten: M. quadrifasciata quadrifasciata und M. quadrifasciata anthidioides. Das hauptsächliche Unterscheidungsmerkmal dieser beiden Unterarten sind gelbe metasomale Streifen, die bei M. q. quadrifasciata durchgehend, und bei M. q. anthidioides unterbrochen sind. Die Art ist entlang der Ostküste Brasiliens verbreitet, wobei M. q. quadrifasciata im Süden und M. q. anthidioides im Norden des Verbreitungsgebiets vorkommt.Vor kurzem wurden zwei Populationen mit zusammenhängenden Streifen beschrieben, die nördlich des von M. q. quadrifasciata besiedelten Gebietes in disjunkten Arealen vorkommen.In dieser Studie untersuchen wir die populationsgenetische Struktur und die historische Demographie von M. quadrifasciata mit Hilfe von Sequenzen der mitochondrialen DNA aus umfangreichem Probenmaterial (Abb. 1 und Tab. S1, online material). Dabei untersuchen wir zwei Hauptfragen: (i) korreliert die morphologische Variation (Verteilung des Streifenmusters auf dem Abdomen) mit der Variation der mitochondrialen DNA? Mit anderen Worten, stammen die Populationen mit zusammenhängenden Streifen aus derselben mütterlichen Linie ab? (ii) Welche demographischen Ereignisse können die beobachteten phylogeographischen Muster erklären?Zur Beantwortung dieser Fragen sequenzierten wir 852 bp der Untereinheit1 des Cytochromoxidase (CoI) Gens aus der mitochondrialen DNA von 145 Völken (1 Arbeiterin pro Volk), die entlang des Verbreitungsgebiets der Art gesammelt worden waren (Abb. 1). Die Ergebnisse von phylogeographischen Tests und Koaleszenzberechnungen machten deutlich, dass es zwei unterschiedliche Gruppen gibt: eine südliche Klade, die aus M. q. quadrifasciata besteht, und eine nördliche, die M. q. anthidioides sowie die beiden disjunkten Populationen mit durchgehenden Streifen beinhaltet. Interessanterweise sind also Formen, die durchgehende Streifen gemeinsam haben, nicht unbedingt monophyletisch; umgekehrt haben Individuen mit verschiedener Morphologie denselben mtDNA Haplotypen gemeinsam (H14 in Abb. 3). Wir schlagen vor, die durchgehenden tergalen Streifen in den diskjunkten Populationen als einen anzestralen Polymorphismus zu betrachten, da nach den Ergebnissen anderer Studien das Muster aus zusammenhängenden Streifen innerhalb der Untergattung Melipona (Melipona) ein plesiomorphes Merkmal zu sein scheint. Der Zeitpunkt der Divergenz der beiden Gruppen wurde auf die Zeit des Pleistozäns, zwischen 0,233 und 0,840 Millionen Jahren vor der Gegenwart bestimmt (online material, Abb. S2 und Tab. S1), die durch Klimaänderungen und geomorphologische Umbildungen in der neotropischen Region charakterisiert war. Ähnliche phylogeographische Muster wurden bei endemischen Wirbeltieren des Atlantischen Regenwaldes nachgewiesen, zum Beispiel bei Schlangen, Vögeln und Amphibien.

A molecular marker distinguishes the subspecies Melipona quadrifasciata quadrifasciata and Melipona quadrifasciata anthidioides (Hymenoptera: Apidae, Meliponinae)

The stingless bee species Melipona quadrifasciata includes two subspecies, Melipona quadrifasciata anthidioids and Melipona quadrifasciata quadrifasciata. The morphological difference between the two subspecies is the presence of three to five continuous yellow stripes on the terga on the 3rd to 6th segments in workers and males of M. q. quadrifasciata, and two to five interrupted bands in M. q. anthidioides. We identified a DNA marker which is present in M. q. quadrifasciata and absent in M. q. anthidioides. Only one among the M. q. quadrifasciata colonies did not present the marker. It was also absent in bees collected in northern Minas Gerais State (Brazil), despite their morphological resemblance to M. q. quadrifasciata. The marker can be used for studying the genetic structure of the hybridization zone formed by the intercrossing of the two subspecies.

Cytogenetic and morphological diversity in populations of Astyanax fasciatus (Teleostei, Characidae) from Brazilian northeastern river basins

In the present work, morphometric and cytogenetic analyses were carried out in populations of the fish Astyanax fasciatus (Characidae) from Contas and Reconcavo Sul River basins (State of Bahia, Brazil), providing new data on the genetic structure of this species along the region. Based on morphologic measurements, we observed that populations from the same hydrographic basin were more similar to each other (Contas and Preto do Costa Rivers), and remarkably divergent from Reconcavo Sul (Mineiro Stream), as indicated by clustering analysis. Cytogenetic data revealed a same diploid number for all populations (2n = 48), but distinct karyotype formulae (8M+24SM+12ST+4A, FN = 92 in Contas River, 8M+24SM+10ST+6A, FN = 90 in Preto do Costa River, and 8M+18SM+16ST+6A, FN = 90 in Mineiro Stream). Ag-NORs were identified at telomeres on a subtelocentric chromosome pair, although multiple ribosomal sites have been detected in some specimens from Contas River. These results show that A. fasciatus populations from northeastern river basins are well differentiated and present peculiar cytogenetic features when compared to populations from other regions. Therefore, the apparent chromosomal plasticity of this species, likely to represent a complex of cryptic forms, is corroborated. Finally, we demonstrated that morphological features can be successfully used to support other sources of genetic information.

Variation and genetic structure of Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) populations based on ISSR pattern

For a study of diversity and genetic structuring in Melipona quadrifasciata, 61 colonies were collected in eight locations in the state of Minas Gerais, Brazil. By means of PCR analysis, 119 ISSR bands were obtained, 80 (68%) being polymorphic. He and H B were 0.20 and 0.16, respectively. Two large groups were obtained by the UPGMA method, one formed by individuals from Januária, Urucuia, Rio Vermelho and Caeté and the other by individuals from São João Del Rei, Barbacena, Ressaquinha and Cristiano Otoni. The Φst and θB values were 0.65 and 0.58, respectively, thereby indicating high population structuring. UPGMA grouping did not reveal genetic structuring of M. quadrifasciata in function of the tergite stripe pattern. The significant correlation between dissimilarity values and geographic distances (r = 0.3998; p < 0.05) implies possible geographic isolation. The genetic differentiation in population grouping was probably the result of an interruption in gene flow, brought about by geographic barriers between mutually close geographical locations. Our results also demonstrate the potential of ISSR markers in the study of Melipona quadrifasciata population structuring, possibly applicable to the studies of other bee species.

New occurrence of B chromosomes in Partamona helleri (Friese, 1900) (Hymenoptera, Meliponini)

Cytogenetic analyses of the stingless bee Partamona helleri collected in the state of Bahia, Northeast Brazil revealed the chromosome numbers n = 18 in the haploid males and 2n = 35 in the diploid females. All karyotypes displayed one large acrocentric B chromosome, which differs from the minute B chromosomes previously described in the populations from southeastern Brazil. Giemsa staining, C-banding and DAPI/CMA3 fluorochrome staining also revealed a remarkable interpopulational divergence regarding both the regular karyotype and the B chromosomes. The B chromosomes found in the samples from Jequié, Bahia, were entirely heterochromatic, while those found in Cravolândia, Bahia, displayed a euchromatic portion at the telomeric end of the long arm. CMA 3 labeling sites varied from seven to eight between the two localities in Bahia, due to the presence of an extra GC-rich block in the karyotype of the samples from Jequié. This is the first report of a large B chromosome in P. helleri and reveals the occurrence of a geographic differentiation within this species.

Genetic Diversity of Melipona mandacaia SMITH 1863 (Hymenoptera, Apidae), an Endemic Bee Species from Brazilian Caatinga, Using ISSR

In order to evaluate the genetic diversity and structure of Melipona mandacaia, we analyzed 104 colonies collected in 12 localities in Bahia state, northeastern Brazil, using ISSR-PCR. A total of 109 bands were obtained with a significant polymorphism of 72.47%. Estimates of genetic diversity indicated low values of heterozygosity ( and values were 0.2616 and 0.2573, resp.). These reduced values have been reported in other studies in stingless bees and maybe justified by dispersion process in the origin of new nests. AMOVA revealed that the higher percentage of variation is within localities (70.39%). The and values were, respectively, 0.2961 and 0.3289, thereby indicating a moderate population structuring. The correlation between genetic and geographic distances (; ) suggests isolation by distance. Our study contributes to describing the genetic diversity of endemic organisms from Caatinga and may help future efforts to preserve this threatened biome.

Variation of fore wing shape in Melipona mandacaia Smith, 1863 (Hymenoptera, Meliponini) along its geographic range

Melipona mandacaia is a stingless bee species responsible for the pollination of many native plants in Brazil, South America. In spite of its ecological and economic importance, natural populations of M. mandacaia have been depleted because of deforestation. In order to evaluate the interpopulation morphometric structure of remaining populations, we carried out geometric morphometric studies based on fore wing shape in this native bee species. The grouping analysis by UPGMA revealed three distinct clusters and significant differences in fore wing size were observed (p<0.001) among populations. The three groups were also reflected in the first two principal components explaining about 60% of the total variation. These results indicate differentiation among populations, which can be regarded as unique management units. Therefore, efforts should be directed to the conservation of local populations of M. mandacaia to avoid the negative impacts of loss in pollination over plant species and environmental services.

Unprecedented record of ten novel B chromosomes in the stingless bee Partamona helleri (Apidae, Meliponini)

Partamona helleri from southeastern Brazil has shown the presence of up to four minute supernumerary chromosomes of the B1 or B2 type and a numeric variation from 2n = 34 to 38 chromosomes. Since the maximum dosage of four of these Bs per individual has been observed, the existence of genetic mechanisms controlling their accumulation has been suggested. In this study, several new types of B chromosomes were found in other localities of the geographic distribution of this species, especially nearby the northern end of the species range, showing a numeric variation of n = 18 to 19 in males and 2n = 34 to 2n = 41 in females. Ten novel types of acrocentric and metacentric B chromosomes were classified as B3 to B12. Eight types were found in the state of Bahia and two types in the state of Espírito Santo. The results suggest that these Bs have had different evolutionary mechanisms involved in their origin and diversification. A variation within and between geographic samples in the number of chromomycin A3 and Ag-NOR markings was observed as a result of the presence or absence of different Bs. Six different B chromosomes showed Ag-NOR markings, which denote presence of gene activity. The karyotype variation and the patterns of B chromosome distribution observed indicate that this species is likely undergoing a process of genetic differentiation.

Bionomics of Melipona mondury Smith 1863 (Hymenoptera: Apidae, Meliponini) in relation to its nesting behavior

Melipona mondury Smith 1863 is an important stingless bee species pollinator and honey/pollen producer, but threatened by fragmentation of habitats throughout its range. This article presents the identification and partial characterization of nesting substrates, bionomic including population features of M. mondury by comparing data between nest architecture of natural beehive and boxes colonies. Nineteen colonies (13 in boxes and six in beehives) were analyzed with regards to substrate nesting, nest characteristics (e.g. length and width of the combs, height and diameter of the pollen and honey jar) and bee population. The average volume and diameter of the trunks cavities used for nesting of M.mondury were18.4 L and 15.8 cm; the average number of combs was 9.26; the average diameter and height of the honey pots were 2.81 cm and 3.29 cm, and the average volume of honey stored was 15.85 mL; the average height and diameter of pollen pots was 3.21 cm and 2.93 cm, respectively, while the average mass of pollen deposited was equal to 12.56 g. The population ranged from 3537 to 10281 individuals between colonies. The results suggest that the conservation of M. mondury should involve reforestation with native species from Atlantic Forest, and the dimensions of boxes should be based on the average size of the nests in the natural environment in order to support strategies of conservation and sustainable management of this species.