Mara Garcia Tavares

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.

Genetic divergence between populations of the stingless bee uruçu amarela (Melipona rufiventris group, Hymenoptera, Meliponini): is there a new Melipona species in the Brazilian state of Minas Gerais?

Allozyme, microsatellite and random amplified polymorphic DNA (RAPD) molecular markers were used to investigate the within and between population genetic variability and between population genetic differentiation of the Brazilian stingless bee urucu amarela (nominally Melipona rufiventris Lepeletier, 1836) present in savanna and Atlantic forest habitats of the Brazilian state of Minas Gerais (MG). We found low levels of within population variability, although there were a large number of private alleles that specifically characterized these populations. The FST values indicated a high level of genetic diversity between populations. Analysis of molecular variance (AMOVA) showed a high degree of population differentiation between the savanna and Atlantic forest habitats, confirmed by population pairwise FST data. Principal coordinates analysis and unweighted pair-group method using an arithmetic average (UPGMA) dendrograms also confirmed that in Minas Gerais the savanna populations (M. rufiventris) were genetically distinct from those present in the Atlantic forest (M. mondury). In addition, populations from locations near the towns of Dom Bosco and Brasilândia de Minas were genetically different from those collected in other localities in the savanna. Our data indicate that populations of urucu amarela found in the savanna and Atlantic forest habitats of Minas Gerais state should be treated separately for conservation purposes and that special attention should be given to the populations found in the region of Dom Bosco and Brasilândia de Minas until their taxonomic status is clarified.

Anthropogenic influence on the distribution, abundance and diversity of sandfly species (Diptera: Phlebotominae: Psychodidae), vectors of cutaneous leishmaniasis in Panama

In Panama, species of the genus Lutzomyia are vectors of American cutaneous leishmaniasis (ACL). There is no recent ecological information that may be used to develop tools for the control of this disease. Thus, the goal of this study was to determine the composition, distribution and diversity of Lutzomyia species that serve as vectors of ACL. Sandfly sampling was conducted in forests, fragmented forests and rural environments, in locations with records of ACL. Lutzomyia gomezi, Lutzomyia panamensis and Lutzomyia trapidoi were the most widely distributed and prevalent species. Analysis of each sampling point showed that the species abundance and diversity were greatest at points located in the fragmented forest landscape. However, when the samples were grouped according to the landscape characteristics of the locations, there was a greater diversity of species in the rural environment locations. The Kruskal Wallis analysis of species abundance found that Lu. gomezi and Lu. trapidoi were associated with fragmented environments, while Lu. panamensis, Lutzomyia olmeca bicolor and Lutzomyia ylephiletor were associated with forested environments. Therefore, we suggest that human activity influences the distribution, composition and diversity of the vector species responsible for leishmaniasis in Panama.

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae)

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

The role of fusion in ant chromosome evolution: insights from cytogenetic analysis using a molecular phylogenetic approach in the genus mycetophylax.

Among insect taxa, ants exhibit one of the most variable chromosome numbers ranging from n = 1 to n = 60. This high karyotype diversity is suggested to be correlated to ants diversification. The karyotype evolution of ants is usually understood in terms of Robertsonian rearrangements towards an increase in chromosome numbers. The ant genus Mycetophylax is a small monogynous basal Attini ant (Formicidae: Myrmicinae), endemic to sand dunes along the Brazilian coastlines. A recent taxonomic revision validates three species, Mycetophylax morschi, M. conformis and M. simplex. In this paper, we cytogenetically characterized all species that belongs to the genus and analyzed the karyotypic evolution of Mycetophylax in the context of a molecular phylogeny and ancestral character state reconstruction. M. morschi showed a polymorphic number of chromosomes, with colonies showing 2n = 26 and 2n = 30 chromosomes. M. conformis presented a diploid chromosome number of 30 chromosomes, while M. simplex showed 36 chromosomes. The probabilistic models suggest that the ancestral haploid chromosome number of Mycetophylax was 17 (Likelihood framework) or 18 (Bayesian framework). The analysis also suggested that fusions were responsible for the evolutionary reduction in chromosome numbers of M. conformis and M. morschi karyotypes whereas fission may determines the M. simplex karyotype. These results obtained show the importance of fusions in chromosome changes towards a chromosome number reduction in Formicidae and how a phylogenetic background can be used to reconstruct hypotheses about chromosomes evolution.

Cytogenetic characterization of Melipona rufiventris Lepeletier 1836 and Melipona mondury Smith 1863 (Hymenoptera, Apidae) by C banding and fluorochromes staining

The stingless bees Melipona rufiventris and M. mondury were analyzed cytogenetically by conventional staining with Giemsa, C-banding and sequential staining with the fluorochromes CMA3/DA/DAPI. Both species presented 2n = 18 and n = 9, except for one colony of M. rufiventris, in which some individuals had 2n = 19 due to the presence of a B chromosome. After Giemsa staining and C-banding the chromosomes appeared very condensed and presented a high heterochromatic content, making it difficult to localize the centromere and therefore to visualize the chromosomes morphology. The constitutive heterochromatin was located in interstitial chromosome regions covering most of the chromosomes extension and consisted mainly of AT, as shown by DAPI staining. The euchromatin was restricted to the chromosome extremities and was GC-rich, as evidenced by CMA3 staining. The B chromosome was CMA3-negative and DAPI-positive, a heterochromatic constitution similar to that of the A genome chromosomes.

Characterization of cuticular hydrocarbons of diploid and haploid males, workers and queens of the stingless bee Melipona quadrifasciata

Males, queens and workers of stingless bees show differences in external morphology, behaviour and roles within a colony. In addition, each individual has a cuticular chemical signature responsible for mutual communication that is essential for maintaining the integrity of the colony. In this paper we characterize the cuticular hydrocarbon composition of newly emerged diploid and haploid males, workers and virgin queens of Melipona quadrifasciata by gas chromatography-mass spectrometry (GC/MS) analysis. This is the first time that the cuticular profile of diploid males in a species of stingless bee has been characterized. We found differences in the cuticular hydrocarbon composition among males, workers and virgin queens, recording both qualitative and quantitative differences among individuals of different phenotypes. However, no compound was found exclusively in diploid males. The cuticular chemical profiles of haploid and diploid males were very similar to those of workers. Moreover, the cuticular lipids of males and workers were significantly different from those of queens. Tricosane, pentacosene-2 and 7-methyl-heptacosane were the compounds responsible for this significant separation. This result correlates with the behavioural and morphological differences among these phenotypes.

Honey physicochemical properties of three species of the brazilian Melipona

Physicochemical analyses were carried out to evaluate 27 samples of honeys from three species of the Brazilian genus Melipona (M. capixaba, M. rufiventris and M. mondury) from Espírito Santo and Minas Gerais States. The parameters water activity (Aw), percentage of soluble solids (Brix %), pH, acidity (meq/Kg) and moisture (%) were evaluated. The honey characteristics obtained from these samples were very similar to the ones from other Melipona species. However, regarding the honey from Apis (honey bee), only the pH values were similar. The low pH value and the high acidity detected in Melipona honey are potential factors for increasing the honey shelf life because they do not provide favorable conditions for the microbial development. On the other hand, the high level of water activity favors the growth of microorganisms, especially yeast, which demands a more careful handled and storage. The observed differences between Melipona and Apis honey reinforce the need for specific quality settings for stingless bee honey.

Pollen Sources for Melipona capixaba Moure & Camargo: An Endangered Brazilian Stingless Bee

Pollen samples were collected in three different periods from 11 Melipona capixaba Moure & Camargo hives and analyzed with melissopalynological methodology. A total of 33 pollen types were identified, of which 23 genera and 15 families were identified. The following families showed the highest pollen richness: Fabaceae (7), Myrtaceae (3), Solanaceae (3), Arecaceae (2), Asteraceae (2), Euphorbiaceae (2), Melastomataceae/Combretaceae (2), Rubiaceae (2), and Sapindaceae (2). The most frequent pollen types (>45%) were Eucalyptus, which generated great similarities between the samples, except one in which the Tibouchina was predominant. Although the majority of the pollen types showed low percentage values, the results demonstrated that M. capixaba has taken advantage of the polliniferous sources available in the Atlantic Rainforest as well as in the “Capoeira” (brushwood, secondary forest) and “ruderal” (field) plants, probably implying its importance as a pollinator of the native flora and of the exotic species.

Microsatellite loci for the stingless bee Melipona rufiventris (Hymenoptera: Apidae).

Eight microsatellite primers were developed from ISSR (intersimple sequence repeats) markers for the stingless bee Melipona rufiventris. These primers were tested in 20 M. rufiventris workers, representing a single population from Minas Gerais state. The number of alleles per locus ranged from 2 to 5 (mean = 2.63) and the observed and expected heterozygosity values ranged from 0.00 to 0.44 (mean = 0.20) and from 0.05 to 0.68 (mean = 0.31), respectively. Several loci were also polymorphic in M. quadrifasciata, M. bicolor, M. mandacaia and Partamona helleri and should prove useful in population studies of other stingless bees.