Fungus-farming ants of the genus Mycetophylax exhibit intra and interspecific chromosome variability, which makes them suitable for testing hypotheses about possible chromosomal rearrangements that endure lineage diversification. We combined cytogenetic and molecular data from Mycetophylax populations from coastal environments to trace the evolutionary history of the clade in light of chromosomal changes under a historical and geographic context. Our cytogenetic analyses revealed chromosomal differences within and among species. M. morschi exhibited three distinct karyotypes and considerable variability in the localization of 45S rDNA clusters. The molecular phylogeny was congruent with our cytogenetic findings. Biogeographical and divergence time dating analyses estimated that the most recent common ancestor of Mycetophylax would have originated at about 30 Ma in an area including the Amazon and Southern Grasslands, and several dispersion and vicariance events may have occurred before the colonization of the Brazilian Atlantic coast. Diversification of the psammophilous Mycetophylax first took place in the Middle Miocene (ca. 18–10 Ma) in the South Atlantic coast, while “M. morschi” lineages diversified during the Pliocene-Pleistocene transition (ca. 3–2 Ma) through founder-event dispersal for the Northern coastal regions. Psammophilous Mycetophylax diversification fits into the major global climatic events that have had a direct impact on the changes in sea level as well as deep ecological impact throughout South America. We assume therefore that putative chromosomal rearrangements correlated with increased ecological stress during the past climatic transitions could have intensified and/or accompanied the divergence of the psammophilous Mycetophylax. We further reiterate that “M. morschi” comprises a complex of at least three well-defined lineages, and we emphasize the role of this integrative approach for the identification and delimitation of evolutionary lineages.

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 uruçu 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 F ST 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 F ST 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 uruçu 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.

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

-The present study standardizes a reproducible flow cytometry (FCM) protocol for DNA content measurement of bee species and applied it to Scaptotrigona and Melipona species. The mean nuclear DNA content value of male and female S. xantotricha was 0.42 pg (410.8 Mbp) and 0.44 pg (430.3 Mbp), respectively, while the mean haploid genome size was determined to be 0.93 pg (909.5 Mbp) for M. rufiventris and 0.95 pg (929.1 Mbp) for M. mondury. The variation observed in this study, albeit in a preliminary way, may be related with the variation in the heterochromatin content in the chromosomes of Scaptotrigona and Melipona species. The results provide a starting point for comparative analysis on the patterns of genome size variation in the stingless bees.flow cytometry / genome size / Hymenoptera / stingless bees

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.