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Leaf-cutting ants are among the New World's most conspicuous and studied ant species due to their notable ecological and economic roles. Cytogenetic studies carried out in Atta show remarkable karyotype conservation among the species. We performed classical cytogenetics and physical mapping of repetitive sequences in the leaf-cutting ant Atta cephalotes (Linnaeus, 1758), the type species of the genus. Our goal was to test the karyotype conservation in Atta and to understand the genomic organization and diversity regarding repetitive sequences in leaf-cutting ants. Atta cephalotes showed 2 n = 22 (18m + 2sm + 2st) chromosomes. The heterochromatin followed a centromeric pattern, and the GC-rich regions and 18S rDNA clusters were co-located interstitially in the 4th metacentric pair. These cytogenetic characteristics were observed in other Atta species that had previously been studied, confirming the karyotype conservation in Atta. Evolutionary implications regarding the conservation of the chromosome number in leaf-cutting ants are discussed. Telomeric motif (TTAGG) n was detected in A. cephalotes as observed in other ants. Five out of the 11 microsatellites showed a scattered distribution exclusively on euchromatic areas of the chromosomes. Repetitive sequences mapped on the chromosomes of A. cephalotes are the first insights into genomic organization and diversity in leaf-cutting ants, useful in further comparative studies.

The fungus-farming ant genus Cyphomyrmex (subtribe Attina, clade Neoattina) comprises 23 described species that are widely distributed throughout the Neotropics. Species within Cyphomyrmex have taxonomic issues such as Cyphomyrmex rimosus (Spinola, 1851) which is likely a species complex. Cytogenetics is a useful tool for evolutionary studies and understanding species with dubious taxonomy. In this study, we characterized the karyotype of C. rimosus from Viçosa, Minas Gerais state, southeastern Brazil using classical and molecular cytogenetic techniques to enrich the chromosomal information about Cyphomyrmex. The karyotype of C. rimosus from the rainforest of southeastern Brazil (2n = 22, 18m + 4sm) notably contrasts with that previously described for this species in Panama (2n = 32). This intraspecific chromosomal variation suggests the existence of a species complex within this taxon according to the previous hypothesis derived from morphological analysis. We detected GC-rich heterochromatic regions in C. rimosus and using repetitive DNA probes showed that this heterochromatin shares repetitive sequences with other Neoattina species already studied, enhancing the importance of this specific genome region in the understanding of Attina evolution. Mapping of microsatellite (GA)15 on C. rimosus was restricted to the euchromatic regions of all chromosomes. The single intrachromosomal rDNA sites observed in C. rimosus follow the general genomic organization trend of ribosomal genes in Formicidae. Our study extends the data of chromosome mapping on Cyphomyrmex and reinforces the importance of cytogenetic studies in different localities to better understand taxonomic issues in widely distributed taxa such as C. rimosus.

Made available in DSpace on 2022-04-28T19:47:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Satellite DNAs (satDNAs) and transposable elements (TEs) are among the main components of constitutive heterochromatin (c-heterochromatin) and are related to their functionality, dynamics, and evolution. A peculiar case regarding the quantity and distribution of c-heterochromatin is observed in the genus of bees, Melipona, with species having a low amount of heterochromatin and species with high amount occupying almost all chromosomes. By combining low-pass genome sequencing and chromosomal analysis, we characterized the satDNAs and TEs of Melipona quadrifasciata (low c-heterochromatin) and Melipona scutellaris (high low c-heterochromatin) to understand c-heterochromatin composition and evolution. We identified 15 satDNA families and 20 TEs for both species. Significant variations in the repeat landscapes were observed between the species. In M. quadrifasciata, the repetitive fraction corresponded to only 3.78% of the genome library studied, whereas in M. scutellaris, it represented 54.95%. Massive quantitative and qualitative changes contributed to the differential amplification of c-heterochromatin, mainly due to the amplification of exclusive repetitions in M. scutellaris, as the satDNA MscuSat01-195 and the TE LTR/Gypsy_1 that represent 38.20 and 14.4% of its genome, respectively. The amplification of these two repeats is evident at the chromosomal level, with observation of their occurrence on most c-heterochromatin. Moreover, we detected repeats shared between species, revealing that they experienced mainly quantitative variations and varied in the organization on chromosomes and evolutionary patterns. Together, our data allow the discussion of patterns of evolution of repetitive DNAs and c-heterochromatin that occurred in a short period of time, after separation of the Michmelia and Melipona subgenera. Laboratório de Citogenética de Insetos Departamento de Biologia Geral Universidade Federal de Viçosa, Campus Viçosa, Av. P.H. Rolfs s/n Departamento de Biologia Geral E Aplicada Instituto de Biociências/IB UNESP–Universidade Estadual Paulista Departamento de Biologia Geral E Aplicada Instituto de Biociências/IB UNESP–Universidade Estadual Paulista

Cytogenetic studies on fungus-farming ants have shown remarkable karyotype diversity, suggesting different chromosomal rearrangements involved in karyotype evolution in some genera. A notable cytogenetic characteristic in this ant group is the presence of GC-rich heterochromatin in the karyotypes of some ancient and derivative species. It was hypothesized that this GC-rich heterochromatin may have a common origin in fungus-farming ants, and the increase in species studied is important for understanding this question. In addition, many genera within the subtribe Attina have few or no cytogenetically studied species; therefore, the processes that shaped their chromosomal evolution remain obscure. Thus, in this study, we karyotyped, through classical and molecular cytogenetic techniques, the fungus-farming ants Cyphomyrmex transversus Emery, 1894, Sericomyrmex maravalhas Ješovnik et Schultz, 2017, and Mycetomoellerius relictus (Borgmeier, 1934), to provide insights into the chromosomal evolution in these genera and to investigate the presence the GC-rich heterochromatin in these species. Cyphomyrmex transversus (2n = 18, 10m + 2sm + 6a) and S. maravalhas (2n = 48, 28m + 20sm) showed karyotypes distinct from other species from their genera. Mycetomoellerius relictus (2n = 20, 20m) presented the same karyotype as the colonies previously studied. Notably, C. transversus presented the lowest chromosomal number for the genus and a distinct karyotype from the other two previously observed for this species, showing the existence of a possible species complex and the need for its taxonomic revision. Chromosomal banding data revealed GC-rich heterochromatin in all three species, which increased the number of genera with this characteristic, supporting the hypothesis of a common origin of GC-rich heterochromatin in Attina. Although a single chromosomal pair carries rDNA genes in all studied species, the positions of these rDNA clusters varied. The rDNA genes were located in the intrachromosomal region in C. transversus and M. relictus, and in the terminal region of S. maravalhas. The combination of our molecular cytogenetic data and observations from previous studies corroborates that a single rDNA site located in the intrachromosomal region is a plesiomorphic condition in Attina. In addition, cytogenetic data obtained suggest centric fission events in Sericomyrmex Mayr, 1865, and the occurrence of inversions as the origin of the location of the ribosomal genes in M. relictus and S. maravalhas. This study provides new insights into the chromosomal evolution of fungus-farming ants.

The Neotropical Meliponini bees, commonly known as stingless bees, are phylogenetically subdivided into three clades in which the chromosome numbers vary from n = 8 to n = 17. The goal of this study was to identify the major chromosomal rearrangements that occurred during the Neotropical Meliponini (Apidae) karyotypic evolution. In this way, we mapped 18S rDNA and five microsatellites in 33 stingless bee species collected from different Brazilian regions. The species belonged to 15 genera and showed six different chromosome numbers: n = 8, n = 9, n = 11, n = 14, n = 15, and n = 17. The 18S rDNA probe showed a variation from 2 to 12 marked chromosomes in different positions (terminal, subterminal, or centromeric), including 2 B chromosomes out of the 7 B found in Tetragonisca fiebrigi. The microsatellite (GA)15, (GAG)10, (CAA)10, and (TCAGG)6 probes formed clusters on the euchromatic regions of the chromosomes and were useful in the identification of putative Robertsonian fusion events that led to the decrease in the chromosome number during the evolution of the Neotropical Meliponini clade. (TTAGG)6 constituted the telomeric sequence of the analyzed species. The ancestral state of the three Neotropical Meliponini clades is difficult to infer, although, the putative ancestral karyotype probably had a single pair of 18S rDNA cistrons, and the decrease in chromosome number and increase in the 18S rDNA sites occurred independently between genera.

Over the past several decades, stingless bee cytogenetic studies have focused on chromosome number and morphology of specific samples, with little emphasis on population variations. More recently, molecular techniques have facilitated analyses of the fine chromosomal structure, allowing the identification of interspecific variations and increasing our knowledge of the karyotype structure. Thus, the present study aimed to analyse the distribution of ribosomal genes and repetitive sequences in the genome of Friesella schrottkyi (Friese 1900), providing relevant information about its genomic organization. Two samples were obtained from Minas Gerais state (Rio Paranaiba and Vicosa) and one from Cunha/Sao Paulo state, Brazil, all of which showed 2n = 34 chromosomes, 2K: 10m + 12sm + 12st. Heterochromatin (C-banding) was detected mainly in the centromeric/pericentromeric region, but some specific patterns were verified in individuals from each location. An intense spreading of the microsatellite (GA)15 was observed, most commonly in the euchromatic areas of the chromosomes. The CMA3+ bands were found in two chromosomes in the samples from Cunha and Vicosa, while the sample from Rio Paranaiba contained three CMA3+ bands. There were also differences in the number and locations of the 18S rDNA clusters among the different samples. Although we found no differences between samples from the same location, the many intraspecific variations founded clearly demonstrate the potential of utilising molecular cytogenetics as a method of improving our understanding of the genetic variability in the Meliponini tribe.

Genome changes, evidenced through karyotype or nuclear genome size data, can result in reproductive isolation, diversification and speciation. The aim of this study was to understand how changes in the karyotype such as chromosome number and nuclear genome size accompanied the evolution of neotropical stingless bees, and to discuss these data in a phylogenetic context focusing on the karyotype evolution of this clade. We sampled 38 species representing the three Neotropical Meliponini groups; 35 for karyotype analyses and 16 for 1C value measurement. The chromosome number varied from 2n = 16 to 2n = 34, with distinct karyotypic formulae and the presence of a few polymorphisms, such as B chromosomes in one species and arm size differences between homologous chromosomes in two species. The mean 1C value varied from 0.31 pg to 0.92 pg. We associated empirical data on chromosome number and mean 1C value to highlight the importance of Robertsonian fusion rearrangements, leading to a decrease in chromosome number during the Neotropical Meliponini evolution. These data also allowed us to infer the independent heterochromatin amplification in several genera. Although less frequent, Melipona species with 2n = 22 represent evidence of Robertsonian fissions. We also pointed out the importance of chromosomal rearrangements that did not alter chromosome number, such as inversions and heterochromatin amplification.

The bee diversity (Apidae) estimative ranges from 18,000 to 20,000 species worldwide. Together, they show an impressive diversity in morphological, ecological, and behavioral traits, and there is still much to be understood about their taxonomy and systematics. Their chromosome count variability and genome biology are also astonishing. To date, around 200 bee species have already been karyotyped, with chromosome numbers varying from n = 3 to n = 28, and nuclear haploid genome sizes are available for approximately 70 species with a variation of 1C = 0.19 pg to 1C = 1.38 pg. The Bee Chromosome database was created ( www.bees.ufop.br ) to summarize the Apidae cytogenetic knowledge by assembling all the cytogenetic information published on bees. Considering the importance of cytogenetic studies for taxonomy, phylogeny, genetics, systematics, conservation, and evolution, the main goal of this database is to outline the advances in the field of bee cytogenetics over the last century.

The species of the four Melipona subgenera can be subdivided into two cytogenetic groups: species belonging to Group I are characterized by having low heterochromatin content in their karyotypes ( 50%). To study the patterns of heterochromatin accumulation in this genus, we isolated moderate to highly repetitive sequences in M. (Michmelia) mondury (Mmon probe) and M. (Melikerria) fasciculata (Mfas probe) obtained based on the renaturation kinetics of C0t − 1 DNA and used these fragments as probes to hybridize in thirteen species belonging to the four Melipona subgenera. The Mmon probe marked all chromosomes in all Michmelia species and additionally the secondary constriction of M. (Eomelipona) puncticollis. The Mfas probe showed positive markings only within Melikerria. These results indicate the independence of the heterochromatin growth between Michmelia and Melikerria. Absence of markings in the regular set of chromosomes in M. (Melikerria) quinquefasciata and positive markings in the Bs, with the Mfas probe, suggests an interspecific origin of these chromosomes. We also hypothesize about the possible scenario that led to the heterochromatin growth in the genus Melipona.

A substantial portion of the eukaryotic genome includes repetitive DNA, which is important for its stability, regulation, and architecture. Fungus-farming ant genomes show remarkable structural rearrangement rates that were necessary for the establishment of their agriculture-based lifestyle, highlighting the relevance of this peculiar group in understanding the repetitive portion of ant genome. Chromosomal banding studies are in accordance with genomic data because they show that repetitive heterochromatic sequences of basal and derivative Attina species are GC-rich, an uncommon trait in Formicidae. To understand the evolutionary dynamics of heterochromatin in Attina, we compared GC-rich heterochromatin patterns between the Paleoattina and Neoattina clades of this subtribe. To this end, we hybridized the Mrel-C

The characterization of karyotypes is an important aspect in understanding the structure and evolution of genomes. Polybia is a genus of social wasps of the family Vespidae. This genus has 58 species, but for only 8 of these chromosome number and morphology have been reported in the literature. The aim of this study was to describe and characterize the Polybia fastidiosuscula Saussure karyotype, presenting the first case of a B chromosome in Vespidae. In addition, we investigated the chromatin composition of this species through C-banding, base-specific fluorochrome staining, and physical mapping of 7 microsatellites and 18S rDNA. Four colonies of P. fastidiosuscula from Minas Gerais and Paraná states, Brazil, were analyzed. The chromosome number identified was 2n = 34, and 2 colonies presented a B chromosome. We characterized the chromatin composition of this species, analyzing the existence of different microsatellite-rich heterochromatic regions which are also enriched with AT or GC base pairs. We suggest an intraspecific origin of the B chromosome based on the homology of the heterochromatic composition with A chromosomes and also verify that the TTAGG and TCAGG sequences are not telomeric, but only microsatellites that occur in the centromeres of most chromosomes, as well as GAG and CGG.

Studies in several organisms have contributed to the understanding of heterochromatin and its biological importance. In bees of the tribe Meliponini, the presence of chromosomes with totally heterochromatic arms has been attributed to the mechanism of karyotype evolution in which this group accumulated heterochromatin to maintain telomere stability after centric fission events. In the present study, the use of classical and molecular cytogenetic techniques as well as automated image analysis software for the description of the karyotypes of Partamonachapadicola and P. nhambiquara bee species revealed variability in the compaction and patterns of chromatin structure. Although both species have the same chromosome number as other species in the genus Partamona (2n = 34), C-banding and image analyses indicated the existence of chromosomes with 3 regions of different staining intensities, suggesting a chromatin structure with distinct patterns and characteristics. Repetitive DNA probes hybridized only in the euchromatic regions, whereas the regions with intermediate staining intensity did not show any hybridization signals. This suggests that these regions present features more similar to heterochromatin. Evidence of the existence of a chromatin class with intermediate condensation compared to euchromatin and heterochromatin indicates a potential mechanism for heterochromatin amplification and demonstrates the need for further studies on this topic. This previously unrecognized class of chromatin should be taken into account in the study of all Meliponini chromosomes.

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.

Stingless bees of the genus Melipona are subdivided into 4 subgenera called Eomelipona, Melikerria, Melipona sensu stricto, and Michmelia according to species morphology. Cytogenetically, the species of the genus Melipona show variation in the amount and distribution of heterochromatin along their chromosomes and can be separated into 2 groups: the first with low content of heterochromatin and the second with high content of heterochromatin. These heterochromatin patterns and the number of chromosomes are characteristics exclusive to Melipona karyotypes that distinguish them from the other genera of the Meliponini. To better understand the karyotype organization in Melipona and the relationship among the subgenera, we mapped repetitive sequences and analyzed previously reported cytogenetic data with the aim to identify cytogenetic markers to be used for investigating the phylogenetic relationships and chromosome evolution in the genus. In general, Melipona species have 2n = 18 chromosomes, and the species of each subgenus share the same characteristics in relation to heterochromatin regions, DAPI/CMA3 fluorophores, and the number and distribution of 18S rDNA sites. Microsatellites were observed only in euchromatin regions, whereas the (TTAGG)6 repeats were found at telomeric sites in both groups. Our data indicate that in addition to the chromosome number, the karyotypes in Melipona could be separated into 2 groups that are characterized by conserved cytogenetic features and patterns that generally are shared by species within each subgenus, which may reflect evolutionary constraints. Our results agree with the morphological separation of the Melipona into 4 subgenera, suggesting that they must be independent evolutionary lineages.

The eukaryote genome is enriched by different types of repetitive DNA sequences and is most abundant in heterochromatin regions. Historically, no function has been assigned to these sequences, which makes them the target of studies that have demonstrated their structural and functional importance in the genome. Despite having a constant chromosome number, the genus Melipona has species with wide variation in heterochromatin content, from 8 to 73%, which is an important feature to be investigated regarding its origin and evolution. In the present study, a repetitive DNA sequence of Melipona mondury was isolated by restriction enzyme digestion. This sequence was used to hybridize chromosomes of eight Melipona species that include representatives of the four subgenera and present divergent characteristics in relation to the heterochromatin content. Considering that rDNA localization has shown differences in Melipona, 16 species of this genus were analyzed with 18S rDNA probe. Our data suggest that heterochromatin growth occurred independently in the Michmelia and Melikerria subgenera, considering that the isolated repetitive DNA sequence was shared only by the Michmelia species. Amplification possibly occurred from the centromeric region, causing the displacement of the rDNA sites to the ends of the chromosomes. The repetitive DNA sequence used is a constituent of Michmelia heterochromatin, which that arose from the common ancestor of the species of this subgenus.

Gnamptogenys includes 138 described species that are widely distributed, with high diversity, in the Neotropics. Some Neotropical species have taxonomic issues, as is the case with Gnamptogenys striatula, for which morphological variations have been observed between different populations. For the ant species with taxonomic issues, classical and molecular cytogenetic studies have assisted in the resolution of these issues. Cytogenetic studies of Gnamptogenys are scarce and have only been reported for 14 taxa. These reports have rarely presented chromosomal morphology. Considering the importance of the taxonomic revision of some species, such as G. striatula, the present study cytogenetically characterized four species of Gnamptogenys: G. striatula, G. moelleri, G. regularis, and G. triangularis, discussing their phylogenetic and biogeographic characteristics. The number of chromosomes ranged from 2n = 26 to 2n = 44, with distinct karyotypes at both species and population levels. All four species presented a pair of 18S rDNA gene markers that coincided with GC-rich regions. In the case of G. striatula from the Atlantic rainforest, a chromosomal polymorphism was observed, with chromosomal translocations being the likely origin of this polymorphism. Two populations of G. striatula showed karyotype differences, thus corroborating previous morphological data indicating the existence of a species complex in this taxon. In addition, G. regularis showed a polymorphism involving a chromosome pair bearing ribosomal genes, possibly caused by unequal crossing-over. Although G. moelleri has a well-defined taxonomy, a population from the eastern Amazon rainforest presented a divergent karyotype from the Atlantic rainforest populations, suggesting the existence of a cryptic species in this taxon.

It is thought that two evolutionary mechanisms gave rise to chromosomal variation in bees: the first one points to polyploidy as the main cause of chromosomal evolution, while the second, Minimum Interaction Theory (MIT), is more frequently used to explain chromosomal changes in Meliponini and suggests that centric fission is responsible for variations in karyotype. However, differences in chromosome number between Meliponini and its sister taxa and in the karyotype patterns of the Melipona genus cannot be explained by MIT, suggesting that other events were involved in chromosomal evolution. Thus, we assembled cytogenetical and molecular information to reconstruct an ancestral chromosome number for Meliponini and its sister group, Bombini, and propose a hypothesis to explain the evolutionary pathways underpinning chromosomal changes in Meliponini. We hypothesize that the common ancestor shared by the Meliponini and Bombini tribes possessed a chromosome number of n = 18. The karyotype with n = 17 chromosomes was maintained in Meliponini, and variations of haploid numbers possibly originated through additional Robertsonian fissions and fusions. Thus, the low chromosome number would not be an ancestral condition, as predicted by MIT. We then conclude that Robertsonian fission and fusions are unlikely to be the cause of chromosomal rearrangements that originated the current karyotypes in Meliponini.

The Brazilian Atlantic rainforest is an endangered biome and biodiversity hotspot. Ant cytogenetic studies from this biome showed remarkable chromosomal diversity among species, and provided useful insights on phylogeny, chromosomal evolution, and taxonomy. In this study, we karyotyped five ant taxa from the Atlantic rainforest. The karyotypes observed were Pheidole germaini 2n=22, Pheidole sp. flavens group 2n=20, Brachymyrmex admotus 2n=18, Camponotus atriceps 2n=40, and Odontomachus haematodus 2n=44. The data obtained for Pheidole spp. represent the first chromosomal record for the genus in Brazil and provide insights on the chromosomal evolution of P. germaini . Karyotypic information from B. admotus brings the genus back to the cytogenetic scenario after decades of neglect. No karyotype variations were observed among the C. atriceps and O. haematodus from different already studied populations, corroborating their status as good species. This study increased the cytogenetic knowledge of ants from the Brazilian Atlantic rainforest.

The genus Melipona is subdivided into four subgenera based on morphological characteristics, and two groups based on cytogenetic patterns. The cytogenetic information on this genus is still scarce, therefore, the goal of this study was to characterize Melipona paraensis, Melipona puncticollis, and Melipona seminigra pernigra using the following techniques: C-banding, DAPI/CMA3 fluorochromes, and FISH with an 18S rDNA probe. Melipona paraensis (2n=18) and M. seminigra pernigra (2n=22) were classified as high heterochromatin content species (Group II). Their euchromatin is restricted to the ends of the chromosomes and is CMA3 +; the 18S rDNA probe marked chromosome pair number 4. Melipona puncticollis (2n=18) is a low heterochromatin content species (Group I) with chromosome pair number 1 marked with CMA3 and 18S rDNA. Low heterochromatin content is a putative ancestral karyotype in this genus and high content is not a monophyletic trait (Melikerria presents species with both patterns). Differences concerning the karyotypic characteristics can be observed among Melipona species, revealing cytogenetic rearrangements that occurred during the evolution of this genus. Studies in other species will allow us to better understand the processes that shaped the chromatin evolution in Melipona.

Over the past several decades, ant cytogenetic studies have focused on chromosome number and morphology; however, recently, additional information concerning heterochromatin composition and 45S rDNA location has become accessible. The fungus-growing ants are a peculiar ant group that cultivates fungus for food, and Trachymyrmex is suspected to be the sister group of leafcutter ants. Cytogenetic data are so far available for sixn Trachymyrmex species. The present study aimed to increase the knowledge about Trachymyrmex cytogenetics by the chromosomal characterization of Trachymyrmex holmgreni including the karyotyping, fluorochromes staining, 18S rDNA, and microsatellite (GA)15 fluorescence in situ hybridization (FISH). Karyotyped samples from four ant colonies showed 2n = 20 metacentric chromosomes. Centromeric heterochromatin rich in GC base pairs was detected in all chromosomes. FISH revealed the presence of rDNA clusters on the fourth chromosome pair, and an intense spreading of the microsatellite (GA)15 including exclusively euchromatic areas of the chromosomes. The GC-rich heterochromatin observed in different ant species may have a common origin and, thus, phylogenetic implication that needs to be further investigated. To the best of our knowledge, this study is the first report of the use of chromosomal physical location of repetitive DNA sequences by means of microsatellite probes in Formicidae.

To date, more than 65 species of Brazilian bees (of the superfamily Apoidea) have been cytogenetically studied, but only a few solitary species have been analyzed. One example is the genus Melitoma Lepeletier & Serville, 1828, for which there is no report in the literature with regard to cytogenetic studies. The objective of the present study is to analyze the chromosome number and morphology of the species Melitoma segmentaria (Fabricius, 1804), as well as to determine the pattern of heterochromatin distribution and identify the adenine–thymine (AT)- and guanine–cytosine (GC)-rich regions. Melitoma segmentaria presents chromosome numbers of 2n=30 (females) and n=15 (males). With C-banding, it is possible to classify the chromosomes into seven pseudo-acrocentric pairs (AM), seven pseudo-acrocentric pairs with interstitial heterochromatin (AMi), and one totally heterochromatic metacentric pair (Mh). Fluorochrome staining has revealed that heterochromatin present in the chromosomal arms is rich in GC base pairs (CMA3+) and the centromeric region is rich in AT base pairs (DAPI+). The composition found for Melitoma diverges from the pattern observed in other bees, in which the heterochromatin is usually rich in AT. In bees, few heterochromatic regions are rich in GC and these are usually associated with or localized close to the nucleolus organizer regions (NORs). Silver nitrate impregnation marks the heterochromatin present in the chromosome arms, which makes identification of the NOR in the chromosomes impossible. As this technique reveals proteins in the NOR, the observation that is made in the present study suggests that the proteins found in the heterochromatin are qualitatively similar to those in the NOR.

Most Meliponini share a distinctive pattern of heterochromatin distribution in relation to other bees. In general, they present one euchromatic and one heterochromatic chromosome arm, a feature explained by minimum interaction theory, which involved centric fissions followed by heterochromatin addition. In this work, two Meliponini with a distinct pattern of heterochromatin distribution, Tetragonisca fiebrigi and Melipona rufiventris, were analyzed using chromosomal microdissection of the heterochromatin region followed by FISH (fluorescent in situ hybridization). Hybridization revealed FISH signals equivalent to location of the isolated fragment that were widespread over heterochromatic portions of other chromosomes. This result showed that the heterochromatic sequences were very similar among chromosomes in the same species. Cross-hybridization of each probe in M. rufiventris and T. fiebrigi yielded no signals, revealing that both species presented differentiated and non-homologous heterochromatin seque...

The present study provides a comprehensive review of cytogenetic data on Meliponini and their chromosomal evolution. The compiled data show that only 104 species of stingless bees, representing 32 of the 54 living genera have been studied cytogenetically and that among these species, it is possible to recognize three main groups with n = 9, 15 and 17, respectively. The first group comprises the species of the genus Melipona, whereas karyotypes with n = 15 and n = 17 have been detected in species from different genera. Karyotypes with n = 17 are the most common among the Meliponini studied to date. Cytogenetic information on Meliponini also shows that although chromosome number, in general, is conserved among species of a certain genus, other aspects, such as chromosome morphology, quantity, distribution and composition of heterochromatin, may vary between them. This reinforces the fact that the variations observed in the karyotypes of different Meliponini groups cannot be explained by a single theory or a single type of structural change. In addition, we present a discussion about how these karyotype variations are related to the phylogenetic relationships among the different genera of this tribe.

In this paper we present, for the first time, a detailed karyotype characterization of a species of the genus Azteca (Dolichoderinae, Formicidae). Cerebral ganglia from Azteca trigona Emery, 1893 were excised and submitted to colchicine hypotonic solution and chromosomal preparations were analyzed through conventional staining with Giemsa, C-banding, silver nitrate staining (AgNO3) and sequential base-specific fluorochromes. The analysis shows that A. trigona has a diploid number of 28 chromosomes. The karyotype consists of five metacentric pairs, seven acrocentric pairs and two pseudo-acrocentric pairs, which represents a karyotype formula 2K= 10M + 14A + 4AM and a diploid number of the arms 2AN = 38. The analysis of heterochromatin distribution revealed a positive block on distal region of the short arm of fourth metacentric pair, which was coincident with Ag-NOR band and CMA3 fluorochrome staining, meaning that rDNA sequences are interspaced by GC-rich base pairs sequences. The C-banding also marked short arms of other chromosomes, indicating centric fissions followed by heterochromatin growth. The karyotype analysis of A. trigona allowed the identification of cytogenetic markers that will be helpful in a difficult taxonomic group as Azteca and discussion about evolutionary aspects of the genome organization.

Due to their high degree of polymorphism, microsatellites are considered useful tools for studying population genetics. Nevertheless, studies of genetic diversity in stingless bees by means of these primers have revealed a low level of polymorphism, possibly the consequence of the heterologous primers used, since in most cases these were not specifically designed for the species under consideration. Herein we compared the number of polymorphic loci and alleles per locus, as well as observed heterozygosity in Melipona rufiventris and M. mondury populations, using specific and heterologous primers. The use of specific primers placed in evidence the greater frequency of polymorphic loci and alleles per locus, besides an expressive increase in observed heterozygosity in M. rufiventris and M. mondury, thereby reinforcing the idea that populational studies should be undertaken by preferably using species-specific microsatellite primers.

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.

Microsatellites are a class of markers much usedin population, phylogenetic and parentage studiesbecause of their high degree of polymorphism, co-dominance and high reproducibility (De la Ruaet al., 2001; Oliveira et al., 2006). Microsatellite se-quences are amplified by PCR, using primers thatflank the repeat sequences of nuclear DNA. The de-velopment of these primers, however, is the lim-iting factor for the use of these markers. Becauseof this difficulty, there are microsatellite primersdeveloped for only three species of stingless bee

O gênero Plebeia não é um grupo taxonômico bem definido e apresenta problemas quanto a sua classificação e sistemática. As ferramentas citogenéticas podem auxiliar no esclarecimento das relações entre as espécies deste gênero. O principal objetivo deste estudo foi caracterizar o cariótipo das espécies de Plebeia lucii Moure, 2004 e Plebeia phrynostoma Moure, 2004. Para isso foram usadas técnicas de coloração convencional, bandeamento C e fluorocromos. O mesmo número cromossômico (2n = 34) foi observado em ambas espécies. A fórmula cariotípica de Plebeia lucii foi 2K = 22AM + 12A. Foi observado um par heteromórfico com as regiões eucromáticas e heterocromáticas localizadas em diferentes porções dos dois homólogos. A presença de uma constrição secundária também foi observada neste mesmo par. Em Plebeia phrynostoma a fórmula cariotípica foi 2K = 18AM + 10A + 6M e não foi observado nem polimorfismos nem contrições secundárias. O fluorocromo DAPI marcou regiões dos braços heterocromáticos e regiões próximas ao centrômero em alguns cromossomos de ambas as espécies. O CMA3 marcou o par heteromórfico de P. lucii e alguns pontos em outros cromossomos, enquanto marcou dois pares de cromossomos em P. phrynostoma. Apesar da similaridade no número cromossômico, estas espécies mostraram variação tanto na morfologia quanto na composição da cromatina o que pode ser reflexo da posição filogenética delas em diferentes clados. Plebeia (Hymenoptera: Apidae) is a poorly defined genus and its classification and systematics are controversial. Tools such as cytogenetics may contribute to clarify the relationships among the species. The aim of this study was to characterize the karyotypes of the species Plebeia lucii Moure, 2004 and Plebeia phrynostoma Moure, 2004. For this purpose conventional staining, C-banding and fluorochrome techniques were performed. The same chromosome number (2n = 34) was observed for both species. The karyotypic formula of P. lucii was 2K = 22 AM + 12A. A heteromorphic pair was observed with euchromatic and heterochromatic regions of different sizes on the 2 homologs. The presence of a secondary constriction was observed in this same pair. In P. phrynostoma the karyotypic formula was 2K = 18AM + 10A + 6M and did not show polymorphisms or secondary constrictions. The DAPI fluorochrome marked portions of the heterochromatic arm and the regions close to the centromere in some chromosomes of both species. CMA3 marked the heteromorphic pair in P. lucii and some points in other chromosomes, while it stained 2 pairs of chromosomes in P. phrynostoma. Despite the similarity in chromosome number, these species show variation both in morphology and in composition of chromatin which may reflect a phylogenetic position in different clades.

Euglossini are solitary bees considered important pollinators of many orchid species. Information regarding chromosome organization is available for only a small number of species in this group. In the present work, the species Euglossa townsendi and E. carolina were analyzed by cytogenetic techniques to collect information that may aid the understanding of their evolution and chromosomal organization. The chromosome number found was n = 21 for males and 2n = 42 for females in the two species. The distribution and amount of heterochromatin regions differed in the two species analyzed, where they were classified as “high” or “low” heterochromatin content, similarly to what has already been performed in social bee species of the genus Melipona. Banding patterns found in this study suggest that other mechanisms may have occurred in the karyotype evolution of this group, unlike those suggested for social bees and ants. Karyotype evolution of solitary bees appears to have occurred as an event separate from other hymenopterans and did not involve chromosome fissions and heterochromatin amplification. Os Euglossini são abelhas solitárias consideradas importantes polinizadores de muitas espécies de orquídeas. Somente um pequeno número de espécies desse grupo possui informações sobre sua organização cromossômica. Neste trabalho as espécies Euglossa townsendi e E. carolina foram analisadas por técnicas citogenéticas a fim de obter informações que auxiliem no entendimento de sua evolução e organização cromossômica. O número cromossômico encontrado foi n = 21 para os machos e 2n = 42 para as fêmeas das duas espécies. A distribuição e a quantidade de heterocromatina diferem nas duas espécies analizadas e podem ser classificadas como alta e baixa quantidade de heterocromatina, similarmente como já foi feito anteriormente para espécies de abelhas sociais do gênero Melipona. O padrão de bandeamento encontrado no presente trabalho sugere que outros mecanismos podem estar envolvidos na evolução cariotípica do grupo, diferente daqueles sugeridos para as abelhas sociais e formigas. A evolução cariotípica de abelhas solitárias parece ter acontecido por eventos distintos que não envolveram fissões cromossômicas e amplificação da heterocromatina.

In most Hymenoptera species the queen mates once but in a small number of species, multiple matings can occur normally. So, in this study, physogastric M. quadrifasciata queens were mated with a second male to investigate how these queens, naturally inseminated and laying eggs, use spermatozoa stored in their spermatheca, when they are mated with a second male. Results demonstrate that spermatozoa of different males mix in the spermatheca of M. quadrifasciata queens and that there is a gradual increase in the utilisation of spermatozoa of the second male, which could be explained by a competition among spermatozoa of different drones over the way in which spermatozoa are stored in the spermatheca.

International audience; The emergence of new molecular biology techniques has provided cytogenetics with tools which allow for the elucidation of questions that classical cytogenetics could not answer. Therefore, the present work standardizes a microdissection protocol for cytogenetic studies in bees. This methodology was first used in these insects and may contribute greatly to studies involving chromosomal rearrangements, heterochromatin composition, B chromosomes and others. For this study, the centromeric region of chromosomes in the stingless bee Tetragonisca fiebrigi was used for probe synthesis. The results demonstrated that the methodology used was efficient, presenting markings in the centromeric regions of several chromosomes. Hybridization in other sites indicates that the probe was able to detect regions that present homology with its sequence. This indicates that the technique is effective to study chromosomal evolution, genome organization and even the origin of B chromosomes.

International audience; 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.

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.