Karyotype description of Cephalotrigona femorata Smith (Hymenoptera: Apidae) and the C-banding pattern as a specific marker for Cephalotrigona

Cephalotrigona femorata (Smith, 1854) was submitted to cytogenetic techniques to study and describe its karyotype. Conventional staining allowed the counting (2n=34) and observation of chromosome morphology. The amount and distribution of heterochromatin in this species was different from Cephalotrigona capitata (Smith, 1854), another species of the genus already analyzed. Our results indicate that heterochromatin a potential marker for the genus, at least for the species found in Brazil. This region was marked by DAPI, revealing a high content of A:T. The CMA3 marked two pairs, and it seems to be  polymorphic in one pair

Karyotypic data of five ant taxa from the Brazilian Atlantic rainforest

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

Cytogenetic studies in Trachymyrmex holmgreni Wheeler, 1925 (Formicidae: Myrmicinae) by conventional and molecular methods

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

Chromosomal dynamics in space and time : evolutionary history of Mycetophylax ants across past climatic changes in the Brazilian Atlantic coast.

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

Cytogenetics of Melitoma segmentaria (Fabricius, 1804) (Hymenoptera, Apidae) reveals differences in the characteristics of heterochromatin in bees.

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 Lepele?tier & 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 dis?tribution 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). Fluo?rochrome 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 impos?sible. 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

Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae)

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

Genetic diversity and structure in Melipona rufiventris and Melipona mondury populations (Hymenoptera: Apidae) by analysis of microsatellites

Melipona rufiventris é uma espécie de meliponíneo que encontra-se ameaçada de extinção no estado de Minas Gerais. Até recentemente, as “formas” encontradas tanto em regiões de mata Atlântica quanto em regiões de cerrado dentro deste estado eram, consideradas uma única espécie. No entanto, devido ao grande número de diferenças morfológicas encontradas entre espécimens provenientes de diferentes regiões, a “forma” encontrada em regiões de mata passou a ser considerada como Melipona mondury e a “forma” presente no cerrado como M. rufiventris. Com o objetivo de estimar a variabilidade genética em populações dessas duas espécies, foram feitas análises com nove “primers” microssatélites em 45 colônias coletadas em diferentes localidades do estado de Minas Gerais. O seqüenciamento de alguns dos locos demonstrou que os fragmentos amplificados realmente continham regiões microssatélites. Considerando todas as amostras analisadas, 7 locos foram polimórficos, sendo que alguns alelos estavam presentes somente em M. rufiventris e outros eram exclusivos de M. mondury. A análise de agrupamento permitiu a formação de dois grupos distintos: o primeiro constituído pelas colônias de M. mondury e o segundo pelas de M. rufiventris. Nesse último observou-se a formação de dois subgrupos, sendo que as colônias de Brasilandia de Minas e Dom Bosco encontraram-se separadas das demais colônias de cerrado. A porcentagem de locos polimórficos foi de 33,3% para as espécies M. mondury e M. rufiventris e 22,2% para as colônias de Brasilandia de Minas/Dom Bosco. A heterozigosidade observada nessas populações foi de 0,1159 para M. mondury, 0,0684 para M. rufiventris e 0,0222 para as colônias de Brasilandia de Minas/ Dom Bosco. M. mondury e M. rufiventris apresentaram valores de F ST próximo de 0,25, mostrando que essas espécies possuem uma elevada estruturação populacional, com grande diferenciação genética entre as subpopulações.Melipona rufiventris is one species of the stingless bee that is becoming very rare in Minas Gerais state. Therefore it was included on the endangered species’ list of Minas Gerais. Until recently, the different "forms" of M. rufiventris founded in the Atlantic forest areas or in the cerrado areas of this state, were considered as a unique species. However, due to the high number of morphological differences founded among specimens from different geographic regions, the "form" found in forest areas passed to be called Melipona mondury while the "form " found in the cerrado was assumed to be M. rufiventris. Aiming to estimate the genetic variability in populations of these two species, we analysed samples from 45 colonies collected at different places of the Minas Gerais state with nine microsatellites primers. The sequencing of some of these loci revealed that the amplified fragments really corresponded to microsatellite areas. Considering all the samples, 7 loci were polymorphic, with some alleles presented only in M. rufiventris while others were exclusive to M. mondury. The Grouping Analysis allowed the establishment of two different groups: the first constituted by the M. mondury’s colonies and the second by the M. rufiventris’ colonies. In the last one, the establishment of two subgroups was observed, being the colonies from Brasilândia de Minas/Dom Bosco separated from the others colonies from the cerrado. The percentage of polymorphic loci was 33.3% to the species M. mondury and M. rufiventris and 22.2% to the colonies from Brasilândia de Minas/Dom Bosco. The observed heterozygosity in these populations was of 0.1159 for M. mondury, 0.0684 for M. rufiventris and 0.0222 for the colonies from Brasilândia de Minas/ Dom Bosco. M. mondury and M. rufiventris presented F ST close to 0.25, indicating that their subpopulations are well structured, with a great genetic differentiation among them.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Molecular analysis in Melipona rufiventris and Melipona mondury (Hymenoptera: Apidae)

Melipona mondury e M. rufiventris são duas espécies de abelhas indígenas sem ferrão morfologicamente bastante semelhantes. Estudos realizados com essas espécies utilizando diferentes tipos de marcadores moleculares mostraram baixa variabilidade genética. Além disso, populações de algumas regiões possuem características que sugerem que mais de uma espécie está recebendo o mesmo nome científico. Neste trabalho procurou-se otimizar ferramentas que pudessem ser utilizadas em estudos populacionais e evolutivos das duas espécies supracitadas, bem como de outras espécies de abelhas. Para isto, foram (1) desenhados primers microssatélites para Melipona mondury e M. rufiventris os quais foram também avaliados em outras espécies de meliponíneos, (2) foram comparados os niveis de polimorfismo encontrado utilizando primers específicos e primers heterólogos, (3) o gene do citocromo B foi parcialmente amplificado e a seqüência do mesmo foi comparada em populações das duas espécies e (4) padronizado um protocolo de citometria de fluxo, para determinação do conteúdo de DNA de abelhas e aplicação do mesmo em M. rufiventris, M. mondury e Scaptotrigona xantotricha. Utilizando-se a técnica de ISSR foram desenhados 10 primers microssatélites para M. rufiventris e 11 para M. mondury e a diversidade genética nessas espécies foi estimada utilizando esses primers e os valores encontrados foram H = 0,47 e H = 0,38, respectivamente. Estes valores são superiores àqueles observados em amplificações nas quais se utilizaram primers desenhados para M. bicolor (H = 0,16 e H = 0,18 para M. mondury e M. rufiventris, respectivamente). Na análise de seqüências do citocromo B encontraram-se onze haplótipos que permitiram a separação de dois grupos distintos, evidenciando a existência de mais de uma espécie nas localidades estudadas. Por fim, a metodologia padronizada para determinação do conteúdo de DNA de abelhas forneceu histogramas, com valores de coeficiente de variação de 2,87 a 4,14%, o que demonstra a utilidade da mesma em estudos envolvendo abelhas. Os valores de conteúdo de DNA 1C encontrados para fêmeas de M. rufiventris, M. mondury e S. xantotricha foram C = 0.93 pg, C = 0,95 pg e C = 0,44 pg, respectivamente, o que demonstra que o tamanho do genoma das duas espécies de Melipona foi cerca de duas vezes maior que o de S. xantotricha. Esta variação pode refletir diferenças na estrutura genômica das espécies analisadas. Entretanto, estudos adicionais deverão ser realizados no sentido de estudar o genoma de outras espécies de Melipona, bem como de outros gêneros de abelhas sem ferrão, a fim de verificar os processos envolvidos na expansão e/ou contração de seus genomas.Melipona mondury and M. rufiventris are two species of indigenous stingless bees which are very similar morphologically. Studies carried out with those species and different kinds of molecular markers revealed low genetic variability. Besides, populations of some regions revealed patterns which suggest that more than one species are receiving the same scientific name. Today, however, several methodologies make it possible to differentiate species and evaluate the genetic structure and the genic flow, besides supplying the basis for the understanding of other important aspects such as the genome evolution of different groups and organisms. So, in this work, it was tried to optimize tools that could help populational and evolutionary studies of the two mentioned species, which could also be used with other bee species. In order to achieve this, several methodologies were used: (1) the drawing of primers microsatellites for Melipona mondury and M. rufiventris and the evaluation of their amplification in other stingless bee species; (2) comparison between the polymorphism found with the specific primers and that of the heterologous primers; (3) analysis of the cytochrome B sequence and (4) standardization of the flow cytometry protocol, in order to determine the DNA content of bees and its use on M. rufiventris, M. mondury and Scaptotrigona xantrotricha. Using the ISSR technique, it was possible to achieve the drawing of 10 primers microsatellites for M. rufiventris and 11 for M. mondury, which presented the following genetic diversity values: H=0,47 and H=0,38, respectively. Those values were higher than those observed in amplifications in which it was used primers drawn for M. bicolor (H=0,16 and H=0,18 for M. mondury and M. rufiventris, respectively). In the analysis of cytochrome B sequences, eleven haplotypes were found which allowed the separation of three different groups, showing that could there are more than one species in these regions. Finally, the standardized methodology for the determination of the bee DNA content furnished good quality histograms, with variation coefficient values of 2,87-4,14%. It demonstrates the usefulness of this methodology in studies involving bees. The DNA 1C content values found for females of M. rufiventris, M. mondury and S. xantotricha were C = 0.93 pg, C = 0,95 pg and C = 0,44 pg, respectively, which reveals that the size of the genome of the two species of Melipona was about twice the size of the one belonging to S. xantotricha. This variation may show differences in the genomic structure of the species analyzed. However, further studies should be carried out to investigate the genome of other species of Melipona and other genders of stingless bees, in order to verify the processes involved in the expansion and contraction of their genomes.Universidade Federal de Viços

An overview of cytogenetics of the tribe Meliponini (Hymenoptera: Apidae)

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

Distribution of GC-rich heterochromatin and ribosomal genes in three fungus-farming ants (Myrmicinae, Attini, Attina): insights on chromosomal evolution

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