RESUMO
Leafcutting ants of the genus Atta are the most conspicuous members of the tribe Attini, the fungus-growing ants. Atta species have long attracted the attention of naturalists, and have since become a common model system for the study of complex insect societies as well as for the study of coevolutionary dynamics due to their numerous interactions with fungi and other microbes. Nevertheless, systematics and taxonomy of the 15 species in the genus Atta have proven challenging, due in part to the extreme levels of worker polymorphism these species display, leading to disagreements about the validity of as many as five different subgenera and calling into question the monophyly of the genus. Here, we use DNA sequence information from fragments of three mitochondrial genes (COI, tRNA leucine and COII) and one nuclear gene (EF1-alphaF1), totaling 1070 base pairs, to reconstruct the phylogenetic relationships of Atta species using maximum parsimony, maximum likelihood and Bayesian inference techniques. Our results provide support for monophyly of the genus Atta, and suggest that the genus is divided into four monophyletic groups, which correspond to four of the five previously erected Atta subgenera: Atta sensu stricto and Archeatta, each with species composition identical to earlier proposals; Neoatta and Epiatta, with major differences in species composition from earlier proposals. The current geographic ranges of these species suggest that the historical separation of South America from Central and North America has played a role in speciation within this genus.
Assuntos
Formigas/genética , Evolução Molecular , Filogenia , Animais , Formigas/classificação , Teorema de Bayes , Núcleo Celular/genética , DNA Mitocondrial/genética , Genes de Insetos , Especiação Genética , Geografia , Funções Verossimilhança , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
PCR-RAPD has been widely used for genetic analysis in several organisms. However, due to complex interactions among the components of the PCR reaction it is unlikely that a single amplification condition can be suitable for all situations. In order to determine the optimum conditions for using PCR-RAPD in taxonomical analyses of the genus Atta, the following factors were tested: concentrations of MgCl2, DNA, BSA, cycling programs and methods of DNA extraction, using Fractional Factorial Design and Central Composite Design. DNA extraction methods had little influence on PCR-RAPD reactions, while the cycling programs showed the largest effect. The MgCl2 concentration was less important than the amount of DNA used in the reaction. Using cycling program P2, a significant improvement in the number of DNA fragments was achieved when MgCl2 concentration was increased to 3.0 mM and the BSA and DNA concentrations were reduced from 0.1 percent to 0.05 percent and from 2 to 1 ng/mul, respectively. The optimum conditions for PCR-RAPD with Atta specimens obtained in this study were: 25 mul reaction with 10 mM Tris-HCl (pH 9.0), 3.0 mM MgCl2, 50 mM KCl, 100 muM of each dNTP, 0.2 muM of primer, 1.0 U of Taq polymerase, 25 ng template DNA (extracted by Cheung's method) and BSA 0.05 percent, using the amplification program which consisted of 3 min at 94°C, 3 min at 35°C, followed by 40 cycles of 1 min at 94°C, 1 min at 36°C and 2 min at 72°C, and a 5 m final extension at 72°C.
A técnica PCR-RAPD tem sido amplamente empregada em estudos genéticos de populações de vários organismos. Entretanto, devido às interações entre os componentes da reação de PCR é improvável que uma única condição de amplificação possa ser adequada para todas as situações. Com o objetivo de determinar as condições ótimas para a utilização da técnica PCR-RAPD em análises taxonômicas do gênero Atta, foram analisados os fatores: concentrações de MgCl2, DNA, BSA, programas de temperaturas e métodos de extração de DNA, utilizando-se os delineamentos Fatorial Fracionado e o Central Composto. Dentre os fatores avaliados, o método de extração de DNA teve pouca influência na reação, enquanto que o programa de temperatura apresentou o maior efeito. Embora a concentração de MgCl2 seja importante para obtenção de um padrão exato de bandas, seu efeito foi menor do que a quantidade de DNA. Com o aumento da concentração de MgCl2 para 3,0 mM e com a diminuição da concentração de BSA de 0,1 por cento para 0,05 por cento e da quantidade de DNA de 2 para 1 ng/mil ocorreu um aumento significativo no número de fragmentos amplificados, sendo esse efeito observado com maior evidência no programa P2. As condições ótimas estabelecidas para as reações PCR-RAPD foram: 25 mil de solução contendo 10 mM Tris-HCl (pH 9,0), 50 mM KCl, 3,0 mM MgCl2, 100 miM de cada dNTP, 0,2 miM de primer, 1,0 U de Taq polimerase, 25 ng DNA (extraído pelo método Cheung) e BSA 0,05 por cento, utilizando-se o programa de amplificação: 3 min. a 94°C, 3 min. a 35°C, seguidos de 40 ciclos de 1 min. a 94°C, 1 min. a 36°C e 2 min. a 72°C, com extensão final de 5 min. a 72°C.