Evidences of differential methylation in the genome during development in the cactophilic Drosophila species.

DNA methylation with 5-methylcytosine (5mC) has been reported in the genome of several eukaryotes, with marked differences between vertebrates and invertebrates. DNA methylation is poorly understood as its role in evolution in insects. Drosophila gouveai (cluster Drosophila buzzatii) presents larvae that develop obligatorily in necrotic tissues of cacti in nature, with the distribution of populations in South America, and plasticity of phenotypes in insect-plant interaction. We characterize organisms at developmental stages and analyze variations at multiple methylation-sensitive loci in pupae, and adult flies using methylation sensitive amplification polymorphism. We obtained 326 loci with CCGG targets in the genome of D. gouveai. Genomic regions with molecular lengths from 100 to 700 pb were most informative about methylation states. Multiple loci show differences in methylation-sensitive sites (MSL) concerning developmental stages, such as in pupae (MSL = 40), female reproductive tissue (MSL = 76), and male reproductive tissues (MSL = 58). Our results are the first evidence of genome-wide methylation in D. gouveai organisms.

Identification of differentially expressed genes in female Drosophila antonietae and Drosophila meridionalis in response to host cactus odor.

BACKGROUND: Studies of insect-plant interactions have provided critical insights into the ecology and evolution of adaptive processes within and among species. Cactophilic Drosophila species have received much attention because larval development occurs in the necrotic tissues of cacti, and both larvae and adults feed on these tissues. Such Drosophila-cactus interactions include effects of the host plant on the physiology and behavior of the flies, especially so their nutritional status, mating condition and reproduction. The aim of this work was to compare the transcriptional responses of two species, Drosophila antonietae and Drosophila meridionalis, and identify genes potentially related to responses to odors released by their host cactus, Cereus hildmannianus. The two fly species are sympatric in most of their populations and use this same host cactus in nature. RESULTS: We obtained 47 unique sequences (USs) for D. antonietae in a suppression subtractive hybridization screen, 30 of these USs had matches with genes predicted for other Drosophila species. For D. meridionalis we obtained 81 USs, 46 of which were orthologous with genes from other Drosophila species. Functional information (Gene Ontology) revealed that these differentially expressed genes are related to metabolic processes, detoxification mechanisms, signaling, response to stimuli, and reproduction. The expression of 13 genes from D. meridionalis and 12 from D. antonietae were further analyzed by quantitative real time-PCR, showing that four genes were significantly overexpressed in D. antonietae and six in D. meridionalis. CONCLUSIONS: Our results revealed the differential expression of genes related to responses to odor stimuli by a cactus, in two associated fly species. Although the majority of activated genes were similar between the two species, we also observed that certain metabolic pathways were specifically activated, especially those related to signaling pathways and detoxification mechanisms. The activation of these genes may reflect different metabolic pathways used by these flies in their interaction with this host cactus. Our findings provide insight into how the use of C. hildmannianus may have arisen independently in the two fly species, through genetic differentiation in metabolic pathways to effectively explore this cactus as a host.

Variations on a theme: diversification of cuticular hydrocarbons in a clade of cactophilic Drosophila.

BACKGROUND: We characterized variation and chemical composition of epicuticular hydrocarbons (CHCs) in the seven species of the Drosophila buzzatii cluster with gas chromatography/mass spectrometry. Despite the critical role of CHCs in providing resistance to desiccation and involvement in communication, such as courtship behavior, mating, and aggregation, few studies have investigated how CHC profiles evolve within and between species in a phylogenetic context. We analyzed quantitative differences in CHC profiles in populations of the D. buzzatii species cluster in order to assess the concordance of CHC differentiation with species divergence. RESULTS: Thirty-six CHC components were scored in single fly extracts with carbon chain lengths ranging from C29 to C39, including methyl-branched alkanes, n-alkenes, and alkadienes. Multivariate analysis of variance revealed that CHC amounts were significantly different among all species and canonical discriminant function (CDF) analysis resolved all species into distinct, non-overlapping groups. Significant intraspecific variation was found in different populations of D. serido suggesting that this taxon is comprised of at least two species. We summarized CHC variation using CDF analysis and mapped the first five CHC canonical variates (CVs) onto an independently derived period (per) gene + chromosome inversion + mtDNA COI gene for each sex. We found that the COI sequences were not phylogenetically informative due to introgression between some species, so only per + inversion data were used. Positive phylogenetic signal was observed mainly for CV1 when parsimony methods and the test for serial independence (TFSI) were used. These results changed when no outgroup species were included in the analysis and phylogenetic signal was then observed for female CV3 and/or CV4 and male CV4 and CV5. Finally, removal of divergent populations of D. serido significantly increased the amount of phylogenetic signal as up to four out of five CVs then displayed positive phylogenetic signal. CONCLUSIONS: CHCs were conserved among species while quantitative differences in CHC profiles between populations and species were statistically significant. Most CHCs were species-, population-, and sex-specific. Mapping CHCs onto an independently derived phylogeny revealed that a significant portion of CHC variation was explained by species' systematic affinities indicating phylogenetic conservatism in the evolution of these hydrocarbon arrays, presumptive waterproofing compounds and courtship signals as in many other drosophilid species.

Phenotypic variation of the Aedeagus of Drosophila serido vilela & sene (Diptera: Drosophilidae).

Drosophila serido Vilela & Sene is a polytypic and cactophilic species with broad geographic distribution in Brazil. The morphology of the aedeagi of eight natural populations of D. serido was analyzed. Based on features of their aedeagi, populations of D. serido were discriminated with an efficiency of nearly 75%. The analysis using the Mantel test suggests that the morphological divergence of D. serido is correlated with the geographic distance among populations. There is no single cause to explain the observed pattern; therefore, the results were discussed considering the three main hypotheses to explain the aedeagus evolution: lock and key, pleiotropy and sexual selection. Alternatively, the aedeagus variability of D. serido might be related to environmental causes, such as temperature and/or host cacti.

Low rates of homogenization of the DBC-150 satellite DNA family restricted to a single pair of microchromosomes in species from the Drosophila buzzatii cluster.

A satellite DNA family, termed DBC-150, comprises slightly GC-rich repeat units of approximately 150 bp that were isolated (by DNA digestions or PCR) from the genome of all seven Drosophila species from the buzzatii cluster (repleta group). The presence of subrepeats suggests that part of the extant DBC-150 monomer originated by the duplication of small sequence motifs. The DBC-150 family is compared to the previously described pBuM satDNA family, an abundant component of the genome of five species of the cluster. The two families are different in several aspects, including primary structure, A + T content, intraspecific and interspecific variability and rates of homogenization (or nucleotide spread). The data indicate a lower rate of homogenization (and absence of complete concerted evolution) of the DBC-150 compared to the pBuM family. FISH on metaphase chromosomes revealed that the DBC-150 family is located exclusively in the microchromosomes. To our knowledge this is the first record of a complex Drosophila satDNA restricted to a single pair of microchromosomes. The observed low rates of homogenization of the DBC-150 family might be related to a presumed reduction or suppression of meiotic recombination in the microchromosomes.

Aedeagus morphology as a discriminant marker in two closely related cactophilic species of Drosophila (Diptera; Drosophilidae) in South America.

Drosophila serido and D. antonietae are sibling species belonging to the Drosophila buzzatii cluster. Morphologically, they can only be discriminated by quantitative traits. In this paper we analyze the length and equalized average curvature of four regions of the aedeagus of D. antonietae and D. serido. Specimens of D. serido and D. antonietae were classified correctly 96.74% of the time. Based only on the variable that most contributed to the discrimination of the groups (equalized average curvature of the arch IV of the aedeagus), we observed significant intraspecific morphological divergence in D. serido in relation to the D. antonietae, in agreement with other markers. The high morphological divergence in equalized average curvature of the arch IV of the aedeagus shows that this region evolved faster than others, since the divergence of the two species. The importance of the present study to the understanding of the genetic basis that controls the formation of the aedeagus, in the species of the Drosophila buzzatii cluster, is discussed.

Nested cladistic analysis of Brazilian populations of Drosophila serido.

The effects of Quaternary climatic cycles were investigated in Drosophila serido, a Brazilian cactophilic fly widely distributed outside the Amazonian region. Previous studies have indicated this species displays remarkable karyotypic, male genitalia, and mtDNA variation, so much so that it has been described as a species complex, or superspecies. In the present study we expand the analysis of the mtDNA COI gene on D. serido populations, particularly in central Brazil, by obtaining DNA sequences from 248 individuals distributed across 47 localities. This allowed us to perform a nested clade analysis to discriminate historical from recurrent forces shaping the evolution of D. serido populations. The nested analysis indicates one event of past fragmentation separating populations from south and central Brazil (referred to as type B) from populations in central and northeast Brazil (type D) and 15 other significant events. The most common outcome of our analysis was contiguous range expansion and we discuss why this was expected in D. serido. Our data indicate that D. serido has been distributed across Brazil at least since the Mid-Pleistocene, which contradicts the hypothesis of current distribution being determined by last glaciation cycle. Nonetheless, we present evidence that climatic cycles during the Quaternary and before have had a significant impact on the differentiation of D. serido in Brazil. Our study confirms the usefulness of the nested clade analysis for disentangling the effects of historical and present-day forces shaping the evolution and distribution of a taxon.

Phenotypic variation of the Aedeagus of Drosophila serido vilela & sene (Diptera: Drosophilidae) / Variação fenotípica do edeago de Drosophila serido vilela & sene (Diptera: Drosophilidae)

Drosophila serido Vilela & Sene é uma espécie cactofílica e politípica, com ampla distribuição geográfica no Brasil. A morfologia do edeago de indivíduos provenientes de oito populações naturais de D. serido foi avaliada. De acordo com as características dos seus edeagos, as populações de D. serido foram discriminadas com eficiência de aproximadamente 75 por cento. O resultado do teste de Mantel sugere que a divergência morfológica de D. serido é correlacionada com a distância geográfica das populações. A explicação para o padrão observado não é única. Por esta razão, os resultados foram discutidos considerando-se as três principais hipóteses para explicar a evolução do edeago: chave-fechadura, pleiotropia e seleção sexual. Alternativamente, a variabilidade encontrada nos edeagos de D. serido poderia estar relacionada a fatores ambientais, tais como temperatura e/ou cacto hospedeiro.

Drosophila serido Vilela & Sene is a polytypic and cactophilic species with broad geographic distribution in Brazil. The morphology of the aedeagi of eight natural populations of D. serido was analyzed. Based on features of their aedeagi, populations of D. serido were discriminated with an efficiency of nearly 75 percent. The analysis using the Mantel test suggests that the morphological divergence of D. serido is correlated with the geographic distance among populations. There is no single cause to explain the observed pattern; therefore, the results were discussed considering the three main hypotheses to explain the aedeagus evolution: lock and key, pleiotropy and sexual selection. Alternatively, the aedeagus variability of D. serido might be related to environmental causes, such as temperature and/or host cacti.

Molecular characterization of SSS139, a new satellite DNA family in sibling species of the Drosophila buzzatii cluster

We characterized sequences of a novel SSS139 RsaI satellite DNA family in Drosophila gouveai and Drosophila seriema, two members of the Drosophila buzzatii cluster (D. repleta group). The sequences were AT-rich (69 percent) with a monomer unit length of about 139 bp and contained two direct subrepeats of 14 bp and 16 bp, suggesting that it might have originated by the duplication of smaller sequences. Southern and dot-blot hybridization analyses also detected SSS139 in other Drosophila buzzatii cluster species (D. koepferae, D. antonietae, D. borborema and D. serido) but not in D. buzzatii. These results agree with the marginal phylogenetic position of D. buzzatii within the D. buzzatii cluster.

Phylogenetic relationships in the Drosophila fasciola species subgroup (Diptera, Drosophilidae) inferred from partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene

The repleta group is one of the largest and most complex species groups in the genus Drosophila, representing an important biological model for the elaboration of evolutionary and biogeographical hypotheses on the American Continent. It is divided into six subgroups, of which the fasciola subgroup is the only one with most of its species found in the humid forests of Central and South America. With the aim of understanding the origin and evolution of the fasciola subgroup, and consequently adding information about the repleta group, we analyzed partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene of nine Drosophila species (D. carolinae, D. coroica, D. ellisoni, D. fascioloides, D. ivai, D. moju, D. onca, D. rosinae and D. senei). The phylogenetic hypotheses obtained neither defined the relationships between the subgroups of the repleta group nor defined all the interspecific relationships in the fasciola subgroup. We found that the species D. carolinae, D. coroica, D. onca, D. rosinae and D. senei comprised a clade in which D. carolinae, D. onca and D. senei appeared together at a polytomy while D. fascioloides and D. ellisoni comprised another clade with a high bootstrap value.

Aedeagus morphology as a discriminant marker in two closely related Cactophilic species of Drosophila (Diptera; Drosophilidae) in South America

Drosophila serido e D. antonietae são espécies crípticas pertencentes ao ''cluster'' Drosophila buzzatii. Morfologicamente, elas podem ser discriminadas apenas por diferenças quantitativas. Neste trabalho, nós analisamos o comprimento e a média da curvatura equalizada de quatro regiões do edeago de indivíduos de D. antonietae e D. serido. Os espécimes de D. serido e D. antonietae foram discriminados com 96,74% de eficiência. Apenas com base na variável que mais contribuiu para discriminação dos grupos (média da curvatura equalizada do arco IV do edeago), nós observamos significativa divergência morfológica intraespecífica em D. serido em relação a D. antonietae, o que está em concordância com outros marcadores. A alta divergência morfológica apenas na média da curvatura equalizada do arco IV do edeago mostra que essa região do edeago evoluiu mais rápido que as demais desde a divergência entre as duas espécies. A importância do presente estudo para o entendimento das bases genética que controlam a formação do edeago, nas espécies do ''cluster'' Drosophila buzzatii, é discutida.

Mitochondrial DNA phylogeography of Brazilian populations of Drosophila buzzatii

The Argentinean Chaco region has been considered the center of origin of Drosophila buzzatii in South America because it contains most of the chromosomal polymorphism detected in natural populations. Two hypotheses have been put forward to explain the distribution of D. buzzatii in Brazil, one proposing that it has only recently passively colonized Brazil via human dispersal and the other suggesting that D. buzzatii has actively migrated to Brazil some time ago. Data from chromosomal inversions support recent colonization, whereas data from allozymes and mtDNA variation indicate that D. buzzatii has been in Brazil longer, favoring an active dispersal hypothesis. In our present work we analyzed data on 56 South American flies, mostly from Brazil, sequenced for the 5Æ end of the mtDNA COI gene. The combined use of many neutrality tests and phylogeographic methods (e.g. nested clade analysis) indicated high gene flow throughout most of the range of D. buzzatii, although significant population structure was still detected. The high nucleotide diversity in the Northeast region of Brazil and the results from the nested clade analysis suggest that D. buzzatii has been in Brazil longer than proposed by the passive dispersal hypothesis. Our data indicate that D. buzzatii has been distributed throughout Brazil and Argentina since the Quaternary, though more data from different localities and markers need to be gathered to determine how the occupation of South America by D. buzzatii has occurred