The willistoni species subgroup has been the subject of several studies since the latter half of the past century and is considered a Neotropical model for evolutionary studies, given the many levels of reproductive isolation and different evolutionary stages occurring within them. Here we present for the first time a phylogenetic reconstruction combining morphological characters and molecular data obtained from 8 gene fragments (COI, COII, Cytb, Adh, Ddc, Hb, kl-3 and per). Some relationships were incongruent when comparing morphological and molecular data. Also, morphological data presented some unresolved polytomies, which could reflect the very recent divergence of the subgroup. The total evidence phylogenetic reconstruction presented well-supported relationships and summarized the results of all analyses. The diversification of the willistoni subgroup began about 7.3 Ma with the split of D. insularis while D.paulistorum complex has a much more recent diversification history, which began about 2.1 Ma and apparently has not completed the speciation process, since the average time to sister species separation is one million years, and some entities of the D. paulistorum complex diverge between 0.3 and 1 Ma. Based on the obtained data, we propose the categorization of the former "semispecies" of D. paulistorum as a subspecies and describe the subspecies D. paulistorum amazonian, D. paulistorum andeanbrazilian, D. paulistorum centroamerican, D. paulistorum interior, D. paulistorum orinocan and D. paulistorum transitional.

Infections by the endosymbiotic bacterium Wolbachia developed a rapid global expansion within Old World Drosophila species, ultimately infecting also Neotropical species. In this sense, screenings are necessary to characterize new variants of Wolbachia or new hosts, and also in order to map the dynamics of already known infections. In this paper, we performed a double screening approach that combined Dot-blot and PCR techniques in order to reevaluate the infection status by Wolbachia in species from the willistoni subgroup of Drosophila. Genomic DNA from isofemale lines descendent from females collected in the Amazonian Rainforest (n=91) were submitted to Dot-blot, and were positive for Wolbachia, producing a gradient of hybridization signals, suggesting different infection levels, which was further confirmed through quantitative PCR. Samples with a strong signal in the Dot-blot easily amplified in the wsp-PCR, unlike most of the samples with a medium to weak signal. It was possible to molecularly characterize three Drosophila equinoxialis isofemale lines that were found to be infected in a low density by a wMel-like Wolbachia strain, which was also verified in a laboratory line of Drosophila paulistorum Amazonian. We also found Drosophila tropicalis to be infected with the wAu strain and a Drosophila paulistorum Andean-Brazilian semispecies laboratory line to be infected with a wAu-like Wolbachia. Moreover, we observed that all Drosophila willistoni samples tested with the VNTR-141 marker harbor the same Wolbachia variant, wWil, either in populations from the South or the North of Brazil. Horizontal transfer events involving species of Old World immigrants and Neotropical species of the willistoni subgroup are discussed.

The prevalence of the endosymbiont Wolbachia pipientis and its effects on mitochondrial genetic diversity were analyzed in natural populations of Drosophila willistoni, a neotropical species recently infected. Total infection rate was 55% and no evidence was found that the Wolbachia infection decreased the diversity of mtDNA. Wolbachia was seen to be associated with different mitochondria, suggesting multiple horizontal transmission events and/or transmission paternal leakage of mitochondrial and/or Wolbachia. These hypotheses are evaluated in the context of the present study and other research.

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