Lizandra J. Robe

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.

Taxonomic boundaries, phylogenetic relationships and biogeography of the Drosophila willistoni subgroup (Diptera: Drosophilidae)

The Drosophila willistoni subgroup represents a complex with varying taxonomic levels. It encompasses D. willistoni and its five sibling species: D. equinoxialis, D. insularis, D. paulistorum, D. pavlovskiana and D. tropicalis. Of these, D. equinoxialis, D. tropicalis and D. willistoni present differentiation at subspecific level, whereas D. paulistorum represents a superspecies, formed by six semispecies. Despite this taxonomic and evolutionary complexity, many of these semi and subspecific taxa have not yet had their phylogenetic status tested in an explicitly molecular study. Aiming to contribute to the understanding of the evolution of this challenging group, we analyzed nucleotide sequences from two mitochondrial and four nuclear datasets, both individually and simultaneously, through different phylogenetic methods. High levels of incongruence were detected among partitions, especially concerning the mitochondrial sequences. As this incongruence was found to be statistically significant and robust to the use of different models and approaches, and basically restricted to mitochondrial loci, we suggest that it may stem mainly from hybridization-mediated asymmetrical introgression. Despite this, our nuclear data finally led to a phylogenetic hypothesis which further refines several aspects related to the willistoni subgroup phylogeny. In this respect, D. insularis, D. tropicalis, D. willistoni and D. equinoxialis successively branched off from the willistoni subgroup main stem, which recently subdivided to produce D. paulistorum and D. pavlovskiana. As regards the semispecies evolution, we found evidence of a recent diversification, which highly influenced the obtained results due to the associated small levels of genetic differentiation, further worsened by the possibly associated incompletely sorted ancestral polymorphisms and by the possibility of introgression. This study also raises the question of whether these semispecies are monophyletic at all. This reasoning is particularly interesting when one considers that similar levels of reproductive isolation could be attained through infection with different Wolbachia strains.

Phylogenetic relationships and macro-evolutionary patterns within the Drosophila tripunctata “radiation” (Diptera: Drosophilidae)

Despite previous efforts, the evolutionary history of the immigrans-tripunctata clade remains obscure in part due to its hypothesized origin through a rapid radiation. We performed a supermatrix analysis (3,243 base pairs) coupled with richness patterns, environmental phylogenetic signal and radiation tests in order to address phylogenetic relationships and macro-evolutionary hypotheses within this complex group of species. We propose a well-supported evolutionary scenario for the immigrans-tripunctata clade species, in which the tripunctata “radiation” was monophyletic and subdivided into three main lineages: the first including D. pallidipennis (pallidipennis group) imbedded among members of the tripunctata group; the second clustering the cardini and guarani groups; and the third grouping representatives from the tripunctata,calloptera and guaramunu groups. Therefore, we hypothesize that the tripunctata group encompasses a diphyletic taxon, with one clade including the pallidipennis group and the other showing a close affinity to the calloptera and guaramunu groups. Our results also suggest that niche evolution seems to have played a central role in the evolutionary history of the tripunctata species “radiation” allowing effective dispersion and diversification in the Neotropics, possibly in a southwards direction. Although the data as a whole support the notion that this occurred through rapid and successive speciation events, the radiation hypothesis remains to be further corroborated.

The DNA barcoding and the caveats with respect to its application to some species of Palaemonidae (Crustacea, Decapoda).

DNA-barcoding has recently attracted considerable attention due to its potential utility in aiding in species identification and discovery through the use of a short standardized sequence of mitochondrial DNA. Nevertheless, despite the fact that this technology has been proven a useful tool in several animal taxa, it also demonstrated limitations that may hinder correct application. Thus, its validity needs to be empirically evaluated in each taxonomic category before forward implementation. As the use of DNA barcoding within Palaemonidae may be of special interest, given its great interspecific morphological conservatism associated with considerable intraspecific morphological variation, we analyze here the potential of this technology in distinguishing and recovering some taxonomic boundaries within this family. We asked whether two GenBank-retrieved sets of COI sequences encompassing the conventional Barcode and Jerry-Pat regions possess the desired properties of reciprocal monophyly among species, and existence of a barcoding gap between intra- and interspecific variations, after performing a careful analysis of numt (nuclear mitochondrial DNA) contamination. These analyses revealed nine non-monophyletic species, with some cases of divergent intraspecific sequences, contrasted with interspecific similarity attained in others. Moreover, we were unable to identify any barcoding gap between intraspecific and interspecific divergences within Palaemonidae, although a threshold of 0.18 substitutions per site would differentiate intraspecific and congeneric divergences in 95% of the cases for the barcoding region. A fraction of the overlap could be certainly attributed to artifacts related to poor taxonomy, but even from this perspective DNA barcoding studies may help to uncover previously disregarded taxonomic and evolutionary issues.

First evidence of methylation in the genome of Drosophila willistoni

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogasterdDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.

The Drosophila flavopilosa species group (Diptera, Drosophilidae): an array of exciting questions.

The D. flavopilosa group encompasses an ecologically restricted set of species strictly adapted to hosting flowers of Cestrum (Solanaceae). This group presents potential to be used as a model to the study of different questions regarding ecologically restricted species macro and microevolutionary responses, geographical vs. ecological speciation and intra and interspecific competition. This review aims to revisit and reanalyze the patterns and processes that are subjacent to the interesting ecological and evolutionary properties of these species. Biotic and abiotic niche properties of some species were reanalyzed in face of ecological niche modeling approaches in order to get some insights into their ecological evolution. A test of the potential of DNA-Barcoding provided evidences that this technology may be a way of overcoming difficulties related to cryptic species differentiation. The new focus replenishes the scenario with new questions, presenting a case where neither geographical nor ecological speciation may be as yet suggested.

Phylogeny of the Drosophila mesophragmatica Group (Diptera, Drosophilidae): An Example of Andean Evolution

Abstract The mesophragmatica group of Drosophila belongs to the virilis-repleta radiation of the Drosophila subgenus. This group comprises 13 Neotropical species that are endemic to the South-American continent and seem to be fundamentally Andean in their distribution. The mesophragmatica-group phylogeny has been inferred previously by other authors based on morphological, cytological, and isozyme analyses. However, the relationships within the group have not yet been completely resolved, although its monophyletic origin has already been confirmed by molecular data. This work attempts to enhance the molecular approach to the relationships among the species of the mesophragmatica group, using both nuclear and mitochondrial markers. Phylogenetic analyses were performed using fragments of the nuclear alcohol dehydrogenase (Adh; 631 bp), alpha-methyldopa (Amd; 1211 bp), dopa-decarboxylase (Ddc; 1105 bp), and hunchback (Hb; 687 bp) genes and the mitochondrial cytochrome oxidase subunit II (COII; 672 bp) gene, and included a total of 4306 bp. The sequences obtained for eight representatives of the mesophragmatica group were analyzed both individually and in combination by distance methods, maximum parsimony, and maximum likelihood. Our results support subdivision of the mesophragmatica group into three main lineages: the first is composed of D. viracochi; the second comprises a clade grouping the sibling species D. pavani and D. gaucha; and the third encompasses D. gasici, D. brncici, and D. mesophragmatica. The best supported scenario suggests that D. viracochi is an early offshoot in the mesophragmatica group, with this and other early branchings occuring in the Pliocene/Pleistocene Epochs, possibly associated with Andean glacial refuges. Also based on the phylogenies obtained, we present a genealogical view of the evolution of previously described characters within the group.

Comparative ecological niche modeling and evolutionary ecology of Neotropical mycophagous Drosophilidae (Diptera) species

This study provides new records on the distribution of 22 species of mycophagous Drosophilidae and reports the southernmost and/or northernmost register points for most of them. Climatic modeling analysis revealed that the sampled species are heterogeneous as concerns the patterns of potential geographical ranges. A regular pattern emerging from the evaluated models is that the minimum temperature of the coldest month is one of the most determinant variables of habitat suitability for most species, although the particular response patterns may be further subdivided. In particular, some putative geographically restricted species appear to be highly susceptible to minor increases in the minimal temperature ranges, indicating that they are potential targets for the temperature rises expected to affect the Neotropics over the next decades and centuries. Moreover, as our analyses detected niche conservatism signals among the studied species, potential range shifts in face of future climatic oscillations may also be questioned.

Microevolutionary analyses of Pythium insidiosum isolates of Brazil and Thailand based on exo-1,3-β-glucanase gene

Pythium insidiosum is an important oomycete due to its ability to infect humans and animals. It causes pythiosis, a disease of difficult treatment that occurs more frequently in humans in Thailand and in horses in Brazil. Since cell-wall components are frequently related to host shifts, we decided here to use sequences from the exo-1,3-β-glucanase gene (exo1), which encodes an immunodominant protein putatively involved in cell wall remodeling, to investigate the microevolutionary relationships of Brazilian and Thai isolates of P. insidiosum. After neutrality ratification, the phylogenetic analyses performed through Maximum parsimony (MP), Neighbor-joining (NJ), Maximum likelihood (ML), and Bayesian analysis (BA) strongly supported Thai isolates being paraphyletic in relation to those from Brazil. The structure recovered by these analyses, as well as by Spatial Analysis of Molecular Variance (SAMOVA), suggests the subdivision of P. insidiosum into three clades or population groups, which are able to explain almost 81% of the variation encountered for exo1. Moreover, the two identified Thai clades were almost as strongly differentiated between each other, as they were from the Brazilian clade, suggesting an ancient Asian subdivision. The derived positioning in the phylogenetic tree, linked to the lower diversity values and the recent expansion signs detected for the Brazilian clade, further support this clade as derived in relation to the Asian populations. Thus, although some patterns presented here are compatible with those recovered with different molecular markers, exo1 was revealed to be a good marker for studying evolution in Pythium, providing robust and strongly supported results with regard to the patterns of origin and diversification of P. insidiosum.

The LTR retrotransposon micropia in the cardini group of Drosophila (Diptera: Drosophilidae): a possible case of horizontal transfer

The presence of the micropia retroelement from the Ty1-copia family of LTR retroelements was investigated in three species of the Drosophilacardini group. Southern blot analysis suggested the existence of at least four micropia copies in the genomes of D. cardinoides, D. neocardini and D. polymorpha populations. The high sequence similarity between dhMiF2 and Dm11 clones (micropia retroelements isolated from D. hydei and D. melanogaster, respectively) with micropia sequences amplified from D.cardini group genome supports the hypothesis that this retroelement plays an active role in horizontal transfer events between D. hydei and the D.cardini group.