Rita Gomes Rocha

Neotropical forest expansion during the last glacial period challenges refuge hypothesis

Significance The tropical forests of South America are among the most diverse and unique habitats in the world in terms of plant and animal species. One of the most popular explanations for this diversity and endemism is the idea that forests retracted and fragmented during glacial periods, forming ecological refuges, surrounded by dry lands or savannas. These historically stable forest refuges would have been responsible for maintaining the pattern of diversity and endemism observed today. Here, we show that the Atlantic Forest of eastern South America probably expanded, rather than contracted, during the last glacial period. In addition, the emerged Brazilian continental shelf played a major, yet neglected, role on the evolution of this biodiversity hotspot during the last glacial period. The forest refuge hypothesis (FRH) has long been a paradigm for explaining the extreme biological diversity of tropical forests. According to this hypothesis, forest retraction and fragmentation during glacial periods would have promoted reproductive isolation and consequently speciation in forest patches (ecological refuges) surrounded by open habitats. The recent use of paleoclimatic models of species and habitat distributions revitalized the FRH, not by considering refuges as the main drivers of allopatric speciation, but instead by suggesting that high contemporary diversity is associated with historically stable forest areas. However, the role of the emerged continental shelf on the Atlantic Forest biodiversity hotspot of eastern South America during glacial periods has been ignored in the literature. Here, we combined results of species distribution models with coalescent simulations based on DNA sequences to explore the congruence between scenarios of forest dynamics through time and the genetic structure of mammal species cooccurring in the central region of the Atlantic Forest. Contrary to the FRH predictions, we found more fragmentation of suitable habitats during the last interglacial (LIG) and the present than in the last glacial maximum (LGM), probably due to topography. We also detected expansion of suitable climatic conditions onto the emerged continental shelf during the LGM, which would have allowed forests and forest-adapted species to expand. The interplay of sea level and land distribution must have been crucial in the biogeographic history of the Atlantic Forest, and forest refuges played only a minor role, if any, in this biodiversity hotspot during glacial periods.

A Genetic Code Alteration Is a Phenotype Diversity Generator in the Human Pathogen Candida albicans

Background The discovery of genetic code alterations and expansions in both prokaryotes and eukaryotes abolished the hypothesis of a frozen and universal genetic code and exposed unanticipated flexibility in codon and amino acid assignments. It is now clear that codon identity alterations involve sense and non-sense codons and can occur in organisms with complex genomes and proteomes. However, the biological functions, the molecular mechanisms of evolution and the diversity of genetic code alterations remain largely unknown. In various species of the genus Candida, the leucine CUG codon is decoded as serine by a unique serine tRNA that contains a leucine 5′-CAG-3′anticodon (tRNACAG Ser). We are using this codon identity redefinition as a model system to elucidate the evolution of genetic code alterations. Methodology/Principal Findings We have reconstructed the early stages of the Candida genetic code alteration by engineering tRNAs that partially reverted the identity of serine CUG codons back to their standard leucine meaning. Such genetic code manipulation had profound cellular consequences as it exposed important morphological variation, altered gene expression, re-arranged the karyotype, increased cell-cell adhesion and secretion of hydrolytic enzymes. Conclusion/Significance Our study provides the first experimental evidence for an important role of genetic code alterations as generators of phenotypic diversity of high selective potential and supports the hypothesis that they speed up evolution of new phenotypes.

Critical roles for a genetic code alteration in the evolution of the genus Candida

During the last 30 years, several alterations to the standard genetic code have been discovered in various bacterial and eukaryotic species. Sense and nonsense codons have been reassigned or reprogrammed to expand the genetic code to selenocysteine and pyrrolysine. These discoveries highlight unexpected flexibility in the genetic code, but do not elucidate how the organisms survived the proteome chaos generated by codon identity redefinition. In order to shed new light on this question, we have reconstructed a Candida genetic code alteration in Saccharomyces cerevisiae and used a combination of DNA microarrays, proteomics and genetics approaches to evaluate its impact on gene expression, adaptation and sexual reproduction. This genetic manipulation blocked mating, locked yeast in a diploid state, remodelled gene expression and created stress cross‐protection that generated adaptive advantages under environmental challenging conditions. This study highlights unanticipated roles for codon identity redefinition during the evolution of the genus Candida, and strongly suggests that genetic code alterations create genetic barriers that speed up speciation.

Unveiling the structural basis for translational ambiguity tolerance in a human fungal pathogen

In a restricted group of opportunistic fungal pathogens the universal leucine CUG codon is translated both as serine (97%) and leucine (3%), challenging the concept that translational ambiguity has a negative impact in living organisms. To elucidate the molecular mechanisms underlying the in vivo tolerance to a nonconserved genetic code alteration, we have undertaken an extensive structural analysis of proteins containing CUG-encoded residues and solved the crystal structures of the two natural isoforms of Candida albicans seryl-tRNA synthetase. We show that codon reassignment resulted in a nonrandom genome-wide CUG redistribution tailored to minimize protein misfolding events induced by the large-scale leucine-to-serine replacement within the CTG clade. Leucine or serine incorporation at the CUG position in C. albicans seryl-tRNA synthetase induces only local structural changes and, although both isoforms display tRNA serylation activity, the leucine-containing isoform is more active. Similarly, codon ambiguity is predicted to shape the function of C. albicans proteins containing CUG-encoded residues in functionally relevant positions, some of which have a key role in signaling cascades associated with morphological changes and pathogenesis. This study provides a first detailed analysis on natural reassignment of codon identity, unveiling a highly dynamic evolutionary pattern of thousands of fungal CUG codons to confer an optimized balance between protein structural robustness and functional plasticity.

FKS2 Mutations Associated with Decreased Echinocandin Susceptibility of Candida glabrata following Anidulafungin Therapy

ABSTRACT This is the first case report of Candida glabrata-disseminated candidiasis describing the acquisition of echinocandin resistance following anidulafungin treatment. The initial isolates recovered were susceptible to echinocandins. However, during 27 days of anidulafungin treatment, two resistant strains were isolated (from the blood and peritoneal fluid). The resistant peritoneal fluid isolate exhibited a Ser663Pro mutation in position 1987 of FKS2 HS1 (hot spot 1), whereas the resistant blood isolate displayed a phenylalanine deletion (Phe659).

Small mammals in the diet of barn owls, Tyto alba (Aves: Strigiformes) along the mid-Araguaia river in central Brazil

We collected and analyzed 286 Barn owl, Tyto alba (Scopoli, 1769), pellets from two nests in different environments along the mid-Araguaia River in central Brazil. Our analyses revealed that these owls feed mainly on small mammals, especially rodents. Owls from the riverbanks at Fazenda Santa Fe had a more diverse diet, preying mainly on rodents that typically inhabit riparian grasslands - Holochilus sciureus Wagner, 1842 - and forests - Hylaeamys megacephalus (Fischer, 1814) and Oecomys spp., which probably also occur in forest borders or clearings. On the other hand, owls from an agroecosystem at Fazenda Lago Verde preyed mostly on rodent species common in these agrarian fields, Calomys tocantinsi Bonvicino, Lima & Almeida, 2003. Additionally, we compared small mammal richness estimates based on the analysis of owl pellets with estimates from live-trapping in the same areas. Owl pellets revealed two rodent species undetected by live traps - Euryoryzomys sp. and Rattus rattus (Linnaeus, 1758) - and four rodent species were trapped, but not found in owl pellets - Oecomys roberti Thomas, 1904, Pseudoryzomys simplex (Winge, 1887), Rhipidomys ipukensis Rocha, B.M.A. Costa & L.P. Costa, 2011, and Makalata didelphoides (Desmarest, 1817). Traps yielded higher species richness, but these two methods complement each other for surveying small rodents.

Synergistic Antimicrobial Action of Chlorhexidine and Ozone in Endodontic Treatment

Objectives. The aim of this study was to determine whether irrigation with sodium hypochlorite, chlorhexidine, and ozone gas, alone or in combination, were effective against Enterococcus faecalis and Candida albicans; these are microorganisms frequently isolated from teeth with periapical lesions resistant to endodontic treatment. Material and Methods. 220 single root teeth, recently extracted, were inoculated with Candida albicans and Enterococcus faecalis. The formulations tested were sodium hypochlorite at 1, 3, and 5% chlorhexidine at 0.2% and 2% and ozone gas applied for different periods of time. The combination of sodium hypochlorite at 5% and chlorhexidine at 2%, with gaseous ozone, were also assessed. For the most active treatments the mechanism of action was assessed through flow cytometry. Results. Sodium hypochlorite, chlorhexidine, and gaseous ozone alone were ineffective in completely eliminating the microorganisms. The association of chlorhexidine at 2% followed by ozone gas for 24 seconds promoted the complete elimination of Candida albicans and Enterococcus faecalis. Flow cytometry shows that ozone and chlorhexidine act differently, which could explain its synergic activity. Conclusions. This new disinfection protocol, combining irrigation with chlorhexidine at 2% and ozone gas for 24 seconds, may be advantageous when treating infected root canals.

DNA from owl pellet bones uncovers hidden biodiversity

Owl pellets are a useful tool for inventorying small mammals and a robust complement to traditional trapping methods. Here we assessed if bone samples recovered from Barn owl pellets in the Neotropics represent a viable source of modern DNA. We used the upper incisor of rodents and jaws of marsupials of pellet material for DNA extraction and amplification. Extraction and amplification success depended on the type of bone element used, being much higher from rodent incisors. The amplification success of small (429 bp) and large (801 bp) mitochondrial cytochrome b fragments was relatively similar, suggesting that DNA was not severely degraded, probably because our pellet samples were less than 8 years old. Eleven species of small mammals were identified, from which two are new to the study area: Gracilinanus emiliae and Marmosops pinheiroi. These records represent the southeastern boundary of both species, and extended their geographic distribution 370 km (G. emiliae) and 700 km (M. pinheiroi) to the south. Moreover, two of three detected species of Oecomys occurring in sympatry may represent undescribed species. The use of molecular analysis as a complement to morphological analysis of pellet bones is very effective in species identification, especially considering poorly known and elusive species, such as those reported here. The search for owl pellets during fieldwork should be a common practice among mammalogists in the Neotropics because this material provides a valuable non-invasive source of DNA, often revealing previously undetected species and sometimes uncovering new species.

Seasonal flooding regime and ecological traits influence genetic structure of two small rodents

Although codistributed species are affected by the same abiotic factors, such as rivers and seasonal flooding regimes, ecological traits, such as locomotion habits and habitat preferences, may also influence differences in levels of genetic diversity and differentiation. We examined population genetic structure and diversity of Hylaeamys megacephalus and Oecomys aff. roberti, two cricetid rodent species from the mid-Araguaia River in central Brazil, using mitochondrial DNA sequence data. Specifically, we aim to test whether the Araguaia River acts as a barrier to the gene flow of these two species and to assess how ecological traits, such as locomotion habits and habitat preferences, may influence differences in levels of genetic diversity and differentiation. As both species occur in flooded forests, neither showed genetic differences related to river banks. Oecomys aff. roberti showed stronger population structure that appears to be associated with isolation by distance. This arboreal species maintained stable populations in the Araguaia River, while the terrestrial H. megacephalus was more affected by seasonal floods, resulting in a genetic signature of population expansion. Our initial predictions were largely supported by our results given that locomotion habits and habitat preferences of each species appears to have played a role on the genetic structure of these two sympatric rodent species.

The Araguaia River as an Important Biogeographical Divide for Didelphid Marsupials in Central Brazil

The riverine barrier model suggests that rivers play a significant role in separating widespread organisms into isolated populations. In this study, we used a comparative approach to investigate the phylogeography of 6 didelphid marsupial species in central Brazil. Specifically, we evaluate the role of the mid-Araguaia River in differentiating populations and estimate divergence time among lineages to assess the timing of differentiation of these species, using mitochondrial DNA sequence data. The 6 didelphid marsupials revealed different intraspecific genetic patterns and structure. The 3 larger and more generalist species, Didelphis albiventris, Didelphis marsupialis, and Philander opossum, showed connectivity across the Araguaia River. In contrast the genetic structure of the 3 smaller and specialist species, Gracilinanus agilis, Marmosa (Marmosa) murina, and Marmosa (Micoureus) demerarae was shaped by the mid-Araguaia. Moreover, the split of eastern and western bank populations of the 2 latter species is consistent with the age of Araguaia River sediments formation. We hypothesize that the role of the Araguaia as a riverine barrier is linked to the level of ecological specialization among the 6 didelphid species and differences in their ability to cross rivers or disperse through the associated habitat types.