Anderson V. Chaves

Exploring the diversity and distribution of neotropical avian malaria parasites--a molecular survey from Southeast Brazil.

Southeast Brazil is a neotropical region composed of a mosaic of different tropical habitats and mountain chains, which allowed for the formation of bird-rich communities with distinct ecological niches. Although this region has the potential to harbor a remarkable variety of avian parasites, there is a lack of information about the diversity of malarial parasites. We used molecular approaches to characterize the lineage diversity of Plasmodium and Haemoproteus in bird communities from three different habitats in southeast Brazil based on the prevalence, richness and composition of lineages. We observed an overall prevalence of 35.3%, with a local prevalence ranging from 17.2% to 54.8%. Moreover, no significant association between prevalence and habitat type could be verified (p>0.05). We identified 89 Plasmodium and 22 Haemoproteus lineages, with 86% of them described for the first time here, including an unusual infection of a non-columbiform host by a Haemoproteus (Haemoproteus) parasite. The composition analyses of the parasite communities showed that the lineage composition from Brazilian savannah and tropical dry forest was similar, but it was different from the lineage composition of Atlantic rainforest, reflecting the greater likeness of the former habitats with respect to seasonality and forest density. No significant effects of habitat type on lineage richness were observed based on GLM analyses. We also found that sites whose samples had a greater diversity of bird species showed a greater diversity of parasite lineages, providing evidence that areas with high bird richness also have high parasite richness. Our findings point to the importance of the neotropical region (southeast Brazil) as a major reservoir of new haemosporidian lineages.

Molecular taxonomy of Brazilian tyrant‐flycatchers (Passeriformes: Tyrannidae)

The tyrannids are one of the most diverse groups of birds in the world, and the most numerous suboscine family in the Neotropics. Reflecting such diversity, many taxonomic issues arise in this group, mainly due to morphological similarities, even among phylogenetically distant species. Other issues appear at higher taxonomic levels, mostly brought up by genetic studies, making systematics a rather inconclusive issue. This study looks into the use of DNA barcodes method to discriminate and identify Tyrannidae species occurring in the Atlantic Forest and Cerrado biomes of Brazil. We analysed 266 individuals of 71 tyrant‐flycatcher species from different geographical locations by sequencing 542 bp of the mtDNA COI gene. The great majority of the analysed species showed exclusive haplotypes, usually displaying low intraspecific diversity and high interspecific divergence. Only Casiornis fuscus and Casiornis rufus, suggested in some studies to belong to a single species, could not be phylogenetically separated. High intraspecific diversity was observed among Elaenia obscura individuals, which can suggest the existence of cryptic species in this taxon. The same was also observed for Suiriri suiriri, considered by some authors to comprise at least two species, and by others to be divided into three subspecies. Additionally, the use of sequences from voucher specimens allowed us to correct four misidentifications that had happened in the field. Our findings suggest a great power of the COI barcodes to discriminate species of the Tyrannidae family that are found in Brazil.

Inaccuracy of enzyme-linked immunosorbent assay using soluble and recombinant antigens to detect asymptomatic infection by Leishmania infantum.

Background One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period. Methodology Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve. Findings The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10). Conclusions Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.

Bacteria and Genes Involved in Arsenic Speciation in Sediment Impacted by Long-Term Gold Mining

The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one of the world’s largest mining regions known as the “Iron Quadrangle”. A total of 123 As-resistant bacteria were recovered from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing (72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA (20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter. Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the molecular diversity of arrA genes is far from being fully explored deserving further attention.

Biogeographic patterns, origin and speciation of the endemic birds from eastern Brazilian mountaintops: a review

We review the main biogeographic and phylogenetic hypotheses about the origin and distribution of the endemic birds from Brazilian mountaintops, a heterogeneous group composed of hummingbirds and passerines living in high-altitude open habitats of campos rupestres, campos de altitude and campos sulinos from southern to north-eastern Brazilian mountaintops (Espinhaço Range, Serra do Mar, Serra da Mantiqueira and Serra Geral). Some authors consider that these birds are probably closely related to other species distributed in cooler areas of South America. We used ranges of these birds, spatial pattern and discontinuities in the distribution of phytophysiognomies to determine potential geographic barriers to dispersal and centres of origin and speciation. We reanalysed biogeographical hypotheses regarding the evolution of the endemic birds from Brazilian mountaintops and their habitats in the light of recent phylogenetic reconstructions that included some of these species. The evolutionary histories of the species Augastes scutatus, Augastes lumachella, Formicivora grantsaui, Polystictus superciliaris and Embernagra longicauda are difficult to assess due to lack of phylogenies that establish their systematic position. For Asthenes luizae, Asthenes moreirae, Cinclodes pabsti and C. espinhacensis, the phylogenies estimate also their lineage divergence times, which allows for inferences concerning connections between the biota of eastern Brazil, southern South America and the Andean mountains. We also identified 26 lowland regions between the mountains that were potentially barriers to dispersal. Phylogenetic relationships strongly highlight the importance of connecting paths and speciation through the south, especially in the cooler areas in South America. These patterns show that eastern Brazil, southern South America and the central and northern Andes are four important centres of origin and radiation for the endemic birds from the eastern Brazilian mountains.

Barcoding Neotropical birds: assessing the impact of nonmonophyly in a highly diverse group

In this study, we verified the power of DNA barcodes to discriminate Neotropical birds using Bayesian tree reconstructions of a total of 7404 COI sequences from 1521 species, including 55 Brazilian species with no previous barcode data. We found that 10.4% of species were nonmonophyletic, most likely due to inaccurate taxonomy, incomplete lineage sorting or hybridization. At least 0.5% of the sequences (2.5% of the sampled species) retrieved from GenBank were associated with database errors (poor‐quality sequences, NuMTs, misidentification or unnoticed hybridization). Paraphyletic species (5.8% of the total) can be related to rapid speciation events leading to nonreciprocal monophyly between recently diverged sister species, or to absence of synapomorphies in the small COI region analysed. We also performed two series of genetic distance calculations under the K2P model for intraspecific and interspecific comparisons: the first included all COI sequences, and the second included only monophyletic taxa observed in the Bayesian trees. As expected, the mean and median pairwise distances were smaller for intraspecific than for interspecific comparisons. However, there was no precise ‘barcode gap’, which was shown to be larger in the monophyletic taxon data set than for the data from all species, as expected. Our results indicated that although database errors may explain some of the difficulties in the species discrimination of Neotropical birds, distance‐based barcode assignment may also be compromised because of the high diversity of bird species and more complex speciation events in the Neotropics.

Genetic Structure Is Associated with Phenotypic Divergence in Floral Traits and Reproductive Investment in a High-Altitude Orchid from the Iron Quadrangle, Southeastern Brazil

Knowledge of the role of Neotropical montane landscapes in shaping genetic connectivity and local adaptation is essential for understanding the evolutionary processes that have shaped the extraordinary species diversity in these regions. In the present study, we examined the landscape genetics, estimated genetic diversity, and explored genetic relationships with morphological variability and reproductive strategies in seven natural populations of Cattleya liliputana (Orchidaceae). Nuclear microsatellite markers were used for genetic analyses. Spatial Bayesian clustering and population-based analyses revealed significant genetic structuring and high genetic diversity (He = 0.733 ± 0.03). Strong differentiation was found between populations over short spatial scales (FST = 0.138, p < 0.001), reflecting the landscape discontinuity and isolation. Monmonier´s maximum difference algorithm, Bayesian analysis on STRUCTURE and principal component analysis identified one major genetic discontinuity between populations. Divergent genetic groups showed phenotypic divergence in flower traits and reproductive strategies. Increased sexual reproductive effort was associated with rock outcrop type and may be a response to adverse conditions for growth and vegetative reproduction. Here we discuss the effect of restricted gene flow, local adaptation and phenotypic plasticity as drivers of population differentiation in Neotropical montane rock outcrops.

Microsatellite markers for Vellozia gigantea (Velloziaceae), a narrowly endemic species to the Brazilian campos rupestres

PREMISE OF THE STUDY Microsatellite primers were developed for the first time in Velloziaceae, in the endangered species Vellozia gigantea. METHODS AND RESULTS Using two different protocols, seven primer sets were characterized in three populations of V. gigantea. The primers amplified di- and trinucleotide repeats with six to 12 alleles per locus. These revealed high levels of genetic variation, presenting an average observed heterozygosity of 0.508 in V. gigantea. The seven primers were tested for cross-amplification in three Vellozia species. All primers successfully amplified in V. auriculata. Six primers amplified in V. compacta and three in V. hirsuta. CONCLUSIONS The new marker set described here will be useful for studies of population genetics of V. gigantea. The cross-amplification results indicate the utility of primers for studies in other Vellozia species.