Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Phylogenetic Analyses Based on Small Subunit rRNA and Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Ultrastructural Characterization of Two Snake Trypanosomes: Trypanosoma serpentis n. sp from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.

A Phylogenetic Lineage of Closely Related Trypanosomes (Trypanosomatidae, Kinetoplastida) of Anurans and Sand Flies (Psychodidae, Diptera) Sharing the Same Ecotopes in Brazilian Amazonia

Analysis of the phylogenetic relationships among trypanosomes from vertebrates and invertebrates disclosed a new lineage of trypanosomes circulating among anurans and sand flies that share the same ecotopes in Brazilian Amazonia. This assemblage of closely related trypanosomes was determined by comparing whole SSU rDNA sequences of anuran trypanosomes from the Brazilian biomes of Amazonia, the Pantanal, and the Atlantic Forest and from Europe, North America, and Africa, and from trypanosomes of sand flies from Amazonia. Phylogenetic trees based on maximum likelihood and parsimony corroborated the positioning of all new anuran trypanosomes in the aquatic clade but did not support the monophyly of anuran trypanosomes. However, all analyses always supported four major clades (An01-04) of anuran trypanosomes. Clade An04 is composed of trypanosomes from exotic anurans. Isolates in clades An01 and An02 were from Brazilian frogs and toads captured in the three biomes studied, Amazonia, the Pantanal and the Atlantic Forest. Clade An01 contains mostly isolates from Hylidae whereas clade An02 comprises mostly isolates from Bufonidae; and clade An03 contains trypanosomes from sand flies and anurans of Bufonidae, Leptodactylidae, and Leiuperidae exclusively from Amazonia. To our knowledge, this is the first study describing morphological and growth features, and molecular phylogenetic affiliation of trypanosomes from anurans and phlebotomines, incriminating these flies as invertebrate hosts and probably also as important vectors of Amazonian terrestrial anuran trypanosomes.

Phylogenetic, morphological and behavioural analyses support host switching of Trypanosoma (Herpetosoma) lewisi from domestic rats to primates

We characterized four Brazilian trypanosomes isolated from domestic rats and three from captive nonhuman primates that were morphologically similar to T. lewisi, a considered non-pathogenic species restricted to rodents and transmitted by fleas, despite its potential pathogenicity for infants. These isolates were identified as T. lewisi by barcoding using V7V8 SSU rDNA sequences. In inferred phylogenetic trees, all isolates clustered tightly with reference T. lewisi and T. lewisi-like trypanosomes from Europe, Asia and Africa and despite their high sequence conservation formed a homogeneous clade separate from other species of the subgenus T. (Herpetosoma). With the aim of clearly resolving the relationships between the Brazilian isolates from domestic rats and primates, we compared sequences from more polymorphic ITS rDNA. Results corroborated that isolates from Brazilian rats and monkeys were indeed of the same species and quite close to T. lewisi isolates of humans and rats from different geographical regions. Morphology of the monkey isolates and their behaviour in culture and in experimentally infected rats were also compatible with T. lewisi. However, infection with T. lewisi is rare among monkeys. We have examined more than 200 free-ranging and 160 captive monkeys and found only three infected individuals among the monkeys held in captivity. The findings of this work suggest that proximity of monkeys and infected rats and their exposure to infected fleas may be responsible for the host switching of T. Iewisi from their natural rodent species to primates. This and previous studies reporting T. lewisi in humans suggest that this trypanosome can cause sporadic and opportunistic fleaborne infection in primates. (C) 2010 Elsevier B.V. All rights reserved.

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.

EthoSeq: A tool for phylogenetic analysis and data mining in behavioral sequences

This article introduces the software program called EthoSeq, which is designed to extract probabilistic behavioral sequences (tree-generated sequences, or TGSs) from observational data and to prepare a TGS-species matrix for phylogenetic analysis. The program uses Graph Theory algorithms to automatically detect behavioral patterns within the observational sessions. It includes filtering tools to adjust the search procedure to user-specified statistical needs. Preliminary analyses of data sets, such as grooming sequences in birds and foraging tactics in spiders, uncover a large number of TGSs which together yield single phylogenetic trees. An example of the use of the program is our analysis of felid grooming sequences, in which we have obtained 1,386 felid grooming TGSs for seven species, resulting in a single phylogeny. These results show that behavior is definitely useful in phylogenetic analysis. EthoSeq simplifies and automates such analyses, uncovers much of the hidden patterns of long behavioral sequences, and prepares this data for further analysis with standard phylogenetic programs. We hope it will encourage many empirical studies on the evolution of behavior.

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion. © The Mycological Society of Japan and Springer-Verlag 2005.

Nucleotide and phylogenetic analysis of human papillomavirus types 6 and 11 isolated from recurrent respiratory papillomatosis in Brazil

There are few studies about the distribution of natural molecular variants of low-risk HPVs. Our aim was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. We also performed a phylogenetic analysis in order to compare nucleotide sequences identified in our study with previously reported isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world to determine the phylogenetic relationships of variants detected in Brazil. The complete coding region of the E6 gene of 25 samples was cloned and sequenced: 18 isolates of HPV-6 (72%) and 7 isolates of HPV-11 (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants was identified. It was not possible to correlate specific variants with disease severity. Phylogenetic trees for both HPV types were constructed enclosing both E6 sequences detected in our study and formerly published sequences. In both phylogenetic trees, the sequences from Brazil did not group together. We could not establish a geographical association between HPV-6 or HPV-11 variants, unlike HPV-16 and HPV-18. © 2013 Elsevier B.V.

Phylogenetic relationships among Brazilian howler monkeys, genus Alouatta (Platyrrhini, Atelidae), based on γ¹-globin pseudogene sequences

Os guaribas, do gênero Alouatta, que são os primatas do Novo Mundo com maior distribuição geográfica, têm sido colocados em três grupos de espécies: o grupo Alouatta palliata da América central, e os grupos sulamericanos Alouatta seniculus e Alouatta caraya. Este último é monotípico, mas o grupo A. seniculus inclui pelo menos três espécies (A. seniculus, A. belzebul e A. fusca). Neste estudo, foram seqüenciados aproximadamente 600 pares de base do pseudogene globina g1 nas quatro espécies brasileiras (A. seniculus, A. belzebul, A. fusca e A. caraya). Os métodos de máxima parcimônia e máxima verossimilhança produziram árvores filogenéticas com o mesmo arranjo: {A. caraya [A. seniculus (A. fusca, A. belzebul)]}. A árvore mais parcimoniosa apresentou valores de bootstrap maiores de 82% para todos os agrupamentos, e valores de força de ligação de pelo menos 2, apoiando o agrupamento irmão de A. fusca e A. belzebul. O estudo também confirmou a presença em A. fusca do elemento de inserção Alu, com 150 pares de base, e uma deleção de 1,8 kb no pseudogene globina g1 já conhecidos nas demais espécies de guaribas. A classificação cladística baseada em dados moleculares é congruente com as de estudos morfológicos, com um isolamento claro do grupo monoespecífico A. caraya em relação ao grupo A. seniculus.

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp nov., from Atlantic Rainforest of southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PHYLOGENETIC STUDY OF GEITLERINEMA AND MICROCYSTIS ( CYANOBACTERIA) USING PC-IGS AND 16S-23S ITS AS MARKERS: INVESTIGATION OF HORIZONTAL GENE TRANSFER

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolutionary Insights from Bat Trypanosomes: Morphological, Developmental and Phylogenetic Evidence of a New Species, Trypanosoma (Schizotrypanum) erneyi sp nov., in African Bats Closely Related to Trypanosoma (Schizotrypanum) cruzi and Allied Species

Parasites of the genus Trypanosoma are common in bats and those of the subgenus Schizotrypanum are restricted to bats throughout the world, with the exception of Trypanosoma (Schizotrypanum) cruzi that also infects other mammals and is restricted to the American Continent. We have characterized trypanosome isolates from Molossidae bats captured in Mozambique, Africa. Morphology and behaviour in culture, supported by phylogenetic inferences using SSU (small subunit) rRNA, gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cyt b (cytochrome b) genes, allowed to classify the isolates as a new Schizotrypanum species named Trypanosoma (Schizotrypanum) erneyi sp. nov. This is the first report of a Schizotrypanum species from African bats cultured, characterized morphologically and biologically, and positioned in phylogenetic trees. The unprecedented finding of a new species of the subgenus Schizotrypanum from Africa that is closest related to the America-restricted Trypanosoma (Schizotrypanum) cruzi marinkellei and T. cruzi provides new insights into the origin and evolutionary history of T. cruzi and closely related bat trypanosomes. Altogether, data from our study support the hypothesis of an ancestor trypanosome parasite of bats evolving to infect other mammals, even humans, and adapted to transmission by triatomine bugs in the evolutionary history of T. cruzi in the New World. (c) 2012 Elsevier GmbH. All rights reserved.

Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

Background: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. Results: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. Conclusion: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.

Phylogenetic relationships between the Acantharea and the Polycystinea: A molecular perspective on Haeckel’s Radiolaria

Polycystine radiolaria are among few protistan groups that possess a comprehensive fossil record available for study by micropaleontologists. The Polycystinea and the Acantharea, whose skeletons do not become fossilized, were once members of the class “Radiolaria” (“Radiolaria” sensu lato: Polycystinea, Phaeodarea, and Acantharea) originally proposed by Haeckel but are now included in the superclass Actinopoda. Phylogenetic relationships within this superclass remain largely enigmatic. We investigated the evolutionary relationship of the Acantharea and the Polycystinea to other protists using phylogenetic analyses of 16S-like ribosomal RNA (rRNA) coding regions. We circumvented the need to culture these organisms by collecting and maintaining reproductive stages that contain many copies of their genomic DNA. This strategy facilitated extraction of genomic DNA and its purification from symbiont and prey DNA. Phylogenetic trees inferred from comparisons of 16S-like coding regions do not support a shared history between the Acantharea and the Polycystinea. However, the monophyly of the Acantharea and the separate monophyly of the Polycystinea (Spumellarida) are well supported by our molecular-based trees. The acantharian lineage branches among crown organisms whereas the polycystine lineage diverges before the radiation of the crown groups. We conclude that the Actinopoda does not represent a monophyletic evolutionary assemblage and recommend that this taxonomic designation be discarded.

Synthesizing and databasing fossil calibrations: divergence dating and beyond

Divergence dating studies, which combine temporal data from the fossil record with branch length data from molecular phylogenetic trees, represent a rapidly expanding approach to understanding the history of life. National Evolutionary Synthesis Center hosted the first Fossil Calibrations Working Group (3–6 March, 2011, Durham, NC, USA), bringing together palaeontologists, molecular evolutionists and bioinformatics experts to present perspectives from disciplines that generate, model and use fossil calibration data. Presentations and discussions focused on channels for interdisciplinary collaboration, best practices for justifying, reporting and using fossil calibrations and roadblocks to synthesis of palaeontological and molecular data. Bioinformatics solutions were proposed, with the primary objective being a new database for vetted fossil calibrations with linkages to existing resources, targeted for a 2012 launch.