Delimiting genetic units in Neotropical toads under incomplete lineage sorting and hybridization

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

Phylogeography of Batrachospermum macrosporum (Batrachospermales, Rhodophyta) from North and South America

Phylogeographic trends in Batrachospermum macrosporum Mont. were investigated using the mitochondrial intergenic spacer between the cytochrome oxidase subunit 2 and 3 genes (cox2-3). A total of 11 stream segments were sampled with seven in the coastal plain of North America and four in tropical areas of South America. Fifteen thalli were sampled from seven streams, 14 thalli from two streams, and eight thalli from two streams. There were 16 haplotypes detected using 149 individuals. of the eight haplotypes from locations in North America, all were 334 base pairs (bp) in length, and of those from South America, five were 344 bp, and three were 348 bp. Two individual networks were produced: one for the haplotypes from North America and another for those from South America, and these could not be joined due to the large number of base pair differences. This split between haplotypes from North and South America was confirmed with sequence data of the rbcL gene. There was very little genetic variation among the haplotypes from the North American locations, leading us to hypothesize that these are fairly recent colonization events along the coastal plain. In contrast, there was high variation among haplotypes from South America, and it would appear that the Amazon serves as a center of diversity. We detected considerable variation in haplotypes among streams, but frequently, a single haplotype in an individual stream segment, which is consistent with data from previous studies of other batrachospermalean taxa, may suggest a single colonization event per stream.

SYSTEMATICS of THE BATRACHOSPERMALES (RHODOPHYTA)-A SYNTHESIS

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phylogenetic relationship of Sirodotia species (Batrachospermales, Rhodophyta) in North and South America

Sequence data from the RUBISCO large subunit (rbcL) plastid gene and nuclear small-subunit ribosomal DNA (SSU rDNA) were examined for five samples of Sirodotia delicatula from southeastern Brazil. Data from six North American samples previously identified as S. huillensis and S. suecica were also included in the analysis. Molecular data supported the continued recognition of these three species as separate entities, although one of the North American collections was misidentified. These results were shown to be congruent with morphology, chromosome number and geographic distribution. S. delicatula is more closely related to S. huillensis, both occurring in tropical-subtropical regions, than either to S. suecica with a temperate-boreal distribution. There was little rbcL variation within S. delicatula from Brazil and Costa Rica (the latter a collection previously identified as S. huillensis), with the six samples sequenced diverging from each other by 0-8 bp (0-0.67%). SSU rDNA data set did not provide sufficient resolution to infer phylogenetic relationships among the species of this group due to the low rates of variation (5 bp). Sirodotia was a well-supported clade (100% bootstrap or 1.00 a posteriori probability) based on rbcL sequences. Thus, the results confirm that Sirodotia is a monophyletic group within the Batrachospermales and we continue to recognize it at the generic level. The species S. delicatula, S. huillensis and S. suecica are morphologically and genetically distinct.

Molecular phylogeny of the green lacewings (Neuroptera : Chrysopidae)

The first quantitative analysis of phylogenetic relationships of green lacewings (Chrysopidae) is presented based on DNA sequence data. A single nuclear and two mitochondrial genes are used in the analysis: carbomoylphosphate synthase (CPS) domain of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) (i.e. rudimentary locus), large subunit ribosomal gene (16S) and cytochrome oxidase I (COI). This study represents the first use of the CAD gene to investigate phylogenetic relationships of the lacewings. DNA sequences for 33 chrysopid species from 18 genera, representing all subfamilies and tribes, were compared with outgroups sampled from families Hemerobiidae, Osmylidae and Polystoechotidae. Parsimony analyses of the combined data set recovered all of the previously established subfamilial and tribal groups as monophyletic clades (although relatively weakly supported) except Apochrysinae sensu lato. The enigmatic Nothancyla verreauxi Navas has historically been difficult to place in a subfamily group based on morphological characteristics; molecular data presented herein do not adequately resolve this problem.

Citrus and coffee strains of Xylella fastidiosa induce Pierce's disease in grapevine

Xylella fastidiosa causes citrus variegated chlorosis (CVC) disease in Brazil and Pierce's disease of grapevines in the United States. Both of these diseases cause significant production problems in the respective industries. The recent establishment of the glassy-winged sharpshooter in California has radically increased the threat posed by Pierces disease to California viticulture. Populations of this insect reach very high levels in citrus groves in California and move from the orchards into the vineyards, where they acquire inoculum and spread Pierce's disease in the vineyards. Here we show that strains of X. fastidiosa isolated from diseased citrus and coffee in Brazil can incite symptoms of Pierce's disease after mechanical inoculation into seven commercial Vitis vinifera varieties grown in Brazil and California. Thus, any future introduction of the CVC strains of X. fastidiosa into the United States would pose a threat to both the sweet orange and grapevine industries. Previous work has clearly shown that the strains of X. fastidiosa isolated from Pierce's disease- and CVC-affected plants are the most distantly related of all strains in the diverse taxon X. fastidiosa. The ability of citrus strains of X. fastidiosa to incite disease in grapevine is therefore surprising and creates an experimental system with which to dissect mechanisms used by X.,fastidiosa in plant colonization and disease development using the full genome sequence data that has recently become available for both the citrus and grapevine strains of this pathogen.

Phylogenetic Relationships in Eleocharis (Cyperaceae): C-4 Photosynthesis Origins and Patterns of Diversification in the Spikerushes

Eleocharis (Cyperaceae) is a morphologically and physiologically diverse lineage of 250 + species with a cosmopolitan distribution. We here explore phylogenetic relationships in this lineage using maximum parsimony and Bayesian inference analyses of nrDNA ITS and cpDNA trnC-ycf6 and ycf6-psbM sequence data with the goals of comparing our phylogenetic hypotheses to previous classifications, morphological variation, and photosynthetic pathway variation. Our results suggest that in Eleocharis C, photosynthesis has been derived at least three times, with several cases of possible reversion to C-3-like or intermediate pathways and several additional origins of C-3-C-4 intermediate photosynthetic pathways, as inferred by carbon isotope ratio measurements. Many classification units currently recognized in Eleocharis are not monophyletic, however, E. subgenus Limnochloa and E. subgenus Scirpidium are monophyletic. Other classification units largely corresponding to clades include E. subgenus Zinserlingia, E. subseries Chaetariae, and E. series Maculosae. Problems with species circumscription and morphological variation in several groups are discussed in light of the phylogeny, particularly in the context of species membership of seven focal clades found in the analyses.

MOLECULAR PHYLOGENY of THE GENUS KUMANOA (BATRACHOSPERMALES, RHODOPHYTA)

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

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

Background: Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.Methods: Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.Results: In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.Conclusion: This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B-bubalis) chromosome 20

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

Molecular phylogeny of microhylid frogs (Anura: Microhylidae) with emphasis on relationships among New World genera

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

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.