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Item Specific boundaries between the causal agents of the soybean stem canker(Tropical Plant Pathology, 2014-07) Oliveira, Luiz Orlando de; Guillin, Eduardo A.; Grijalba, Pablo E.; Gottlieb, Alexandra M.Pathogens within the Diaporthe complex cause seed decay, stem blight and stem canker on soybean, representing a serious threat for this crop species. We herein utilize worldwide sequence data retrieved from Genbank in order to assess the species boundaries between the soybean stem canker causal agents, and define whether or not they should be regarded as members of the same biological species. These studies were complemented with compatibility tests, in order to validate our findings from a biological standpoint. Species delimitation assays supported the occurrence of a speciation event between D. caulivora and D. phaseolourm var. meridionalis. A speciation hypothesis between D. aspalathi and D. phaseolourm var. meridionalis was also supported, based on three reciprocally monophyletic substitutions at locus EF1-α. Compatibility tests further validated species delimitation assays indicating that D. caulivora has developed barriers to gene exchange with D. phaseolorum var. meridionalis. Clarification of the specific boundaries of the SSC pathogens and related entities will be an important asset to future research in soybean pathology, epidemiology and breeding.Item Specific boundaries between the causal agents of the soybean stem canker(Tropical Plant Pathology, 2014-07) Oliveira, Luiz Orlando de; Guillin, Eduardo A.; Grijalba, Pablo E.; Gottlieb, Alexandra M.Pathogens within the Diaporthe complex cause seed decay, stem blight and stem canker on soybean, representing a serious threat for this crop species. We herein utilize worldwide sequence data retrieved from Genbank in order to assess the species boundaries between the soybean stem canker causal agents, and define whether or not they should be regarded as members of the same biological species. These studies were complemented with compatibility tests, in order to validate our findings from a biological standpoint. Species delimitation assays supported the occurrence of a speciation event between D. caulivora and D. phaseolourm var. meridionalis. A speciation hypothesis between D. aspalathi and D. phaseolourm var. meridionalis was also supported, based on three reciprocally monophyletic substitutions at locus EF1-α. Compatibility tests further validated species delimitation assays indicating that D. caulivora has developed barriers to gene exchange with D. phaseolorum var. meridionalis. Clarification of the specific boundaries of the SSC pathogens and related entities will be an important asset to future research in soybean pathology, epidemiology and breeding.Item Diversidade genética estimada com marcadores entre sequências simples repetidas em cultivos comerciais de Cupuaçuzeiro(Ciência Rural, 2016-01) Oliveira, Luiz Orlando de; Silva, Bruna Mezzalira da; Rossi, Ana Aparecida Bandini; Dardengo, Juliana de Freitas Encinas; Araujo, Vitor Arreguy Amado Correa de; Rossi, Fernanda Saragosa; Clarindo, Wellington RonildoQuinze primers ISSR (entre sequências simples repetidas) foram utilizados para avaliar a diversidade genética entre e dentro de pomares comerciais de Theobroma grandiflorum (Willd. ex Spreng.) K. Schum. Para isso, foram analisados sessenta indivíduos, distribuídos nos três cultivos. Um total de 102 bandas foi amplificado, com uma porcentagem de 52,0% de polimorfismo em nível de espécie e média de 6,8 alelos por primer ISSR. A média do Índice de Conteúdo Polimórfico (PIC) foi de 0,55. Em relação aos índices de diversidade gênica de Nei (H) e de Shannon (I), os cultivos analisados apresentaram os valores: SAR H = 0,114 e I = 0,177; SSL H = 0,108 e I = 0,162 e SEC H = 0,104 e I = 0,156, considerados valores de moderados a baixos. A AMOVA revelou 34,91% da variância total entre os cultivos e 65,09% dentro deles. Os marcadores moleculares ISSR revelaram que há diversidade genética dentro de cada cultivo comercial estudado, portanto é possível selecionar genótipos superiores que poderão ser utilizados para originar cultivos mais uniformes. Esse resultado tem sido considerado de grande relevância, por fornecer ferramentas para a implementação de programas de melhoramento e delineamento de estratégias de conservação ex situ e in situ.Item More Cercospora species infect soybeans across the Americas than meets the eye(Plos One, 2015-08-07) Soares, Ana Paula Gomes; Guillin, Eduardo A.; Borges, Leandro Luiz; Silva, Amanda C. T. da; Almeida, Álvaro M. R. de; Grijalba, Pablo E.; Gottlieb, Alexandra M.; Bluhm, Burton H.; Oliveira, Luiz Orlando deDiseases of soybean caused by Cercospora spp. are endemic throughout the world’s soybean production regions. Species diversity in the genus Cercospora has been underestimated due to overdependence on morphological characteristics, symptoms, and host associations. Currently, only two species (Cercospora kikuchii and C. sojina) are recognized to infect soybean; C. kikuchii causes Cercospora leaf blight (CLB) and purple seed stain (PSS), whereas C. sojina causes frogeye leaf spot. To assess cryptic speciation among pathogens causing CLB and PSS, phylogenetic and phylogeographic analyses were performed with isolates from the top three soybean producing countries (USA, Brazil, and Argentina; collectively accounting for ~80% of global production). Eight nuclear genes and one mitochondrial gene were partially sequenced and analyzed. Additionally, amino acid substitutions conferring fungicide resistance were surveyed, and the production of cercosporin (a polyketide toxin produced by many Cercospora spp.) was assessed. From these analyses, the long-held assumption of C. kikuchii as the single causal agent of CLB and PSS was rejected experimentally. Four cercosporin-producing lineages were uncovered with origins (about 1 Mya) predicted to predate agriculture. Some of the Cercospora spp. newly associated with CLB and PSS appear to represent undescribed species; others were not previously reported to infect soybeans. Lineage 1, which contained the ex-type strain of C. kikuchii, was monophyletic and occurred in Argentina and Brazil. In contrast, lineages 2 and 3 were polyphyletic and contained wide-host range species complexes. Lineage 4 was monophyletic, thrived in Argentina and the USA, and included the generalist Cercospora cf. flagellaris. Interlineage recombination was detected, along with a high frequency of mutations linked to fungicide resistance in lineages 2 and 3. These findings point to cryptic Cercospora species as underappreciated global considerations for soybean production and phytosanitary vigilance, and urge a reassessment of host-specificity as a diagnostic tool for Cercospora.Item Molecular evidence of cryptic speciation, historical range expansion, and recent intraspecific hybridization in the Neotropical seasonal forest tree Cedrela fissilis (Meliaceae)(Elsevier Molecular Phylogenetics and Evolution, 2011-09-10) Garcia, Magali Gonçalves; Silva, Roberta Santos; Carniello, Maria Antonia; Veldman, Joseph William; Rossi, Ana Aparecida Bandini; Oliveira, Luiz Orlando deMolecular phylogeography can lead to a better understanding of the interaction between past climate events, large-scale vegetation shifts, and the evolutionary history of Neotropical seasonal forests. The endangered timber tree species Cedrela fissilis is associated with seasonal forests and occurs throughout South America. We sampled C. fissilis from 56 sites across the species’ range in Brazil and Bolivia and obtained sequence data for nuclear and chloroplast DNA. Most specimens (149 out of 169) exhibited intraindividual polymorphism for the nuclear internal transcribed spacer (ITS). Cloning and an array of complementary sequence analyses indicated that the multiple copies of ITS were functional paralogs – concerted evolution in C. fissilis appeared to be incomplete. Independent Bayesian analyses using either ITS or cpDNA data revealed two separate phylogenetic lineages within C. fissilis that corresponded to populations located in separate geographic regions. The divergence occurred in the Early Pliocene and Late Miocene. We argue that climate-mediated events triggered dispersal events and split ancestral populations into at least two large refugial areas of seasonal forest that were located to the east and west of the present day Cerrado. Upon recent climate amelioration, formerly isolated lineages reconnected and intraspecific hybridization gave rise to intraindividual polymorphism and incomplete concerted evolution in C. fissilis.Item Molecular evidence of hybrid zones of Cedrela (Meliaceae) in the Yungas of Northwestern Argentina(Molecular Phylogenetics and Evolution, 2016-05-19) Zelener, Noga; Tosto, Daniela; Oliveira, Luiz Orlando de; Soldati, María Cristina; Inza, María Virginia; Fornes, Luis FernandoIn the Yungas of Northwestern Argentina, three endangered species of Cedrela (C. angustifolia, C. saltensis, and C. balansae) follow altitudinal gradients of distribution with contact zones between them. We sampled 210 individuals from 20 populations that spanned most of Cedrela’s geographical range in the Yungas, and used Amplified Fragment Length Polymorphism (AFLP) markers and DNA sequences of the nuclear Internal Transcribed Spacer (ITS) to investigate hybrid zones. Data analyses employed an array of complementary methods, including principal coordinate analyses, Bayesian clustering analyses, maximum likelihood tree-building, and network techniques. Both nuclear molecular systems – AFLP and ITS – provided insights into the evolutionary history of Cedrela in the Yungas in a congruent manner. We uncovered strong support for the occurrence of natural hybridization between C. balansae and C. saltensis. Additionally, we identified hybrid zones in areas of sympatry (at both the Calilegua National Park and the San Andrés farm) and in transition zones from 820 to 1100 meters above sea level (localities of Pintascayo and Acambuco). There was no evidence for hybridization of either C. balansae or C. saltensis with C. angustifolia. The role of hybrid populations in conservation and use of genetic resources in the Yungas were discussed.Item Molecular phylogeography of Carapichea ipecacuanha, an amphitropical shrub that occurs in the understory of both semideciduous and evergreen forests(Molecular Ecology, 2010-01-24) Oliveira, Luiz Orlando de; Rossi, Ana Aparecida Bandini; Martins, Ernane Ronie; Batista, Flávia Reis de Carvalho; Silva, Roberta SantosThe medicinal shrub Carapichea ipecacuanha (ipecac) is an amphitropic species with three disjunct areas of distribution. In the Brazilian Atlantic and Amazonian ranges, the species was associated mostly with the understory of seasonal semideciduous forests, whereas in the Central American–Colombian range, the species occurred in the understory of moist evergreen forests. We examined the phylogeographic structure of ipecac using chloroplast trnT-trnL and nuclear internal transcribed spacer (ITS) sequences from 120 and 46 specimens, respectively. To complement existing data on root alkaloid profiles, we used high-performance liquid chromatography to assess the levels of emetine and cephaeline in 33 specimens from the two Brazilian ranges. The three ranges shared neither nuclear nor chloroplast haplotypes. The phylogeographic structures showed an uneven distribution of genetic diversity, sharp breaks and high levels of genetic differentiation among ranges. Our results suggest that the extant populations are descendents of at least four distinct ancestral lineages. The Atlantic ipecacs showed higher levels of genetic diversity than ipecacs from the other two ranges; it is likely that they derive from two ancestral lineages, with long-term persistence in that region. The Amazonian ipecacs were monomorphic with respect to the ITS and cpDNA sequences, which supports the view that there was a recent expansion from a single parental source after a strong genetic bottleneck. The existence of a fourth distinct lineage is apparent from the high levels of genetic and chemical differentiation that we identified in the Central American–Columbian ipecacs.Item Mesoamerican origin and pre- and post-columbian expansions of the ranges of Acanthoscelides obtectus say, a cosmopolitan insect pest of the common bean(PLOS ONE, 2013-07-31) Oliveira, Márcia Rodrigues Carvalho; Corrêa, Alberto Soares; Souza, Giselle Anselmo de; Guedes, Raul Narciso Carvalho; Oliveira, Luiz Orlando deAn unprecedented global transfer of agricultural resources followed the discovery of the New World; one consequence of this process was that staple food plants of Neotropical origin, such as the common bean (Phaseolus vulgaris), soon expanded their ranges overseas. Yet many pests and diseases were also transported. Acanthoscelides obtectus is a cosmopolitan seed predator associated with P. vulgaris. Codispersal within the host seed seems to be an important determinant of the ability of A. obtectus to expand its range over long distances. We examined the phylogeographic structure of A. obtectus by (a) sampling three mitochondrial gene sequences (12s rRNA, 16s rRNA, and the gene that encodes cytochrome c oxidase subunit I (COI)) throughout most of the species’ range and (b) exploring its late evolutionary history. Our findings indicate a Mesoamerican origin for the current genealogical lineages of A. obtectus. Each of the two major centers of genetic diversity of P. vulgaris (the Andes and Mesoamerica) contains a highly differentiated lineage of the bean beetle. Brazil has two additional, closely related lineages, both of which predate the Andean lineage and have the Mesoamerican lineage as their ancestor. The cosmopolitan distribution of A. obtectus has resulted from recent expansions of the two Brazilian lineages. We present additional evidence for both pre-Columbian and post-Columbian range expansions as likely events that shaped the current distribution of A. obtectus worldwide.Item Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust(Genetics and Molecular Biology, 2010-06-01) Maciel, Talles Eduardo Ferreira; Freire, Maíra Cristina Menezes; Almeida, Álvaro M.R. de; Oliveira, Luiz Orlando deβ-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His 6 , Glu 198 and Phe 200 , were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.