DendroSSR: SSRs and sequence alignment as tools for building phylogeny trees / DendroSSR: SSRs e alinhamento de sequência como ferramentas para a construção de árvores filogenéticas

Abstract This study introduces a new method to construct phylogenetic trees by combining both of the Simple Sequence Repeats (SSRs) and sequence alignments. The purpose of this work is to present the DendroSSR program and show it via a case study involving diverse Aspergillus species. To show how the DendroSSR program works to resolve complicated species relationships in phylogenetic trees, we employed the Aspergillus species as an example of a research case. The DendroSSR employs a technique containing multiple phases beginning with, detecting SSRs, computing SSRs similarities, sequences alignment, building a distance matrix based on SSRs similarity and sequences alignments, and then hierarchical clustering, and presenting the findings in a dendrogram. Sometimes sequence alignments alone may not give adequate information to generate a phylogenetic tree to resolve complicated species relationships. Therefore, establishing a distance matrix that is formed of addition of SSRs similarity across sequences to the traditional sequence alignment helps the process substantially and resolves the connections of complex species on phylogenetic trees. Additionally, it may be hard to distinguish complex relationships across species when studying conserved sequences, which could lead to an incomplete representation of their evolutionary relationships. These limitations are addressed by DendroSSR, which offers a technique to produce phylogenetic trees by incorporating SSRs similarity across species into the approach of generating phylogenetic trees. As it is known, SSRs are extensively scattered across the genomes of species and exhibit a great variation. Therefore, SSRs may support the knowledge gathered from sequence alignments by providing more information on genetic variation and even evolutionary relationships. The use of DendroSSR analysis might be considered for creating phylogenetic trees as a complementary or secondary strategy among the species under examination in circumstances where traditional phylogenetic analysis fails to clarify the species complex phylogenetic relationships.

Resumo Este estudo apresenta um novo método para construir árvores filogenéticas, combinando tanto as Sequências Repetitivas Simples (SSRs) quanto os alinhamentos de sequência. O objetivo deste trabalho é apresentar o programa DendroSSR por meio de um estudo de caso envolvendo diversas espécies de Aspergillus. Para mostrar como o programa DendroSSR funciona a fim de resolver relações complicadas de espécies em árvores filogenéticas, empregamos a espécie Aspergillus como exemplo de caso de pesquisa. O DendroSSR utiliza uma técnica contendo várias fases, começando com detecção de SSRs, computação de similaridades de SSRs, alinhamento de sequências, construção de uma matriz de distância baseada na similaridade de SSRs e alinhamentos de sequências e, em seguida, agrupamento hierárquico e apresentação das descobertas em um dendrograma. Às vezes, os alinhamentos de sequência sozinhos podem não fornecer informações adequadas para gerar uma árvore filogenética a fim de resolver relações complicadas de espécies. Portanto, estabelecer uma matriz de distância, que é formada pela adição de similaridade de SSRs entre sequências ao alinhamento de sequência tradicional, ajuda substancialmente o processo e resolve as conexões de espécies complexas em árvores filogenéticas. Além disso, pode ser difícil distinguir relações complexas entre espécies ao estudar sequências conservadas, o que pode levar a uma representação incompleta de suas relações evolutivas. Essas limitações são abordadas pelo DendroSSR, que oferece uma técnica para produzir árvores filogenéticas ao incorporar a similaridade de SSRs entre as espécies na abordagem de geração de árvores filogenéticas. Como se sabe, os SSRs estão amplamente dispersos nos genomas das espécies e exibem grande variação. Portanto, os SSRs podem apoiar o conhecimento obtido a partir de alinhamentos de sequências, fornecendo mais informações sobre variação genética e até mesmo relações evolutivas. O uso da análise DendroSSR pode ser considerado para a criação de árvores filogenéticas como uma estratégia complementar ou secundária entre as espécies sob exame em circunstâncias em que a análise filogenética tradicional falha em esclarecer as complexas relações filogenéticas das espécies.

Insights about the epidemiology of Salmonella Typhimurium isolates from different sources in Brazil using comparative genomics.

BACKGROUND: Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic agent worldwide. The aim of this work was to compare genetically 117 S. Typhimurium isolated from different sources over 30 years in Brazil using different genomics strategies. RESULTS: The majority of the 117 S. Typhimurium strains studied were grouped into a single cluster (≅ 90%) by the core genome multilocus sequence typing and (≅ 77%) by single copy marker genes. The phylogenetic analysis based on single nucleotide polymorphism (SNP) grouped most strains from humans into a single cluster (≅ 93%), while the strains isolated from food and swine were alocated into three clusters. The different orthologous protein clusters found for some S. Typhimurium isolated from humans and food are involved in metabolic and regulatory processes. For 26 isolates from swine the sequence types (ST) 19 and ST1921 were the most prevalent ones, and the ST14, ST64, ST516 and ST639 were also detected. Previous results typed the 91 S. Typhimurium isolates from humans and foods as ST19, ST313, ST1921, ST3343 and ST1649. The main prophages detected were: Gifsy-2 in 79 (67.5%) and Gifsy-1 in 63 (54%) strains. All of the S. Typhimurium isolates contained the acrA, acrB, macA, macB, mdtK, emrA, emrB, emrR and tolC efflux pump genes. CONCLUSIONS: The phylogenetic trees grouped the majority of the S. Typhimurium isolates from humans into a single cluster suggesting that there is one prevalent subtype in Brazil. Regarding strains isolated from food and swine, the SNPs' results suggested the circulation of more than one subtype over 30 years in this country. The orthologous protein clusters analysis revealed unique genes in the strains studied mainly related to bacterial metabolism. S. Typhimurium strains from swine showed greater diversity of STs and prophages in comparison to strains isolated from humans and foods. The pathogenic potential of S. Typhimurium strains was corroborated by the presence of exclusive prophages of this serovar involved in its virulence. The high number of resistance genes related to efflux pumps is worrying and may lead to therapeutic failures when clinical treatment is needed.

Bootstrap and Rogue Identification Tests for Phylogenetic Analyses.

Most phylogenetic tree-generating programs produce a fully dichotomous phylogenetic tree. However, as different markers may produce distinct topologies for the same set of organisms, topological tests are used to estimate the statistical reliability of the clades. In this protocol, we provide step-by-step instructions on how to perform the widely used bootstrap test using MEGA. However, a single unstable lineage, also known as a rogue lineage, may decrease the bootstrap proportions in many branches of the tree. This occurs because rogue taxa tend to bounce between clades from one pseudo-replicate to the next, lowering bootstrap proportions for many correct clades. Thus, it is important to identify and exclude rogue taxa before initiating a final phylogenetic analysis; here, we provide this protocol using the RogueNaRok platform.

The Reliability and Stability of an Inferred Phylogenetic Tree from Empirical Data.

The reliability of a phylogenetic tree obtained from empirical data is usually measured by the bootstrap probability (Pb) of interior branches of the tree. If the bootstrap probability is high for most branches, the tree is considered to be reliable. If some interior branches show relatively low bootstrap probabilities, we are not sure that the inferred tree is really reliable. Here, we propose another quantity measuring the reliability of the tree called the stability of a subtree. This quantity refers to the probability of obtaining a subtree (Ps) of an inferred tree obtained. We then show that if the tree is to be reliable, both Pb and Ps must be high. We also show that Ps is given by a bootstrap probability of the subtree with the closest outgroup sequence, and computer program RESTA for computing the Pb and Ps values will be presented.

Morphometric and molecular differentiation between quetzal subspecies of Pharomachrus mocinno (Trogoniformes: Trogonidae)

The resplendent Quetzal (Pharomachrus mocinno) is an endemic Mesoamerican bird species of conservation concern. Within this species, the subspecies P. m. costaricensis and P. m. mocinno, have been recognized by apparent morphometric differences; however, presently there is no sufficient data for confirmation. We analyzed eight morphometric attributes of the body from 41 quetzals: body length, tarsus and cord wing, as well as the length, wide and depth of the bill, body weight; and in the case of the males, the length of the long upper-tail cover feathers. We used multivariate analyses to discriminate morphometric differences between subspecies and contrasted each morphometric attribute between and within subspecies with paired non-parametric Wilcoxon test. In order to review the intraspecific taxonomic status of this bird, we added phylogenetic analysis, and genetic divergence and differentiation based on nucleotide variations in four sequences of mtDNA. The nucleotide variation was estimated in control region, subunit NDH6, and tRNA Glu and tRNA Phe in 26 quetzals from eight localities distributed in five countries. We estimated the genetic divergence and differentiation between subspecies according to a mutation-drift equilibrium model. We obtained the best mutation nucleotide model following the procedure implemented in model test program. We constructed the phylogenetic relationships between subspecies by maximum parsimony and maximum likelihood using PAUP, as well as with Bayesian statistics. The multivariate analyses showed two different morphometric groups, and individuals clustered according to the subspecies that they belong. The paired comparisons between subspecies showed strong differences in most of the attributes analyzed. Along the four mtDNA sequences, we identified 32 nucleotide positions that have a particular nucleotide according to the quetzals subspecies. The genetic divergence and the differentiation was strong and markedly showed two groups within P. mocinno that corresponded to the quetzals subspecies. The model selected for our data was TVM+G. The three phylogenetic methods here used recovered two clear monophyletic clades corresponding to each subspecies, and evidenced a significant and true partition of P. mocinno species into two different genetic, morphometric and ecologic groups. Additionally, according to our calculations, the gene flow between subspecies is interrupted at least from three million years ago. Thus we propose that P. mocinno be divided in two independent species: P. mocinno (Northern species, from Mexico to Nicaragua) and in P. costaricensis (Southern species, Costa Rica and Panama). This new taxonomic classification of the quetzal subspecies allows us to get well conservation achievements because the evaluation about the kind and magnitude of the threats could be more precise. Rev. Biol. Trop. 58 (1): 357-371. Epub 2010 March 01.

El Quetzal (Pharomachrus mocinno) es un ave endémica mesoamericana de interés en conservación. Dentro de esta especie, se reconocen a las subespecies P. m. costaricensis y P. m. mocinno por aparentes diferencias morfométricas, sin embargo, hasta el momento no hay datos suficientes que las confirmen. En este estudio, analizamos ocho rasgos morfométricos de 41 quetzales: la longitud del cuerpo, del tarso y de la cuerda alar, así como la longitud, el ancho y la profundidad del pico, el peso corporal, y en el caso de los machos, la longitud de las plumas cobertoras supracaudales. Usamos análisis multivariados para discriminar diferencias morfométricas entre las subespecies. Comparamos cada rasgo morfométrico dentro y entre las subespecies a partir de comparaciones pareadas con el análisis no-paramétrico de Wilcoxon. Realizamos análisis filogenéticos, y de diferenciación y divergencia genéticas fundamentados en las variaciones nucleotídicas de cuatro secuencias de ADNm con la finalidad de revisar el estatus taxonómico de esta ave. La variación nucleotídica fue estimada en la región control, la subunidad NDH6 y los tRNA Glu y tRNA Phe en 26 quetzales de ocho localidades de cinco países. Estimamos la divergencia y la diferenciación genética entre subespecies con base en el modelo de equilibrio mutación-deriva. Obtuvimos el mejor modelo de mutación nucleotídica siguiendo el procedimiento implementado en el programa Model test. Construimos las relaciones filogenéticas entre las subespecies con máxima parsimonia y máxima verosimilitud usando PAUP, así con estadística Bayesiana. Los análisis multivariados discriminaron dos grupos morfométricos, y los individuos se agruparon de acuerdo con la subespecie a la que pertenecen. Las comparaciones pareadas entre las subespecies mostraron fuertes diferencias en la mayoría de los rasgos analizados. En las cuatro secuencias de ADNmt identificamos 32 posiciones nucleotídicas que tienen un nucleótido particular de acuerdo con la subespecie de quetzal. La divergencia genética y la diferenciación fueron marcadas y mostraron dos grupos dentro de P. mocinno que correspondieron a las subespecies de quetzales. El modelo seleccionado para nuestros datos fue el TVM+G. Los tres métodos filogenéticos usados recuperaron dos clados monofiléticos robustos correspondiendo a cada una de las subespecies. Consideramos que nuestros resultados muestran una significativa y real división de P. mocinno en dos grupos genéticos, morfométricos y ecológicos. Además de acuerdo con nuestras estimaciones, el flujo génico está interrumpido entre las subespecies desde al menos hace tres millones de años. Por ello, proponemos que P. mocinno sea dividido en dos especies independientes: P. mocinno (especie norteña, desde México hasta Nicaragua) y P. costaricensis (especie sureña, Costa Rica y Panamá). Esta nueva clasificación de las subespecies de quetzal permitirá mejores logros en su conservación, dado que la evaluación de la clase y magnitud de las amenazas serán más precisas.

Construção de uma biblioteca de módulos para utilização na composição de workflows para filogenia / Construction of a library of modules for use in the composition of workflows for phylogeny

Com o advento da era pós-genômica, ocorreu uma explosão de informações onde inúmeras descobertas geraram grande quantidade de dados biológicos, que para serem analisados, necessitavam da cooperação de várias áreas de conhecimento. Inicialmente, as atividades de análises destes dados são suportadas por programas que constituem um fluxo de trabalho, baseado em scripts, que normalmente são executados por linha de comando, obrigando os seus usuários a terem domínio de algoritmos e lógica de programação. Tais scripts auxiliam muito na entrada, processamento e resultado final da análise, mas ainda apresentam dificuldades em interferir, coletar e armazenar dados ao longo de sua execução. Além disso, dependendo da especificidade do script, o seu uso pode ser muito complexo, em função da dificuldade da implementação, manutenção e reuso. Também, neste tipo de ambiente, o registro de execução das atividades do fluxo, da origem dos dados utilizados e das transformações aplicadas aos dados, geralmente, não são mantidos. Para tanto, tem havido o crescente uso de workflows científicos na execução e condução de experimentos científicos. Os workflows científicos pressupõem a resolução de problemas científicos através das técnicas de composição do fluxo de atividades, onde os passos normalmente são compostos por programas de bioinformática que recebem, processam e geram um conjunto de dados que podem ser repassados aos demais passos do workflow. Toda a estrutura de desenvolvimento e execução desses workflows é apoiada por sistemas específicos, conhecidos como Sistemas de Gerenciamento de Workflows Científicos (SGWfC), que possuem seus próprios mecanismos de gerência e linguagem. Considerando as vantagens de uso dos SGWfC no cenário da Bioinformática, este trabalho apresenta o workflow científico para reconstrução filogenética denominado PHYLO...

With the advent of post-genomic era, there was an explosion of information where many discoveries have generated large amounts of biological data, which, to be analyzed, needed the cooperation of various fields of knowledge. Initially, the to industrial activities of analysis of these data are supported by programs that constitute the workflow, based on scripts, that normally run from command line, forcing users to algorithms and programming logic. Such scripts help much the input, processing and outcome of the analysis, but still present difficulties for usersto interfere, collect and store data throughout their implementation. Also, according to the specific use of the script, it can be very complex, depending on the difficultyof implementation, maintenance and reuse. Also, in this type of environment, the registration of the execution of the activities of the flow, the source of data use d and the transformations applied to the data are generally not retained. For these, there has been the growing use of scientific workflows for the implementation and execution of scientific experiments. Scientific workflows assume scientificproblems solving through techniques of composition of the flow of activities, where the steps are usually composed of bioinformatics programs that receive, process and generate a data set that can be passed on to other steps of the workflow. The structure of development and implementation of these workflows is supported by specific systems, known as Scientific Workflows Management Systems (SGWfC), which have their own management mechanisms and language. Considering theadvantages of using the scenario SWfMS in the scientific bioinformatics, this work presents the scientific workflow PHYLO for phylogenetic reconstruction...

An empirical test of the midpoint rooting method.

The outgroup method is widely used to root phylogenetic trees. An accurate root indication, however, strongly depends on the availability of a proper outgroup. An alternate rooting method is the midpoint rooting (MPR). In this case, the root is set at the midpoint between the two most divergent operational taxonomic units. Although the midpoint rooting algorithm has been extensively used, the efficiency of this method in retrieving the correct root remains untested. In the present study, we empirically tested the success rate of the MPR in obtaining the outgroup root for a given phylogenetic tree. This was carried out by eliminating outgroups in 50 selected data sets from 33 papers and rooting the trees with the midpoint method. We were thus able to compare the root position retrieved by each method. Data sets were separated into three categories with different root consistencies: data sets with a single outgroup taxon (54% success rate for MPR), data sets with multiple outgroup taxa that showed inconsistency in root position (82% success rate), and data sets with multiple outgroup taxa in which root position was consistent (94% success rate). Interestingly, the more consistent the outgroup root is, the more successful MPR appears to be. This is a strong indication that the MPR method is valuable, particularly for cases where a proper outgroup is unavailable.

PHYLOGENETIC ESTIMATION OF PLASMID EXCHANGE IN BACTERIA.

The existence of differential horizontal gene transfer may be assessed by comparing the phylogenetic trees derived from two different genes. We use this concept to estimate quantitatively the amount of plasmid exchange that has occurred in a bacterial population. By means of computer simulations we studied the effect of gene transfer on the topological distortion between two phylogenetic trees: one obtained from an euchromosomal gene and another from a plasmid-borne sequence, which may be subjected to horizontal transfer. The basic assumptions of our simulations were (a) that plasmid exchange had occurred recently (after the last population split); and (b) that either the amount of chromosomal horizontal exchange was negligible or that it was only a fraction of the amount of plasmid exchange in which case we will be estimating relative amounts of plasmid transfer. We found that the topological difference between two such trees is a function of the number of plasmid exchange events that have occurred. It can be explained by a logistic model that relates the average distortion index between two trees (dT ) to the number of transfer events (x). The behavior remains the same under different conditions that were tested (symmetry of the topology, number of taxa in the tree, effect of reconstruction errors, mutation after plasmid transfer). We have also tried our method on empirical data from the literature and estimated the amount of gene transfer that may have occurred among Sym plasmids in agricultural field populations of Rhizobium leguminosarum biovar phaseoli. We found that between 15.77 to 29.98% of all genetic types in these populations have been either the source or the target of a plasmid transfer event. When the comparisons were made among trees derived exclusively from plasmid probes this value dropped to 2.00%. Phylogenetic trees derived from symbiotic and nonsymbiotic sequences were also used to infer the number of gene transfer events among 11 isolates from R. galegae. The estimated number of transfer events of symbiotic sequences was 10.515 (although we do not know out of how many genetic types). We concluded that intraspecific transfer of symbiotic sequences is widespread in these two species of the genus Rhizobium.