Effects of Sequence Alignment and Structural Domains of Ribosomal DNA on Phylogeny Reconstruction for the Protozoan Family Sarcocystidae

Molecular Biology and Evolution, Dec 2000

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.

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Effects of Sequence Alignment and Structural Domains of Ribosomal DNA on Phylogeny Reconstruction for the Protozoan Family Sarcocystidae

Effects of Sequence Alignment and Structural Domains of Ribosomal DNA on Phylogeny Reconstruction for the Protozoan Family Sarcocystidae N. B. Mugridge 2 3 D. A. Morrison 2 3 T. Ja¨ kel 0 2 A. R. Heckeroth 1 2 A. M. Tenter 1 2 A. M. Johnson 2 3 0 Division of Parasitology, University of Hohenheim , Stuttgart , Germany 1 Institut fu ̈r Parasitologie, Tiera ̈rztliche Hochschule Hannover , Hannover , Germany 2 ular Parasitology Unit, University of Technology , Sydney, Westbourne Street, Gore Hill, NSW 2065 , Australia 3 Molecular Parasitology Unit, University of Technology , Sydney, New South Wales , Australia Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis. Hammondia; large-subunit ribosomal DNA; Neospora; nucleotide sequence alignment; phylogeny; Sarcocystidae; Sarcocystis; Toxoplasma Introduction For taxa that cannot be unequivocally classified based on phenotypic markers, DNA sequencing provides another marker for the investigation of phylogenetic relationships (Olsen and Woese 1993) . The relative ease with which nucleotide sequences can be obtained today allows one to compare a large number of taxa based on nucleotide variations; hence, the phylogenetic informativeness of DNA now has the potential to outweigh that of the morphology or biology of an organism. However, the informativeness of the gene being used requires consideration if one is to make assumptions regarding species relationships (Olsen and Woese 1993) . One way of ensuring a robust phylogeny in which the species signal, rather than the gene signal, emerges is to analyze numerous genes. Consequently, the DNA sequence chosen for the construction of a phylogeny is a very important factor, due to the varying phylogenetic content among genes. The phylogenetic informativeness of a gene can be a factor of the function of its product or a result of its length. In some cases, such as that of the stems and loops of the ribosomal RNA, different parts of the gene can have varying information contents due to evolutionary selection pressures on secondary structure and function (Sogin and Gunderson 1986; Olsen 1987, 1988; Olsen and Woese 1993) . The different methods available for building a phylogenetic tree can also affect the outcome of the analysis. Tree-building algorithms have different aim (...truncated)


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N. B. Mugridge, D. A. Morrison, T. Jäkel, A. R. Heckeroth, A. M. Tenter, A. M. Johnson. Effects of Sequence Alignment and Structural Domains of Ribosomal DNA on Phylogeny Reconstruction for the Protozoan Family Sarcocystidae, Molecular Biology and Evolution, 2000, pp. 1842-1853, 17/12,