Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family Sarcocystidae

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 infe rred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny to ols 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 g roup for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particu larly based on small-subunit ribosomal DNA, have not resolved all of the re lationships 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 th is in mind, we tested the informativeness of our chosen molecule, the large -subunit ribosomal DNA (Isu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The s egments of sequence applied correspond to areas of higher nucleotide variat ion in a secondary-structure alignment involving 21 taxa. We found that sub division of the entire Isu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the Isu rDNA for certain clades, such as the one encompassing the subfamily To xoplasmatinae. Consequently, the removal of any segment of the alignment co mpromises the final tree topology. We also tested the effect of using two d ifferent 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 alignme nt based on primary structure may not be comparing homologous nucleotides e ven though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammon dia and again bring to question the relationships of Sarcocystis muris, Iso spora felis, and Neospora caninum. In addition, results based on phylogenet ic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monop hyletic with Sarcocystis species using mammalian definitive hosts if the ge nus Frenkelia is synonymized with Sarcocystis.