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

Abstract

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.

Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences

“…as sister group of T. gondii/H. hammondi was also difficult to demonstrate with Hsp70-and 18S rRNA phylogenies (ELLIS et al, 1999;MUGRIDGE et al, 2000;MONTEIRO et al, 2007).…”

“…The evolutionary history of Sarcocystidae have been extensively study using 18S rRNA and 28S rRNA (ELLIS et al, 1999;MUGRIDGE et al, 1999MUGRIDGE et al, , 2000MORRISON et al, 2004). However, few nucleotide differences among species of the Toxoplasma-Neospora-Hammondia at these loci make difficult to resolve consistently their phylogenetic relationships.…”

“…In fact, in order to be comparable to the rest of the coccidia ToxoplasmaNeospora-Hammondia would be separate species of a single genus based only on 18S rRNA (MORRISON et al, 2004). rRNA sequences cannot always be unambiguously aligned and details of the alignment are known to greatly affect the results of phylogenetic analyses involving these sequences (MUGRIDGE et al, 2000;MORRISON et al, 2004). Thus, other universal markers are required to resolve phylogenies especially those that codes for proteins, which allow for robust alignments (MONTEIRO et al, 2007).…”

“…Thus, other universal markers are required to resolve phylogenies especially those that codes for proteins, which allow for robust alignments (MONTEIRO et al, 2007). Moreover, analysis of more than one gene is necessary in order to obtain a robust species signal (MUGRIDGE et al, 2000).…”

Studies on Serological Cross-Reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni.

“…While the sequence analyses of the large subunit of ribosomal RNA and the internal transcribed spacer-1 revealed a closer relationship between N. caninum and H. heydorni than with either H. hammondi or T. gondii [3,14], H. heydorni genomic DNA could not be amplified in the PCR assay targeting the N. caninum-specific Nc5 genomic sequence [6]. Several reports on similarities in the life cycle and morphology of N. caninum and H. heydorni suggest a closer relationship between them than with T. gondii.…”