Long branch attraction effects and the status of "basal eukaryotes": phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis

The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species. and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.     

The largest subunit of RNA polymerase II from the Glaucocystophyta: functional constraint and short-branch exclusion in deep eukaryotic phylogeny

Background  

Evolutionary analyses of the largest subunit of RNA polymerase II (RPB1) have yielded important and at times provocative results. One particularly troublesome outcome is the consistent inference of independent origins of red algae and green plants, at odds with the more widely accepted view of a monophyletic Plantae comprising all eukaryotes with primary plastids. If the hypothesis of a broader kingdom Plantae is correct, then RPB1 trees likely reflect a persistent phylogenetic artifact. To gain a better understanding of RNAP II evolution, and the presumed artifact relating to green plants and red algae, we isolated and analyzed RPB 1 from representatives of Glaucocystophyta, the third eukaryotic group with primary plastids.     

Taming of the shrewd: novel eukaryotic genes from RNA viruses

Genomes of several yeast species contain integrated DNA copies of complete genomes or individual genes of non-retroviral double-strand RNA viruses as reported in a recent BMC Biology article by Taylor and Bruenn. The integrated virus-specific sequences are at least partially expressed and seem to evolve under pressure of purifying selection, indicating that these are functional genes. Together with similar reports on integrated copies of some animal RNA viruses, these results suggest that integration of DNA copies of non-reverse-transcribing RNA viruses might be much more common than previously thought. The integrated copies could contribute to acquired immunity to the respective viruses.     

Molecular phylogeny of protists: origin and evolution of the Dinoflagellates

Abstract

Molecular sequence data have become prominent tools for phylogenetic relationship inference, particularly useful in the analysis of highly diverse taxonomic orders. Ribosomal RNA sequences provide markers that can be used in the study of phylogeny, because their function and structure have been conserved to a large extent throughout the evolutionary history of organisms. These sequences are inferred from cloned or enzymatically amplified gene sequences, or determined by direct RNA sequencing. The first step of the phylogenetic interpretation of nucleic acid sequence variations implies proper alignment of corresponding sequences from various organisms. Best alignment based on similarity criteria is greatly reinforced, in the case of ribosomal RNAs, by secondary structure homologies. Distance matrix methods to infer evolutionary trees are based on the assumption that the phylogenetic distance between each pair of organisms is proportional to the number of nucleotide substitution events. Computed tree inference methods usually take into consideration the possibility of unequal mutation rates among lineages. Divergence times can be estimated on the tree, provided that at least one lineage has been dated by fossil records. We have utilized this approach based on ribosomal RNA sequence comparison to investigate the phylogenetic relationship between dinoflagellated and other eukaryote protists, and to refine controverse phylogenies of the class Dinophycae.     

Last of the human protists: the phylogeny and genetic diversity of Iodamoeba

Iodamoeba is the last genus of obligately parasitic human protist whose phylogenetic position is unknown. Iodamoeba small subunit ribosomal DNA sequences were obtained using samples from three host species, and phylogenetic analyses convincingly placed Iodamoeba as a sister taxon to Endolimax. This clade in turn branches among free-living amoeboflagellates of the genus Mastigamoeba. Two Iodamoeba ribosomal lineages (RL1 and RL2) were detected whose sequences differ by 31%, each of which is found in both human and nonhuman hosts.     

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD''s overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism.     

Hox genes and the parasitic flatworms: new opportunities, challenges and lessons from the free-living

Research into the roles played by Hox and related homeotic gene families in the diverse and complex developmental programmes exhibited by parasitic flatworms (Platyhelminthes) can hardly be said to have begun, and thus presents considerable opportunity for new research. Although featured in some of the earliest screens for homeotic genes outside Drosophila and mice, surveys in parasitic flatworms are few in number and almost nothing is yet known of where or when the genes are expressed during ontogeny. This contrasts sharply with a significant body of literature concerning Hox genes in free-living flatworms which have long served as models for the study of regeneration and the maintenance of omnipotent cell lines. Nevertheless, available information suggests that the complement of Hox genes and other classes of homeobox-containing genes in parasitic flatworms is typical of their free-living cousins and of other members of the Lophotrochozoa. Recent work on Schistosoma combined with information on Hox gene expression in planarians indicates that at least some disruption of the clustered genomic arrangement of the genes, as well as of the strict spatial and temporal colinear patterns of expression typical in other groups, may be characteristic of flatworms. However, available data on the genomic arrangement and expression of flatworm Hox genes is so limited at present that such generalities are highly tenuous. Moreover, a basic underlying pattern of colinearity is still observed in their spatial expression patterns making them suitable as cell or region-specific markers. I discuss a number of fundamental developmental questions and some of the challenges to addressing them in relation to each of the major parasitic lineages. In addition, I present newly characterized Hox genes from the model tapeworm Hymenolepis and analyze these by Bayesian inference together with >100 Hox and ParaHox homeodomains of flatworms and select lophotrochozoan taxa, providing a phylogenetic scaffold for their identification.     

Ribosomal RNA genes and deuterostome phylogeny revisited: more cyclostomes, elasmobranchs, reptiles, and a brittle star

This is an expanded study of the relationships among the deuterostome animals based on combined, nearly complete 28S and 18S rRNA genes (>3925 nt.). It adds sequences from 20 more taxa to the approximately 45 sequences used in past studies. Seven of the new taxa were sequenced here (brittle star Ophiomyxa, lizard Anolis, turtle Chrysemys, sixgill shark Hexanchus, electric ray Narcine, Southern Hemisphere lamprey Geotria, and Atlantic hagfish Myxine for 28S), and the other 13 were from GenBank and the literature (from a chicken, dog, rat, human, three lungfishes, and several ray-finned fishes, or Actinopterygii). As before, our alignments were based on secondary structure but did not account for base pairing in the stems of rRNA. The new findings, derived from likelihood-based tree-reconstruction methods and by testing hypotheses with parametric bootstrapping, include: (1) brittle star joins with sea star in the echinoderm clade, Asterozoa; (2) with two hagfishes and two lampreys now available, the cyclostome (jawless) fishes remain monophyletic; (3) Hexanchiform sharks are monophyletic, as Hexanchus groups with the frilled shark, Chlamydoselachus; (4) turtle is the sister taxon of all other amniotes; (5) bird is closer to the lizard than to the mammals; (6) the bichir Polypterus is in a monophyletic Actinopterygii; (7) Zebrafish Danio is the sister taxon of the other two teleosts we examined (trout and perch); (8) the South American and African lungfishes group together to the exclusion of the Australian lungfish. Other findings either upheld those of the previous rRNA-based studies (e.g., echinoderms and hemichordates group as Ambulacraria; orbitostylic sharks; batoids are not derived from any living lineage of sharks) or were obvious (monophyly of mammals, gnathostomes, vertebrates, echinoderms, etc.). Despite all these findings, the rRNA data still fail to resolve the relations among the major groups of deuterostomes (tunicates, Ambulacraria, cephalochordates and vertebrates) and of gnathostomes (chondrichthyans, lungfishes, coelacanth, actinopterygians, amphibians, and amniotes), partly because tunicates and lungfishes are rogue taxa that disrupt the tree. Nonetheless, parametric bootstrapping showed our RNA-gene data are only consistent with these dominant hypotheses: (1) deuterostomes consist of Ambulacraria plus Chordata, with Chordata consisting of tunicates and 'vertebrates plus cephalochordates'; and (2) lungfishes are the closest living relatives of tetrapods.     

Distribution and consensus of branch point signals in eukaryotic genes: a computerized statistical analysis.

An intermediate stage in the process of eukaryotic RNA splicing is the formation of a lariat structure. It is anchored at an adenosine residue in intron between 10 and 50 nucleotides upstream of the 3' splice site. A short conserved sequence (the branch point sequence) functions as the recognition signal for the site of lariat formation. It has been generally assumed that the branch point is recognized mainly by the presence of its unique sequence where the lariat is formed. However, the known branch point consensus sequence is found to be distributed nearly randomly throughout the gene sequence with only a slightly higher frequency in the expected lariat region. Further, the known consensus sequence is found to be clearly inadequate to specify branch points. These observations have implications for understanding the mechanism of branch point recognition in the process of splicing, and the possible evolution of the branch point signal.     

Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene

Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.     

The Use of the Nuclear Protein-Encoding Gene, RNA Polymerase II, for Tick Molecular Systematics

Phylogenetic studies of ticks have been increasing in recent years, particularly in the use of molecular data. However, all of the studies to date are either limited to the mitochondrial genome or to a few nuclear ribosomal genes. There is a need to explore the use of nuclear protein-encoding genes because these genes direct most aspects of the phenotypic traits in the development of an organism. We report here the test of a nuclear protein-encoding gene, RNA polymerase II, for the phylogenetic study of ticks. Thirty-eight ticks representing 26 species of hard and soft ticks were chosen for the study. The pairwise divergences among sampled species are ranged from 0.3 to 15.2% and most of the substitutions are transitions. In addition, the nucleotide composition is not obviously biased in POL II gene. The trees inferred from the POL II sequences using maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) by PAUP* and MrBayes are largely concordant with the existing phylogenies. Our study demonstrated that POL II gene sequences contain strong phylogenetic signals in ticks at the generic and higher levels. POL II has proven to be a useful gene for resolving tick phylogeny. This revised version was published online in July 2006 with corrections to the Cover Date.