Characterization of Mitochondrial Small-Subunit Ribosomal RNAs from Holoparasitic Plants

Mitochondrial small-subunit (19S) rDNA sequences were obtained from 10 angiosperms to further characterize sequence divergence levels and structural variation in this molecule. These sequences were derived from seven holoparasitic (nonphotosynthetic) angiosperms as well as three photosynthetic plants. 19S rRNA is composed of a conservative core region (ca. 1450 nucleotides) as well as two variable regions (V1 and V7). In pairwise comparisons of photosynthetic angiosperms to Glycine, the core 19S rDNA sequences differed by less than 1.4%, thus supporting the observation that variation in mitochondrial rDNA is 3–4 times lower than seen in protein coding and rDNA genes of other subcellular organelles. Sequences representing four distinct lineages of nonasterid holoparasites showed significantly increased numbers of substitutions in their core 19S rDNA sequences (2.3–7.6%), thus paralleling previous findings that showed accelerated rates in nuclear (18S) and plastid (16S) rDNA from the same plants. Relative rate tests confirmed the accelerated nucleotide substitution rates in the holoparasites whereas rates in nonparasitic plants were not significantly increased. Among comparisons of both parasitic and nonparasitic plants, transversions outnumbered transitions, in many cases more than two to one. The core 19S rRNA is conserved in sequence and structure among all nonparasitic angiosperms whereas 19S rRNA from members of holoparasitic Balanophoraceae have unique extensions to the V5 and V6 variable domains. Substitution and insertion/deletion mutations characterized the V1 and V7 regions of the nonasterid holoparasites. The V7 sequence of one holoparasite (Scybalium) contained repeat motifs. The cause of substitution rate increases in the holoparasites does not appear to be a result of RNA editing, hence the underlying molecular mechanism remains to be fully documented. Received: 18 May 1997 / Accepted: 11 July 1997     

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed. Received: 4 September 1996 / Accepted: 7 April 1997     

Evolution of 28S Ribosomal DNA in Chaetognaths: Duplicate Genes and Molecular Phylogeny

The chaetognaths are an extraordinarily homogeneous phylum of animals at the morphological level, with a bauplan that can be traced back to the Cambrian. Despite the attention of zoologists for over two centuries, there is little agreement on classification within the phylum. We have used a molecular biological approach to investigate the phylogeny of extant chaetognaths. A rapidly evolving expansion segment toward the 5′ end of 28S ribosomal DNA (rDNA) was amplified using the polymerase chain reaction (PCR), cloned, and sequenced from 26 chaetognath samples representing 18 species. An unusual finding was the presence of two distinct classes of 28S rDNA gene in chaetognaths; our analyses suggest these arose by a gene (or gene cluster) duplication in a common ancestor of extant chaetognaths. The two classes of chaetognath 28S rDNA have been subject to different rates of molecular evolution; we present evidence that both are expressed and functional. In phylogenetic reconstructions, the two classes of 28S rDNA yield trees that root each other; these clearly demonstrate that the Aphragmophora and Phragmophora are natural groups. Within the Aphragmophora, we find good support for the groupings denoted Solidosagitta, Parasagitta, and Pseudosagitta. The relationships between several well-supported groups within the Aphragmophora are uncertain; we suggest this reflects rapid, recent radiation during chaetognath evolution. Received: 19 March 1996 / Accepted: 5 August 1996     

Origin and Evolution of Paralogous rRNA Gene Clusters Within the Flatworm Family Dugesiidae (Platyhelminthes, Tricladida)

Analysis of the 18S rDNA sequences of five species of the family Dugesiidae (phylum Platyhelminthes, suborder Tricladida, infraorder Paludicola) and eight species belonging to families Dendrocoelidae and Planaridae and to the infraorder Maricola showed that members of the family Dugesiidae have two types of 18S rDNA genes, while the rest of the species have only one. The duplication event also affected the ITS-1, 5.8S, ITS-2 region and probably the 28S gene. The mean divergence value between the type I and the type II sequences is 9% and type II 18S rDNA genes are evolving 2.3 times more rapidly than type I. The evolutionary rates of type I and type II genes were calibrated from biogeographical data, and an approximate date for the duplication event of 80–120 million years ago was calculated. The type II gene was shown, by RT-PCR, to be transcribed in adult individuals of Schmidtea polychroa, though at very low levels. This result, together with the fact that most of the functionally important positions for small-subunit rRNA in prokaryotes have been conserved, indicates that the type II gene is probably functional. Received: 24 March 1998 / Accepted: 17 March 1999     

Ancyromonadida: A New Phylogenetic Lineage Among the Protozoa Closely Related to the Common Ancestor of Metazoans, Fungi, and Choanoflagellates (Opisthokonta)

Molecular and morphological evidence points to the ancyromonad Ancyromonas as a plausible candidate for the closest relative to the common ancestor of metazoans, fungi, and choanoflagellates (the Opisthokonta). Using 18S rDNA sequences from most of the major eukaryotic lineages, maximum-likelihood, minimum-evolution, and maximum-parsimony analyses yielded congruent phylogenies supporting this hypothesis. Combined with ultrastructural similarities between Ancyromonas and opisthokonts, the evidence presented here suggests that Ancyromonas may form an independent lineage, the Ancyromonadida Cavalier-Smith 1997, closer in its relationship to the opisthokonts than is its nearest protist relatives, the Apusomonadida. However, the very low bootstrap support for deep nodes and hypothesis testing indicate that the resolving power of 18S rDNA sequences is limited for examining this aspect of eukaryotic phylogeny. Alternate branching positions for the Ancyromonas lineage cannot be robustly rejected, revealing the importance of ultrastructure when examining the origins of multicellularity. The future use of a multigene approach may additionally be needed to resolve this aspect of eukaryotic phylogeny. Received: 27 March 2000 / Accepted: 12 June 2000     

Molecular Evolution of Cytochrome c Oxidase Subunit IV: Evidence for Positive Selection in Simian Primates

Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of ``neutral DNA' nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages. Received: 22 May 1996 / Accepted: 24 November 1996     

The Small-Subunit rRNA Gene Sequences of Venerids and the Phylogeny of Bivalvia

The complete nucleotide sequence of the 18S subunit of ribosomal DNA (rDNA) was determined for the venerid clams Callista chione (Pitarinae) and Venus verrucosa (Venerinae). Comparison of the new sequences with the published sequences of 1 annelid, 2 gastropods, 2 polyplacophorans, and 19 bivalves showed that when the annelids are used as outgroup the gastropods diverge from the bivalves, which form a cluster including the polyplacophorans. When the gastropods alone were compared with the bivalves, the latter split in two groups corresponding to the two subclasses of Heterodonta and Pteriomorpha. The former include two taxa that diverged early, Galeomma and Tridacna, while the Veneridae and Mactridae form two sister groups. In contrast to previous reports and in line with morphological data, the Ostreidae are included in the Pteriomorphia and form a monophyletic group. Received: 16 May 1998 / Accepted: 11 August 1998     

Secondary structure and patterns of evolution among mammalian mitochondrial 12S rRNA molecules

Forty-nine complete 12S ribosomal RNA (rRNA) gene sequences from a diverse assortment of mammals (one monotreme, 11 marsupials, 37 placentals), including 11 new sequences, were employed to establish a ``core' secondary structure model for mammalian 12S rRNA. Base-pairing interactions were assessed according to the criteria of potential base-pairing as well as evidence for base-pairing in the form of compensatory mutations. In cases where compensatory evidence was not available among mammalian sequences, we evaluated evidence among other vertebrate 12S rRNAs. Our results suggest a core model for secondary structure in mammalian 12S rRNAs with deletions as well as additions to the Gutell (1994: Nucleic Acids Res. 22) models for Bos and Homo. In all, we recognize 40 stems, 34 of which are supported by at least some compensatory evidence within Mammalia. We also investigated the occurrence and conservation in mammalian 12S rRNAs of nucleotide positions that are known to participate in the decoding site in E. coli. Twenty-four nucleotide positions known to participate in the decoding site in E. coli also occur among mammalian 12S rRNAs and 17 are invariant for the same base as in E. coli. Patterns of nucleotide substitution were assessed based on our secondary structure model. Transitions in loops become saturated by approximately 10–20 million years. Transitions in stems, in turn, show partial saturation at 20 million years but divergence continues to increase beyond 100 million years. Transversions accumulate linearly beyond 100 million years in both stems and loops although the rate of accumulation of transversions is three- to fourfold higher in loops. Presumably, this difference results from constraints to maintain pairing in stems. Received: 21 June 1995 / Accepted: 25 March 1996     

Characteristics of Nucleotide Substitution in the Hepatitis C Virus Genome: Constraints on Sequence Change in Coding Regions at Both Ends of the Genome

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5′ and 3′ ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T ↔ C transitions were twice as frequent as A ↔ G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies. Received: 9 September 1996 / Accepted: 20 April 1997     

Limitations of Metazoan 18S rRNA Sequence Data: Implications for Reconstructing a Phylogeny of the Animal Kingdom and Inferring the Reality of the Cambrian Explosion

We document the phylogenetic behavior of the 18S rRNA molecule in 67 taxa from 28 metazoan phyla and assess the effects of among-site rate variation on reconstructing phylogenies of the animal kingdom. This empirical assessment was undertaken to clarify further the limits of resolution of the 18S rRNA gene as a phylogenetic marker and to address the question of whether 18S rRNA phylogenies can be used as a source of evidence to infer the reality of a Cambrian explosion. A notable degree of among-site rate variation exists between different regions of the 18S rRNA molecule, as well as within all classes of secondary structure. There is a significant negative correlation between inferred number of nucleotide substitutions and phylogenetic information, as well as with the degree of substitutional saturation within the molecule. Base compositional differences both within and between taxa exist and, in certain lineages, may be associated with long branches and phylogenetic position. Importantly, excluding sites with different degrees of nucleotide substitution significantly influences the topology and degree of resolution of maximum-parsimony phylogenies as well as neighbor-joining phylogenies (corrected and uncorrected for among-site rate variation) reconstructed at the metazoan scale. Together, these data indicate that the 18S rRNA molecule is an unsuitable candidate for reconstructing the evolutionary history of all metazoan phyla, and that the polytomies, i.e., unresolved nodes within 18S rRNA phylogenies, cannot be used as a single or reliable source of evidence to support the hypothesis of a Cambrian explosion. Received: 9 December 1997 / Accepted: 23 March 1998     

Phylogeny Derived from Coding Retroviral Genome Organization

When divergence between viral species is large, the analysis and comparison of nucleotide or protein sequences are dependent on mutation biases and multiple substitutions per site leading, among other things, to the underestimation of branch lengths in phylogenetic trees. To avoid the problem of multiply substituted sites, a method not directly based on the nucleic or protein sequences has been applied to retroviruses. It consisted of asking questions about genome structure or organization, and gene function, the series of answers creating coded sequences analyzed by phylogenic software. This method recovered the principal retroviral groups such as the lentiviruses and spumaviruses and highlighted questions and answers characteristic of each group of retroviruses. In general, there was reasonable concordance between the coded genome methodology and that based on conventional phylogeny of the integrase protein sequence, indicating that integrase was fixing mutations slowly enough to marginalize the problem of multiple substitutions at sites. To a first approximation, this suggests that the acquisition of novel genetic features generally parallels the fixation of amino acid substitutions. Received: 18 May 2001 / Accepted: 7 September 2001     

Molecular Phylogeny of Metazoan Intermediate Filament Proteins

We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia. Received: 2 April 1998 / Accepted: 19 June 1998     

A Group I Intron in the 18S Ribosomal DNA from the Parasitic Fungus Isaria japonica

The nucleotide sequence of the 18S rDNA coding gene in the ascomycetes parasitic fungus Isaria japonica contains a group I intron with a length of 379 nucleotides. The identification of the DNA sequence as a group I intron is based on its sequence homology to other fungal group I introns. Its group I intron contained the highly conserved sequence elements P, Q, R, and S found in other group I introns. Surprisingly, the intron sequence of I. japonica is more similar to that of Ustilago maydis than to the one found in Sclerotinia sclerotiorum. This is in contrast to the sequence identity found on the neighboring rDNA. This is an interesting finding and suggests a horizontal transfer of group I intron sequences. Received: 19 September 1997 / Accepted: 10 September 1998     

Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

This study provides a phylogenetic/comparative approach to deciphering the processes underlying the evolution of plastid rRNA genes in genomes under relaxed functional constraints. Nonphotosynthetic green algal taxa that belong to two distinct classes, Chlorophyceae (Polytoma) and Trebouxiophyceae (Prototheca), were investigated. Similar to the situation described previously for plastid 16S rRNA genes in nonphotosynthetic land plants, nucleotide substitution levels, extent of structural variations, and percentage AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives. However, the mutational processes appear to be different in many respects. First, with the increase in AT content, more transversions are noted in Polytoma and holoparasite angiosperms, while more transitions characterize the evolution of the 16S rDNA sequences in Prototheca. Second, although structural variations do accumulate in both Polytoma and Prototheca (as well as holoparasitic plastid 16S rRNAs), insertions as large as 1.6 kb characterize the plastid 16S rRNA genes in the former, whereas significantly smaller indels (not exceeding 24 bp) seem to be more prevalent in the latter group. The differences in evolutionary rates and patterns within and between lineages might be due to mutations in replication/repair-related genes; slipped-strand mispairing is likely the mechanism responsible for the expansion of insertions in Polytoma plastid 16S rRNA genes. Received: 29 December 2000 / Accepted: 18 May 2001     

Estimating the Transition/Transversion Ratio from Independent Pairwise Comparisons with an Assumed Phylogeny

A method is presented for estimating the transition/transversion ratio (TI/TV), based on phylogenetically independent comparisons. TI/TV is a parameter of some models used in phylogeny estimation intended to reflect the fact that nucleotide substitutions are not all equally likely. Previous attempts to estimate TI/TV have commonly faced three problems: (1) few taxa; (2) nonindependence among pairwise comparisons; and (3) multiple hits make the apparent TI/TV between two sequences decrease over time since their divergence, giving a misleading impression of relative substitution probabilities. We have made use of the time dependency, modeling how the observed TI/TV changes over time and extrapolating to estimate the ``instantaneous' TI/TV—the relevant parameter for phylogenetic inference. To illustrate our method, TI/TV was estimated for two mammalian mitochondrial genes. For 26 pairs of cytochrome b sequences, the estimate of TI/TV was 5.5; 16 pairs of 12s rRNA yielded an estimate of 9.5. These estimates are higher than those given by the maximum likelihood method and than those obtained by averaging all possible pairwise comparisons (with or without a two-parameter correction for multiple substitutions). We discuss strengths, weaknesses, and further uses of our method. Received: 22 August 1995 / Accepted: 26 July 1996     

Horizontal transfer of genes coding for the photosynthetic reaction centers of purple bacteria

Phylogenetic trees were drawn and analyzed based on the nucleotide sequences of the 1.5-kb gene fragment coding for the L and M subunits of the photochemical reaction center of various purple photosynthetic bacteria. These trees are mostly consistent with phylogenetic trees based on 16S rRNA and soluble cytochrome c, but differ in some significant details. This inconsistency implies horizontal transfer of the genes that code for the photosynthetic apparatus in purple bacteria. Possibilities of similar transfers of photosynthesis genes during the evolution of photosynthesis are discussed especially for the establishment of oxygenic photosynthesis. Received: 8 July 1996 / Accepted: 12 March 1997     

Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Evolution of the mitochondrial DNA control region in the mbuna (Cichlidae) species flock of lake Malawi, East Africa

Considerable controversy has surrounded the application of mitochondrial DNA data to reconstruction of evolutionary relationships among the endemic cichlids of Lake Malawi. Central to this debate has been the issue of whether lineage sorting is complete, and thus whether these data actually reflect species phylogeny, or simply gene genealogy. Review of all mtDNA control region sequences available for members of one monophyletic subset of this species flock, the Malawi rockfishes, or mbuna, strongly indicates that lineage sorting is incomplete: Character-based analyses of these sequences reconstruct gene, not species, interrelationships. Analysis of the pattern of nucleotide substitutions differentiating these mtDNA alleles suggests that pyrimidine residues undergo transition substitutions more often than do purines. Estimation of the magnitude of derived sequence differentiation in light of the reconstructed gene genealogy suggests that the mbuna may be of considerably more recent vintage than previous molecular characterizations have indicated. Received: 6 April 1996 / Accepted: 3 March 1997     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996