Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

A Molecular Phylogeny of Lilium in the Internal Transcribed Spacer Region of Nuclear Ribosomal DNA

Phylogenetic relationships among 55 species of Lilium, Cardiocrinum giganteum, and Nomocharis saluenensis were inferred from nucleotide sequence variations in the internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA. The phylogeny derived from ITS sequences estimated using maximum-likelihood methods indicated that (1) most of the species construct their own clade according to the classification based on morphological features at the section level; (2) section Daurolirion is not independent of Sinomartagon, and it is appropriate to integrate two sections as Sinomartagon; (3) it is appropriate that L. henryi and L. bulbiferum are classified into subsection 6a and Sinomartagon–Daurolirion, respectively; (4) subsection 6b is much closer to Sinomartagon than subsection 6a and Archelirion, and it arose directly from Sinomartagon; and (5) Lilium is much closer to Nomocharis than Cardiocrinum. Phylogenetic estimation using sequences of the ITS region is suitable at the levels of genus, section, and most of subsection. Received: 18 December 1998 / Accepted: 14 March 1999     

A Common Structural Motif in Elongation Factor Ts and Ribosomal Protein L7/12 May Be Involved in the Interaction with Elongation Factor Tu

Elongation factor (EF) Tu alternates between two interaction partners, EF-Ts and the ribosome, during its functional cycle. On the ribosome, the interaction involves, among others, ribosomal protein L7/12. Here we compare EF-Ts and L7/12 with respect to the conservation of sequence and structure. There is significant conservation of functionally important residues in the N-terminal domain of EF-Ts and in the C-terminal domain of L7/12. The structure alignment based on the crystal structures of the two domains suggests a high degree of similarity between the αA–βD–αB motif in L7/12 and the h1–turn–h2 motif in EF-Ts which defines a common structural motif. The motif is remarkably similar with respect to fold, bulkiness, and charge distribution of the solution surface, suggesting that it has a common function in binding EF-Tu. Received: 12 June 2000 / Accepted: 10 October 2000     

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     

Taxonomy of 5 S Ribosomal RNA by the Linguistic Technique: Probing with Mitochondrial and Mammalian Sequences

Linguistic similarities and dissimilarities between 5 S rRNA sequences allowed taxonomical separation of species and classes. Comparisons with the molecule from mammals distinguished fungi and plants from protists and animals. Similarities to mammalians progressively increased from protists to invertebrates and to somatic-type molecules of the vertebrates lineage. In this, deviations were detected in avian, oocyte type, and pseudogene sequences. Among bacteria, actinobacteria were most similar to the mammalians, which could be related to the high frequency of associations among members of these groups. Some archaebacterial species most similar to the mammalians belonged to the Thermoproteales and Halobacteria groups. Comparisons with the soybean mitochondrial molecule revealed high internal homogeneity among plant mitochondria. The eubacterial groups most similar to it were Thermus and Rhodobacteria γ-1 and α-2. Other procedures have already indicated similarities of Rhodobacteria α to mitochondria but the linguistic similarities were on the average higher with the first two groups. Received: 5 August 1996 / Accepted: 9 April 1997     

Analysis of the Cluster of Ribosomal Protein Genes in the Plastid Genome of a Unicellular Red Alga Cyanidioschyzon merolae: Translocation of the str Cluster as an Early Event in the Rhodophyte-Chromophyte Lineage of Plastid Evolution

The nucleotide sequence of a cluster of ribosomal protein genes in the plastid genome of a unicellular red alga, Cyanidioschyzon merolae, which has been supposed to be the most primitive alga, was determined. The phylogenetic tree inferred from the amino acid sequence of ribosomal proteins of two rhodophytes, a chromophyte, a glaucophyte, two chlorophytes (land plants), a cyanobacterium, and three eubacteria suggested a close relationship between the cyanobacterium Synechocystis PCC6803 and the plastids of various species in the kingdom Plantae, which is consistent with the hypothesis of the endosymbiotic origin of plastids. In this tree, the two species of rhodophytes were grouped with the chromophyte, and the glaucophyte was grouped with the chlorophytes. Analysis of the organization of the genes encoding the ribosomal proteins suggested that the translocation of the str cluster occurred early in the lineage of rhodophytes and chromophytes after these groups had been separated from chlorophytes and glaucophytes. Received: 2 June 1997 / Accepted: 15 July 1997     

Variability of Ribosomal DNA ITS-2 and Its Utility in Detecting Genetic Relatedness of Pearl Oyster

The objective of this study was to detect interspecific and intraspecific genetic variations of the second internal transcribed spacer of ribosomal DNA (ITS-2), and explore the feasibility of using it as a molecular marker phylogenetic analyses and species identification among pearl oysters. ITS-2 sequences of 6 pearl oysters were amplified via polymerase chain reaction. The amplified DNA fragments were about 500 bp, spanning the partial sequences of 5.8S and 28S rRNA genes. The GC contents of all species used in this study were higher than the AT contents. The variations of sequences involved substitutions as well as insertions/deletions and were mainly concentrated in spacer regions. Sequences of about 30-bp in spacer regions showed no variations among 5 Pincatda species. Intraindividual and intraspecific polymorphisms of ITS-2 sequences were detected in some species; the interspecific variability was significantly larger than the variability within species, and the variability at the genus level was higher than that at the species level. Both neighbor-joining and parsimony analyses of ITS-2 sequences revealed the distinguishable species boundary of 6 pearl oysters, and indicated that P. chemnitzi and P. nigra were the closely related species, as were P. maxima and P. margaritifera. The findings revealed that ITS-2 sequences could be an appropriate tool for phylogenetic study of pearl oysters.     

Trematode and Monogenean rRNA ITS2 Secondary Structures Support a Four-Domain Model

The secondary structure of rRNA internal transcribed spacer 2 is important in the process of ribosomal biogenesis. Trematode ITS sequences are poorly conserved and difficult to align for phylogenetic comparisons above a family level. If a conserved secondary structure can be identified, it can be used to guide primary sequence alignments. ITS2 sequences from 39 species were compared. These species span four orders of trematodes (Echinostomiformes, Plagiorchiformes, Strigeiformes, and Paramphistomiformes) and one monogenean (Gyrodactyliformes). The sequences vary in length from 251 to 431 bases, with an average GC content of 48%. The monogenean sequence could not be aligned with confidence to the trematodes. Above the family level trematode sequences were alignable from the 5′ end for 139 bases. Secondary structure foldings predicted a four-domain model. Three folding patterns were required for the apex of domain B. The folding pattern of domains C and D varies for each family. The structures display a high GC content within stems. Bases A and U are favored in unpaired regions and variable sites cluster. This produces a mosaic of conserved and variable regions with a structural conformation resistant to change. Two conserved strings were identified, one in domain B and the other in domain C. The first site can be aligned to a processing site identified in yeast and rat. The second site has been found in plants, and structural location appears to be important. A phylogenetic tree of the trematode sequences, aligned with the aid of secondary structures, distinguishes the four recognized orders. Received: 21 November 1997 / Accepted: 9 February 1998     

Phylogenetic Relationships of Fungi, Plantae, and Animalia Inferred from Homologous Comparison of Ribosomal Proteins

The complete set of available ribosomal proteins was utilized, at both the peptidic and the nucleotidic level, to establish that plants and metazoans form two sister clades relative to fungi. Different phylogenetic inference methods are applied to the sequence data, using archeans as the outgroup. The evolutionary length of the internal branch within the eukaryotic crown trichotomy is demonstrated to be, at most, one-tenth of the evolutionary length of the branch leading to the cenancester of these three kingdoms. Received: 1 November 1997 / Accepted: 7 January 1998     

Phylogeny of Organisms Investigated by the Base-Pair Changes in the Stem Regions of Small and Large Ribosomal Subunit RNAs

In order to obtain the evolutionary distance data that are as purely additive as possible, we have developed a novel method for evaluating the evolutionary distances from the base-pair changes in stem regions of ribosomal RNAs (rRNAs). The application of this method to small-subunit (SSU) and large-subunit (LSU) rRNAs provides the distance data, with which both the unweighted pair group method of analysis and the neighbor-joining method give almost the same tree topology of most organisms except for some Protoctista, thermophilic bacteria, parasitic organisms, and endosymbionts. Although the evolutionary distances calculated with LSU rRNAs are somewhat longer than those with SSU rRNAs, the difference, probably due to a slight difference in functional constraint, is substantially decreased when the distances are converted into the divergence times of organisms by the measure of the time scale estimated in each type of rRNAs. The divergence times of main branches agree fairly well with the geological record of organisms, at least after the appearance of oxygen-releasing photosynthesis, although the divergence times of Eukaryota, Archaebacteria, and Eubacteria are somewhat overestimated in comparison with the geological record of Earth formation. This result is explained by considering that the mutation rate is determined by the accumulation of misrepairs for DNA damage caused by radiation and that the effect of radiation had been stronger before the oxygen molecules became abundant in the atmosphere of the Earth. Received: 23 October 1997 / Accepted: 12 August 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     

Restriction Endonuclease Analysis of Highly Repetitive DNA as a Phylogenetic Tool

Multiple band patterns of DNA repeats in the 20–500-nucleotide range can be detected by digesting genomic DNA with short—cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA ``taxonprints' (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects—altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies. Received: 10 February 1997 / Accepted: 10 March 1997     

The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs

The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5′-CACACCCA and (b) 5′-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA genes of the donkey and the horse, Equus caballus, were compared in detail. Total nucleotide difference outside the control region was 6.9%. Nucleotide difference between peptide-coding genes ranged from 6.4% to 9.4% with a mean of 8.0%. In the inferred protein sequences of the 13 peptide-coding genes the amino acid difference was 0.2–8.8%, and the mean for the 13 concatenated amino acid sequences was 1.9%. In the 22 tRNA genes, the mean difference was 3.5%, and that in the two rRNA genes was 4.1%. The mtDNA differences between the donkey and the horse suggest that the evolutionary separation of the two species occurred ≈9 million years ago. Analyses of differences among the mtDNAs of three other species-pairs, harbor seal/grey seal, fin whale/blue whale, and Homo/common chimpanzee, showed that the relative evolutionary rate of individual peptide-coding genes varies among different species-pairs and modes of comparison. The findings show that the superimposition of sequence data of one lineage for resolving and dating evolutionary divergences of other lineages should be performed with caution unless based on comprehensive data. Received: 15 October 1995 / Accepted: 15 April 1996     

Phylogenetic Depth of the Bacterial Genera Aquifex and Thermotoga Inferred from Analysis of Ribosomal Protein, Elongation Factor, and RNA Polymerase Subunit Sequences

The phylogenetic placement of the Aquifex and Thermotoga lineages has been inferred from (i) the concatenated ribosomal proteins S10, L3, L4, L23, L2, S19, L22, and S3 encoded in the S10 operon (833 aa positions); (ii) the joint sequences of the elongation factors Tu(1α) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase β- and β′-type subunits encoded in the rpoBC operon (1130 aa positions). Phylogenies of r-protein and EF sequences support with moderate (r-proteins) to high statistical confidence (EFs) the placement of the two hyperthermophiles at the base of the bacterial clade in agreement with phylogenies of rRNA sequences. In the more robust EF-based phylogenies, the branching of Aquifex and Thermotoga below the successive bacterial lineages is given at bootstrap proportions of 82% (maximum likelihood; ML) and 85% (maximum parsimony; MP), in contrast to the trees inferred from the separate EF-Tu(1α) and EF-G(2) data sets, which lack both resolution and statistical robustness. In the EF analysis MP outperforms ML in discriminating (at the 0.05 level) trees having A. pyrophilus and T. maritima as the most basal lineages from competing alternatives that have (i) mesophiles, or the Thermus genus, as the deepest bacterial radiation and (ii) a monophyletic A. pyrophilus–T. maritima cluster situated at the base of the bacterial clade. RNAP-based phylogenies are equivocal with respect to the Aquifex and Thermotoga placements. The two hyperthermophiles fall basal to all other bacterial phyla when potential artifacts contributed by the compositionally biased and fast-evolving Mycoplasma genitalium and Mycoplasma pneumoniae sequences are eschewed. However, the branching order of the phyla is tenuously supported in ML trees inferred by the exhaustive search method and is unresolved in ML trees inferred by the quartet puzzling algorithm. A rooting of the RNA polymerase-subunit tree at the mycoplasma level seen in both the MP trees and the ML trees reconstructed with suboptimal amino acid substitution models is not supported by the EF-based phylogenies which robustly affiliate mycoplasmas with low-G+C gram-positives and, most probably, reflects a ``long branch attraction' artifact. Received: 22 September 1999 / Accepted: 11 January 2000     

Use of a deviant mitochondrial genetic code in yellow-green algae as a landmark for segregating members within the phylum

Several algae that were previously classified in the phylum Xanthophyta (yellow-green algae) were assigned in 1971 to a new phylum, Eustigmatophyta. It was anticipated that the number of algae reclassified to Eustigmatophyta would increase. However, due to the fact that the morphological characteristics that segregate eustigmatophytes from other closely related algae can be only obtained through laborious electron microscopic techniques, the number of members in this phylum have increased rather slowly. We attempted, therefore, to segregate two closely related groups of algae, eustigmatophytes and yellow-green algae, on the basis of a molecular phylogenetic tree as a means of providing an alternative method of distinguishing these phyla. We analyzed the mitochondrial cytochrome oxidase subunit I (COXI) gene sequences of eight algae classified as xanthophyceans and found that six manifested the expected deviant genetic code where AUA codes for methionine (AUA/Met), but not for isoleucine (AUA/Ile) as in the universal genetic code. The other two, Monodus sp. (CCMP 505) and Ophiocytium majus (CCAP 855/1), which were presumed to be yellow-green algae, and all the examined eustigmatophytes utilized AUA for Ile. In addition, the phylogenetic tree of COXI gene sequences showed that the six yellow-green algae bearing the AUA/Met deviant code composed a tight clade with a bootstrap value of 100%. The phylogenetic tree of the corresponding sequences from Monodus sp. and Ophiocytium majus and the eustigmatophytes also composed a tight cluster, but with a bootstrap value of 92%. These results strongly suggest that two previously classified members of yellow-green algae belong to the phylum Eustigmatophyta. Therefore, examination of the mitochondrial genetic code in algae appears to be a potentially very useful genetic marker for classifying these organisms, especially when it is considered with the results obtained through a molecular phylogenetic tree. Received: 14 December 1996 / Accepted: 3 April 1997     

Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Concordant Evolution of a Symbiont with Its Host Insect Species: Molecular Phylogeny of Genus Glossina and Its Bacteriome-Associated Endosymbiont, Wigglesworthia glossinidia

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the γ subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species. Received: 17 March 1998 / Accepted: 1 May 1998     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998