Transition and transversion rate in the evolution of animal mitochondrial DNA

We present a further application of the stochastic model previously described (Lanave et al., 1984, 1985) for measuring the nucleotide substitution rate in the mammalian evolution of the mitochondrial DNA (mtDNA). The applicability of this method depends on the validity of "stationarity conditions" (equal nucleotide frequencies at first, second and third silent codon positions in homologous protein coding genes). In the comparison of homologous sequences satisfying the stationarity condition at the silent sites, only the four codon families (quartets) for which both transitions and transversions are silent at the third position are considered here. This has allowed us to estimate the transition and transversion rates for any pair of species. We have analyzed the third silent codon position of the triplet rat-mouse-cow, of a series of slightly divergent primates and of two Drosophila species. In terms of two external dating input we have then determined the phylogenetic trees for rat, mouse, and cow as well as for a number of primates including man. The phylogenetic tree that we have derived for the triplet rat, mouse and cow agrees with that we had previously determined by analyzing the first, second and third silent codon positions (in both duets and quartets) of mt genes (Lanave et al., 1985). For primates our method leads to the following branching order from the oldest to the most recent: Gibbon, Orangutan, Gorilla, Chimpanzee and Man. In absolute time, fixing the distance Chimpanzee-Man as 5 million years (Myr) we estimate the dating of the divergence nodes as: Gorilla 7 Myr; Orangutan 16 Myr; Gibbon 20 Myr. In all cases analyzed, the transition rate has been found to be substantially higher than the transversion rate. Moreover we have found that the transition/transversion ratio is different in the various lineages. We suggest that this fact is probably related to the nucleotide frequencies at the third silent codon position.     

Preliminary analysis of the mitochondrial genome evolutionary pattern in primates

Since the birth of molecular evolutionary analysis,primates have been a central focus of study and mitochondrial DNA is well suited to these endeavors because of its unique features.Surprisingly,to date no comprehensive evaluation of the nucleotide substitution patterns has been conducted on the mitochondrial genome of primates.Here,we analyzed the evolutionary patterns and evaluated selection and recombination in the mitochondrial genomes of 44 Primates species downloaded from GenBank.The results revealed that a strong rate heterogeneity occurred among sites and genes in all comparisons.Likewise,an obvious decline in primate nucleotide diversity was noted in the subunit rRNAs and tRNAs as compared to the protein-coding genes.Within 13 protein-coding genes,the pattern of nonsynonymous divergence was similar to that of overall nucleotide divergence,while synonymous changes differed only for individual genes,indicating that the rate heterogeneity may result from the rate of change at nonsynonymous sites.Codon usage analysis revealed that there was intermediate codon usage bias in primate protein-coding genes,and supported the idea that GC mutation pressure might determine codon usage and that positive selection is not the driving force for the codon usage bias.Neutrality tests using site-specific positive selection from a Bayesian framework indicated no sites were under positive selection for any gene,consistent with near neutrality.Recombination tests based on the pairwise homoplasy test statistic supported complete linkage even for much older divergent primate species.Thus,with the exception of rate heterogeneity among mitochondrial genes,evaluating the validity assumed complete linkage and selective neutrality in primates prior to phylogenetic or phylogeographic analysis seems unnecessary.     

Preliminary analysis of the mitochondrial genome evolutionary pattern in primates

Since the birth of molecular evolutionary analysis, primates have been a central focus of study and mitochondrial DNA is well suited to these endeavors because of its unique features. Surprisingly, to date no comprehensive evaluation of the nucleotide substitution patterns has been conducted on the mitochondrial genome of primates. Here, we analyzed the evolutionary patterns and evaluated selection and recombination in the mitochondrial genomes of 44 Primates species downloaded from GenBank. The results revealed that a strong rate heterogeneity occurred among sites and genes in all comparisons. Likewise, an obvious decline in primate nucleotide diversity was noted in the subunit rRNAs and tRNAs as compared to the protein-coding genes. Within 13 protein-coding genes, the pattern of nonsynonymous divergence was similar to that of overall nucleotide divergence, while synonymous changes differed only for individual genes, indicating that the rate heterogeneity may result from the rate of change at nonsynonymous sites. Codon usage analysis revealed that there was intermediate codon usage bias in primate protein-coding genes, and supported the idea that GC mutation pressure might determine codon usage and that positive selection is not the driving force for the codon usage bias. Neutrality tests using site-specific positive selection from a Bayesian framework indicated no sites were under positive selection for any gene, consistent with near neutrality. Recombination tests based on the pairwise homoplasy test statistic supported complete linkage even for much older divergent primate species. Thus, with the exception of rate heterogeneity among mitochondrial genes, evaluating the validity assumed complete linkage and selective neutrality in primates prior to phylogenetic or phylogeographic analysis seems unnecessary.     

Forty Million Years of Independent Evolution: A Mitochondrial Gene and Its Corresponding Nuclear Pseudogene in Primates

Sequences from nuclear mitochondrial pseudogenes (numts) that originated by transfer of genetic information from mitochondria to the nucleus offer a unique opportunity to compare different regimes of molecular evolution. Analyzing a 1621-nt-long numt of the rRNA specifying mitochondrial DNA residing on human chromosome 3 and its corresponding mitochondrial gene in 18 anthropoid primates, we were able to retrace about 40 MY of primate rDNA evolutionary history. The results illustrate strengths and weaknesses of mtDNA data sets in reconstructing and dating the phylogenetic history of primates. We were able to show the following. In contrast to numt-DNA, (1) the nucleotide composition of mtDNA changed dramatically in the different primate lineages. This is assumed to lead to significant misinterpretations of the mitochondrial evolutionary history. (2) Due to the nucleotide compositional plasticity of primate mtDNA, the phylogenetic reconstruction combining mitochondrial and nuclear sequences is unlikely to yield reliable information for either tree topologies or branch lengths. This is because a major part of the underlying sequence evolution model — the nucleotide composition — is undergoing dramatic change in different mitochondrial lineages. We propose that this problem is also expressed in the occasional unexpected long branches leading to the “common ancestor” of orthologous numt sequences of different primate taxa. (3) The heterogeneous and lineage-specific evolution of mitochondrial sequences in primates renders molecular dating based on primate mtDNA problematic, whereas the numt sequences provide a much more reliable base for dating.[Reviewing Editor: Dr. Rafael Zardoya]     

A molecular view of primate phylogeny and important systematic and evolutionary questions

Phylogenetic analysis of extensive nucleotide sequence data from primate beta-globin gene clusters elucidates the systematics and evolution of the order Primates and reveals that rates of accumulation of mutations vary by as much as a factor of seven among different primate lineages. The picture of primate phylogeny from DNA sequences clarifies many ambiguities of the morphological picture. In the molecular picture, dwarf and brown lemurs group together into superfamily Lemuroidea, Lemuroidea and Lorisoidea into suborder Strepsirhini, and Tarsius and Anthropoidea into suborder Haplorhini. The molecular picture also provides both significant evidence for a human-chimpanzee clade that narrowly excludes gorilla and overwhelming evidence for the gorilla-chimpanzee-human clade within Hominoidea. Rates of DNA sequence evolution appear to have been fastest in the early primates ancestral to Anthropoidea and next fastest on the lorisoid branch. Rates were slowest over the past 25 Myr of hominoid descent, suggesting that mechanisms lowering the mutation rate evolved in correlation with lengthened life spans.     

Oesophagostomum dentatum and Oesophagostomum quadrispinulatum: characterization of the complete mitochondrial genome sequences of the two pig nodule worms

In the present study, the complete mitochondrial DNA (mtDNA) sequences of the pig nodule worm Oesophagostomum quadrispinulatum were determined for the first time, and the mt genome of Oesophagostomum dentatum from China was also sequenced for comparative analysis of their gene contents and genome organizations. The mtDNA sequences of O. dentatum China isolate and O. quadrispinulatum were 13,752 and 13,681 bp in size, respectively. Each of the two mt genomes comprises 36 genes, including 12 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, but lacks the ATP synthetase subunit 8 gene. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T are 75.79% and 77.52% for the mt genomes of O. dentatum and O. quadrispinulatum, respectively. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), all revealed that O. dentatum and O. quadrispinulatum represent distinct but closely-related species. These data provide novel and useful markers for studying the systematics, population genetics and molecular diagnosis of the two pig nodule worms.     

Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens

Chicken coccidiosis caused by members of the genus Eimeria causes significant economic losses worldwide. In the present study we sequenced the complete mitochondrial DNA (mtDNA) sequences of six Eimeria species and analyzed features of their gene contents and genome organizations. The complete mt genomes of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. tenella and E. praecox were 6179bp, 6148bp, 6169bp, 6214bp, 6213bp and 6174bp in size, respectively. All of the mt genomes consist of 3 genes for proteins (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes. The organization of the mt genomes is similar to that of Plasmodium, but distinct from Babesia and Theileria. The putative direction of translation for 3 genes (cox1, cox3, and cytb) was the same in all six Eimeria species. The contents of A+T of the mt genomes were 65.35% for E. acervulina, 65.43% for E. brunetti, 64.53% for E. maxima, 65.04% for E. necatrix, 64.98% for E. tenella and 65.59% for E. praecox. The AT bias has a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses using concatenated nucleotide sequences of the 2 protein-coding genes (cytb and cox1), with three different computational algorithms (Bayesian analysis, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support, indicating that the six Eimeria spp. represent six distinct but closely-related species. These data provide novel mtDNA markers for studying the molecular epidemiology and population genetics of the six Eimeria spp., and should have implications for the molecular diagnosis, prevention and control of coccidiosis in domestic chickens.     

The complete mitochondrial genome of the Asiatic cavity-nesting honeybee Apis cerana (Hymenoptera: Apidae)

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequence of Apis cerana, the Asiatic cavity-nesting honeybee. We present here an analysis of features of its gene content and genome organization in comparison with Apis mellifera to assess the variation within the genus Apis and among main groups of Hymenoptera. The size of the entire mt genome of A. cerana is 15,895 bp, containing 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes and one control region. These genes are transcribed from both strands and have a nucleotide composition high in A and T. The contents of A+T of the complete genomes are 83.96% for A. cerana. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. There are a total of 3672 codons in all 13 protein-coding genes, excluding termination codons. The most frequently used amino acid is Leu (15.52%), followed by Ile (12.85%), Phe (10.10%), Ser (9.15%) and Met (8.96%). Intergenic regions in the mt genome of A. cerana are 705 bp in total. The order and orientation of the gene arrangement pattern is identical to that of A. mellifera, except for the position of the tRNA-Ser(AGN) gene. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes, with three different computational algorithms (NJ, MP and ML), all revealed two distinct groups with high statistical support, indicating that A. cerana and A. mellifera are two separate species, consistent with results of previous morphological and molecular studies. The complete mtDNA sequence of A. cerana provides additional genetic markers for studying population genetics, systematics and phylogeographics of honeybees.     

The complete sequence of the mitochondrial genome of Daphnia pulex (Cladocera: Crustacea).

The sequence of the mitochondrial DNA (mtDNA) of the branchiopod crustacean Daphnia pulex has been completed. It is 15333bp with an A+T content of 62.3%, and contains the typical complement of 13 protein-coding, 22 transfer RNA (tRNA) and two ribosomal RNA (rRNA) genes. Comparison of this sequence with the sequences of the other eight completely sequenced arthropod mtDNAs showed that gene order and orientation are identical to that of Drosophila but different from Artemia due to the rearrangement of two tRNA genes. Nucleotide composition, codon usage, and amino acid composition are very similar in the crustaceans, but divergent from insects and chelicerates which show a much higher bias towards A+T. However, with few exceptions, the mitochondrial proteins of Daphnia are more similar to those of the dipteran insects (Drosophila and Anopheles) than to those of Artemia, at both the nucleotide and amino acid levels, suggesting that Artemia mtDNA is evolving at an accelerated rate. These results also show that sequence evolution and the evolution of nucleotide composition can be decoupled. Analysis of nucleotide substitution patterns in COII showed that there has been an unbiased acceleration of the overall substitution rate in Artemia. In contrast, the accelerated substitution rate in Apis is due partly to extreme A+T mutation pressure. Secondary structures are proposed for the Daphnia tRNAs and rRNAs. The tRNAs are similar to those of other arthropods but tend to have TPsiC arms that are only 4bp long. The rRNA secondary structures are similar to those proposed for insects except for the absence of a small number of helices in Daphnia. Phylogenetic analysis of second codon positions grouped Daphnia with Artemia, as expected, despite the latter's accelerated divergence rate. In contrast, the unusual pattern of mtDNA divergence in Apis led to a topology in which the holometabolous insects (Anopheles, Drosophila, Apis) appeared to be paraphyletic with respect to the hemimetabolous insect, Locusta, due to the early branching of Apis.     

Adaptive threonine increase in transmembrane regions of mitochondrial proteins in higher primates

Background

The mitochondrial (mt) gene tree of placental mammals reveals a very strong acceleration of the amino acid (AA) replacement rate and a change in AA compositional bias in the lineage leading to the higher primates (simians), in contrast to the nuclear gene tree. Whether this acceleration and compositional bias were caused by adaptive evolution at the AA level or directional mutation pressure at the DNA level has been vigorously debated.

Methodology/Principal Findings

Our phylogenetic analysis indicates that the rate acceleration in the simian lineage is accompanied by a marked increase in threonine (Thr) residues in the transmembrane helix regions of mt DNA-encoded proteins. This Thr increase involved the replacement of hydrophobic AAs in the membrane interior. Even after accounting for lack of independence due to phylogeny, a regression analysis reveals a statistical significant positive correlation between Thr composition and longevity in primates.

Conclusion/Significance

Because crucial roles of Thr and Ser in membrane proteins have been proposed to be the formation of hydrogen bonds enhancing helix-helix interactions, the Thr increase detected in the higher primates might be adaptive by serving to reinforce stability of mt proteins in the inner membrane. The correlation between Thr composition in the membrane interior and the longevity of animals is striking, especially because some mt functions are thought to be involved in aging.     

The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella)

The phylogenetic relationships of primates have been extensively investigated, but key issues remain unresolved. Complete mitochondrial genome (mitogenome) data have many advantages in phylogenetic analyses, but such data are available for only 46 primate species. In this work, we determined the complete mitogenome sequence of the black-capped capuchin (Cebus apella). The genome was 16,538 bp in size and consisted of 13 protein-coding genes, 22 tRNAs, two rRNAs and a control region. The genome organization, nucleotide composition and codon usage did not differ significantly from those of other primates. The control region contained several distinct repeat motifs, including a putative termination-associated sequence (TAS) and several conserved sequence blocks (CSB-F, E, D, C, B and 1). Among the protein-coding genes, the COII gene had lower nonsynonymous and synonymous substitutions rates while the ATP8 and ND4 genes had higher rates. A phylogenetic analysis using Maximum likelihood and Bayesian methods and the complete mitogenome data for platyrrhine species confirmed the basal position of the Callicebinae and the sister relationship between Atelinae and Cebidae, as well as the sister relationship between Aotinae (Aotus) and Cebinae (Cebus/Saimiri) in Cebidae. These conclusions agreed with the most recent molecular phylogenetic investigations on primates. This work provides a framework for the use of complete mitogenome information in phylogenetic analyses of the Platyrrhini and primates in general.     

Analysis of directional mutation pressure and nucleotide content in mitochondrial cytochrome b genes

We present a new approach for analyzing directional mutation pressure and nucleotide content in protein-coding genes. Directional mutation pressure, the heterogeneity in the likelihood of different nucleotide substitutions, is used to explain the increasing or decreasing guanine-cytosine content (GC%) in DNA and is represented by µ_D, in agreement with Sueoka (1962, Proc Natl Acad Sci USA 48:582–592). The new method uses simulation to facilitate identification of significant A + T or G + C pressure as well as the comparison of directional mutation pressure among genes, even when they are translated by different genetic codes. We use the method to analyze the evolution of directional mutation pressure and nucleotide content of mitochondrial cytochrome b genes. Results from a survey of 110 taxa indicate that the cytochrome b genes of most taxa are subjected to significant directional mutation pressure and that the gene is subject to A + T pressure in most cases. Only in the anseriform bird Cairina moschata is the cytochrome b gene subject to significant G + C pressure. The GC% at nonsynonymous codon sites decreases proportionately with increasing A + T pressure, and with a slope less than one, indicating a presence of selective constraints. The cytochrome b genes of insects, nematodes, and eumycotes are subject to extreme A + T pressures (µ_D = 0.123, 0.224, and 0.130) and, in parallel, the GC% of the nonsynonymous codon sites has decreased from about 0.44 in organisms that are not subjected to A + T or G + C pressure to about 0.332, 0.323, and 0.367, respectively. The distribution of taxa according to the GC% at nonsynonymous codon sites and directional mutation pressure supports the notion that variation in these parameters is a phylogenetic component.     

Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex

Many vectors of human malaria belong to complexes of morphologically indistinguishable cryptic species. Here we report the analysis of the newly sequenced complete mitochondrial DNA molecules from six recognized or putative species of one such group, the Neotropical Anopheles albitarsis complex. The molecular evolution of these genomes had been driven by purifying selection, particularly strongly acting on the RNA genes. Directional mutation pressure associated with the strand-asynchronous asymmetric mtDNA replication mechanism may have shaped a pronounced DNA strand asymmetry in the nucleotide composition in these and other Anopheles species. The distribution of sequence polymorphism, coupled with the conflicting phylogenetic trees inferred from the mitochondrial DNA and from the published white gene fragment sequences, indicates that the evolution of the complex may have involved ancient mtDNA introgression. Six protein coding genes (nad5, nad4, cox3, atp6, cox1 and nad2) have high levels of sequence divergence and are likely informative for population genetics studies. Finally, the extent of the mitochondrial DNA variation within the complex supports the notion that the complex consists of a larger number of species than until recently believed.     

The complete nucleotide sequence and gene organization of the mitochondrial genome of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae)

The complete nucleotide sequences of the mitochondrial genome (mitogenome) of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae), were determined. The 15,521-bp-long G. orientalis mitogenome contains typical gene complement, base composition, and codon usage found in metazoan mitogenomes. The G. orientalis mitogenome contains the third lowest A+T content (70.5%) among the complete insects mt genome sequences. The initiation codon for the G. orientalis COI gene appears to be ATG, instead of the tetranucleotides, which have been postulated to act as initiation codon for Locusta migratoria and some lepidopteran COI genes. The initiation codon for ND2 appears to be GTG, which is rare, but has been designated as an initiator of Tricholepidion gertschi ND2. All anticodons of G. orientalis tRNAs were identical to Drosophila yakuba and L. migratoria. The tRNA(Ser)(AGN) could not form a stable stem loop structure in the DHU arm as shown in many other insect tRNA(Ser)(AGN). Phylogenetic analysis of nucleotide sequence information from all mt genes supported a monophyletic Diptera, a monophyletic Lepidoptera, a monophyletic Coleoptera, a monophyletic Mecopterida (Diptera+Lepidoptera), and a monophyletic Endopterygota (Diptera+Lepidoptera+Coleoptera), suggesting that the complete insect mitogenome sequence has a resolving power to the diversification events within Endopterygota. However, the relationships of ancient insect orders were unstable, indicating the limited use of mitogenome information at deeper phylogenetic depth.     

Ancestral sequence reconstruction in primate mitochondrial DNA: compositional bias and effect on functional inference

Reconstruction of ancestral DNA and amino acid sequences is an important means of inferring information about past evolutionary events. Such reconstructions suggest changes in molecular function and evolutionary processes over the course of evolution and are used to infer adaptation and convergence. Maximum likelihood (ML) is generally thought to provide relatively accurate reconstructed sequences compared to parsimony, but both methods lead to the inference of multiple directional changes in nucleotide frequencies in primate mitochondrial DNA (mtDNA). To better understand this surprising result, as well as to better understand how parsimony and ML differ, we constructed a series of computationally simple "conditional pathway" methods that differed in the number of substitutions allowed per site along each branch, and we also evaluated the entire Bayesian posterior frequency distribution of reconstructed ancestral states. We analyzed primate mitochondrial cytochrome b (Cyt-b) and cytochrome oxidase subunit I (COI) genes and found that ML reconstructs ancestral frequencies that are often more different from tip sequences than are parsimony reconstructions. In contrast, frequency reconstructions based on the posterior ensemble more closely resemble extant nucleotide frequencies. Simulations indicate that these differences in ancestral sequence inference are probably due to deterministic bias caused by high uncertainty in the optimization-based ancestral reconstruction methods (parsimony, ML, Bayesian maximum a posteriori). In contrast, ancestral nucleotide frequencies based on an average of the Bayesian set of credible ancestral sequences are much less biased. The methods involving simpler conditional pathway calculations have slightly reduced likelihood values compared to full likelihood calculations, but they can provide fairly unbiased nucleotide reconstructions and may be useful in more complex phylogenetic analyses than considered here due to their speed and flexibility. To determine whether biased reconstructions using optimization methods might affect inferences of functional properties, ancestral primate mitochondrial tRNA sequences were inferred and helix-forming propensities for conserved pairs were evaluated in silico. For ambiguously reconstructed nucleotides at sites with high base composition variability, ancestral tRNA sequences from Bayesian analyses were more compatible with canonical base pairing than were those inferred by other methods. Thus, nucleotide bias in reconstructed sequences apparently can lead to serious bias and inaccuracies in functional predictions.     

Phylogenetic relationships of discoglossid frogs (Amphibia:Anura:Discoglossidae) based on complete mitochondrial genomes and nuclear genes

The complete nucleotide sequence of the mitochondrial (mt) genome was determined for three species of discoglossid frogs (Amphibia:Anura:Discoglossidae), representing three of the four recognized genera: Alytes obstetricans, Bombina orientalis, and Discoglossus galganoi. The organization and size of these newly determined mt genomes are similar to those previously reported for other vertebrates. Phylogenetic analyses (maximum likelihood, Bayesian inference, minimum evolution, and maximum parsimony) of mt protein-coding genes at the amino acid level were performed in combination with already published mt genome sequence data of three species of Neobatrachia, one of Pipoidea, and four of Caudata. Phylogenetic analyses based on the deduced amino acid sequences of all mt protein-coding genes arrived at the same topology. The monophyly of Discoglossidae is strongly supported. Within the Discoglossidae, Alytes is consistently recovered as sister group of Discoglossus, to the exclusion of Bombina. The three species representing Neobatrachia exhibited extremely long branches irrespective of the phylogenetic inference method used, and hence their relative position with respect to Discoglossidae and Xenopus may be artefactual due to a severe long branch attraction effect. To further investigate the phylogenetic intrarelationships of discoglossids, nucleotide sequences of four nuclear protein-coding genes (CXCR4, RAG1, RAG2, and Rhodopsin) with sequences available for the three discoglossid genera and Xenopus were retrieved from GenBank, and together with a concatenated nucleotide sequence data set containing all mt protein-coding genes except ND6 were subjected to separate and combined phylogenetic analyses. In all cases, a sister group relationship between Alytes and Discoglossus was recovered with high statistical support.     

Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis

There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences.     

Contracaecum rudolphii B: gene content, arrangement and composition of its complete mitochondrial genome compared with Anisakis simplex s.l

In the present study, we sequenced the complete mt genome (14,022 bp) of parasitic nematode Contracaecum rudolphii B and its structure and organization compared with Anisakis simplex s.l. The mt genome of C. rudolphii B is slightly longer than that of A. simplex s.l. (13,916 bp). C. rudolphii B mt genome is circular, and consists of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA. This genome contains a high A+T (70.5%) content. The mt gene order for C. rudolphii B is the same as those for A. simplex s.l., but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of C. rudolphii B are TTG and ATT. Six protein-coding genes use TAA as a stop codon whereas five genes use T and one genes use TAG as a termination codon. This pattern of codon usage reflects the strong bias for A and T in the mt genome of C. rudolphii B. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (Bayes, ML and MP), all revealed distinct groups with high statistical support, indicating that C. rudolphii B and A. simplex s.l. is distinct but closely related species. These data provide additional novel mtDNA markers for studying the molecular epidemiology and population genetics of the C. rudolphii B, and should have implications for the molecular diagnosis, prevention and control of anisakidosis in humans and animals.     

The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.