Evolution of hominoid mitochondrial DNA with special reference to the silent substitution rate over the genome

Summary Focusing on the synonymous substitution rate, we carried out detailed sequence analyses of hominoid mitochondrial (mt) DNAs of ca. 5-kb length. Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low saturation level. The extent of the compositional biases differs from gene to gene. Such changes in base compositions, even if small, can bring about considerable variation in observed synonymous differences and may result in the region-dependent estimate of the synonymous substitution rate. We demonstrate that such a region dependency is due to a failure to take proper account of heterogeneous compositional biases from gene to gene but that the actual synonymous substitution rate is rather uniform. The synonymous substitution rate thus estimated is 2.37 ± 0.11 × 10^–8 per site per year and comparable to the overall rate for the noncoding region. On the other hand, the rate of nonsynonymous substitutions differs considerably from gene to gene, as expected under the neutral theory of molecular evolution. The lowest rate is 0.8 × 10^–9 per site per year forCOI and the highest rate is 4.5 × 10^–9 forATPase 8, the degree of functional constraints (measured by the ratio of the nonsynonymous to the synonymous substitution rate) being 0.03 and 0.19, respectively. Transfer RNA (tRNA) genes also show variability in the base contents and thus in the nucleotide differences. The average rate for 11 tRNAs contained in the 5-kb region is 3.9 × 10^–9 per site per year. The nucleotide substitutions in the genome suggest that the transition rate is about 17 times faster than the transversion rate.     

A method for estimating the numbers of synonymous and nonsynonymous substitutions per site

A method for estimating the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site is proposed. The method is based on the Li's (J Mol. Evol. 36:96–99, 1993) and Pamilo and Bianchi's (Mol. Biol. Evol. 10:271–281, 1993) method, but a putative source of bias is solved. It is proposed that the number of synonymous substitutions that are actually transitions or transversions should be computed by separating the twofold degenerate sites into two types of sites, 2S-fold and 2V-fold, where only transitional and transversional substitutions are synonymous, respectively. Kimura's (J. Mol. Evol. 16:111–120, 1980) two-parameter correcting method for multiple substitutions at a site is then applied using the overall observed synonymous transversion frequency to estimate both the numbers of synonymous transversional (Bs) and transitional (As) substitutions per site. This approach, therefore, also minimizes stochastic errors. Computer simulations indicate that the method presented gives more accurate Ks and Ka estimates than the aforementioned methods. Furthermore, the obtention of confidence intervals for divergence estimates by computer simulation is proposed.     

The origin and evolution of Ebola and Marburg viruses

Molecular evolutionary analyses for Ebola and Marburg viruses were conducted with the aim of elucidating evolutionary features of these viruses. In particular, the rate of nonsynonymous substitutions for the glycoprotein gene of Ebola virus was estimated to be, on the average, 3.6 x 10(-5) per site per year. Marburg virus was also suggested to be evolving at a similar rate. Those rates were a hundred times slower than those of retroviruses and human influenza A virus, but were of the same order of magnitude as that of the hepatitis B virus. When these rates were applied to the degree of sequence divergence, the divergence time between Ebola and Marburg viruses was estimated to be more than several thousand years ago. Moreover, most of the nucleotide substitutions were transitions and synonymous for Marburg virus. This suggests that purifying selection has operated on Marburg virus during evolution.      

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.     

Selective processes and adaptive evolution of the cytochrome b gene in salamanders of the genus Salamandrella

Sequence analysis of the cytochrome b gene fragment in the salamanders of the genus Salamandrella, Siberian salamander and Schrenk salamander was performed with the purpose to elucidate the effect of natural selection on the evolution of mitochondrial DNA (mtDNA) in these species. It was demonstrated that despite of notable influence of negative selection (expressed as very low dN/dS values), speciation and intraspecific divergence in salamanders was accompanied by the appearance of radical amino acid substitutions, caused by the influence of positive (directional) selection. To examine the evolutionary pattern of synonymous mtDNA sites, distribution of conservative and non-conservative substitutions was analyzed. The rates of conservative and non-conservative substitutions were nearly equal, pointing to neutrality of mutation process at synonymous mtDNA sites of salamanders. Analysis of conservative and non-conservative synonymous substitution distributions in different parts of phylogenetic trees showed that the differences between the synonymous groups compared were statistically significant only in one phylogenetic group of Siberian salamander (haplogroup C) (P = 0.02). In the group of single substitutions, located at terminal phylogenetic branches of Siberian salamanders from group C, increased rate of conservative substitutions was observed. Based on these findings, it was suggested that selective processes could have an influence on the formation of the synonymous substitution profile in the Siberian salamander mtDNA fragment examined.     

Crassostrea angulata Bindin Gene and the Divergence of Fucose-Binding Lectin Repeats Among Three Species of Crassostrea

Bindin is a major protein for species-specific recognition between sperm and congenetic egg in many free-spawning marine invertebrates. We cloned a novel bindin gene from the oyster Crassostrea angulata by 3′ and 5′ rapid amplification of cDNA ends. The full-length bindin cDNA was 1,049 bp with a 771-bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA was 8,508 bp containing four exons and three introns. Three haplotypes of F-lectin repeat were detected from seven sequences of F-lectin repeat of six male oysters. Both neighbor-joining and minimum-evolution phylogenetic trees show that haplotype an1 was close to Crassostrea gigas while an2 and an3 were close to Crassostrea sikamea. Intron-4 in the middle of F-lectin repeat is highly variable in both size and sequence. We classified intron-4 into three types according to their size and the F-lectin repeat they were located in. Intron-4 may play an important role in recombination. We compared the number of nonsynonymous substitutions (Dn) and synonymous substitutions (Ds) per nucleotide site among 19 F-lectin haplotypes of the three species. Dn/Ds ratios suggested that positive selection occurred between C. gigas and C. sikamea and between C. gigas and C. angulata. Nine positive selected positions (p > 90%) are identified among 19 haplotypes of three species. They are located on the F-lectin binding face around the three recognition motif residues. We assume that these nine clustered amino acids are related with species-specific recognition.     

Nucleotide Sequence Analyses and Predicted Coding of Bunyavirus Genome RNA Species

We performed 3′ RNA sequence analyses of [³²P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3′-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3′-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3′-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3′-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.     

Multiple L1 progenitors in prosimian primates: Phylogenetic evidence from ORF1 sequences

One of the uncertainties regarding the evolution of L1 elements is whether there are numerous progenitor genes. We present phylogenetic evidence from ORF1 sequences of slow loris (Nycticebus coucang) and galago (Galago crassicaudatus) that there were at least two distinct progenitors, active at the same time, in the ancestor of this family of prosimian primates. A maximum parsimony analysis that included representative L1s from human, rabbit, and rodents, along with the prosimian sequences, revealed that one of the galago L1s (Gc11) grouped very strongly with the slow loris sequences. The remaining galago elements formed their own unique and strongly supported clade. An analysis of replacement and silent site changes for each link of the most parsimonious tree indicated that during the descent of the Gc11 sequence approximately two times more synonymous than nonsynonymous substitutions had occurred, implying that the Gc11 founder was functional for some time after the split of galago and slow loris. Strong purifying selection was also evident on the galago branch of the tree. These data indicate that there were two distinct and contemporaneous L1 progenitors in the lorisoid ancestor, evolving under purifying selection, that were retained as functional L1s in the galago lineage (and presumably also in the slow loris). The prosimian ORF1 sequences could be further subdivided into subfamilies. ORF1 sequences from both the galago and slow loris have a premature termination codon near the 3′ end, not shared by the other mammalian sequences, that shortens the open reading frame by 288 bp. An analysis of synonymous and nonsynonymous substitutions for the 5′ and 3′ portions, that included intra- and inter-subfamily comparisons, as well as comparisons among the other mammalian sequences, suggested that this premature stop codon is a prosimian acquisition that has rendered the 3′ portion of ORF1 in these primates noncoding. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Molecular considerations in the evolution of bacterial genes

Summary Synonymous and nonsynonymous substitution rates at the loci encoding glyceraldehyde-3-phosphate dehydrogenase (gap) and outer membrane protein 3A (ompA) were examined in 12 species of enteric bacteria. By examining homologous sequences in species of varying degrees of relatedness and of known phylogenetic relationships, we analyzed the patterns of synonymous and nonsynonymous substitutions within and among these genes. Although both loci accumulate synonymous substitutions at reduced rates due to codon usage bias, portions of thegap andompA reading frames show significant deviation in synonymous substitution rates not attributable to local codon bias. A paucity of synonymous substitutions in portions of theompA gene may reflect selection for a novel mRNA secondary structure. In addition, these studies allow comparisons of homologous protein-coding sequences (gap) in plants, animals, and bacteria, revealing differences in evolutionary constraints on this glycolytic enzyme in these lineages.     

Phylogenetic Analysis Reveals Rapid Evolutionary Dynamics in the Plant RNA Virus Genus Tobamovirus

Early studies on the evolutionary dynamics of plant RNA viruses suggested that they may evolve more slowly than their animal counterparts, sometimes dramatically so. However, these estimates were often based on an assumption of virus–host codivergence over time-scales of many millions of years that is difficult to verify. An important example are viruses of the genus Tobamovirus, where the assumption of host–virus codivergence over 100 million years has led to rate estimates in the range of ~1 × 10⁻⁸ nucleotide substitutions per site, per year. Such a low evolutionary rate is in apparent contradiction with the ability of some tobamoviruses to quickly overcome inbred genetic resistance. To resolve how rapidly molecular evolution proceeds in the tobomaviruses, we estimated rates of nucleotide substitution, times to common ancestry, and the extent of congruence between virus and host phylogenies. Using Bayesian coalescent methods applied to time-stamped sequences, we estimated mean evolutionary rates at the nucleotide and amino acid levels of between 1 × 10⁻⁵ and 1.3 × 10⁻³ substitutions per site, per year, and hence similar to those seen in a broad range of animal and plant RNA viruses. Under these rates, a conservative estimate for the time of origin of the sampled tobamoviruses is within the last 100,000 years, and hence a far more recently than proposed assuming codivergence. This is supported by our cophylogeny analysis which revealed significantly discordant evolutionary histories between the tobamoviruses and the plant families they infect.     

Rates of synonymous substitution in plant nuclear genes

Summary The rate of synonymous nucleotide substitution in nuclear genes of higher plants has been estimated. The rate varies among genes by a factor of up to two, in a manner that is not immediately explicable in terms of base composition or codon usage bias. The average rate, in both monocots and dicots, is about four times higher than that in chloroplast genes. This leads to an estimated absolute silent substitution rate of 6 × 10^–9 substitutions per site per year that falls within the range of average rates (2–8 × 10^–9) seen in different mammalian nuclear genomes.     

Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae)

Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the rbcL-462 intron in cases of spontaneous intron loss. On the other hand, the presence of intact to largely degenerate ORFs of the rbcL-462 introns within the three isolates of Gonium viridistellatum and the rare occurrence of the ORF-lacking rbcL-462 intron suggested that the ORFs might degenerate to result in the spontaneous intron loss during a very short evolutionary time following the loss of the ORF function. Thus, the sporadic distribution of the rbcL-462 introns within the colonial Volvocales can be largely explained by an equilibrium between maintenance of the introns by the intron ORF and spontaneous loss of introns when the introns do not have a functional ORF.     

Molecular evolution of two paralogous tandemly repeated heterochromatic gene clusters linked to the X and Y chromosomes of Drosophila melanogaster

Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene betaCK2tes encoding the putative testis-specific beta-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3' splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5-10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions.     

Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses

Because most extant viruses mutate rapidly and lack a true fossil record, their deep evolution and long-term substitution rates remain poorly understood. In addition to retroviruses, which rely on chromosomal integration for their replication, many other viruses replicate in the nucleus of their host''s cells and are therefore prone to endogenization, a process that involves integration of viral DNA into the host''s germline genome followed by long-term vertical inheritance. Such endogenous viruses are highly valuable as they provide a molecular fossil record of past viral invasions, which may be used to decipher the origins and long-term evolutionary characteristics of modern pathogenic viruses. Hepadnaviruses (Hepadnaviridae) are a family of small, partially double-stranded DNA viruses that include hepatitis B viruses. Here we report the discovery of endogenous hepadnaviruses in the genome of the zebra finch. We used a combination of cross-species analysis of orthologous insertions, molecular dating, and phylogenetic analyses to demonstrate that hepadnaviruses infiltrated repeatedly the germline genome of passerine birds. We provide evidence that some of the avian hepadnavirus integration events are at least 19 My old, which reveals a much deeper ancestry of Hepadnaviridae than could be inferred based on the coalescence times of modern hepadnaviruses. Furthermore, the remarkable sequence similarity between endogenous and extant avian hepadnaviruses (up to 75% identity) suggests that long-term substitution rates for these viruses are on the order of 10⁻⁸ substitutions per site per year, which is a 1,000-fold slower than short-term rates estimated based on the sequences of circulating hepadnaviruses. Together, these results imply a drastic shift in our understanding of the time scale of hepadnavirus evolution, and suggest that the rapid evolutionary dynamics characterizing modern avian hepadnaviruses do not reflect their mode of evolution on a deep time scale.     

Evolution of Rh Blood Group Genes Have Experienced Gene Conversions and Positive Selection

There are two tightly linked loci (D and CE) for the human Rh blood group. Their gene products are membrane proteins having 12 transmembrane domains and form a complex with Rh50 glycoprotein on erythrocytes. We constructed phylogenetic networks of human and nonhuman primate Rh genes, and the network patterns suggested the occurrences of gene conversions. We therefore used a modified site-by-site reconstruction method by using two assumed gene trees and detected 9 or 11 converted regions. After eliminating the effect of gene conversions, we estimated numbers of nonsynonymous and synonymous substitutions for each branch of both trees. Whichever gene tree we selected the branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions, an indication of positive selection. Many other branches also showed higher nonsynonymous than synonymous substitutions; this suggests that the Rh genes have experienced some kind of positive selection. Received: 16 March 1999 / Accepted: 17 June 1999     

Detection of Signature Sequences in Overlapping Genes and Prediction of a Novel Overlapping Gene in Hepatitis G Virus

In viruses an increased coding ability is provided by overlapping genes, in which two alternative open reading frames (ORFs) may be translated to yield two distinct proteins. The identification of signature sequences in overlapping genes is a topic of particular interest, since additional out-of-frame coding regions can be nested within known genes. In this work, a novel feature peculiar to overlapping coding regions is presented. It was detected by analysis of a sample set of 21 virus genomic sequences and consisted in the repeated occurrence of a cluster of basic amino acid residues, encoded by a frame, combined to a stretch of acidic residues, encoded by the corresponding overlapping frame. A computer scan of an additional set of virus sequences demonstrated that this feature is common to several other known overlapping ORFs and led to prediction of a novel overlapping gene in hepatitis G virus (HGV). The occurrence of a bifunctional coding region in HGV was also supported by its extremely lower rate of synonymous nucleotide substitutions compared to that observed in the other gene regions of the HGV genome. Analysis of the amino acid sequence that was deduced from the putative overlapping gene revealed a high content of basic residues and the presence of a nuclear targeting signal; these characteristics suggest that a core-like protein may be expressed by this novel ORF. Received: 21 July 1999 / Accepted: 26 October 1999     

Bayesian Coalescent Analysis Reveals a High Rate of Molecular Evolution in GB Virus C

GB virus C/hepatitis G (GBV-C) is an RNA virus of the family Flaviviridae. Despite replicating with an RNA-dependent RNA polymerase, some previous estimates of rates of evolutionary change in GBV-C suggest that it fixes mutations at the anomalously low rate of ∼10⁻⁷ nucleotide substitution per site, per year. However, these estimates were largely based on the assumption that GBV-C and its close relative GBV-A (New World monkey GB viruses) codiverged with their primate hosts over millions of years. Herein, we estimated the substitution rate of GBV-C using the largest set of dated GBV-C isolates compiled to date and a Bayesian coalescent approach that utilizes the year of sampling and so is independent of the assumption of codivergence. This revealed a rate of evolutionary change approximately four orders of magnitude higher than that estimated previously, in the range of 10⁻² to 10⁻³ sub/site/year, and hence in line with those previously determined for RNA viruses in general and the Flaviviridae in particular. In addition, we tested the assumption of host-virus codivergence in GBV-A by performing a reconciliation analysis of host and virus phylogenies. Strikingly, we found no statistical evidence for host-virus codivergence in GBV-A, indicating that substitution rates in the GB viruses should not be estimated from host divergence times.     

The fitness of defective interfering murine coronavirus DI-a and its derivatives is decreased by nonsense and frameshift mutations.

The genome of the defective interfering (DI) mouse hepatitis virus DI-a carries a large open reading frame (ORF) consisting of ORF1a, ORF1b, and nucleocapsid sequences. To test whether this fusion ORF is important for DI virus replication, we constructed derivatives of the DI-a genome in which the reading frame was truncated by a nonsense codon or a frameshift mutation. In vitro-transcribed DI RNAs were transfected into mouse hepatitis virus-infected cells followed by undiluted passage of the resulting virus-DI virus stocks. The following observations were made. (i) Truncation of the fusion ORF was not lethal but led to reduced accumulation of DI RNA. (ii) When pairs of nearly identical in-frame and out-of-frame DI RNAs were directly compared by cotransfection, DI viruses containing in-frame genomic RNAs prevailed within three successive passage even when the out-of-frame RNAs were transfected in 10-fold molar excess. (iii) When DI viruses containing out-of-frame genomic RNAs were passaged, mutants emerged and were selected for that had restored the reading frame. We conclude that translation of the fusion ORF is indeed required for efficient propagation of DI-a and its derivatives.