Evolutionary change of codon usage for the histone gene family in Drosophila melanogaster and Drosophila hydei

The nucleotide divergence in the protein-coding region for replication-dependent and replication-independent histone 3 and 4 genes of Drosophila melanogaster and Drosophila hydei occurred mostly at the synonymous site. Therefore, the pattern of codon usage was analyzed in the two species, considering the genomic codon bias, which is proposed for estimating the genomic composition pressure in the protein-coding regions. The results indicated that the codon usage in the histone gene family could be explained mostly by the genomic codon bias. However, biases for Ala and Arg were commonly observed for the histone 3 and histone 4 gene families, and biases for Ser, Leu, and Glu were observed in a gene-specific manner. This suggests that both genomic codon bias and gene- or codon-specific bias are responsible for the nucleotide differentiation in the protein-coding region of the histone genes.     

Divergence and heterogeneity of the histone gene repeating units in the Drosophila melanogaster species subgroup

The repeating units of the histone gene cluster containing the H1, H2A, H2B and H4 genes were amplified by PCR from the Drosophila melanogaster species subgroup, i.e., D. yakuba, D. erecta, D. sechellia, D. mauritiana, D. teissieri and D. orena. The PCR products were cloned and their nucleotide sequences of about 4.6-4.8kbp were determined to elucidate the mechanism of molecular evolution of the histone gene family. The heterogeneity among the histone gene repeating units was 0.6% and 0.7% for D. yakuba and D. sechellia, respectively, indicating the same level of heterogeneity as in the H3 gene region of D. melanogaster. Divergence of the genes among species even in the most closely related ones was much greater than the heterogeneity among family members, indicating a concerted mode of evolution for the histone gene repeating units. Among the species in the D. melanogaster species subgroup, the histone gene regions as well as 3rd codon position of the coding region showed nearly the same GC contents. These results suggested that the previous conclusion on analysis of the H3 gene regions, the gene family evolution in a concerted fashion, holds true for the whole histone gene repeating unit.     

Molecular evolutionary analysis of a histone gene repeating unit from Drosophila simulans

A repeating unit of the histone gene cluster from Drosophila simulans containing the H1, H2A, H2B and H4 genes (the H3 gene region has already been analyzed) was cloned and analyzed. A nucleotide sequence of about 4.6 kbp was determined to study the nucleotide divergence and molecular evolution of the histone gene cluster. Comparison of the structure and nucleotide sequence with those of Drosophila melanogaster showed that the four histone genes were located at identical positions and in the same directions. The proportion of different nucleotide sites was 6.3% in total. The amino acid sequence of H1 was divergent, with a 5.1% difference. However, no amino acid change has been observed for the other three histone proteins. Analysis of the GC contents and the base substitution patterns in the two lineages, D. melanogaster and D. simulans, with a common ancestor showed the following. 1) A strong negative correlation was found between the GC content and the nucleotide divergence in the whole repeating unit. 2) The mode of molecular evolution previously found for the H3 gene was also observed for the whole repeating unit of histone genes; the nucleotide substitutions were stationary in the 3' and spacer regions, and there was a directional change of the codon usage to the AT-rich codons. 3) No distinct difference in the mode or pattern of molecular evolution was detected for the histone gene repeating unit in the D. melanogaster and D. simulans lineages. These results suggest that selectional pressure for the coding regions of histones, which eliminate A and T, is less effective in the D. melanogaster and D. simulans lineages than in the other GC-rich species.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Nucleotide Variation and Divergence in the Histone Multigene Family in Drosophila Melanogaster

Nucleotide differences in the histone H3 gene family in Drosophila melanogaster were studied on three levels: (1) within a chromosome, (2) within a population and (3) between species (D. melanogaster and Drosophila simulans). The average difference within the H3 gene within a chromosome was 0.0040 per nucleotide site, about 52% of that within a population (0.0077). The proportion of divergent sites between the two species was 0.0575, which is about 8.5 times the difference within a species. The distribution of divergence between species was similar to that of variation within a species. Divergence and variation were noted to be greatest in the 3' noncoding region and least in the coding region. Values intermediate between these were found for the 5' noncoding region. Divergence and variation in silent sites exceeded those in the total coding region, thus indicating possible purifying selection for amino-acid-altering change. Phylogenetic relations among H3 genes and genetic differences on these three levels are evidence for the concerted evolution of the histone gene family. The molecular mechanism by which variation is produced and maintained is discussed.     

Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by using Drosophila pseudoobscura as the out group. Our results show that the melanogaster species group clustered in three main lineages: (1). montium subgroup; (2). ananassae subgroup; and (3). the seven oriental subgroups, among which the montium subgroup diverged first. In the third main lineage, suzukii and takahashii subgroups formed a clade, while eugracilis, melanogaster, elegans, ficusphila, and rhopaloa subgroups formed another clade. The bootstrap values at subgroup levels are high. The phylogenetic relationships of these species subgroups derived from our data are very different from those based on some other DNA data and morphology data.     

Ancestral inference and the study of codon bias evolution: implications for molecular evolutionary analyses of the Drosophila melanogaster subgroup

Reliable inference of ancestral sequences can be critical to identifying both patterns and causes of molecular evolution. Robustness of ancestral inference is often assumed among closely related species, but tests of this assumption have been limited. Here, we examine the performance of inference methods for data simulated under scenarios of codon bias evolution within the Drosophila melanogaster subgroup. Genome sequence data for multiple, closely related species within this subgroup make it an important system for studying molecular evolutionary genetics. The effects of asymmetric and lineage-specific substitution rates (i.e., varying levels of codon usage bias and departures from equilibrium) on the reliability of ancestral codon usage was investigated. Maximum parsimony inference, which has been widely employed in analyses of Drosophila codon bias evolution, was compared to an approach that attempts to account for uncertainty in ancestral inference by weighting ancestral reconstructions by their posterior probabilities. The latter approach employs maximum likelihood estimation of rate and base composition parameters. For equilibrium and most non-equilibrium scenarios that were investigated, the probabilistic method appears to generate reliable ancestral codon bias inferences for molecular evolutionary studies within the D. melanogaster subgroup. These reconstructions are more reliable than parsimony inference, especially when codon usage is strongly skewed. However, inference biases are considerable for both methods under particular departures from stationarity (i.e., when adaptive evolution is prevalent). Reliability of inference can be sensitive to branch lengths, asymmetry in substitution rates, and the locations and nature of lineage-specific processes within a gene tree. Inference reliability, even among closely related species, can be strongly affected by (potentially unknown) patterns of molecular evolution in lineages ancestral to those of interest.     

Changes in the recombinational environment affect divergence in the yellow gene of Drosophila

The complete coding region of the yellow (y) gene was sequenced in different Drosophila species. In the species of the melanogaster subgroup (D. melanogaster, D. simulans, D. mauritiana, D. yakuba, and D. erecta), this gene is located at the tip of the X chromosome in a region with a strong reduction in recombination rate. In contrast, in D. ananassae (included in the ananassae subgroup of the melanogaster group) and in the obscura group species (D. subobscura, D. madeirensis, D. guanche, and D. pseudoobscura), the y gene is located in regions with normal recombination rates. As predicted by the hitchhiking and background selection models, this change in the recombinational environment affected synonymous divergence in the y-gene-coding region. Estimates of the number of synonymous substitutions per site were much lower between the obscura group species and D. ananassae than between the species of the obscura group and the melanogaster subgroup. In fact, a highly significant increase in the rate of synonymous substitution was detected in all lineages leading to the species of the melanogaster subgroup relative to the D. ananassae lineage. This increase can be explained by a higher fixation rate of mutations from preferred to unpreferred codons (slightly deleterious mutations). The lower codon bias detected in all species of the melanogaster subgroup relative to D. ananassae (or to the obscura group species) would be consistent with this proposal. Therefore, at least in Drosophila, changes in the recombination rate in different lineages might cause deviations of the molecular-clock hypothesis and contribute to the overdispersion of the rate of synonymous substitution. In contrast, the change in the recombinational environment of the y gene has no detectable effect on the rate of amino acid replacement in the Yellow protein.     

Evolution of the GC content of the histone 3 gene in seven Drosophila species

The molecular evolution of the histone multigene family was studied by cloning and determining the nucleotide sequences of the histone 3 genes in seven Drosophila species, D. takahashii, D. lutescens, D. ficusphila, D. persimilis, D.pseudoobscura, D. americana and D. immigrans. CT repeats, a TATA box and an AGTG motif in the 5' region, and a hairpin loop and purine-rich motifs (CAA(T/G)GAGA) in the 3' region were conserved even in distantly related species. In D. hydei and D.americana, the GC content at the third codon position in the protein coding region was relatively low (49% and 45%), while in D. takahashii and D. lutescens it was relatively high (64% and 65%). The non- significant correlation between the GC contents in the 3' region and at the third codon position as well as the evidence of less constraint in the 3' region suggested that mutational bias may not be the major mechanism responsible for the biased nucleotide change at the third codon position or for codon usage bias.     

Patterns of nucleotide polymorphism and divergence in the odorant-binding protein genes OS-E and OS-F: analysis in the melanogaster species subgroup of Drosophila

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.     

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.     

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.     

Basal relationships in the Drosophila melanogaster species group

The Drosophila melanogaster species group is a popular model for evolutionary studies due to its morphological and ecological diversity and its inclusion of the model species D. melanogaster. However, phylogenetic relationships among major lineages within this species group remain controversial. In this report, the phylogeny of 10 species representing each of the well-supported monophyletic clades in the melanogaster group was studied using the sequences of 14 loci that together comprise 9493 nucleotide positions. Combined Bayesian analysis using gene-specific substitution models produced a 100% credible set of two trees. In the strict consensus of these trees, the ananassae subgroup branches first in the melanogaster species group, followed by the montium subgroup. The remaining lineages form a monophyletic clade in which D. ficusphila and D. elegans branch first, followed by D. biarmipes, D. eugracilis, and the melanogaster subgroup. This strongly supported phylogeny resolves most basal relationships in the melanogaster species group, and provides a framework that can be extended in the future to encompass more species.     

Drosophila Melanogaster Mitochondrial DNA: Gene Organization and Evolutionary Considerations

The sequence of a 8351-nucleotide mitochondrial DNA (mtDNA) fragment has been obtained extending the knowledge of the Drosophila melanogaster mitochondrial genome to 90% of its coding region. The sequence encodes seven polypeptides, 12 tRNAs and the 3' end of the 16S rRNA and CO III genes. The gene organization is strictly conserved with respect to the Drosophila yakuba mitochondrial genome, and different from that found in mammals and Xenopus. The high A + T content of D. melanogaster mitochondrial DNA is reflected in a reiterative codon usage, with more than 90% of the codons ending in T or A, G + C rich codons being practically absent. The average level of homology between the D. melanogaster and D. yakuba sequences is very high (roughly 94%), although insertion and deletions have been detected in protein, tRNA and large ribosomal genes. The analysis of nucleotide changes reveals a similar frequency for transitions and transversions, and reflects a strong bias against G + C on both strands. The predominant type of transition is strand specific.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons

The nucleotide sequence of the Xdh region of Drosophila pseudoobscura is presented. The Xdh gene structure and organization are compared with the homologous region in D. melanogaster. This locus is shown to have similar organization in the two species, although an additional intron and three insertion/deletion events are described for the D. pseudoobscura coding region. The encoded proteins are predicted to have very similar charges and hydrophobic/hydrophilic domains even though 11% of the amino acids are different. A gene 5' to Xdh, putative l(3)s12, is suggested from sequence similarity between the species. Synonymous differences at the Xdh locus between the two species are analyzed using a new method described in the preceding paper by Lewontin. This analysis shows that synonymous positions within the Xdh locus are evolving at very different rates, being dependent on level of codon redundancy. A comparison of synonymous divergence between D. melanogaster and D. pseudoobscura in five additional genes reveals variation in the level of synonymous substitution.      

A phylogeny of Drosophilidae using the Amyrel gene: questioning the Drosophila melanogaster species group boundaries

In this study, the phylogenetic relationships of 164 species of the family Drosophilidae are discussed, using the Amyrel gene, a member of the α -amylase multigene family. This study focuses on numerous species groups in the subgenera Sophophora and Drosophila of the genus Drosophila but also includes other closely related genera. Nucleotide data were analysed by several methods: maximum parsimony, neighbour joining, maximum likelihood and Bayesian inference. Heterogeneity of base composition (mainly low GC contents in the species groups willistoni and saltans ) has been addressed. In all analyses, the genus Drosophila appeared paraphyletic. The subgenus Sophophora clearly appeared to be a monophyletic group, showing well-resolved clades, with the Neotropical groups arising in a basal position. Here, it is proposed to raise the species subgroups ananassae and montium to the rank of species group, and to restrict the melanogaster species group to the melanogaster subgroup plus the 'Oriental' subgroups, among which the suzukii subgroup is polyphyletic. Some related genera such as Zaprionus , Liodrosophila , Scaptomyza and Hirtodrosophila are clustered with, or inside the subgenus Drosophila , which is therefore paraphyletic and should be reviewed.     

Molecular Evolution between Drosophila Melanogaster and D. Simulans: Reduced Codon Bias, Faster Rates of Amino Acid Substitution, and Larger Proteins in D. Melanogaster

Both natural selection and mutational biases contribute to variation in codon usage bias within Drosophila species. This study addresses the cause of codon bias differences between the sibling species, Drosophila melanogaster and D. simulans. Under a model of mutation-selection-drift, variation in mutational processes between species predicts greater base composition differences in neutrally evolving regions than in highly biased genes. Variation in selection intensity, however, predicts larger base composition differences in highly biased loci. Greater differences in the G+C content of 34 coding regions than 46 intron sequences between D. melanogaster and D. simulans suggest that D. melanogaster has undergone a reduction in selection intensity for codon bias. Computer simulations suggest at least a fivefold reduction in N(e)s at silent sites in this lineage. Other classes of molecular change show lineage effects between these species. Rates of amino acid substitution are higher in the D. melanogaster lineage than in D. simulans in 14 genes for which outgroup sequences are available. Surprisingly, protein sizes are larger in D. melanogaster than in D. simulans in the 34 genes compared between the two species. A substantial fraction of silent, replacement, and insertion/deletion mutations in coding regions may be weakly selected in Drosophila.     

Molecular Evolution of the Duplicated Amy Locus in the Drosophila Melanogaster Species Subgroup: Concerted Evolution Only in the Coding Region and an Excess of Nonsynonymous Substitutions in Speciation

From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a ``specialist' species for host plants and probably also occurs in the process of speciation in general.