We have previously reported 19 the identification of 285 D. melanogaster nuclear genes encoding mitochondrial proteins that represent the counterparts of human peptides annotated in the Swiss-Prot database as mitochondrial 20. On the basis of comparative evidence obtained by BLASTP analysis, 78 of these genes are involved in the OXPHOS system, encoding 66 proteins known to be components of the five large respiratory complexes and 12 proteins involved in oxidative phosphorylation as accessory proteins. To identify the putative counterparts of the D. melanogaster OXPHOS genes in D. pseudoobscura and A. gambiae we performed a TBLASTN search 1321 on the whole genome sequences of these species using the amino-acid sequences of the 78 D. melanogaster peptides as queries. Sequences giving the best reciprocal BLAST hits were tentatively assumed to identify functional counterparts in two species if they could be aligned over at least 60% of the gene length and the BLAST E-score was less than 10^-30. By these criteria, all the 78 D. melanogaster OXPHOS genes investigated have a counterpart both in D. pseudoobscura and in A. gambiae. To better compare the structure of the OXPHOS genes in the three dipteran species, we used the predicted coding sequences as queries for a search of expressed sequence tags (EST) 21, and used the retrieved sequences to annotate the transcribed noncoding sequences of the A. gambiae genes investigated. Although little EST information is available for D. pseudoobscura, it was still possible to predict unambiguously the exon-intron gene structure of the OXPHOS genes in this species, as well as the amino-acid sequence of their full-length products, by exploiting the high level of similarity with D. melanogaster. The results of BLAST analysis, together with the construction of phylogenetic trees that also include other genes that show lesser but still significant sequence similarity to the 78 genes assumed to be one-to-one orthologs in the three species investigated (see below), strongly suggest that the newly identified D. pseudoobscura and A. gambiae genes are the functional counterparts of the 78 D. melanogaster genes used as probes.

Table 1 lists the 78 putative orthologous OXPHOS genes in the three dipteran genomes and their cytological location. For each gene, a record showing the gene map and reporting the annotated genomic sequences as well as the mRNA and protein sequences is available and can be queried at the MitoComp website 22 (see also Additional data files). MitoComp also compares the structure of the D. melanogaster, D. pseudoobscura and A. gambiae putative orthologous genes and their duplications when present (see below), and aligns the orthologous coding sequences (CDS), and also aligns their deduced amino-acid products with the corresponding human protein.

For the products of the OXPHOS genes investigated, the D. melanogaster/D. pseudoobscura average amino-acid sequence identity is 88%, compared to 64% between D. melanogaster and A. gambiae. Figure 1 shows the frequency distribution of sequence identities, and Additional data file 1 lists all pairwise identity values between the products of the 78 OXPHOS genes when orthologous D. melanogaster/D. pseudoobscura, D. melanogaster/A. gambiae and D. melanogaster/human gene products are compared. A multiple alignment of each cluster of homologous proteins is shown at the MitoComp website 22.

It should be kept in mind that identity values reported in Figure 1 and in the table in Additional data file 1 were calculated on the whole sequence of the predicted unprocessed proteins; they are much higher if the putative amino-terminal pre-sequences are excluded, since such sequences, possessed by most mitochondrion-targeted products, show little amino-acid sequence conservation 2324, although they do share specific physicochemical properties 2526. When only the predicted mature protein is considered, the average percentage identity increases to 90% between D. melanogaster and D. pseudoobscura, and to 70% between D. melanogaster and A. gambiae.

A striking example of evolutionary conservation is provided by the genes encoding cytochrome c (an essential and ubiquitous protein found in all organisms) in the three dipteran species: the amino-acid sequences of the gene products are identical in D. melanogaster and D. pseudoobscura, whereas 96% identity is preserved between Drosophila and Anopheles. Coding sequences are also extremely conserved, suggesting that the nucleotide sequence itself is subject to strong evolutionary constraints, maybe due to codon usage bias. Only synonymous substitutions (21 out of 108 codons) were found on comparing D. melanogaster and D. pseudoobscura cytochrome c coding sequences, whereas 28 synonymous substitutions and only four nonsynonymous substitutions were observed between D. melanogaster and A. gambiae (see MitoComp website 22).

It is well known that a given function may be supplied in different species by genes that are not directly derived from a common ancestor, that is, by paralogous, not orthologous, genes. Therefore, we thought it would be interesting to compare the structural organization of the OXPHOS genes in the three species investigated, on the principle that it should be possible to infer derivation from a common ancestor, that is, 'structural orthology', if an identical or very similar overall structure was preserved. As the introns of the putative orthologous OXPHOS genes in the three species are, as expected, too divergent in DNA sequence to be aligned, we used conservation of number of introns, conservation of their location in the coding sequence, and preservation of the reading frame with respect to the flanking exons as our primary criteria.

With the only exception of Dpse\CG5037, putatively encoding protoheme IX farnesyltransferase, whose 5' genomic sequence was impossible to find in the relevant contig assembly, all other investigated D. pseudoobscura genes show a structural organization almost identical to that of their D. melanogaster counterparts. Of the 78 Anopheles genes studied, 39 maintain the structural organization observed in Drosophila, whereas gain or loss of introns occurred in 33, and in six the location of introns is not preserved at all. In agreement with a previous report 4, the intron-exon structure of the gene appears to be conserved in all three dipteran species when splicing of alternative coding exons occurs: the alternative splice forms of both the Drosophila NADH-ubiquinone oxidoreductase acyl carrier protein (mtacp1, CG9160) 27 and the Drosophila ATP synthase epsilon chain (sun, CG9032) 19 have very similar counterparts in Anopheles, as shown by genomic structure comparison, alignment of splice variants and EST mapping (Figure 2).

Genes encoding the acyl carrier protein (mtacp1) in the three species are characterized by the mutually exclusive use of homologous exons that are repeated in tandem (Figure 2a). The duplicate exons occur at the same location in the aligned amino-acid sequences, and are flanked on both sides by a phase 1 intron. When the sequences of the duplicated exons are compared, they show the expected divergence pattern (that is, the similarity between duplicate exons within a gene is less than the similarity of each exon to its equivalent in the orthologous gene). Evidence from genomic and transcribed sequences (GenBank accession numbers BI510891 and BI508135) shows that the duplicated mtacp1exons are also preserved in the more distantly related insect Apis mellifera (honeybee) (Figure 2c,d), indicating a specific adaptive benefit for this gene structure, as also suggested by the evolutionary convergence leading to the occurrence of alternative splicing in members of three different ion-channel gene families from Drosophila to humans 28. However, there is no evidence from ESTs that duplicated mtacp1 exons undergo alternative splicing in vertebrates and nematodes.

Interspecies comparison of the introns of putative orthologous genes indicates that there is little constraint on their nucleotide sequence, which undergoes nucleotide substitutions at a rate comparable to that of pseudogenes 29. However, several observations suggest that intron size is subject to natural selection. For example, in D. melanogaster and several other organisms the distribution of intron length has been shown to be asymmetrical, with a large group of introns falling into a narrow distribution around a 'minimal' length and the remaining showing a much broader length distribution, ranging from hundreds to thousands of base-pairs 303132.

Of the introns that interrupt the coding sequence in the 78 OXPHOS genes investigated in the present study, 88 (64.7%) of 136 in D. melanogaster, 96 (70.5%) of 136 in D. pseudoobscura and 87 (67.9%) of 128 in A. gambiae fall into the short-size class (Figure 3a). However, in A. gambiae the length distribution of these introns appears slightly broader (62-150 bp, compared with 51-100 bp in both Drosophila species). The remaining introns show a broad length distribution, ranging from 151 to 4,702 bp with no clear boundary between classes.

A comparison of the length of introns in corresponding positions in the putative D. melanogaster, D. pseudoobscura and A. gambiae orthologs suggests that changes from the short-size to the long-size (more than 300 bp) intron class, or the converse, have been rare in the evolutionary history of these species: only seven class changes were observed comparing D. melanogaster and D. pseudoobscura introns, and six between D. melanogaster and A. gambiae (Figure 3b). On the whole, our data confirm the highly asymmetrical intron length distribution in D. melanogaster and extend this finding to the introns of the D. pseudoobscura and A. gambiae OXPHOS genes.

It is generally accepted that gene duplication is the basic process that underlies the diversification of genes and the origination of novel gene functions 33; however, many features of this process are still elusive. To obtain more information on the molecular evolution of the genes involved in the OXPHOS system, we searched the genomes of D. melanogaster, D. pseudoobscura and A. gambiae for duplications of the 78 OXPHOS genes whose orthologs we have identified in the three species.

Duplicate gene pairs were tentatively identified within each genome as best reciprocal hits with an E-value of less than 10^-20 in both directions in a TBLASTN search using the default parameters. Deciding whether two proteins may be considered homologous becomes difficult when their sequence identity is within the 20-30% range (the so-called 'twilight zone' 34), and so the following additional criteria were used: first, the two sequences could be aligned over more than 60% of their length; second, the putative processed proteins encoded had to have more than 40% identity; and third, amino-acid percentage similarity had to be larger than percentage identity 35. Even if meeting these criteria and reported as different genes in the ENSEMBL database 36, identical Anopheles nucleotide sequences were excluded from further analysis, as they are likely to reflect annotation artifacts.

Duplications, or in some instances triplications, of 24 OXPHOS genes were found. Overall, we identified 47 genes (20 in D. melanogaster, 19 in D. pseudoobscura and eight in A. gambiae) each of which shows significant similarity with one of the 78 OXPHOS genes reported above. When the structure of a member of a paralogous gene set indicates that it has been produced by retroposition, it seems reasonable to assume that it is derived from a pre-existing 'parent' gene. For duplicates not clearly originating by retroposition, we also assume, on the basis of the much higher level of conservation and expression, that the genes we find to be the structural orthologs in all three species are the parent ones, and in this case also we will henceforth refer to their paralogs as OXPHOS gene duplicates. The amino-acid percentage identity between the products of duplicate gene pairs ranges from 40% to 85%. For each of the OXPHOS gene duplicates, cytological localization, number of exons interrupting the coding sequence, and number of ESTs found in the D. melanogaster and A. gambiae EST databases are reported in Table 2. Neighbor-joining trees derived from distance matrix analysis and showing the inferred evolutionary relationship between members of each gene cluster are available at the MitoComp website 22.

Duplications (or triplications) of 16 of the 78 OXPHOS genes investigated were found in both D. melanogaster and D. pseudoobscura. In such cases, to assign pairwise orthology, besides taking into account conservation of structural organization, given the general conservation of microsyntenic gene order in the two species, we used the products of D. melanogaster genes flanking the duplicate loci to search for homologous sequences also flanking the same genes in the D. pseudoobscura genome.

The genomic organization of many OXPHOS duplicates shows that they were originated by retropositional events, because they are intronless, or have only very few introns that are likely to have been inserted into the coding sequence after the duplication event. In other cases, duplication apparently resulted from transposition of genomic DNA sequences or from recombinational events, as duplicate genes maintain an identical or very similar structural organization.

On the basis of the presence of the duplication in both species, supported by evidence from evolutionary trees and conservation of microsyntenic gene order, it can be inferred that 15 of the duplications identified occurred before the D. melanogaster/D. pseudoobscura divergence (about 46 million years ago). On the other hand, five duplications were found only in D. melanogaster and four only in D. pseudoobscura; in these instances, if the duplication occurred before the divergence of the two species, it has been followed by loss of one of the copies in the lineage leading to the species in which the gene is no longer duplicated. On the assumption that the rate of gene duplication is constant over time, this translates to approximately 0.0014 duplications per gene per million years (4 or 5 duplications per 78 genes per 46 million years) that achieved fixation and long-term preservation in the genome. This value is about twofold lower than the 0.0023 value calculated by Lynch and Conery 37 for the 13,601 genes of the whole genome of D. melanogaster. However, it can be argued that the rate of long-term preservation in the genome of OXPHOS gene duplicates cannot be meaningfully compared with the general rate of preservation of duplicates in the whole genome since, while recent data suggest that in eukaryotic genomes there is preferential duplication of conserved proteins 38, duplicates of genes that encode subunits of multiprotein complexes, as most of the genes we have investigated do, negatively influence the fitness of an organism 39, and are therefore unlikely to become fixed in the population. In summary, it appears reasonable to assume that the preservation in the genome of OXPHOS gene duplicates should occur very infrequently, unless special mechanisms allowing their fixation in the population are present (see the next section).

In A. gambiae we found only four duplications and two triplications of the OXPHOS genes analyzed; of these, four involve genes also duplicated in one or both Drosophila species (Table 2). Pairwise orthology could not be assigned between Drosophila and Anopheles gene duplicates as neither microsynteny nor evolutionary trees provide sufficient evidence for the origin of the gene pairs from a single-copy gene before the Drosophila/Anopheles divergence.

The relative abundance of ESTs in a EST library may be assumed roughly to reflect the level of expression of each mRNA in the tissues from which the library was prepared. We therefore used the mRNA sequences predicted in silico to be transcribed from the OXPHOS duplicate genes investigated in this work as queries in a search of the public D. melanogaster and A. gambiae EST databases to infer the relative abundance of the mRNA copies from the hits scored. For each gene, the number of ESTs found in the databases is detailed in Table 2. With the exception of one of the paralogs of the A. gambiae gene encoding ubiquinol-cytochrome c reductase core protein 1, in all cases the search found the number of ESTs originating from the duplicate gene was strikingly lower than that originating from the putative parent gene, in both D. melanogaster and A. gambiae (in total, 100 versus 1,747 in D. melanogaster and 60 versus 687 in A. gambiae). A smaller number of ESTs originating from the OXPHOS gene duplicates was observed even in A. gambiae EST libraries that are normalized. Remarkably, and regardless of the mechanism of the duplication, in D. melanogaster, in which several organ-specific or developmental stage specific libraries are available, the search showed that the expression of the OXPHOS gene duplicates is strongly testis-biased, as 97 out of the 100 ESTs originating from them were found in testis-derived libraries, while only 27 out of the 1,769 ESTs originating from the parent genes were found in such libraries, the bulk of them being instead found in libraries derived from embryos or somatic tissues.

Our finding that the expression of the OXPHOS gene originated by duplication is strongly testis-biased is validated by the data obtained by Parisi et al. 40 using the FlyGEM microarray to identify D. melanogaster genes showing ovary-, testis- or soma-biased expression. With the exception of CG7349, CG30354, CG30093 and CG12810, for which no data were presented by Parisi et al. 40, all other genes reported in this work as OXPHOS gene duplicates were found in the genomic fraction showing testis-biased expression, whereas all the parent genes present in the dataset showed soma-biased expression. Additional data file 2 summarizes the relevant data extracted from Parisi et al. 40.

The pattern of strongly testis-biased expression of OXPHOS gene duplicates holds for a further sample of 40 duplications of genes annotated in the MitoDrome database 19 as encoding products that are mitochondrion-targeted but not involved in the OXPHOS system. For 15 of these no data are provided by Parisi et al. 40, but all the remaining 25 genes show a testes-biased expression (data not shown).

Duplications of genes encoding OXPHOS subunits, for which stoichiometry is important, are likely to be strongly deleterious owing to the negative consequences of an imbalance in the concentration of the respiratory complex constituents, unless, as proposed by Lynch and Force 41, 'subfunctionalization' and/or a differential expression pattern of duplicate copies occurs. In this case, the duplicate OXPHOS genes would have a reduced or absent capacity to functionally complement mutations in their parent genes, in contrast to what is generally assumed to be the main short-term advantage of gene duplication. In D. melanogaster at least there is evidence for this, as FlyBase 42 and BDGP P-Element Gene Disruption Project 43 searches for P-insertion mutants in the D. melanogaster OXPHOS genes found that lethal alleles for 11 out of 19 D. melanogaster parent genes are known (see the MitoComp website 22), indicating that loss-of-function of the parent gene cannot be compensated for by the presence of the gene duplicate. P-insertion mutants with an abnormal phenotype, indicating a functional divergence, are known for only one of the D. melanogaster OXPHOS gene duplicates - Cyt-c-d, encoding cytochrome c). Interestingly, although Cyt-c-d is adjacent to its putative parent gene, Cyt-c-p, it shows a different pattern of expression, suggesting that the two genes must be regulated at individual gene level and not at chromatin domain level (see Table 2).

A systematic investigation of the expression pattern of other D. melanogaster duplicate genes will be necessary to answer the question of whether the testis-biased expression pattern reported here is specific to the duplicates of genes encoding mitochondrial proteins, or is a more general phenomenon. According to the balance hypothesis, validated by experimental results obtained on yeast 39, single gene duplications involving genes encoding components of multiprotein complexes are expected to severely affect fitness. Therefore, the expression pattern we have observed could be a necessary condition to maintain some gene duplicates in the D. melanogaster genome, at least until they evolve a new useful function. Finally, as nothing is known about the tissue-specific pattern of expression of the genes investigated in D. pseudoobscura and Anopheles, it also remains unclear whether the testis-biased expression of gene copies originated by duplication is specific to D. melanogaster, or is also to be found in other dipterans, and possibly in other organisms.

Because of the preferential use of codons ending in C or G, the D. melanogaster coding sequences have an average GC content higher than the genomic average 4445. This is also true for the 78 D. melanogaster OXPHOS coding sequences reported in this work and for their D. pseudoobscura and A. gambiae counterparts (68% of the codons in the OXPHOS genes end in C or G in D. pseudoobscura and 77% in A. gambiae, compared to 74% in D. melanogaster). In all three species, the coding sequences of OXPHOS gene duplicates show a lower percentage of codons ending in C or G, when compared to both the entire set of 78 orthologous OXPHOS genes and the gene subset including only their parent genes. In samples including all the OXPHOS gene duplicates annotated in this paper the aggregate percentage of C- or G-ending codons is 63%, 46% and 73% in D. melanogaster, D. pseudobscura and A. gambiae respectively, as compared with 70%, 64% and 88% in their parent genes. In D. pseudoobscura, the shift toward a higher percentage of A- or T-ending codons is also detected in the pattern of synonymous codon usage; for 12 of the 18 amino acids that are encoded by more than one codon, the most frequently used codon in the D. pseudoobscura gene duplicates is different from the one used in their parent genes (see Additional data file 3).

It has been reported that in many eukaryotes including yeast 46, C. elegans 47, D. melanogaster 4849 and humans 50, genes with related functions and similar expression patterns tend to be clustered, suggesting that they share aspects of transcriptional regulation depending on their inclusion in the same chromatin domain. In particular, Boutanaev et al. 48 reported that in D. melanogaster clusters of three or more testis-specific genes are much more frequent than expected by chance. Therefore, we investigated the chromosomal distribution of the OXPHOS genes to determine whether clustering could be detected. In all three dipteran species considered, the 78 OXPHOS orthologous genes are randomly distributed on all chromosomal arms (Table 1). Two D. melanogaster genes (Ucrh, encoding the 11 kDa subunit of ubiquinol-cytochrome c reductase, and CG40002, encoding the AGGG subunit of NADH-ubiquinone oxidoreductase) have a heterochromatic location.

No evidence of OXPHOS gene duplicate clustering was found either, despite the common testis-biased expression of such genes. Moreover, no evidence of clustering with other testis-specific genes was found when an EST database search for such genes was performed in the regions flanking the investigated gene duplicates.

However, in accord with two studies reporting a significant deficit of genes with a male-biased expression on the D. melanogaster X chromosome 5152, only one out of the 20 D. melanogaster OXPHOS gene duplicates, two out of 19 in D. pseudoobscura and none (out of eight) in A. gambiae were found to be X-linked (Table 2). It may be that duplications of X-linked genes encoding OXPHOS subunits would be especially deleterious because of the male X chromosome transcriptional hyperactivity, which allows dosage compensation.

In all three dipteran species, a disproportionately high fraction of OXPHOS gene duplicates appears to be constituted of autosomal genes derived from parent genes located on the X chromosome (Table 2). As suggested by recent work on the generation and preservation of functional genes produced by retroposition both in Drosophila 53 and in the human and mouse genomes 54, this may be explained by a selective advantage for duplicates of X-linked genes that move to an autosomal location and so escape the X inactivation in early spermatogenesis that occurs both in Drosophila 55 and in mammals 56.

We would like to speculate that such selective advantage may be especially significant for duplicates of OXPHOS genes, given the heavy reliance of sperm on mitochondrial function. In fact, the excess of autosomal duplicates of X-linked genes is not observed for MitoDrome annotated genes not involved in the OXPHOS system (see above). However, as the general pattern of much lower, testis-biased expression holds even for OXPHOS and other mitochondrial gene duplicates that apparently derive from autosomal parental genes, and even for X-linked duplicates, this pattern (and the explanation of the evolutionary preservation of such genes) cannot only be due to the selective advantage of escaping X inactivation during spermatogenesis.

With the exception of CG9603, all euchromatic D. melanogaster orthologs maintain their localization on the homologuos D. pseudoobscura chromosomal arm (Table 3). CG9603, encoding the VIIa polypeptide of cytochrome c oxidase, is located on the 3R chromosomal arm in D. melanogaster, whereas Dpse\CG9603, its counterpart in D. pseudoobscura, is located on XR; microsyntenic gene order with the flanking genes is conserved in both species, suggesting that a chromosomal rearrangement occurred after their divergence.

OXPHOS gene duplicates also almost always maintain the same chromosomal location and microsyntenic gene order in D. melanogaster and in D. pseudoobscura. However, a more complex situation was observed with regard to the gene encoding subunit IV of cytochrome c oxidase, which is duplicated in D. melanogaster and triplicated in D. pseudoobscura (Table 2). On the basis of identical genomic organization, conserved chromosomal location and mycrosyntenic gene order Dpse\CG10664 is inferred to be the ortholog of D. melanogaster CG10664. Dm CG10396, Dpse\CG10396.1 and Dpse\CG10396.2 are intronless, and neither interarm homology nor microsyntenic order offer any clue to their phylogenetic relationship. The dendrogram based on sequence divergence (see the MitoComp website 22, complex IV, subunit IV) suggests, however, that a duplication event occurred before the D. melanogaster/D. pseudobscura speciation, originating the CG10664-CG10396 gene pair (Dpse\CG10664-Dpse\CG10396 in D. pseudoobscura). A further duplication event, occurring in the D. pseudoobscura lineage after the D. melanogaster/D. pseudoobscura divergence, probably created the Dpse\CG10396.1-Dpse\CG10396.2 gene pair.

In contrast to the maintained location of almost all investigated genes on homologous chromosomal arms in the two Drosophila species, when D. melanogaster and A. gambiae are compared the only meaningful correspondence found concerns the genes on the D. melanogaster 2L and the A. gambiae 3R chromosomal arms (Table 3). This result is consistent with previous reports that compared the location of homologous genes in D. melanogaster and A. gambiae, concluding that extensive reshuffling both within and between chromosomal regions has occurred since the divergence of the two species 417.