Codon Bias Differentiates Between the Duplicated Amylase Loci Following Gene Duplication in Drosophila

Corresponding author: Nobuyuki Inomata, Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan., ninomscb{at}mbox.nc.kyushu-u.ac.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We examined the pattern of synonymous substitutions in the duplicated Amylase (Amy) genes (called the Amy1- and Amy3-type genes, respectively) in the Drosophila montium species subgroup. The GC content at the third synonymous codon sites of the Amy1-type genes was higher than that of the Amy3-type genes, while the GC content in the 5'-flanking region was the same in both genes. This suggests that the difference in the GC content at third synonymous sites between the duplicated genes is not due to the temporal or regional changes in mutation bias. We inferred the direction of synonymous substitutions along branches of a phylogeny. In most lineages, there were more synonymous substitutions from G/C (G or C) to A/T (A or T) than from A/T to G/C. However, in one lineage leading to the Amy1-type genes, which is immediately after gene duplication but before speciation of the montium species, synonymous substitutions from A/T to G/C were predominant. According to a simple model of synonymous DNA evolution in which major codons are selectively advantageous within each codon family, we estimated the selection intensity for specific lineages in a phylogeny on the basis of inferred patterns of synonymous substitutions. Our result suggested that the difference in GC content at synonymous sites between the two Amy-type genes was due to the change of selection intensity immediately after gene duplication but before speciation of the montium species.

ADAPTIVE evolution of amino acid substitutions caused by positive Darwinian selection is one of the most important mechanisms for the functional divergence between members of a multigene family (HUGHES 2000 ). Therefore, previous studies have concentrated on detecting an excess of (amino acid) replacement substitutions by comparing the patterns of replacement and synonymous substitutions. This procedure has been used to infer evolutionary forces and provided evidence for adaptive amino acid evolution after gene duplication (MESSIER and STEWART 1997 ; ZHANG et al. 1998 ; HUGHES et al. 2000 ).

Increasingly more findings suggest that most synonymous changes in unicellular organisms and Drosophila are not neutral. Indeed, synonymous codon usage bias is ubiquitous in Escherichia coli, Saccharomyces cerevisiae, and Drosophila. In these organisms, codon usage is biased toward a subset of major codons (G- or C-ending codons), which generally code for the most abundant tRNA(s) (IKEMURA 1981 , IKEMURA 1982 ; BENNETZEN and HALL 1982 ; GROSJEAN and FIERS 1982 ; SHIELDS et al. 1988 ; MORIYAMA and POWELL 1997 ). In addition, the positive relationship between expression levels and codon bias was observed. That is, highly expressed genes show greater codon bias than genes with limited or low expression (GOUY and GAUTIER 1982 ; SHIELDS et al. 1988 ). Furthermore, the efficacy of natural selection on codon usage is a function of recombination rate (KLIMAN and HEY 1993 ; COMERON et al. 1999 ; TAKANO 1999 ). In Drosophila, on the basis of patterns of polymorphism and divergence at synonymous sites, it has been found that synonymous substitutions were subject to weak selection against major and nonmajor codons (AKASHI 1995 ). All of these studies have suggested the action of natural selection on synonymous (silent) sites in Drosophila. However, the possible role of synonymous substitutions following gene duplication has been scarcely evaluated. In particular, very few cases of weak selection causing divergence at the synonymous sites between members of multigenes have been documented so far.

The Amy genes of Drosophila encoding -amylase proteins, which break starch into maltose and glucose and interact directly with food environments, constitute a relatively small multigene family with two to seven copies (BAHN 1967 ; BROWN et al. 1990 ; DA LAGE et al. 1992 ; SHIBATA and YAMAZAKI 1995 ; POPADIC et al. 1996 ; STEINEMANN and STEINEMANN 1999 ; INOMATA and YAMAZAKI 2000 ). The organization and molecular evolution of the Amy multigene family in the melanogaster species subgroup and several other species have been well characterized. Previous studies showed that the members of the Amy multigene family have evolved in a concerted manner (HICKEY et al. 1991 ; POPADIC and ANDERSON 1995 ; SHIBATA and YAMAZAKI 1995 ; INOMATA and YAMAZAKI 2000 ). On the other hand, Drosophila kikkawai and its sibling species were found to have two types of very diverged Amy genes (the Amy1- and Amy3-type genes) encoding active amylase isozymes (INOMATA and YAMAZAKI 2000 ). Their expression patterns in different food environments diverged after gene duplication but before speciation. The Amy1-type genes have higher GC content at the third position of codons and more biased codon usage than do the Amy3-type genes. These results suggest the presence of some relationship between regulatory and synonymous evolution after gene duplication.

To elucidate what evolutionary forces have acted on the Amy1- and Amy3-type genes, we sequenced the full length of both genes of the montium species. Here, we describe evolutionary patterns of the two Amy-type genes and propose that the divergence at the synonymous sites between them is due to the change of selection intensity immediately after gene duplication but before speciation of the montium species.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA sequences:
Genomic DNA libraries of D. nagarholensis (strain name: PGE in Centre National de la Recherche Scientifique), D. punjabiensis (strain name: 14028-0531.0 in Bowling Green State University), and D. watanabei (strain name: SWB248 in Tokyo Metropolitan University) were constructed. It should be noted that on the basis of a morphological analysis the species of the stock number 14028-0531.0 at Bowling Green State University was regarded as D. punjabiensis, although it is described as D. jambulina. The Amy1, Amy2 (Amy1-type), and Amy3 (Amy3-type) genes were isolated from the genomic libraries by plaque hybridization using recombinant plasmids with the PCR product containing the partial Amy1- or Amy3-type gene from each species as probes. They were sequenced on both strands of DNA using ABI automated sequencer Model 377 and a DNA sequencing kit (BigDye terminator cycle sequencing ready reaction, ABI) with the synthetic oligonucleotide primers. The new sequences obtained in this study were deposited in the DNA Data Bank of Japan (DDBJ) and their accession numbers are AB078765–AB078773. All other Amy sequences of D. kikkawai, D. bocki, D. leontia, and D. lini (accession nos. AB035055–AB035069), which came from the genomic libraries (INOMATA and YAMAZAKI 2000 ) were obtained from the DDBJ. The Amy sequences of D. virilis (accession no. U02029) and Scaptodrosophila lebanonensis (accession no. AB078774) were used as outgroups in the phylogenetic tree reconstruction and in the inference of patterns of synonymous substitutions. The Amy sequences of D. pseudoobscura (accession no. X76240) and D. melanogaster (accession no. L22730) were also included in the phylogenetic tree.

AMY protein electrophoresis:
The samples for AMY protein electrophoresis were collected as follows. Adult flies of the three montium species were transferred to the two test foods, glucose medium [10% glucose (w/v), 5% killed yeast (w/v), 0.6% agar (w/v), and 0.4% propionic acid (v/v) in distilled water] and starch medium [10% soluble starch (w/v), 5% killed yeast (w/v), 0.6% agar (w/v), and 0.4% propionic acid (v/v) in distilled water]. They laid eggs for 3 days at 22°. After laying eggs, 10 adult flies were randomly collected without distinguishing sexes and frozen at -70°. Ten third instar larvae grown on glucose medium and an additional 10 third instar larvae grown on starch medium were also randomly collected without distinguishing sexes. Larvae were washed with distilled water and then stored at -70°.

The samples were homogenized by sonication in a buffer [pH 8.9; 0.1 M Tris-borate, 5 mM MgCl₂, and 10% sucrose (w/v)]. Before electrophoresis the protein content of each sample was measured by the BCA protein assay reagent (Pierce, Rockford, IL). Then, the samples with the equal protein content were applied to a polyacrylamide gel [5% acrylamide (w/v), 0.2% bis-acrylamide (w/v), 20 mM CaCl₂, and 0.1 M Tris-borate] in a 0.1 M Tris-borate (pH 8.9) buffer. After running for 3 hr at 4° and 300 V, the gel was incubated at 37° in starch solution [1% soluble starch (w/v), 0.1 M Tris-HCl (pH 7.4), and 20 mM CaCl₂] for 1 hr. The gels were then washed with water and stained in I₂-KI solution. The band mobility was referred to as AMY1 and AMY3 isozymes in D. melanogaster (INOMATA et al. 1995 ).

Data analysis:
Alignment of DNA sequences was performed using the CLUSTAL W program (THOMSON et al. 1994 ). Gap alignment in the 5'-flanking regions was corrected by hand. Codon usage bias (effective number of codons [ENC]; WRIGHT 1990 ) and GC content at synonymous third codon position was computed using the DnaSP program, version 3.50 (ROZAS and ROZAS 1999 ). A neighbor-joining (NJ) tree from the 1000-bootstrap resampling with JUKES and CANTOR's (1969) distance was produced by using the CLUSTAL W. The PAML program, version 3.0 (YANG 2000 ), employing the maximum likelihood (ML) method, and the PAUP program, version 4.0 (SWOFFORD 1998 ), employing the maximum parsimony (MP) method, were also used to construct phylogenetic trees.

The likelihood ratio test was used to test two evolutionary models. The null hypothesis was that there would be no lineage-specific effects of evolutionary rate with constant dN/dS ratio throughout lineages, and the alternative hypothesis was that there would be an independent dN/dS ratio for every lineage. We incorporated transition/transversion bias and biased codon frequencies into the models. Estimation of dN/dS ratio was performed by the ML method using the PAML program. According to the best model obtained by the likelihood ratio test, the ancestral sequences at the nodes were estimated by the ML method using the PAML program. Then their GC content and the number of nucleotide substitutions at synonymous third position along branches were counted.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Phylogenetic tree of the Amy genes:
In D. kikkawai and its sibling species, there were three or four paralogous genes. On the basis of the restriction maps and subsequent sequencing, the Amy1 gene was distinguished from the Amy2, although they were similar to each other, while the sequences of the Amy3 and Amy4 genes were identical. Therefore, the numbering of Amy3 and Amy4 was arbitrary (INOMATA and YAMAZAKI 2000 ). The Amy1 and Amy2 genes and the Amy3 and Amy4 genes are called the Amy1-type gene and the Amy3-type gene, respectively (INOMATA and YAMAZAKI 2000 ). The Amy genes cloned from genomic libraries of the three montium species, D. nagarholensis, D. punjabiensis, and D. watanabei, could be assigned to each gene type on the basis of their flanking sequences. Fig 1 shows an NJ tree constructed using coding regions of the Amy1- and Amy3-type genes in D. bocki, D. kikkawai, D. leontia, D. lini, D. nagarholensis, D. punjabiensis, and D. watanabei. The Amy3-type genes were outside of the Amy genes of D. melanogaster and D. pseudoobscura, but the bootstrap value for the Amy gene of D. pseudoobscura was not high (79%). The ML method supported the branching pattern of the NJ tree. However, the Amy gene of D. pseudoobscura was outside of the two Amy gene types when the MP method was used (data not shown). Furthermore, although the location of the Amy gene of D. melanogaster in the MP tree was the same as that in the NJ tree, its bootstrap value was not high (75%). Therefore, the placement of the Amy genes of D. melanogaster and D. pseudoobscura is not clear. In both types of Amy genes the branching pattern in the kikkawai complex (D. bocki, D. kikkawai, D. leontia, and D. lini) was consistent with the previous report (INOMATA and YAMAZAKI 2000 ) and those four species clustered together with D. nagarholensis, D. punjabiensis, and D. watanabei. For the Amy3-type gene D. punjabiensis clustered with D. watanabei and then with D. nagarholensis. On the other hand, for the Amy1-type gene the branching pattern of those three species was not clear (see Fig 1). Therefore, for further analyses, we used, for simplicity, the Amy1 and Amy3 genes as representatives of each gene type, and to exclude the uncertainty of the topology we chose the four montium species (D. kikkawai, D. lini, D. nagarholensis, and D. watanabei) and two outgroup species (D. virilis and S. lebanonensis). Their topology is shown in Fig 4. However, including other species (D. punjabiensis, D. pseudoobscura, and D. melanogaster), as in Fig 1, did not change our results fundamentally (data not shown).

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. A neighbor-joining tree for the two Amy-type genes of the seven montium species. Bootstrap value from 1000 replications is shown along each branch. The Amy sequences of D. virilis and S. lebanonensis were used as an outgroup. The Amylase gene locus is indicated in parentheses.

View larger version (76K): In this window In a new window Download PPT slide   Figure 2. Electrophoretic pattern of AMY isozymes at two stages, larval (L) and adult (A), on two test media in D. punjabiensis, D. watanabei, and D. nagarholensis. G and S indicate glucose and starch media. White numbers indicate AMY isozymes encoded by the Amy1, Amy2, and Amy3 genes. U indicates an unassigned isozyme. The AMY1 and AMY3 isozymes of D. melanogaster were used as mobility markers (M).

View larger version (35K): In this window In a new window Download PPT slide   Figure 3. Average GC content at different sites of codons for the two Amy (Amy1 and Amy3)-type genes in the montium species. GC1, GC2, GC3, and GC4 refer to GC content at the first, second, and third codon position and fourfold degenerate sites of codons, respectively.

View larger version (12K): In this window In a new window Download PPT slide   Figure 4. Amylase gene tree used for estimation of the pattern of substitutions. The numbers in parentheses represent the Amy gene type. GC3s indicates GC content at synonymous third codon position.

Electrophoretic pattern of AMY isozymes:
Fig 2 shows electrophoretic pattern of AMY isozymes on two media (glucose and starch) at two stages (larval and adult). In Drosophila the mobility of AMY isozymes is determined mostly by the charge differences of putative mature proteins (INOMATA et al. 1995 ; MATSUO et al. 1999 ). Therefore, we scored -1, 0, or +1 for each amino acid with negative, neutral, or positive charges, respectively. On the basis of the charge differences, we inferred which gene copy encodes an AMY isozyme. The net charges of the Amy1, Amy2, and Amy3 genes were, respectively, -6, -6, and -7 in D. nagarholensis; -9, -9, and -7 in D. punjabiensis; and -9, -10, and -6 in D. watanabei. However, there was an exception for the relationship between the mobility and the charge. Although the Amy3 gene in D. watanabei has the same charge as the Amy1 and Amy2 genes in D. nagarholensis, we regarded the slowest isozyme in D. watanabei as the product of the Amy3 gene and the slowest isozyme in D. nagarholensis as the product of the Amy1 and Amy2 genes. This exception might be due to the difference in the three-dimensional structure of the AMY proteins. In D. watanabei an additional isozyme was observed. It could be encoded by the fourth Amy copy or by the allele of Amy1, Amy2, or Amy3 genes. Hereafter, we call the AMY isozymes encoded by the Amy1-type gene and Amy3-type gene the AMY1 isozymes and AMY3 isozymes, respectively.

As previously reported in D. bocki, D. kikkawai, D. leontia, and D. lini (INOMATA and YAMAZAKI 2000 ), the AMY3 isozymes of the three species analyzed in this study, D. nagarholensis, D. punjabiensis, and D. watanabei, were observed at both larval and adult stages, and their activities were lower on glucose medium than on starch medium. In contrast, the AMY1 isozymes were observed only in larvae and their activities were higher on starch medium. This indicates that expression of the Amy1-type gene is more regulated than that of the Amy3-type gene at the transcriptional level, since amylase activity is mostly determined by the amount of mRNA (BENKEL and HICKEY 1986 ; YAMATE and YAMAZAKI 1999 ).

Base composition of the two Amy-type genes:
Codon bias and GC content are summarized in Table 1. Fig 3 shows average GC content in the two Amy-type genes. As demonstrated in previous studies (INOMATA and YAMAZAKI 2000 ), the Amy1-type genes have higher GC content at synonymous third codon positions. In addition, GC content at fourfold degenerate sites is also higher in the Amy1-type genes than in the Amy3-type genes. On the other hand, there were few differences in GC content at the first and second codon positions between the two Amy-type genes (Fig 3). Therefore, the differences in base composition between them can be attributed to base composition at synonymous sites.

In the Amy1- and Amy3-type genes the average GC content at synonymous third codon position was 88.7 and 69.8%, respectively, and the average codon usage bias measured by ENC (WRIGHT 1990 ) was 29.9 and 41.8, respectively (see Table 1). On the other hand, the average GC content of the intron of the Amy1- and Amy3-type genes was 42.8 and 35.2%, respectively, and that of the 5'-flanking region was 46.1 and 45.0%, respectively (see Table 1). The difference in GC content between the two Amy-type genes in the noncoding regions is relatively small compared with that in the coding regions, suggesting the similar mutational bias between the two Amy-type genes in the noncoding regions.

Patterns of synonymous substitutions:
For further examination of the difference in base composition, we estimated the ancestral sequences at each node by the maximum likelihood method. Then we computed their GC content at synonymous third codon positions and divided synonymous substitutions into G/C (G or C) A/T (A or T) and A/T G/C substitution along each branch. Before estimation, we tested the constancy of the lineage-specific dN/dS ratio on the topology shown in Fig 4 using the likelihood ratio test. Twice the difference in log-likelihood scores between the null hypothesis (constant ratio) and the alternative (lineage-specific ratio) was 49.30 and then the ratio constancy was rejected (d.f. = 17, P < 0.005). Therefore, we employed the model with the lineage-specific ratio for estimation of the ancestral sequences. The estimated number of synonymous and replacement substitutions and direction of synonymous substitutions at third codon positions for each branch are summarized in Table 2. The total number of synonymous substitutions along branches leading to the Amy1- and Amy3-type genes was 60 and 85, respectively. The estimates of dS along branches leading to the Amy1- and Amy3-type genes were 0.5816 and 0.6348, respectively, and then the total number of synonymous substitutions estimated using these values reached 140 and 150, respectively. Therefore, the total number of synonymous substitutions was underestimated. This is because no multiple-hit correction was made. However, for our analysis direction of substitutions or substitutional bias, rather than their total number, is important. After Amy1/Amy3 duplication but before montium speciation, there was a highly significant difference in the G/C (G or C) A/T (A or T) substitution pattern between the Amy1- and Amy3-type genes (see Table 3, G with Williams' correction = 59.16, d.f. = 1, P >> 0.01). On the other hand, after montium speciation there was no difference in the G/C A/T substitution pattern between the two Amy-type genes (see Table 4, G with Williams' correction = 0.72, d.f. = 1, P > 0.5), although substitutions from G/C to A/T were predominant in both Amy-type genes (G with Williams' correction = 7.24, d.f. = 1, P < 0.01 for Amy1-type gene; G with Williams' correction = 5.85, d.f. = 1, P < 0.05 for Amy3-type genes). These observations indicate that the direction of synonymous substitutions has changed between the two Amy-type genes immediately after gene duplication but before speciation of the montium species. That is, an excess of synonymous substitutions from A/T to G/C has occurred only in the lineage leading to the Amy1-type genes, whereas the G/C to A/T substitutions have been generally predominant throughout all other lineages (see Table 2).

Divergent evolution at synonymous sites:
To infer the possible causes for synonymous changes, we consider the simplest model for major codon preference. Assuming the mutation rate is constant for the two Amy-type genes, it is very likely that changes in the pattern of synonymous substitutions are due to the fluctuation of selective constraint after gene duplication. To investigate the dynamics of the fluctuation of selective constraint, consider a population of N diploid individuals at mutation-selection-drift equilibrium, assuming that the internal nodes 1, 3, and 6 in Fig 3 are at statistical equilibrium. For simplicity, assume two states, major and nonmajor codon, and that the actual population size is equal to the effective size (N). Here, G- or C-ending codons and A- or T-ending codons are defined as major and nonmajor codons, respectively, and their frequencies are equal to GC content at synonymous third positions. This assumption could be reasonable, since codon preference pattern is very similar among Drosophila species examined (AKASHI 1994 , AKASHI 1995 ; AKASHI and SCHAEFFER 1997 ). And let s be the selective advantage of major codons over nonmajor codons under semidominance. The genetic model is

(LI 1987 ; BULMER 1991 ), where u is the mutation rate from a major codon to a nonmajor codon and v is the reverse mutation rate. The number of synonymous substitutions from G/C to A/T, k_AT, from the ancestral node (e.g., node 1 in Fig 4) to the second node (e.g., node 3 in Fig 4) is given by

(1)

where q is the frequency of major codons in the ancestral node (e.g., node 1 in Fig 4), S = 4Ns, -S/(2N(1 - e^S)) is the ultimate fixation probability of nonmajor codons whose initial frequency is ¹/₂N, and t is the number of generations from the ancestral node to the second node. Similarly, the number of synonymous substitutions from A/T to G/C, k_GC, from the ancestral node (e.g., node 1 in Fig 4) to the second node (e.g., node 3 in Fig 4) is given by

(2)

where (1 - q) is the frequency of nonmajor codons in the ancestral node, and S/(2N(1 - e^-S)) is the ultimate fixation probability of major codons whose initial frequency is ¹/₂N. On the basis of Equation 1 and Equation 2, N and t should be canceled out by taking the ratio k_AT/k_GC, in the case of the duplicated genes, and then we have

(3)

where S is a function of mutation bias (u/v). For a given lineage, q and k_AT/k_GC can be estimated by the ML method. Suppose the estimates of k_AT/k_GC for the Amy1- and Amy3-type gene lineages are (k_AT/k_GC)₁ and (k_AT/k_GC)₃, respectively. Here, estimates of q at node 1, (k_AT/k_GC)₁, and (k_AT/k_GC)₃, were 0.846, 0.270, and 8.857, respectively. Note that, on the basis of Equation 3, the difference in selection intensities at the same u/v between the two lineages is

(4)

We estimated the changes of selection intensity (S = 4Ns) with the increase of mutation bias (u/v) for the two lineages, nodes 1–3 and nodes 1–6, respectively. Fig 5 shows that selection intensity (Ns) of the Amy1-type gene lineage is always larger than that of the Amy3-type gene lineage under the same u/v and that their difference is 1. Furthermore, selection intensity of the Amy1-type gene lineage was Ns > ¹/₂ in any u/v, suggesting that the major codons have been preferred.

View larger version (12K): In this window In a new window Download PPT slide   Figure 5. Estimates of selection intensity (Ns) with different mutation biases (u/v) for the Amy1 and Amy3 lineages.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We could not estimate the direction of substitutions between the two Amy-type genes in the noncoding regions by the ML method because the two regions were too diverged and could not be aligned. Therefore, we cannot directly infer what changes have occurred in the noncoding regions after Amy gene duplication before speciation of the montium species. However, differences in base composition between the coding and noncoding regions of the two Amy-type genes are striking, especially those between synonymous sites and the 5'-flanking region. This result indicated that an excess of synonymous substitutions from AT to GC has occurred in the Amy1 gene lineage after gene duplication but before speciation of the montium species and that this has resulted in higher GC content in the Amy1-type genes. One of the plausible explanations for the excess of synonymous substitutions from AT to GC is the temporal or regional changes in mutation bias. If the excess of synonymous substitutions is due to the changes in mutation bias after gene duplication but before speciation of the montium species, GC content in the noncoding regions should differ between the two Amy-type genes. However, the GC content in the 5'-flanking region was the same in both genes (Table 1). Although GC content of the intron was higher in the Amy1-type genes than in the Amy3-type genes, the difference was smaller than that in GC content at synonymous sites (27.1% increase in Amy1 synonymous sites, while 9.7% increase in the Amy1 intron). This observation is not likely to support the temporal or regional changes in mutation bias. Alternatively, it suggests that a small difference in selection intensity has caused the synonymous divergence between the two Amy-type genes.

Here, consider the plausible selection intensity in the two Amy gene lineages. At equilibrium and s = 0 in the model described above, (SUEOKA 1962 ); then mutation bias (the ratio of mutation rates), u/v, is equal to the ratio of AT to GC content. Assuming equilibrium and neutral evolution in the noncoding regions, mutation bias is 1.2–1.6 in the two Amy gene regions. In D. melanogaster, mutation bias is suggested to be 1.5 (AKASHI 1996 ); thus those values are consistent with the previous study. Therefore, roughly speaking, selection intensity, Ns, could be 1 and 0 in the Amy1 and Amy3 lineages, respectively (see Fig 5). That is, after gene duplication but before speciation, synonymous sites of the Amy1-type gene have been under weak selection where major codons have selective advantage over nonmajor codons (e.g., AKASHI 1995 ), while those of the Amy3-type gene have evolved in neutral fashion.

The difference in the selection intensity between the two Amy-type genes could result from an increase in the magnitude of selection in the Amy1-type gene lineage, a decrease in the Amy3-type gene lineage, or both, comparing to the ancestral state. At present, we cannot estimate the ancestral state and our present results give only weak support for the increase in the selection intensity in the Amy1-type gene lineage. On the other hand, since synonymous sites in Drosophila genes have been under weak selection (e.g., AKASHI 1995 ), the weakened selection in the Amy3-type gene lineage is plausible.

The weakened selection intensity in the Amy3-type gene lineage is likely to be caused by the relaxation of selective constraint following gene duplication, the difference in recombination environment, or both. In D. kikkawai the Amy1-type genes reside in a chromosomal arm, suggesting a normal recombination rate, whereas the Amy3-type genes are located near the centromere, suggesting a low recombination rate (INOMATA and YAMAZAKI 2000 ). A lower recombination rate leads to reduction of selective efficacy (HILL and ROBERTSON 1966 ; BEGUN and AQUADRO 1992 ; COMERON et al. 1999 ; MCVEAN and CHARLESWORTH 2000 ). Therefore, assuming that the chromosomal locations of the two Amy-type genes in the ancestral montium species were the same, the difference in recombination environment might result in the different selection intensity between the Amy1- and Amy3-type genes. However, in the case of the duplicated genes the weakened selection intensity is likely to be more easily explained by a fluctuation of selection coefficient, s, itself because of functional redundancy following gene duplication.

As shown in the present and previous studies (INOMATA and YAMAZAKI 2000 ), the montium species shows more biased codon usage in the Amy1-type genes than in the Amy3-type genes. The degree to which codon usage is biased toward major codons is associated with gene expression levels (GOUY and GAUTIER 1982 ; GROSJEAN and FIERS 1982 ; SHIELDS et al. 1988 ). Amylase activity encoded by the Amy1-type genes strikingly differed in response to food environments and developmental stages, where activity level at the larval stage was highest on a starch (substrate) food environment. On the other hand, activity of amylase isozymes that are encoded by the Amy3-type genes was almost the same (see Fig 2; INOMATA and YAMAZAKI 2000 ). Since amylase activity is determined mostly by the amount of mRNA (BENKEL and HICKEY 1986 ; YAMATE and YAMAZAKI 1999 ), patterns of amylase activity reflect the mRNA expression profile. These quantitative differences of mRNAs between the two type genes could be due to the changes of the regulatory elements such as cis-sequences and/or trans-acting elements. Similar to the four species in the kikkawai complex (INOMATA and YAMAZAKI 2000 ), only some cis-regulatory sequences were found in the 5'-flanking region of the Amy3-type genes, whereas the cis-regulatory sequences of the Amy1-type genes were well conserved compared with other Drosophila Amy genes (data not shown). Together with those observations, the most plausible scenario is as follows: After gene duplication, the duplicated genes could have functional redundancy, resulting in the weakened selection. Therefore, one of the duplicated genes, the Amy3-type gene, lost the ancient function, probably in the cis-regulatory regions. This caused a lower level of expression, resulting in neutral evolution at synonymous sites. However, we still do not know why the Amy3-type genes have not lost their function completely. More studies are needed to address this interesting question.

	FOOTNOTES

¹ Present address: Laboratory of Digital Agriculture and Bioinformatics, Southwest Agricultural University, Chongqing 400716, People's Republic of China.

	ACKNOWLEDGMENTS

We thank Drs. T. Ohta, H. Tachida, T. S. Takano, and A. E. Szmidt for fruitful discussion. This work was supported by research grants to N.I. and T.Y. and by a research fellowship to Z.Z. from the Ministry of Education, Science and Culture of Japan and by a research cooperative program (PICS 607) to M.-L.C. from the CNRS.

Manuscript received July 26, 2001; Accepted for publication April 18, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AKASHI, H., 1994  Synonymous codon usage in Drosophila melanogaster: natural selection and translational accuracy. Genetics 136:927-935.[Abstract]

AKASHI, H., 1995  Inferring weak selection from patterns of polymorphism and divergence at "silent" sites in Drosophila DNA. Genetics 139:1067-1076.[Abstract]

AKASHI, H., 1996  Molecular evolution between Drosophila melanogaster and Drosophila simulans: reduced codon bias, faster rates of amino acid substitution, and larger proteins in D. melanogaster.. Genetics 144:1297-1307.[Abstract]

AKASHI, H. and S. W. SCHAEFFER, 1997  Natural selection and the frequency distributions of "silent" DNA polymorphism in Drosophila. Genetics 146:295-307.[Abstract]

BAHN, E., 1967  Crossing over in the chromosomal region determining amylase isozymes in Drosophila melanogaster.. Hereditas 58:1-12.[Medline]

BEGUN, D. J. and C. F. AQUADRO, 1992  Levels of naturally occurring DNA polymorphism correlate with recombination rates in D. melanogaster.. Nature 356:519-520.[Medline]

BENKEL, B. F. and D. A. HICKEY, 1986  The interaction of genetic and environmental factors in the control of amylase gene expression in Drosophila melanogaster.. Genetics 114:943-954.[Abstract/Free Full Text]

BENNETZEN, J. L. and B. D. HALL, 1982  Codon selection in yeast. J. Biol. Chem. 257:3026-3031.[Abstract/Free Full Text]

BROWN, C. J., C. F. AQUADRO, and W. W. ANDERSON, 1990  DNA sequence evolution of the amylase multigene family in Drosophila pseudoobscura.. Genetics 126:131-138.[Abstract]

BULMER, M., 1991  The selection-mutation-drift theory of synonymous codon usage. Genetics 129:897-907.[Abstract]

COMERON, J. M., M. KREITMAN, and M. AGUADE, 1999  Natural selection on synonymous sites is correlated with gene length and recombination in Drosophila. Genetics 151:239-249.[Abstract/Free Full Text]

DA LAGE, J.-L., F. LEMEUNIER, M.-L. CARIOU, and J. R. DAVID, 1992  Multiple amylase genes in Drosophila ananassae and related species. Genet. Res. Comb. 59:85-92.

GOUY, M. and C. GAUTIER, 1982  Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids Res. 10:7055-7064.[Abstract/Free Full Text]

GROSJEAN, H. and W. FIERS, 1982  Preferential codon usage in prokaryotic genes: the optimal codon-anti-codon interaction energy and selective codon usage in efficiently expressed genes. Gene 18:199-209.[Medline]

HICKEY, D. A., L. BALLY-CUIF, S. ABUKASHAWA, V. PAYANT, and B. F. BENKEL, 1991  Concerted evolution of duplicated protein-coding genes in Drosophila. Proc. Natl. Acad. Sci. USA 88:1611-1615.[Abstract/Free Full Text]

HILL, W. G. and A. ROBERTSON, 1966  The effect of linkage on limits to artificial selection. Genet. Res. 8:269-294.[Medline]

HUGHES, A. L., 2000 Adaptive Evolution of Genes and Genomes. Oxford University Press, New York.

HUGHES, A. L., J. A. GREEN, J. M. GARBAYO, and R. M. ROBERTS, 2000  Adaptive diversification within a large family of recently duplicated, placentally expressed genes. Proc. Natl. Acad. Sci. USA 97:3319-3323.[Abstract/Free Full Text]

IKEMURA, T., 1981  Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translation system. J. Mol. Biol. 151:389-409.[Medline]

IKEMURA, T., 1982  Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes: differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs. J. Mol. Biol. 158:573-597.[Medline]

INOMATA, N. and T. YAMAZAKI, 2000  Evolution of nucleotide substitutions and gene regulation in the amylase multigenes in Drosophila kikkawai and its sibling species. Mol. Biol. Evol. 17:601-615.[Abstract/Free Full Text]

INOMATA, N., H. SHIBATA, E. OKUYAMA, and T. YAMAZAKI, 1995  Evolutionary relationships and sequence variation of -amylase variants encoded by duplicated genes in the Amy locus of Drosophila melanogaster.. Genetics 141:237-244.[Abstract]

JUKES, T. H., and C. R. CANTOR, 1969 Evolution of protein molecules, pp. 21–131 in Mammalian Protein Metabolism, edited by H. N. MUNRO. Academic Press, New York.

KLIMAN, R. M. and J. HEY, 1993  Reduced natural selection associated with low recombination in Drosophila melanogaster.. Mol. Biol. Evol. 10:1239-1258.[Abstract]

LI, W.-H., 1987  Models of nearly neutral mutations with particular implications for nonrandom usage of synonymous codons. J. Mol. Evol. 24:337-345.[Medline]

MATSUO, Y., N. INOMATA, and T. YAMAZAKI, 1999  Evolution of the amylase isozymes in Drosophila melanogaster species subgroup. Biochem. Genet. 37:289-300.[Medline]

MCVEAN, G. A. T. and B. CHARLESWORTH, 2000  The effects of Hill-Robertson interference between weakly selected mutations on patterns of molecular evolution and variation. Genetics 155:929-944.[Abstract/Free Full Text]

MESSIER, W. and C.-B. STEWART, 1997  Episodic adaptive evolution of primate lysozymes. Nature 385:151-154.[Medline]

MORIYAMA, E. N. and J. R. POWELL, 1997  Codon usage bias and tRNA abundance in Drosophila.. J. Mol. Evol. 45:514-523.[Medline]

POPADIC, A. and W. W. ANDERSON, 1995  Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura.. Mol. Biol. Evol. 12:564-572.[Abstract]

POPADIC, A., R. A. NORMAN, W. W. DOANE, and W. W. ANDERSON, 1996  The evolutionary history of the amylase multigene family in Drosophila pseudoobscura.. Mol. Biol. Evol. 13:883-888.[Abstract]

ROZAS, J. and R. ROZAS, 1999  DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174-175.[Abstract/Free Full Text]

SHIBATA, H. and T. YAMAZAKI, 1995  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. Genetics 141:223-236.[Abstract]

SHIELDS, D. C., P. M. SHARP, D. G. HIGGINS, and F. WRIGHT, 1988  "Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons. Mol. Biol. Evol. 5:704-716.[Abstract]

STEINEMANN, S. and M. STEINEMANN, 1999  The amylase gene cluster on the evolving sex chromosomes of Drosophila miranda.. Genetics 151:151-161.[Abstract/Free Full Text]

SUEOKA, N., 1962  On the genetic basis of variation and heterogeneity of DNA base composition. Proc. Natl. Acad. Sci. USA 48:582-592.[Free Full Text]

SWOFFORD, D. L., 1998 PAUP* (phylogenetic analysis using parsimony *and other methods), version 4. Sinauer Associates, Sunderland, MA.

TAKANO, T. S., 1999  Local recombination and mutation effects on molecular evolution in Drosophila. Genetics 153:1285-1296.[Abstract/Free Full Text]

THOMSON, J. D., D. G. HIGGINS, and T. J. GIBSON, 1994  CLUSTAL W: improving the sensitivity of progressive sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]

WRIGHT, F., 1990  The ‘effective number of codons’ used in a gene. Gene 87:23-29.[Medline]

YAMATE, N. and T. YAMAZAKI, 1999  Is the difference in -amylase activity in the strains of Drosophila melanogaster with different allozymes due to transcriptional control? Biochem. Genet. 37:345-356.[Medline]

YANG, Z., 2000 Phylogenetic analysis by maximum likelihood (PAML), version 3.0. University College, London.

ZHANG, J., H. F. ROSENBERG, and M. NEI, 1998  Positive Darwinian selection after gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci. USA 95:3708-3713.[Abstract/Free Full Text]

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