Nucleotide Variation of the Duplicated Amylase Genes in Drosophila kikkawai

Nobuyuki Inomata2 and Tsuneyuki Yamazaki

Laboratory of Molecular Population Genetics, Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We examined levels and patterns of the nucleotide polymorphism of the Amylase genes with a head-to-head duplication in Drosophila kikkawai. The levels of variation in D. kikkawai were comparable to those in Drosophila melanogaster. Tajima's test, Fu and Li's test, HKA test, and MK test did not show significant departure from neutrality. We found an excess of replacement changes in the within-locus class, representing polymorphism in one of the duplicated genes, compared with the between-locus class, representing polymorphism shared between the duplicated genes. Most replacement changes in the within-locus class were singletons. These results suggest that most replacement changes are deleterious. A contrasting evolutionary pattern, involving concerted evolution in the coding regions but differential evolution in the 5'-flanking regions, was observed. However, unlike the duplicated Amy genes of D. melanogaster, the coding regions of the duplicated genes in D. kikkawai tended to diverge. Using Ohta's model of the small multigene family, we found that recombination (interchromosomal equal crossing-over) rate was one order higher than gene conversion (unequal crossing-over) rate, resulting in a considerable but incomplete homogenization of the duplicated coding regions. Linkage disequilibria were found in the intron as well as within and around the regulatory cis-element sequences of one of the duplicated genes (Amy1). The possible causes of these linkage disequilibria were discussed.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Levels of polymorphism and patterns of substitutions enable us to infer as to what evolutionary forces have acted on genes. Population genetic surveys provide information about molecular characterization of fitness-related genes upon which natural selection acts. The alpha-amylase system of Drosophila is one of the extensively examined systems in the field of evolutionary research. The alpha-amylase (EC 3.2.1.1, alpha-1,4-glucan-4-glucanohydrolase) is a digestive enzyme, which breaks down starch into glucose and maltose to produce life energy. Six major and many minor isozymes of amylase have been found in natural populations of Drosophila melanogaster (Kikkawa 1964Citation ; Doane 1969Citation ; Dainou et al. 1987Citation ; Inomata et al. 1995bCitation ). The activity of amylase is repressed by the products (glucose and maltose) (Hoorn and Scharloo 1978Citation ; Hickey and Benkel 1982Citation ; Inomata et al. 1995aCitation ) and induced by the substrate (starch) (Inomata et al. 1995aCitation ). Variation in activity levels and the food-environment-response ability (inducibility) was observed both within (Matsuo and Yamazaki 1984Citation ; Yamazaki and Matsuo 1984Citation ) and between species (Inomata et al. 1995aCitation ). This variation is mainly because of the mRNA abundance (Benkel and Hickey 1986Citation ; Yamate and Yamazaki 1999Citation ), although the difference in catalytic efficiency of individual isozymes has also been suggested to contribute to some extent to the differences in activity (Yamate and Yamazaki 1999Citation ). In addition to variation in the food-environment-response ability (inducibility), variation in developmental- (Yamazaki 1986Citation ; Da Lage and Cariou 1993Citation ; Inomata and Yamazaki 2000Citation ) and tissue-specific (Abraham and Doane 1978Citation ; Klarenberg et al. 1986Citation ) expression has been reported. These observations can be more easily interpreted in terms of adaptation because amylase directly interacts with environments through foods. Actually, the food-environment-response ability (inducibility) is related to fitness (Matsuo and Yamazaki 1984Citation ; Yamazaki and Matsuo 1984Citation ).

The Amylase (Amy) gene of Drosophila is a member of a multigene family (Payant et al. 1988Citation ; Shibata and Yamazaki 1995Citation ; Popadic et al. 1996Citation ; Inomata, Tachida, and Yamazaki 1997Citation ; Da Lage et al. 1998Citation ; Inomata and Yamazaki 2000Citation ). The closely linked (duplicated) Amy genes show a contrasting evolutionary pattern, involving concerted evolution in coding regions and differential evolution in the flanking regions (Hickey et al. 1991Citation ; Popadic and Anderson 1995Citation ; Shibata and Yamazaki 1995Citation ; Inomata and Yamazaki 2000Citation ; Araki, Inomata, and Yamazaki 2001Citation ). The latter suggests that differential selection has acted on each flanking region (Shibata and Yamazaki 1995Citation ; Okuyama et al. 1996Citation ; Inomata and Yamazaki 2000Citation ; Araki, Inomata, and Yamazaki 2001Citation ). Moreover, adaptive evolution of amylase protein during speciation has also been suggested (Shibata and Yamazaki 1995Citation ; Araki, Inomata, and Yamazaki 2001Citation ). Other lines of evidence for differential selection come from the findings of divergent paralogous Amy genes. One is the Amyrel gene in the Sophophora subgenus, which encodes divergent proteins and shows expression patterns different from the Amy genes (Da Lage et al. 1998Citation ). The other involves the presence of two duplication groups of Amy genes in Drosophila kikkawai and its sibling species (Inomata and Yamazaki 2000Citation ). The first group includes head-to-head duplicated genes (Amy1 and Amy2) and the second group includes tail-to-tail duplicated genes (Amy3 and Amy4). The Amy1 and Amy2 genes cluster with the Amy genes of D. melanogaster, rather than with the Amy3 and Amy4 genes of D. kikkawai. Both coding and flanking regions are very divergent between the two duplication groups. In particular, the Amy1 and Amy2 genes have higher GC content at third positions of codons and more biased codon usage than the Amy3 and Amy4 genes. In the previous study based on the between-species comparison, we found that the Amy1 and Amy2 genes show a contrasting evolutionary pattern involving concerted evolution in coding regions and differential evolution in the flanking regions (Inomata and Yamazaki 2000Citation ). However, we still do not know the detailed mechanism that produced such a contrasting pattern. Therefore, in this study we examined levels and patterns of intraspecific variation of the Amy1 and Amy2 genes in D. kikkawai.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Fly Strains
Twenty-three isofemale lines of D. kikkawai and three isofemale lines of Drosophila bocki were supplied by the Tokyo Metropolitan University. Drosophila kikkawai strains are as follows: Naha-2, Naha-3, Naha-4, Nago-1, Nago-2, Nago-3, Nago-4 (Okinawa Island, Japan); Miyako-2, Miyako-4 (Miyako Island, Japan); MDL172, MDL173, MDL178, SWB174, SWB184, RGN31 (Myanmar); HNL122, HNL124, HNL126, HNL202 (Hawaii); HYD101, HYD102, CJB202, CJB216 (India). Drosophila bocki strains are A94 (Taiwan), AO-1 (Thailand), and IRO2-37 (Iriomote Island, Japan). Together with the published data (accession numbers, AB035055 and AB035056 for Naha-1 strain in D. kikkawai, AB035059 and AB035060 for A65 strain in D. bocki), 24 lines of D. kikkawai and four of D. bocki were used in this study.

Genomic DNA Extraction, Polymerase Chain Reaction Amplification and Sequencing
Genomic DNA from a single fly was extracted according to a standard procedure (Ashburner 1989Citation , pp. 108–109). Polymerase chain reaction (PCR) amplification was performed using the gene-specific primers. They were kik1f5 (5'-TAAATATCTGACCACCAAGGAG-3') and kik12f3 (5'-CTACATTATCTGCCTGAATCCCT-3') for the Amy1 gene and kik2f5 (5'-CCTAACATCGGCAGATATCAGC-3') and kik12f3 for the Amy2 gene. PCR conditions were as follows: 50 µl of the reaction mix was preheated at 95°C for 3 min. The reaction condition for 32 cycles was denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and polymerization at 72°C for 1 min; after 32 cycles, additional polymerization at 72°C for 7 min was performed. Both 5'-flanking and coding regions were sequenced. The PCR products were directly sequenced. The sequence of both strands was determined using ABI Model 377 automatic sequencer and a DNA sequencing kit (BigDye terminator cycle sequencing ready reaction, ABI) with the PCR primers and the internal primers. After direct sequencing, several PCR products were found to be heterozygous because isofemale lines were used. The PCR products of the heterozygous lines were subcloned into pGEM-T Easy Vector (promega), and the sequence of one of the alleles was determined.

Data Analysis
DNA sequences were initially aligned by the CLUSTAL W program (Thompson, Higgins, and Gibson 1994Citation ) and then further aligned by hand. Neighbor-joining (NJ) trees (Saitou and Nei 1987Citation ) with the bootstrap values based on nucleotide substitutions were constructed using the CLUSTAL W program. Correction for multiple hits was performed by the Jukes and Cantor's method (1969)Citation . The Amy genes of Drosophila lini (14028-0581.0 strain, accession numbers AB035067 and AB035068) were used as outgroup. Molecular population analyses were done using the DnaSP program, Version 3.14 (Rozas and Rozas 1999Citation ). Nucleotide sequences obtained in this study were deposited in the DNA Data Bank of Japan with the following accession numbers: AB077388–AB077436.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Nucleotide Variation
We examined nucleotide variation of the duplicated Amy (Amy1 and Amy2) genes containing head-to-head structure from 24 strains of D. kikkawai. For the between-species comparisons, we also sequenced the duplicated Amy genes in four strains of D. bocki. Our PCR primer sets amplify about 1.8 kbp Amy gene regions, including about 300 bp of the 5'-flanking region, the entire coding region (1,482 bp), and a few bases of the 3'-flanking region. The Amy1 gene of the Naha-3, Naha-4, and CJB202 strains could not be amplified with the Amy1-specific primers kik1f5 and kik12f3. Similarly, it could not be amplified with the internal Amy1-specific primers (5'-TTCTGACCAAGAGCATCGTA-3' and 5'-TCAGGAACTCGGCAATCTTT-3'). These results suggest that the Amy1 gene is either deleted in those strains or the corresponding regions cannot be amplified because of insufficient homology of the primers used in this study. In the following analyses, 21 Amy1 and 24 Amy2 genes of D. kikkawai, together with the four Amy1 and Amy2 genes of D. bocki, were used. Polymorphic sites in D. kikkawai are shown in figure 1 . There were 62 polymorphic sites and one indel at the -195 position in the entire Amy1 gene region and 53 polymorphic sites and one indel at the -67 position in the entire Amy2 gene region. Together with the duplicated genes, there were 115 polymorphic sites and 13 indels. There were seven singleton synonymous changes, seven non-singleton synonymous changes, and eight singleton replacement changes within the Amy1 gene. The Amy2 gene had one singleton synonymous change, four non-singleton synonymous changes, and five singleton replacement changes. We found one non-singleton synonymous change within the Amy1 gene and one singleton replacement change within the Amy2 gene at the 1062 position and two singleton replacement changes within the Amy1 gene at the 1478 position. Twenty-one synonymous and three replacement changes were common to the Amy1 and Amy2 genes. In D. bocki, there were 73 polymorphic sites and no indels in the Amy1 gene region and 71 polymorphic sites in the Amy2 gene region. One indel was found in an intron of the Amy2 gene. Together with the duplicated genes, there were 148 polymorphic sites and 12 indels. Nucleotide diversity measures ({pi}, Nei 1987Citation , pp. 261–263) and 4Nµ ({theta}, Watterson 1975Citation ) are summarized in table 1 . The levels of variation in D. kikkawai were comparable to those in D. melanogaster (Araki, Inomata, and Yamazaki 2001Citation ).



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Fig. 1.—Segregating sites of the duplicated Amy1 and Amy2 genes in D. kikkawai. A dot indicates the same nucleotide as consensus one and a dash indicates deletion. A dot of the Amy2 consensus sequence indicates an identical nucleotide to the Amy1 consensus sequence. An asterisk above the position indicates replacement substitutions. The I and II following the strain names indicate the Amy1 and Amy2 genes, respectively. An adenine of the first codon (Met) is numbered as +1, and intron sequences were represented by i

 

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Table 1 Nucleotide Variation of the Duplicated Amy Genes in Drosophila kikkawai and Drosophila bocki

 
Amino Acid Variation
Amino acid segregating sites in D. kikkawai are shown in figure 2 . There were 14 amino acid segregating sites in D. kikkawai and nine sites in D. bocki. Four sites were common between the two species. At 7 of the 14 sites in D. kikkawai and at one of nine sites in D. bocki, amino acid substitutions cause charge differences, resulting in four and two isozymes in D. kikkawai and D. bocki, respectively. In the case of Amylase proteins, the electrophoretic mobility was determined by the charge differences rather than by the molecular weight differences (Matsuo, Inomata, and Yamazaki 1999Citation ). The frequencies of individual isozymes in our sample were 0.044 (AMY5.5), 0.289 (AMY6.5), 0.600 (AMY7.5), and 0.067 (AMY8.5). There were several amino acid substitutions within the same isozyme. Moreover, different combinations of amino acid substitutions with the charge change resulted in the same isozyme. For example, the AMY6.5 isozyme of the Amy1 gene in the Nago-1 strain has Leu, Asn, and Arg at positions 220, 278, and 328, respectively, whereas the HYD101 strain has Arg, Asp, and Lys. It is noteworthy that the AMY6.5 isozyme of the Amy1 gene in the Nago-3 strain shows a combination of amino acid substitutions between the Amy1 gene of the Nago-1 and HYD101 strains; that is, it has Arg, Asn, and Lys at positions 220, 278, and 328, respectively. In addition to those substitutions, it has a Lys-Met substitution at position 493, resulting in the same AMY6.5 isozyme. These observations suggest a complex history of AMY isozymes, probably caused by the past genetic exchanges such as gene conversion. The evidence of genetic exchanges at the isozyme level was also found in D. melanogaster (Inomata et al. 1995bCitation ).



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Fig. 2.—Amino acid segregating sites in D. kikkawai. The I and II following the strain names indicate the Amy1 and Amy2 genes, respectively. Relative mobilities to the AMY1 through AMY6 isozymes of D. melanogaster were used for isozyme names and are shown in the third column. Gray color columns indicate the amino acid substitutions with charge changes. The correspondence between the amino acid position and the nucleotide position is as follows (the nucleotide position[s] is indicated in parentheses)—4 (10), 8 (22), 121 (361), 156 (468), 180 (538), 220 (659), 278 (832 and 833), 328 (983), 338 (1012), 354 (1060 and 1062), 391 (1171), 478 (1432), and 493 (1477 and 1478)

 
Neutrality Tests
First, we examined the neutrality using the Tajima's D (1989)Citation statistic and Fu and Li's D* and F* (1993)Citation test statistics without outgroup species (table 2 ). Tajima's D and Fu and Li's F* were positive (P < 0.05) in the Amy1 intron of D. kikkawai, and the three statistics were negative in the Amy2 intron of D. kikkawai. However, they were not significant after the Bonferroni correction for multiple comparisons. None of these statistics were significant in other regions analyzed in this study.


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Table 2 Summary of Tajima's D and Fu and Li's D* and F* Statistics

 
The HKA test (1987)Citation was also conducted. Because no polymorphism data other than that for the Amy gene regions in D. kikkawai are available, six combinations of the 5'-flanking and coding regions, 5'-flanking and 5'-flanking regions, and coding and coding regions from each of the duplicated genes were used for regional comparisons, and D. lini was used for between-species comparisons. For all combinations, we could not reject the neutral hypothesis (data not shown). For the HKA test, the gene should be divided into two regions. The tests for regional heterogeneity detailed in McDonald (1998)Citation were performed using the DNA Slider program (McDonald 1998Citation ). Here, unlike the HKA test, regional heterogeneity in the ratio of polymorphism to divergence was tested without any a priori division of regions, with D. lini as an outgroup. Statistical significance of these tests was estimated by computer simulations, with 1,000 replicates for each recombination value. The largest P values were used, and the Bonferroni correction was applied. Only one of the obtained statistics, the maximum sliding G statistic, in the Amy2 gene region was significant at the 5% level (data not shown).

We also performed the McDonald and Kreitman test (1991)Citation , which examines the imbalance of the ratio of the number of replacements to synonymous substitutions between polymorphic and fixed classes. In this study, alleles from both Amy loci were pooled together and regarded as alleles of a single gene. We classified substitutions into the fixed class, which included nucleotides that were identical within alleles but differed between species, and the polymorphic class, which included other substitutions. The purpose of this classification was to take into account the effects of genetic exchanges such as gene conversion. Fixed substitutions between species should result from genetic exchanges between the duplicated genes. Assuming selective neutrality, this classification does not affect the results of the test because the ratio of the number of replacements to synonymous substitutions is the same between the polymorphic and fixed classes in any clade of the phylogenetic tree. We found that there are no fixed differences between D. kikkawai and D. bocki in replacement or synonymous classes (see table 3 ). This was probably because of the close relationship between the two species. Actually, the two species are morphologically very similar (Lemeunier et al. 1986Citation ), and females of D. kikkawai and males of D. bocki can cross and produce fertile F1 flies (Kim, Watanabe, and Kitagawa 1989Citation ). Thus, we could not perform the test. We therefore used D. lini for a between-species comparison. The results obtained did not reject the neutral hypothesis (see table 3 ).


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Table 3 The Modified McDonald and Kreitman Test

 
The independence test for the duplicated genes was conducted using only intraspecific variation. We classified nucleotide changes into two classes. One class included nucleotide changes observed within one locus; the other class included nucleotide changes shared between the two loci. They are hereafter referred to as within-locus class and between-locus class, respectively. The nucleotide changes can also be classified into replacement and synonymous classes. Even if the genetic exchanges such as gene conversion occur between duplicated genes, the ratio of replacement to synonymous changes should be the same between the two classes whenever the selective neutrality is held. We found an excess of replacement changes in the within-locus class (GWill = 4.81, P < 0.05) (table 4 ).


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Table 4 A 2 x 2 Contingency Table for Independence Test

 
Estimation of Population Parameters for the Duplicated Genes Evolving in Concerted Fashion
On the basis of the between-species comparisons in the four species of the kikkawai complex, the contrasting evolutionary pattern, that is, a concerted evolution in the coding regions but differential evolution in the 5'-flanking regions, of the duplicated Amy1 and Amy2 genes was found (Inomata and Yamazaki 2000Citation ). In the present within-species comparison we found a similar contrasting pattern. Figure 3 shows a NJ tree of exon sequences of the duplicated Amy gene in D. kikkawai and D. bocki. Duplication of the Amy1 and Amy2 genes has taken place before speciation of the four species of the kikkawai complex (Inomata and Yamazaki 2000Citation ). Therefore, in the absence of genetic exchanges, the genes from each locus should cluster separately. However, the duplicated genes of D. kikkawai clustered together with a high bootstrap value (97%), suggesting the occurrence of genetic exchanges such as gene conversion. This clustering pattern is similar to that found in D. melanogaster (Araki, Inomata, and Yamazaki 2001Citation ). However, unlike the duplicated genes of D. melanogaster (Araki, Inomata, and Yamazaki 2001Citation ), the duplicated genes of D. kikkawai were not nested in the tree, although the bootstrap value was not high. In other words, the alleles from each locus tend to cluster together, implying a weaker effect of genetic exchanges between the duplicated Amy genes than in D. melanogaster. On the other hand, a NJ tree of the 5'-flanking regions of D. kikkawai and D. bocki (fig. 4 ) shows tight clustering of genes from each locus, reflecting a history of the duplication event, which occurred before speciation of the species from the kikkawai complex. As far as the closely linked Amy genes are concerned, this contrasting pattern of molecular evolution between the coding and 5'-flanking regions was also observed in Drosophila (Hickey et al. 1991Citation ; Popadic and Anderson 1995Citation ; Shibata and Yamazaki 1995Citation ; Araki, Inomata, and Yamazaki 2001Citation ).



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Fig. 3.—A NJ tree of exon sequences of the duplicated Amy1 and Amy2 genes in D. kikkawai and D. bocki. Over 50% bootstrap probability from 1,000 replications is shown along each branch. Drosophila lini is used as an outgroup

 


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Fig. 4.—A NJ tree of 5'-flanking sequences without gaps of the duplicated Amy1 and Amy2 genes in D. kikkawai and D. bocki. Over 50% bootstrap probability from 1,000 replications is shown along each branch. Drosophila lini is used as an outgroup

 
Using the intraspecific variation, we can estimate the population parameters for the duplicated Amy genes. Assuming selective neutrality, we applied Ohta's model (1983)Citation for the small multigene family to the duplicated Amy genes. In this model three identity coefficients are defined as follows: f is the average identity probability between the genes at the same locus, c1 is the average identity probability between the duplicated genes on the same chromosome, and c2 is the average identity probability between the duplicated genes on different chromosomes. Because the linkage between Amy1 and Amy2 was uncertain in the heterozygotes, only 15 homozygous chromosomes (strains) with the Amy loci whose sequences can be directly determined were analyzed. Applying the observed f, c1, and c2 values to Ohta's formulas, we estimated the population parameters 4N{lambda}, 4Nµ, and 2Nß at the equilibrium state, where N, {lambda}, µ, and ß are the effective population size, (unequal) crossing-over (probably gene conversion) rate between the loci, mutation rate, and interchromosomal (equal) crossing-over rate, respectively. The observed f, c1, and c2 values were 0.9927, 0.9903, and 0.9897, respectively. The estimated 4N{lambda} 4Nµ, and 2Nß values were 1.005, 4.410 x 10-3 and 8.551, respectively. Assuming N = 106, gene conversion rate is 3.72 x 10-4 per gene per generation, mutation rate is 1.10 x 10-9 per site, and interchromosomal crossing-over rate within a locus is 6.34 x 10-3 per gene.

Linkage Disequilibrium
Linkage disequilibrium between polymorphic DNA sites was investigated (fig. 5 ). We found highly significant linkage disequilibria (P < 0.001) after the Bonferroni correction for multiple comparisons; for example, a pair of sites +1332 and +1335 in the Amy1 exon and the pair +690 and +708 in the Amy2 exon. Only in one of the duplicated genes, the Amy1 gene, highly significant linkage disequilibria (P < 0.001, after the Bonferroni correction) were observed in the 5'-flanking region and in the intron. The pairs of sites with the significant linkage disequilibria were A-C and T-A at -180 and -178 immediately upstream of the glucose repressible element (Boer and Hickey 1986Citation ) and C-T at +138, A-G, A-T, C-T, and G-A at 3rd, 9th, 18th, and 20th in the anterior part of the intron sequences. The pairs of sites, G-A, C-T, A-C, and A-C at -111, -105, -104, and -101, near the CAAT motif were also highly significant (P < 0.001), although they were not significant after the Bonferroni correction.



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Fig. 5.—Linkage disequilibrium between polymorphic DNA sites in the Amy1 gene and Amy2 gene. Only the informative sites were used for Fisher's exact test. Black, dark gray, and gray colors indicate the significance levels at 0.1%, 1%, and 5%, respectively. Shadowed boxes indicate the significance comparisons after the Bonferroni correction

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Mode of Concerted Evolution
The contrasting evolutionary pattern involving differential evolution in the 5'-flanking regions and the concerted evolution in the coding regions was reported in the Amy genes in Drosophila (Hickey et al. 1991Citation ; Popadic and Anderson 1995Citation ; Shibata and Yamazaki 1995Citation ; Inomata and Yamazaki 2000Citation ; Araki, Inomata, and Yamazaki 2001Citation ). In this study we confirmed the contrasting evolutionary pattern previously deduced from the between-species comparison of the kikkawai complex (Inomata and Yamazaki 2000Citation ). However, on the basis of a population survey, we found that the mode of concerted evolution in D. kikkawai was different from that of D. melanogaster. The duplicated Amy1 and Amy2 genes of D. kikkawai have evolved in a concerted fashion but tend to diverge (see fig. 3 ), whereas the Amy gene tree of D. melanogaster showed nested topology of the duplicated proximal and distal Amy genes (Araki, Inomata, and Yamazaki 2001Citation ). The occurrence of frequent gene conversion between the duplicated Amy genes could cause concerted evolution in the coding regions (Inomata et al. 1995bCitation ). In the case of D. melanogaster, assuming that mutation rate is of order of 10-9 per site per generation, the gene conversion rate was estimated to be 1.22 x 10-4 and 9.73 x 10-5 per gene per generation in Japan and Kenya, respectively (Araki, Inomata, and Yamazaki 2001Citation ). These values appear to be comparable to our present estimate of gene conversion rate in D. kikkawai (3.72 x 10-4 per gene per generation) because mutation rate estimated in D. kikkawai (1.10 x 10-9) was of the same order as the one assumed in D. melanogaster. Therefore, the observed difference in the mode of evolution does not seem to be because of gene conversion rate itself. The interchromosomal crossing-over rate could not be computed in D. melanogaster because c1 <= c2. Therefore, we cannot directly compare the level of the interchromosomal crossing-over between the two species. Similarly, we cannot assess the effect of the interchromosomal crossing-over upon the concerted evolution in D. melanogaster. On the other hand, in D. kikkawai we could compute the interchromosomal crossing-over rate. The estimated value of the interchromosomal crossing-over rate was one order higher than that of the gene conversion rate. Our assumption of population size and estimates of population parameters, {lambda} and ß, seem to be valid because the order of our estimated mutation rate (10-9) is plausible. Because the same population size is used for estimating both {lambda} and ß, one order difference should hold regardless of the assumed order of population size. The one order higher rate of the interchromosomal crossing-over found in our study could result in a considerable but incomplete homogenization of the duplicated coding regions in D. kikkawai.

We found an excess of replacement polymorphic sites in the within-locus class. Furthermore, most replacement changes in this class (11 out of 12) are singletons. These results indicate that most replacement changes are deleterious. The fate of neutral variants is determined by random genetic drift. Moreover, in the case of duplicated genes, neutral variants which have occurred in one gene could spread to the other locus by gene conversion. On the other hand, advantageous variants go to fixation, whereas deleterious variants should be immediately eliminated by purifying selection. Therefore, a newly arisen deleterious variant is unlikely to be distributed to both loci. In this study, there was no supportive evidence that the shared replacement changes between the duplicated genes were adaptive.

Differential Evolution in the 5'-Flanking and Intron Regions
The Amy-coding region is suggested to have duplicated together with 450 bp of the 5'-flanking region (Okuyama et al. 1996Citation ). Therefore, if the same evolutionary force acts on the 5'-flanking regions as on the coding regions, we should observe concerted pattern in the 5'-flanking regions. However, the 5'-flanking regions analyzed in our study have evolved divergently, indicating that they are affected by different selective forces.

We observed highly significant linkage disequilibria in the 5'-flanking region and intron of the Amy1 gene. One possible explanation is low recombination rate. However, the duplicated Amy1 and Amy2 genes are located in the center of the chromosome arm (Inomata and Yamazaki 2000Citation ), suggesting a normal recombination rate. Furthermore, our estimate of the recombination rate is three orders larger than the mutation rate. Thus, the linkage does not appear to be responsible for the observed disequilibria. The observed linkage disequilibria may also reflect the genetic differentiation caused by the population structure because our samples come from geographically different locations. If this is true, we should observe a characteristic haplotype for each population throughout the genomic regions. However, we could not find such a tendency. For example, the highly significant pair of sites (AACG-GTTA) in the intron was found in all populations. Furthermore, most of the significant pairs of sites were observed in only one of the duplicated genes, the Amy1 gene. These observations do not support the hypothesis that population structure contributed to the observed linkage disequilibria.

Another possible explanation for the observed linkage disequilibria is epistatic selection between sites. The pairs of sites showing linkage disequilibria were found in a region immediately upstream of the putative glucose repressible element (Boer and Hickey 1986Citation ) and within and around the CAAT motif, although most of them were not significant after the Bonferroni correction. The regulatory sequences found in the 5'-flanking region appear to be important for amylase expression (Magoulas et al. 1993Citation ; Choi and Yamazaki 1994Citation ). In a population cage experiment with D. melanogaster using different food environments, H. Araki et al. (personal communication) found that selection acted on the interaction between the Amy locus and its genetic background. If the interaction between the cis-regulatory sequences and trans-acting elements is necessary to optimize the levels of gene expression, a specific linkage between sites within the cis-regulatory sequences may be maintained to interact with trans-acting elements. In this case, the pairs of closely located sites may show linkage disequilibria because the cis-regulatory sequences are generally very short. Significant linkage disequilibria found in the Amy1 intron involved two haplotypes, AACG and GTTA. One GTTA haplotype in the Amy2 intron of the Nago-4 strain is probably caused by gene conversion between the duplicated genes, because we detected a gene conversion track from +138 to 20th position of the intron in the Nago-4 strain using an algorithm of Betran et al. (1997)Citation . Linkage disequilibria in the Amy1 intron may be caused by compensatory nucleotide changes to maintain the secondary structure of mRNAs. Linkage disequilibria observed in the Adh gene introns of Drosophila were suggested to be caused by epistatic selection maintaining precursor mRNA secondary structure rather than subpopulation structure (Schaeffer and Miller 1993Citation ). Kirby, Muse, and Stephan (1995)Citation actually showed that the linkage disequilibria can be caused by epistatic selection maintaining precursor mRNA secondary structure. Furthermore, compensatory interactions between the 5' and 3' ends of the Adh mRNA were confirmed experimentally (Parsch, Tanda, and Stephan 1997Citation ). However, at present we do not have any information on the secondary structure of amylase mRNAs. A population survey of random samples from a single natural population and molecular analyses are needed to clarify the selective significance in those pairs of sites.

Patterns of Molecular Evolution
In this study selective neutrality could not be rejected by the tests. Most of the tests for neutrality assume no recombination. Apparently genetic exchanges (recombination and gene conversion) have occurred in the Amy-coding regions of D. kikkawai. Although some researchers pointed out that the occurrence of recombination is conservative for the tests (Hudson, Kreitman, and Aguade 1987Citation ; Tajima 1989Citation ; Fu and Li 1993Citation ) and its effect is also partly understood (Wall 1999Citation ), the results of the tests may still be biased by genetic exchanges between loci. Nevertheless, our results are not likely to reject the neutrality. On the other hand, the tests should yield unbiased results when applied to the 5'-flanking regions because these regions have evolved independently like a single locus. Although the differential selection on the 5'-flanking regions was suggested (Shibata and Yamazaki 1995Citation ; Okuyama et al. 1996Citation ; Inomata and Yamazaki 2000Citation ; Araki, Inomata, and Yamazaki 2001Citation ), no significant departure from the neutrality was found by the tests. The reason is probably that the power of the statistical tests is not high in most cases (Wall 1999Citation ).

Although the results of the sliding window plot analyses cannot be tested statistically, the within- and between-species comparisons of the pattern of substitutions along the sequences enable us to infer as to what evolutionary forces have acted on the genes. A region immediately upstream of exon 1 in the 5'-flanking regions indicated by an arrow diverged between D. kikkawai and D. lini, whereas the variation of the same region was reduced within D. kikkawai (see figs. 6A, 6B, and 7 ). This region is downstream of the region where linkage disequilibria were observed. The low polymorphism and high divergence are not predicted by simple neutral hypothesis. In the corresponding region, the level of variation within D. bocki was not reduced compared with that between D. bocki and D. lini (data not shown). This is consistent with the neutral prediction. Thus, the observed pattern is specific to D. kikkawai. One of the plausible explanations for this pattern is selective sweep (Maynard Smith and Haigh 1974Citation ; Kaplan, Hudson, and Langley 1989Citation ). During fixation, newly arising advantageous mutations sweep neutral alleles at linked sites. This results in the reduction of polymorphism without affecting neutral divergence. Another explanation is background selection (Charlesworth, Morgan, and Charlesworth 1993Citation ). Chromosomes with deleterious mutations are eliminated from the population and do not contribute to the next generation, resulting in a reduction of linked neutral variation. In this sense its effect is identical to that of a population size reduction. These two mechanisms have been proposed to explain low variation in low recombination regions. It is difficult to detect their effects in the regions with normal recombination rate. Recombination rate in the Amy1 and Amy2 gene regions is unlikely to be low because they are located in the center of the chromosomal arm (Inomata and Yamazaki 2000Citation ). Even if the regions with low polymorphism and high divergence are found, the two models are not easily distinguished. However, Tajima's D is expected to be significantly negative in the selective sweep model (Braverman et al. 1995Citation ), whereas significant Tajima's D values are not likely to be detected in the background selection model (Charlesworth, Charlesworth, and Morgan 1995Citation ). Tajima's D in the region of the Amy1 gene was negative (-1.514), although it was unlikely to be significant (see fig. 7). Furthermore, in the Amy2 gene Tajima's D was zero. At face value, Tajima's D values obtained in the present study seem to be consistent with each model. However, we still cannot decide which mechanism is more plausible. This puzzling observation remains to be solved in future studies.



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Fig. 6.—Sliding window plot of the nucleotide diversity in D. kikkawai and the nucleotide divergence between D. kikkawai and D. lini in the (A) Amy1 gene and (B) Amy2 gene. 5f, e1, i, and e2 indicate the 5'-flanking region, exon 1, intron, and exon 2, respectively. An arrow indicates the region with low polymorphism and high divergence. The window size is 100 bp, and the step size is 10 bp

 



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Fig. 7.—Sliding window plot of Tajima's D. 5f, e1, i, and e2 indicate the 5'-flanking region, exon 1, intron, and exon 2, respectively. An arrow indicates the region with low polymorphism and high divergence. The window size is 100 bp, and the step size is 10 bp

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank A. E. Szmidt and H. Tachida for useful discussions. This work was supported by research grants to N. I. and T. Y. from the Ministry of Education, Science and Culture of Japan.


    Footnotes
 
Wolfgang Stephann, Reviewing Editor

Keywords: DNA polymorphism Amylase (Amy) multigenes linkage disequilibrium adaptive evolution Drosophila Back

Address for correspondence and reprints: Nobuyuki Inomata, Laboratory of Molecular Population Genetics, Department of Biology, Faculty of Sciences, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. ninomscb{at}mbox.nc.kyushu-u.ac.jp . Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication January 2, 2002.