Tracing the History of an Enzyme Polymorphism: The Case of Alcohol Dehydrogenase-2 (Adh-2) of the Olive Fruit Fly Bactrocera oleae

George N. Goulielmos*,, Nickolaos Cosmidis*, Marianna E. Theodorakopoulou{dagger}, Michael Loukas* and Eleftherios Zouros{dagger},{ddagger}

* Department of Genetics, Agricultural University of Athens, Athens, Greece
{dagger} Department of Biology, University of Crete, Crete, Greece
{ddagger} Institute of Marine Biology of Crete, Crete, Greece


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
In the olive fruit fly Bactrocera oleae, previous studies have described a one-locus three-allele electrophoretic polymorphism of the enzyme alcohol dehydrogenase and provided evidence that the polymorphism is under the influence of selection. A recent study has shown that this species carries a two-locus duplication for alcohol dehydrogenase. Here, we show that the polymorphism maps at one of the duplicated loci, Adh2, and identify the nucleotide and, therefore, the inferred amino acid differences among the three allozymes. At the amino acid level, the polymorphism is of the simplest possible form: there is no intra-allozyme variation, and interallozyme differences are restricted to one amino acid for two pairs of alleles and to two amino acids for the third pair. Consideration of the amino acid residues at the sites that segregate in B. oleae in four congeneric species and the phylogenetic trees produced from the nucleotide sequences of the Adh2 gene of these species point to the same allozyme as the ancestral form of the polymorphism. Interestingly, this allozyme comprises less than 1% of the gene pool of present-day natural populations of B. oleae, where the other two allozymes appear to form a stable polymorphism. Previous studies have shown that the frequency of the rare allozyme rises rapidly in laboratory colonies maintained on artificial diet and declines again when the artificial diet is replaced with olive fruit, the natural substrate of B. oleae. The geographical distribution of several congeneric species suggests that B. oleae originated in the Indian subcontinent, where the olive tree is practically absent. The poor performance of the ancestral allele on the olive fruit suggests the possibility that the decline of this allele and the concomitant rise of the presently common alleles might be associated with the expansion of the insect's geographical distribution to areas where the olive tree has become its main and perhaps sole host. The estimated age of the polymorphism is compatible with this hypothesis, but firmer support could be difficult to obtain.

Key Words: Alcohol dehydrogenase • olive fruit fly • amino acid polymorphism


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The use of electrophoresis for the detection of protein variability has led to the conclusion that natural populations contain large amounts of variation at the single-locus level (Lewontin 1974). In turn, this has fueled discussion on what are the relative roles of deterministic and stochastic forces in generating and maintaining this variation (Kimura 1983; Gillespie 1991). The search for the answer to this question has produced several examples of enzyme polymorphisms that are selectively maintained and has illuminated the mechanism through which this selection may operate. One such example is the alcohol dehydrogenase (ADH) polymorphism of Drosophila melanogaster. The case is notable for two reasons. First, a large body of evidence at the biochemical (Thatcher 1980; Juan and Gonzalez-Duarte 1981), organismal (van Delden 1982) and population (Gibson and Oakeshott 1982; Bodmer and Ashburner 1984; David and Capy 1988) levels has virtually eliminated any doubt that the polymorphism is under the influence of selection. Of special interest is the pattern of allele frequency change with distance from the equator in all major continents (Singh, Hickey, and David 1982; Berry and Kreitman 1993; Begun et al. 1999). Second, this was the first polymorphism whose degree and nature of variation was studied at the nucleotide level (Kreitman 1983) and whose selective history in natural populations could be deduced from the pattern of nucleotide variation along its coding region (Kreitman and Hudson, l991).

Another Adh polymorphism for which there is strong evidence for selection is that of the olive fruit fly Bactrocera oleae. Because this point is of central importance to the work we present here, we defer its discussion to a later section of the paper (see Discussion) and provide here only a short description of the enzyme and its polymorphism. The principal function of the ADH enzyme in insects is the catalysis of the reversible conversion of the various alcohols generated by microbial fermentation of larval and adult food to aldehydes and ketones (David et al. 1976). The ADH enzyme of B. oleae has been biochemically characterized and found to have a dimeric structure with a subunit mass of 26 kDa (Mazi et al. 1998). The first study of natural variation of the enzyme produced two electrophoretic alleles, F and S (for fast and slow mobility, respectively) (Bush and Kitto 1979). Shortly afterward, Zouros et al. (1982) reported a third allele, I, with intermediate mobility. This third allele is rare in natural populations but was found in high frequencies in laboratory colonies maintained on an artificial food specially devised for the rearing of the larva of olive fruit fly (Tsitsipis 1983). Repeated initiations of colonies with samples in which the I allele was in low frequency showed that the allele increased its frequency to more than 30% in less than five generations (Zouros et al. 1982). Also, multiple samplings showed little variation among natural populations in the frequencies of F and S alleles, which were maintained around 0.4 and 0.6, respectively.

The use of genetic variation for historical inference may vary on whether or not the variation could be assumed to be neutral to the forces of natural selection. Neutral variation is most suitable for phylogenetic and phylogeographic inference and for deducing the breeding structure of populations. This application comprises the vast share of studies that have used allozymes, microsatellites, or other types of molecular variants as genetic markers (Avise 2000). On the other hand, selective polymorphic states or replacements could be useful when attempting to read the adaptational history of the species. Examples of this type can be found in histocompatibility gene polymorphisms that are shared by several species (Hughes et al. 1994) and are obvious examples of balancing selection (Hedrick and Thomson 1988). Enzyme polymorphisms whose interactions with the natural environment are fairly well understood may also shed light on current selection forces acting on populations. An example of the latter type can be found in the leucine aminopeptidase enzyme of Mytilus edulis (Koehn, Newell, and Immermen 1980; Hibish and Koehn 1985). The polymorphism of the Adh gene within the D. melanogaster species subgroup may also serve as an example of how selective forces may differ among closely related species. The extensive studies by Singh, Hickey, and David (1982) and Choudhary and Singh (1987) revealed that D. simulans is globally monomorphic for the S allele of Adh, whereas most D. melanogaster populations contain both the S and the F alleles. There is clear evidence that the S allele is the ancestral allele and that the appearance of the F allele and its selective drive to the F/S polymorphism is an evolutionary feature of D. melanogaster (Kreitman 1983; Bodmer and Ashburner 1984; Veuille et al. 1998).

In addition to being a prototype for the study of polymorphism and associated selection, loci coding for the ADH enzyme have been used as a case study for molecular evolution in Drosophilidae (see reviews by Sullivan, Atkinson, and Starmer 1989; Ashburner 1998). These studies cover a wide spectrum of topics, including the molecular organization and regulation of gene expression (Benyajati et al. 1983; Savakis and Ashburner 1985; Savakis, Ashburner, and Willis 1986), the dynamics and fate of duplicated genes (Fischer and Maniatis 1985, 1986), the role of enzyme functionality in evolution (Atrian et al. 1998), and the dating of speciation and duplication events (Sullivan, Atkinson, and Starmer 1989; Rowan and Hunt 1991; Thomas and Hunt 1991; Russo, Takezaki, and Nei 1995). Similar, though less extensive, studies exist in Tephritidae, another family of dipteran insects that includes some of the world's most important agricultural pests. Most of these studies refer to the Mediterranean fly (medfly), Ceratitis capitata. The ADH system of this species is of interest because of its potential use in the biological control of the insect (Robinson and MacLeod 1993). C. capitata is known to possess two Adh genes, Adh1 and Adh2, tightly linked (0.49 centimorgans [cM]) at the end of the left arm of the second chromosome (Malacrida et al. 1992). This suggests that the two loci were produced by gene duplication and subsequent divergence. Adh1 is expressed mainly in muscle and Adh2 is expressed mainly in fat body and ovary, respectively (Gasperi et al. 1992; Gasperi et al. 1994; Benos et al. 2000). Brogna et al. (2001) observed that the amino acid sequence divergence of medfly Adh1 or Adh2 genes from the Adh gene of Sarcophaga peregrina is smaller than the divergence of the medfly genes from the Adh gene of D. melanogaster. S. peregrina belongs to the Calyptrata series of the dipteran division of Schizophora (Griffiths 1972), whereas both the tephritids and the drosophilids belong to the Acalyptrata series of the same division. Thus, the clustering of the tephritid with the sarcophagid genes in this three-way comparison is an anomaly for which Brogna et al. (2001) proposed two possibilities: (1) the Adh gene of D. melanogaster is not orthologous to the tephritid-sarcophagid pair (which would be assumed to be orthologous), or (2) the Adh of drosophilids has experienced an accelerated rate of evolution. The latter hypothesis has also been suggested for the glycerol-3-phosphate dehydrogenase of Drosophila (Kwiatowski et al. 1997).

Goulielmos et al. (2001) have shown that Bactrocra oleae has also a duplicate Adh system and obtained the full genomic sequence of these loci. By making use of the available information on cDNA sequences from the medfly, they attempted to answer the question of whether in tephritids a single prespeciation duplication event is a more likely alternative to the hypothesis of two separate postspeciation duplication events. Interestingly, amino acid and nucleotide sequence comparisons provided opposite answers, with the former supporting one duplication event and the latter supporting two separate duplications. The knowledge of the B. oleae Adh system at the molecular level has made it possible to ask some specific questions about the system's enzyme polymorphism: To which of the two Adh loci, Adh1 or Adh2, does the enzyme polymorphism map? What is the molecular basis of the enzyme polymorphism? Would it be possible to infer the phylogenetic relationship of the three electrophoretic alleles? How old may the electrophoretic variation be in comparison with the age of the species? Do the molecular data produce information about the role of selection in maintaining the polymorphism? Would it be possible to link, even in a tentative way, the history and present state of the polymorphism to key events in the history of the species?

The existing studies of the Adh loci in drosophilids and in two tephritid species provide valuable information with which to approach these questions. Yet, more informative answers could be obtained by including additional species that are evolutionary closer to B. oleae. For this reason we have obtained and report here the cDNA sequence of the Adh2 locus (which we show here as the locus that codes of the enzyme polymorphism of B. oleae) from four other Bactrocera species.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Artificial Rearing of Bactrocera oleae
Details of the method of artificial rearing of B. oleae are described by Tsitsipis (1983). Larvae were reared on a cellulose-based artificial diet containing yeast, soy hydrolyzate, sugar, and olive oil, as nutrient sources; polyoxyethylene-sorbitan-monooleate (Tween 80) as homogenizer; and hydrochloric acid, sodium sorbate, and p-hydroxy-benzoic acid methyl ester (Nipagin) as antimicrobial agents. Adults were kept on yeast hydrolyzate, sugar, and egg yolk as a nutrient source and on streptomycin as an antimicrobial agent (Tzanakakis 1989).

Sources of Bactrocera oleae DNA
For this study we used flies from six laboratory strains that are described in detail by Cosmidis, Loukas, and Zouros (1997). We refer to these strains as F.M, I.M, S.M, F.T, I.T and S.T. F, I, and S indicate that the strain was fixed for the F, the I or the S allele, respectively, and M or T indicate that the strain was obtained by pair-mating of flies from a laboratory colony that was established from wild flies from the locality of Marathon (M), Attica, Greece or from Tatoi (T), Attica, Greece. The different geographic origin of the flies assured that any two strains that were homozygous for the same allele were extracted from different genetic backgrounds. Samples were removed and examined electrophoretically on regular time intervals to assure the strains remained pure for their respective alleles. For the purpose of this study, we removed 50 pupae from the F.M, I.T, and S.T strains. Pupae from each strain were pooled together and used for the extraction of genomic DNA (we refer to this as "pooled lab-fly DNA"). Two samples from each of these DNA homogenates were used for separate PCR amplifications of the Adh2 locus, and the PCR products were cloned. Three randomly picked clones from each PCR reaction were eventually sequenced. This generated six sequences for the F allele, six for the S allele, and five for the I allele (we failed to obtain the sequence of one clone from the I.T strain). In addition, DNA was extracted separately from two individual pupae from each of the six strains (we refer to these preparations as "single lab-fly DNA"). Finally, we obtained B. oleae pupae from infected olives collected from an orchard in the western part of Crete, Greece. Each pupa was cut into two parts, of which one was used to determine the individual's allozyme genotype by starch gel electrophoresis (see below). The other half was used to extract DNA (we refer to this as "single wild-fly DNA"). This was done for two pupae found to be homozygous for the F allele and for two pupae homozygous for the S allele. None of the 34 wild pupae tested was either homozygous or heterozygous for the I allele. In total, we obtained 12 F sequences, 12 S sequences and nine I sequences (figure 1). The pooled lab-fly DNA was also used to amplify, clone, and sequence the Adh1 locus. In total, five Adh1 sequences were produced from the F.M strain, five from the S.T, and six from the I.T (data not shown).



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FIG. 1. Variable nucleotides at 33 genomic sequences (total length 988 bp) of Adh2 of B. oleae. The sequences are shown in blocks corresponding to the exon/intron organization of the gene (exon-1, intron-1, exon-2, intron-2, exon-3). Nucleotide substitutions leading to amino acid replacement are marked with an asterisk. The designation in the first column is as follows: I, F, or S correspond to the three allozymes; T, M, or C indicate localities from which the insects originated (T = Tatoi, Attica; M = Marathon, Attica; C = Chania, Crete); M or S indicate multiple or single pupa extraction; numbers identify the particular sequence

 
Sources of DNA from Other Bactrocera Species
The samples of B. dorsalis, B. cucurbitae, B. scutellatus, and B. tryoni that were used in this study were collected from different countries. The B. dorsalis and B. cucurbitae flies used (15 adults for each species) were obtained from a laboratory strain, originated from flies collected in Amami Oshima (Kagoshima, Japan), in 1979 and reared in the laboratory since then. The B. scutellatus sample used represented by 15 adults, collected in May 2000 in Tsukuba (Ibaraki, Japan). A total of 12 adult individuals of B. tryoni was used in this study, collected from Australian tropical rainforests and kept as laboratory colony in the University of Sydney. All specimens of the same species were used together for extraction of DNA.

Scoring of ADH Allozyme Polymorphism
A detailed description of the ADH polymorphism (which is controlled by an autosomal locus) and the method of its detection are given by Zouros et al. (1982). Even though ADH activity is low in adults, the polymorphism can be detected at all stages of the life cycle of the insect except in larvae less than 4 days old. The highest concentration of ADH was found in the pupa, and, therefore, this was the insect stage used for electrophoresis. In short, ADH allozymes were resolved by starch gel electrophoresis using Poulik's (1957) discontinuous buffer system. Gel slabs of 10% in starch and of 0.5 x 18 x 20 cm in dimensions were run at 500V/150 mA for about 2 h. The cathodal part of the gel was stained for ADH using isopropanol as a substrate. The sample from the wild population consisted of pupae that were collected from Crete at the end of the summer from unsprayed olives. Half of the pupa was homogenized and used for scoring the allozyme polymorphism, and the remaining material was used for DNA extraction, as described below.

Cloning of the Adh1 and Adh2 Genes
Preparation of genomic DNA, the design of the primers, and the PCR amplification of the corresponding genomic fragments of the Adh1 and Adh2 genes were done according to the protocols described by Goulielmos et al. (2001). The upstream primers 5'-ACGCGTCGACGAATTCATGAG(C/T)TTGGCIGGIAAAAA(C/T)G-3' and 5'-ACGCGTCGACGAATTCATGGGTTTGAGCGGCAAAAAT-3' and the downstream 5'-ACGGA-GCTC(G/A)TAIGTGGG(T/C)TCCCA(G/A)TAIAC-3' and 5'-CCGAGCTCGGATCCCTAGTTGAATGTGGGTTGCCA-3' were used for the amplification of Adh1 and Adh2 products, respectively. The resulting PCR products contained EcoRI and SalI overhangs, which allowed their directional cloning into the plasmid vector pBluescript II KS (Stratagene). Because of an EcoRI internal site in the fragment that corresponded to the Adh1 gene of B. oleae, the pGEM (Promega) vector was used for the cloning of the Adh1 fragments. In both cases, standard PCR amplification procedures were followed (Sambrook, Fritsch, and Maniatis 1989). Restriction and DNA modification enzymes were provided from MinoTech and New England Biolabs. Pwo polymerase (a proof-reading enzyme, Boehringer-Mannheim) was used to get amplification products of high fidelity. Sequencing of the double-stranded plasmids carrying the Adh2 genomic products was carried out according to the di-deoxy-chain termination method following the manufactures protocol (Sequenase, USB), using vector specific (T3, T7, SP6) primers. Custom gene-specific primers were used for sequencing the double-stranded plasmids carrying the Adh1 genomic fragments in combination to the aforementioned vector-specific primers. For each genomic region, both strands were completely sequenced. Agarose gel electrophoresis and other recombinant DNA methods were performed essentially as described by Sambrook, Fritsch, and Maniatis (1989).

The orthology of Adh1 and Adh2 genes across species was based on the length of intron-1 and on the number of amino acids of the predicted polypeptide. In all species for which both genes were detected (C. capitata [Gomulski et al. 1998], B. oleae [Goulielmos et al. 2001], C. cosyra, and B. cucurbitae [our data, unpublished]), the intron-1 of Adh1 was longer than the intron-1 of Adh2 by more than 900 bp, and the polypeptide of Adh1 was smaller by one amino acid than that of Adh2. The PCR products we obtained from other Bactrocera species using the primers that were designed for the Adh2 locus had all the above properties of Adh2 and were therefore assumed to belong to this locus.

DNA Sequence Analysis
The DNA sequences were analyzed with the GCG Sequence Analysis computer software package. The alignment of the sequences was done using the ClustalX program (Thompson et al. 1997). The nucleotide sequences used in this study have the following GenBank accession numbers: AJ277835(B. oleae Adh1-F), AJ488561 (B. oleae Adh1-I), AJ488562 (B. oleae Adh1-S), AJ277834 (B. oleae Adh2-F), AJ488559 (B. oleae Adh2-I), AJ488560 (B. oleae Adh2-S), AJ488557 (B. cucurbitae Adh2), AJ488556 (B. tryoni Adh2), AJ488555 (B. scutellatus Adh2), and AJ488554 (B. dorsalis Adh2). The rates of synonymous (Ks) and nonsynonymous (Ka) substitutions were estimated using the DnaSP computer program (Rozas and Rozas 1999).

Phylogenetic Tree Construction
Neighbor-Joining trees were constructed using the Kimura two-parameter model for the estimation of distances with C. capitata as outgroup and ran using the MEGA-2 computer package (Kumar et al. 2001). Exhaustive maximum-parsimony trees were run using the PAUP* version 4.0b10 (Swofford 1998). Maximum-likelihood trees were constructed using the Tree-Puzzle 5.0 (Schmidt et al. 2002). Bootstrap values were obtained with 1,000 replications (Felsenstein 1985).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The Molecular Basis of the ADH Allozyme Polymorphism
All Adh1 and Adh2 DNA sequences of B. oleae obtained in this study conformed to the genomic organization described by Goulielmos et al. (2001): Adh1 consisted of three exons of 31, 134, and 92 amino acids interrupted by two introns of 1,071 and 136 nucleotides. Adh2 consisted also of three exons of 31, 134, and 93 amino acids interrupted by two introns of 125 and 82 nucleotides.

The predicted amino acid sequences obtained from the Adh1 clones were all identical, regardless of whether the clones originated from the F, I, or S strain (data not shown). Nucleotide differences for all B. oleae Adh2 locus sequences obtained for this study are given in figure 1. The figure includes 33 sequences, nine from strains homozygous for the I allele, 12 from strains or wild individuals homozygous for the F allele, and 12 from strains or wild individuals homozygous for the S allele. Even though there was a certain amount of intra-allelic variation in intronic and third codon positions, an allele-specific DNA consensus sequence could be easily deduced. The amino acid sequences of the Adh2 clones were also identical except at positions 74 and 126, both in the second exon. All sequences extracted from pupae from the I strains had a leucine residue at amino acid position 74 and a lysine residue at position 126. All sequences from pupae from the F strains and from two wild pupae with the FF genotype had the residues phenylalanine and lysine. Finally, all sequences from pupae from the S strains and from the two wild pupae with the SS genotype had the residues plenylalanine and asparagine (fig. 2). This gives the answer to three of our questions. The first is that the ADH allozyme polymorphism maps on the Adh2 locus rather than on the Adh1 locus. The second is that the polymorphism can be explained parsimoniously by assuming only two mutational events, a thymine/cytocine transition in nucleotide position 346, resulting in a leucine/phenylalaline replacement, and a guanine/cytocine transversion in nucleotide position 504, resulting in a lysine/asparagine replacement. The third is that there is not much, if any, cryptic amino acid variation within allozyme types.



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FIG. 2. Variable nucleotides at the consensus Adh2 cDNA sequences (total length 774 bp) of the three B. oleae allozymes, four other Bactrocera species and the medfly Ceratitis capitata. Sequences are divided according to the exon to which they belong. Note that cDNA numbering of nucleotide sites does not correspond to the numbering of genomic sequence sites (fig. 1)

 
Deducing the Time Sequence of Amino Acid Replacements
Assuming that one of the three allozymes is the ancestral and that from this allozyme the others were derived by two mutation events, there would be nine possibilities for the sequence of events that could lead to present-day polymorphism. From the information of amino acid sharing alone, alleles I and S are the more removed from each other (they differ by two amino acid residues, when each differs by one from F). Thus, the following alternatives emerge as the more likely ones: (1) I produced F, F produced S, (2) S produced F, F produced I, or (3) F produced I and S. The question, then, is whether the DNA sequences could provide support for one of these three alternatives, could point to another alternative, or would be unable to provide any information at all. To gain more information on this point, we have obtained cDNA sequences of the Adh2 locus from four congeneric species: B. dorsalis, B. scutellatus, B. cucurbitae, and B. tryoni.

Figure 3 provides the cDNA sequences of these four species together with the consensus sequences of the three B. oleae allozymes and the corresponding sequence of the medfly Ceratitis capitata. As in figure 1, only variable sites are shown. The full cDNA sequences were used for phylogenetic analysis. The rooted Neighbor-Joining tree (fig. 4A) joins the F with the S allele of B. oleae, in support of alternative (1) above. However, this grouping has only 46% bootstrap support. This tree also identifies B. dorsalis as the closest relative to B. oleae. Maximum parsimony with C. capitata as an outgroup produced four equally parsimonious trees, of which one (fig. 4B) agrees with the NJ tree. The other three trees differ either by producing S as basal to F and I or by producing B. dorsalis as basal to all other Bactrocera species. The maximum likelihood tree (fig. 4C) joins F with S with weak support and puts B. dorsalis as basal to other Bactrocera species, but this placement has less than 50% support. Neighbor-Joining, maximum-parsimony, and maximum likelihood trees produced from the amino acid sequences (fig. 2) were consistent in grouping the F and S alleles together and in placing B. dorsalis as basal to other Bactrocera species (fig. 5). All cDNA and amino acid sequences were individually tested for departure from stationarity using the TreePuzzle option (Schmidt et al. 2002), and no departure was detected.



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FIG. 3. Variable amino acid sites of the Adh2 gene of the three B. oleae allozymes, four other Bactrocera species, and the medfly Ceratitis capitata presented according to the exon they belong. Amino acid differences among B. oleae allozymes are marked with an asterisk

 


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FIG. 4. Neighbor-Joining (A), maximum-parsimony (B), and maximum likelihood (C) trees obtained from cDNA sequences (fig. 3). The tree in (B) is one of four equally parsimonious trees (see text for explanation). Numbers in (A) and (B) are bootstrap confidence values. Numbers in (C) are the percentage of support for the internal branches of the quartet puzzling tree topology

 


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FIG. 5. Neighbor-Joining (A), maximum-parsimony (B), and maximum likelihood trees obtained from amino acid sequences (fig. 2). Numbers in (A) and (B) are bootstrap confidence values. Numbers in (C) are the percentage of support for the internal branches of the quartet puzzling tree topology

 
The overall picture from the phylogenetic analysis is that the grouping of F and S alleles is more likely than any of the other two alternative groupings. Both distance and maximum-parsimony trees support this grouping when using amino acid sequences. The weak bootstrap support is to be expected from the fact that there are only two variable sites in the amino acid sequences. The distance and maximum likelihood methods of nucleotide analysis also support this grouping of alleles, but, again, weakly, given the small number of nucleotide differences in the consensus sequences, whereas the maximum-parsimony analysis produces FS and IF as equally likely groups. With regard to B. dorsalis, in unrooted nucleotide or amino acid trees (with C. capitata removed) this species shows a smaller distance from B. oleae than from any other Bactrocera species (data not shown). A possible reason for the fact that in amino acid rooted trees B. dorsalis appears basal to other Bactrocera species might be its high number of unique amino acid replacements (fig. 2). This number is 0 for B. cucurbitae, 2 for B. scutellatus, 4 for B. oleae (not counting the polymorphism), 6 for B. tryoni, and 12 for B. dorsalis (chi-square 17.667 on 4 df, P < 0.005). The overall evidence suggests, therefore, that B. dorsalis is basal to the other four Bactrocera species, but this cannot be considered as an established result.

Consideration of individual amino acid replacements provides additional support for the ancestry of allele I and also points to the sequence of events that produced the F and S alleles. At position 126, lysine is shared by alleles I and F of B. oleae and by B. dorsalis, whereas asparagine is shared by allele S of B. oleae and the other three Bactrocera species. This pattern can be explained by assuming that lysine was the ancestral amino acid and was replaced by asparagine in allele S as well as in the common ancestor of B. scutellatus, B. cucurbitae, and B. tryoni (figs. 4 and 5). Alternatively, we may assume that asparagine was the ancestral amino acid in the genus Bactrocera and was replaced by lysine in the pair of alleles I and F of B. oleae as well as in B. dorsalis. According to the first hypothesis, the ancestral allele of the B. oleae allozyme polymorphism would be allele I. This hypothesis is further supported by the fact that lysine is the residue in the corresponding position of Adh genes of Drosophila (Atkinson et al. 1988; Kreitman and Hudson 1991; Menotti-Raymond, Starmer, and Sullivan 1991; Nurminsky et al. 1996). According to the second hypothesis, the ancestral allele would be allele S. We conclude that the polymorphism at position 126 is uninformative in this respect. At amino acid position 74 (fig. 2), leucine is most likely the ancestral residue in B. oleae, given that it is present in all Bactrocera species examined, as well as in Ceratitis capitata. The corresponding amino acid position of Adh genes of Drosophilidae is also occupied by leucine (Atkinson et al. 1988; Kreitman and Hudson 1991; Menotti-Raymond et al. 1991; Nurminsky et al. 1996). This argues against the ancestry of F or S, both of which have phenylalanine in this position. Thus, position 74 provides strong support for the ancestry of allele I, which is also supported by the phylogenetic analysis of amino acid sequences and, to a lesser extent, by the phylogenetic analysis of the nucleotide sequences. From this we conclude that the most likely sequence of events is alternative (1) above (i.e., I produced F, F produced S).

The Age of the Polymorphism
We may use the amount of divergence among the three alleles of B. oleae and the divergence of B. oleae from B. dorsalis to obtain an estimate of the age of the polymorphism relative to the age of the split of these two species. The Kimura two-parameter Ka and Ks values, as well as the Ktotal from the cDNA sequences for these comparisons, are shown in table 1. The mean Ks value of B. dorsalis from the three alleles of B. oleae is 0.344 and that of allele I from F and S 0.039, suggesting that the polymorphism is about one tenth as old as the species itself. The same calculation suggests that the emergence of S from the F is about 85% as old as the emergence of F from I.


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Table 1 Estimates of Divergence Time (MYA) of B. oleae Allozymes, Two Bactrocera Species, Two Tephritid Genera, and Tephritids from Drosophilids.

 
The Adh gene was used extensively to obtain times of divergence among species of Drosophila. Using the time of the emergence of the Hawaiian Islands, Rowan and Hunt (1991) suggested a rate of 1.5 x 10-8 synonymous substitutions per year (Ks) or 0.5 x 10-8 for the total coding region (both synonymous and nonsynonymous sites) of Adh of Hawaiian Drosophilids. Russo, Takezaki, and Nei (1995) considered only the total coding region substitution rate, which they revised to 1 x 10-8. Table 1 gives Ks, Ka, and Ktotal values for various taxonomic comparisons and the estimated times of divergence. The upper estimate obtained by the method of Rowan and Hunt (1991) is the mean of the two estimates they proposed, one based on Ks and the other based on Ktotal. To see whether the Drosophila rate can be used as a first approximation to estimate rates of divergence in tephritids, we compared the cDNA of the Adh2 gene of Ceratitis capitata with the consensus cDNA sequence of the Adh2 gene of five Drosophila species (D. montana, D. virilis, D. hydei, D. mojavensis, and D. buzzatii) used in Goulielmos et al. (2001), which are known to have two active duplicate Adh genes. The mean Ks and Ktotal values produce a divergence time for the two families between 83 and 123 Myr. The upper estimate is nearly identical to one given by Beverley and Wilson (1984) that was based on immunological reaction of larval serum proteins. Using the same rates, the split of B. oleae from B. dorsalis is placed between 11 and 22 Myr and the age of the B. oleae polymorphism between 1.1 and 2.4 Myr.

We may compare this age of enzyme polymorphism with the ADH polymorphism of D. melanogaster. Because intronic sequences are of different length in the two species, we have confined this comparison to cDNA sequences. Exempting the nucleotide substitution that generates the F/S polymorphism, the Ks value between the consensus S of D. melanogaster and the S of D. simulans is 0.022, and the Ks value between the consensus of F and S of D. melanogaster is 0.011. The best estimate for the time of the D. melanogaster/D. simulans split is 2.3 MYA (Russo, Takezaki, and Nei 1995), which makes the age of the melanogaster polymorphism 1.2 Myr.

Molecular Evidence for Selection
In the following section, we review the experimental evidence for selection on the ADH allozyme polymorphism of B. oleae. Here we examine whether the molecular data we have obtained for the purposes of this paper provide support for selection. Kreitman and Hudson (1991) have used the distribution of silent nucleotide polymorphism along the Adh-F and Adh-S alleles of D. melanogaster to ask if it were compatible with the neutral model of molecular variation. They observed an excess of polymorphic sites located within a stretch of less than 600 bp around the nucleotide position that is responsible for the allozyme polymorphism. This was interpreted as evidence for balancing selection acting on the two allozyme forms. Our data set is not suitable for this analysis. Most sequences are derived from colonies maintained in the laboratory for a large number of generations and thus cannot be considered to be representative of the nucleotide variation of allozymes in natural populations. Moreover, a proper application of the Kreitman and Hudson (1991) method would require extraction of isofemale lines from natural populations, which is not readily feasible for B. oleae.

One question that may be asked, however, from our data set is whether the amino acid and nucleotide differences among B. oleae allozymes are consistent with differences that have been observed for other allozyme polymorphisms. Table 2 summarizes the information about nucleotide and amino acid variation at five loci, including the one presented in this study. For comparability across loci and species, we have included only protein-coding sequences. By its nature, this information is sensitive to the number of independent lines that were sequenced in each case, as a larger number is likely to lead to higher levels of variation, mainly at the nucleotide and secondarily at the amino acid level. In the cases presented in table 2, this number is comparable across cases, allowing for a valid comparison of observed levels of variation. The minimum number of 10 independent lines for the Adh of B. oleae is derived by assuming at least one independent chromosome from each laboratory population and one for each wild-caught individual (see Materials and Methods). Statistics for the data of table 2 are given in table 3. The first observation is that the proportion of polymorphic nucleotide sites is comparable and statistically not different in four loci. The Pgd locus of D. melanogaster stands out as exceptionally poor in variation (Begun and Aquadro 1994). When the variation was partitioned between synonymous and nonsynonymous sites, again the Pgd locus was found to contain a much lower level of variation at synonymous sites. This, however, was not so for nonsynonymous sites, where the five loci appear to split into two groups: (1) Est6 + Xdh and (2) Pgd + Adh of D. melanogaster + Adh of B. oleae (table 3). The homogeneity of the first group is barely acceptable, however, so one may claim that the loci cannot be grouped into discrete classes with regard to constraint for variation at nonsynonymous sites.


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Table 2 Levels of Nucleotide and Amino Acid Variation at Five Loci.

 

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Table 3 Results from Chi-Square Tests.

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The information we have obtained in this study reveals a simple molecular basis for the ADH allozyme polymorphism of B. oleae. First, each of the three allozymes was found to represent a single amino acid sequence (i.e., there is no intra-allelic amino acid variation). This conclusion is based on the observation that all homozygous individuals for the I allele produced cDNAs whose nucleotide sequences corresponded to the same amino acid sequence. These individuals were drawn from two cages, each of which was established with pupae collected from a different wild population from the province of Attica, Greece. Exactly the same was observed for homozygous individuals for the F or the S allele drawn from these cages. In addition, we examined four individuals drawn directly from a wild population from the island of Crete. Two of these were homozygous for the F allele and two were homozygous for the S allele. All four individuals produced the amino acid sequence that was obtained from laboratory animals of the same allozyme genotype. We cannot rule out the possibility that some intra-allozyme amino acid variation would be found if a larger number of animals were examined, but this appears unlikely given the substantial within-allozyme synonymous variation we have seen. The second observation underlying the molecular simplicity of the polymorphism is that interallelic differences in amino acid sequence are of the simplest possible degree: two of the three pairwise combinations of alleles differ by one amino acid and the third differs by two amino acids.

Interestingly, the only other case of ADH allozyme polymorphism that has been studied at the nucleotide and amino acid level, the ADH polymorphism of D. melanogaster, shares these characteristics of the B. oleae polymorphism: the two allozymes differ by only one amino acid and there is no amino acid variation within alleles. The parallels become even stronger if one ignores the I allele of B. oleae, which is practically absent in natural populations. In D. melanogaster, the ancestral allele is the S, judging from the fact that all other species of the subgroup have the S allele (Bodmer and Ashburner 1984). The emergence of F from S involved the replacement of the basic amino acid lysine by the neutral amino acid threonine. In B. oleae, we have argued that the S allele resulted from the F, also through a replacement of a lysine residue by a neutral amino acid, in this case asparagine.

Table 2 extends the similarities of the two Adh genes at the level of variation and, by extension, to the type of selection that might be acting upon these loci. The table includes three more enzyme loci, two from D. melanogaster and one from D. pseudoobscura. The proportion of segregating nucleotide sites appears to be the same for four loci. The same can be said for the proportion of the segregating synonymous sites. Pgd is a strong exception. The variation appears to be very low at this locus, even at the third codon position, an observation that the original authors attributed to the X-linked condition of the locus, resulting in very low recombination rate, or to the restricted geographical region from which the sample was taken (Begun and Aquadro 1994). The consideration of the proportion of segregating nonsynonymous sites produces a different result. The two Adh loci are in this respect more closely related to the Pgd locus than to Est6 or Xdh. This means that whereas the Adh loci are as free to vary at the synonymous sites as Est6 and Xdh, they are under a severe constraint for variation at nonsynonymous sites. On the face of this type of selection acting on the entirety of the coding part of the gene, the presence of two alleles in high frequencies suggests the action of strong balancing selection for the amino acid difference that characterizes the allozymes of the two loci. As noted, there is good evidence from field, laboratory, and molecular studies for this type of selection at the Adh polymorphism of D. melanogaster. The evidence for the Adh of B. oleae is given below.

In natural populations of B. oleae, alleles S and F occur in frequencies of about 65% and 35%, respectively, and allele I is either absent or in very low frequencies (fig. 6A). In contrast, laboratory colonies that were established from natural populations in which the I allele was present were found to contain the I allele in a frequency of about 30%. Three types of experiments have shown that this difference between natural and laboratory populations is not accidental. In the first type of experiments (Zouros et al. 1982) new laboratory populations were established using samples from natural populations, and allele frequencies were monitored for several generations. In all cases, the frequency of allele I increased from less than 0.01 to 0.30 within the first four generations (fig. 6B). The second type of experiments were "perturbation" experiments in which flies homozygous for the I, the F, and the S allele derived from laboratory colonies were used to establish new laboratory populations with an input frequency of the I allele at 5%. Again, this experiment was repeated several times (Cosmidis 1995), and the result was the same as in experiments of the first type: the frequency of allele I increased to about 30% in a few generations (fig. 6C). The third type was a food-reversing experiment in which adult flies raised in a population cage with artificial food were forced to lay eggs on olive fruit, the natural substrate for the insect's larval stage (Economopoulos and Loukas 1986). Emerging flies were again forced to lay eggs on olive fruit. The experiment lasted for three generations, after which no fresh olives could be obtained from nature and refrigerated olives had become sensitive to fungal infection when placed in the cage for oviposition. Within the first generation, the I allele dropped from 0.28 to 0.11 and remained at this level until the termination of the experiment (fig. 6D).



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FIG. 6. Evidence for selection at the ADH enzyme of B. oleae. (A) Frequencies of allozymes I (squares), F (circles), and S (triangles) in seven samples from natural populations from Greece. T = Tatoi, Attica; A = Aghia Triada, Attica; M = Marathon, Attica; E = Eretria, Evia Island; C = Chania, Crete Island. B, C, D = Change in frequency of allele I in consecutive generations (numbers in abscissa). (B) Under artificial rearing when sample originated from a natural population. (C) Under artificial rearing when sample originated from a colony kept on artificial medium. (D) Under rearing on olive fruits when sample originated from a colony kept on artificial medium (the reverse experiment). Figure based on the following references: Economopoulos and Loukas 1982; Zouros et al. 1982; Economopoulos and Loukas 1986; Loukas et al. 1985; Cosmidis 1995

 
Cosmidis (1995) and Cosmidis, Loukas, and Zouros (1997) have used FF, II, and SS homozygous lines and their F1 progeny to measure viability in the larval, pupal, and adult stage and developmental time, female fecundity, male mating propensity, and mating preferences for all six allozyme genotypes under artificial rearing. Several significant differences were observed, with the most significant being the egg-to-pupa developmental time. In a similar experiment, Konstantopoulou, Economopoulos, and Manoukas (1996) compared the three homozygous types and observed that type II had higher pupal weight and higher pupa-to-adult viability. Mazi et al. (1998) obtained highly purified preparations of the ADH enzyme from the three homozygous lines and showed that these preparations had significant differences in the catalysis of a number of alcohols. All this evidence converges on the larval food as the selective agent. The larval food, whether it is the olive fruit itself or an artificial diet, is an environment that undergoes dramatic changes through fermentation, accumulation of metabolic products, and composition of the associated flora. This suggests that no single genotype might be best for the duration of larval development. Cosmidis, Loukas, and Zouros (1999) focused on developmental time (days from egg to adult eclosion) as the most important trait under selection. They observed that the performance of a genotype on the artificial food depended not only on the genotype's frequency but also on the frequencies of the other genotypes in the cohort of larvae that grew together. In single-genotype cohorts, developmental times were longer than in mixed-genotype cohorts. In the later cohorts, a genotype's developmental time was worse the further its frequency was from the equilibrium frequency. Moreover, in newly established colonies, the genotype-specific developmental times converged to the same value as the colony approached gene frequency equilibrium. These observations can be best explained by frequency-dependent selection induced by changes in the larval food substrate.

The results from all these experiments can be summarized as follows: (1) In natural populations, the F and S alleles are under some form of balancing selection; (2) the I allele is disfavored in natural populations, possibly because larvae with this allele cannot efficiently use the olive fruit as a diet; (3) the I allele is selected for when larvae are grown on artificial food; and (4) the behavior of the three alleles under artificial rearing is under frequency-dependent selection.

How might this knowledge about the molecular phylogeny of the Adh alleles and their response to selection be used to understand the historical aspects of the polymorphism? B. oleae is known to occur in temperate and subtropical regions of all continents of the Old World (Hardy 1977). A few years ago, it was reported in California, apparently as a result of human-mediated transfer (Economopoulos, personal communication). The highest concentration of populations occurs in the Mediterranean basin and surrounding areas (Middle East and North Africa), where the world's largest concentrations of olive trees also occur. The insect is present in the Western Cape Province of South Africa, where it infests cultivated and wild varieties of olives and extends its distribution northward to Ethiopia (Hancock 1989). In South Africa, wild olives are also attacked by the congeneric species B. biguttulus, which, however, is not known to infest cultivated varieties of olives (Munro 1924). In Asia, B. oleae has been reported in Cherat, in northwestern Pakistan and in northwest India as a pest of wild and cultivated olive trees (Fletcher 1919; Pruthi and Batra 1938).

The northwest part of the Indian subcontinent is an area of sympatry of B. oleae with three other closely related Bactrocera species: B. cucurbitae, D. scutellatus, and B. dorsalis (Kapoor 1989). It is, therefore, reasonable to assume that B. oleae originated in this part of the world and spread westward to the Middle East, the Mediterranean, and the African continent. The plant family Oleaceae is represented in central and east Asia by many native species that include about 40 species of the genus Oleae but not Olea europaea, the species whose the wild variety O. europaea oleaster or sylvestris is considered to be the forebear of the cultivated variety O. europaea sativa (Wallander and Albert 2000). Fossil records of Olea europaea in the Mediterranean region go as back as far as the Upper Meiocene (30 to 7 Myr before present) (Kavadas 1956). Our dating of the B. oleae/B. dorsalis split is between 11 and 22 MYA (table 1), which is compatible with the hypothesis that the species arrived in the Mediterranean region well after its present host, O. europeae, had established itself as a main component of the region's dry land vegetation. The appearance of the F allele is placed between 1 and 2.4 MYA (table 1). This involved a replacement of leucine by phenylalanine in amino acid position 74. As noted, this position is occupied by leucine in all tephritid and drosophilid Adh sequences that are presently known and must, therefore, represent one of the most conservative sites in the amino acid sequence of the enzyme. In view of this, the drive of the leucine-to-phenylalanine mutation to high frequencies in the B. oleae gene pool was most likely the result of active selection rather than random drift. The causal factor for this selection remains unknown. We may use the observation that the I allele has a poor performance on the olive fruit to hypothesize that the emergence of the F/S polymorphism and the concomitant decline of the I allele resulted from the species' specialization on the olive fruit. Today B. oleae is considered as a strictly monophagous insect, whose larva can grow only in the olive fruit. The S allele must have emerged from the F between 0.9 and 2 MYA and established a balanced polymorphism, for reasons that are not well understood.

The hypothesis we have outlined above about the origin of the Adh2 polymorphism of B. oleae has two parts. The first refers to the sequence of events that generated the polymorphism, (i.e., that I was the original allele, that F originated from I, and S originated from F). This part of the hypothesis appears well supported by the evidence. The second part of the hypothesis refers to the time and place of origin of the species and to ecological factors that may relate to the history and present role of the polymorphism. The evidence is much weaker for this part of the hypothesis. One weak point, for example, is the presence of allele I in natural populations, even in the low frequencies in which it is found. If the relative performance of allele I against alleles F and S on the olive fruit is as poor as the experimental evidence indicates, and if the F/S polymorphism is as old as the molecular evidence suggests, then allele I ought to have been driven to extinction. That this has not happened leads to the suspicion that heterozygotes for allele I may have an occasional advantage. There are several testable aspects about the hypothesis that I was the original allele and that the F/S polymorphism followed as an adaptation to specialization to olive fruit. The most appealing of these would be testing predictions about the early history of the species. If samples of B. oleae could be collected from its presumed native place (i.e., northwest India or Pakistan), and if they were found to contain the I allele in higher frequencies than the Mediterranean samples, this would provide support for the hypothesis.

In summary, our study provides a second case of an Adh polymorphism studied at the molecular level, after that of D. melanogaster (Kreitman 1983). As such, it supplements and provides a useful parallel to one of the most well-known cases of molecular variation. The two cases have several important similarities that set them apart from other polymorphisms. Both are sufficiently old to have accumulated several amino acid variants. The fact that they do not have more amino acid variation is strong evidence that the polypeptide is under strong purifying selection. The fact that the two allozymes for which the loci segregate are old and present in all natural populations and that they respond to various tests of selection suggest that the polymorphism is maintained by some form of balancing selection. It remains to be seen whether the same pattern of amino acid variation will be found in other Adh loci, at least within dipteran insects. In the case of the D. melanogaster Adh polymorphism, examination of other closely related species helped to identify the S allele as the ancestral allozyme. Following the same strategy, we were able to trace the sequence of events that generated the B. oleae Adh2 polymorphism and provide time estimates about these events. Interestingly, the molecular reading of these events when coupled with the insect's present-day distribution and host association provides further insights about the time, the geography, and the ecological frame under which the Adh2 polymorphism of B. oleae might have evolved.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Prof. A. Economopoulos for sharing with us his knowledge about tephritids. We thank Dr. Masahiko Muraji (National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan), Dr. Merryl Robson (Fruit Fly Research Center, The University of Sydney, Australia), and Dr. Susan McCobs (Department of Entomology, University of Hawaii) for providing us with B. scutellatus, B. tryoni and B. cucurbitae, and B. dorsalis, respectively. We also thank the associate editor, Professor R. Crozier, and Drs. M. Mateos, E. Ladoukakis, and A. Parmakelis for help with the phylogenetic analysis.


    Footnotes
 
E-mail: goulielmos{at}imbb.forth.gr. Back

Ross Crozier, Associate Editor


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 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication September 16, 2002.





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