The Drosophila Alcohol Dehydrogenase Gene May Have Evolved Independently of the Functionally Homologous Medfly, Olive Fly, and Flesh Fly Genes

Saverio Brogna1,*, Panayiotis V. Benos2,*, Giuliano Gasperi3,* and Charalambos SavakisGo,*{dagger}

*Institute of Molecular Biology and Biotechnology, Research Center of Crete, Foundation for Research and Technology, Heraklion, Crete, Greece; and
{dagger}Division of Medical Sciences, University of Crete Medical School, Heraklion, Crete, Greece


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
cDNAs for alcohol dehydrogenase (ADH) isozymes were cloned and sequenced from two tephritid fruit flies, the medfly Ceratitis capitata and the olive fly Bactrocera oleae. Because of the high sequence divergence compared with the Drosophila sequences, the medfly cDNAs were cloned using sequence information from the purified proteins, and the olive fly cDNAs were cloned by functional complementation in yeast. The medfly peptide sequences are about 83% identical to each other, and the corresponding mRNAs have the tissue distribution shown by the corresponding isozymes, ADH-1 and ADH-2. The olive fly peptide sequence is more closely related to medfly ADH-2. The tephritid ADHs share less than 40% sequence identity with Drosophila ADH and ADH-related genes but are >57% identical to the ADH of the flesh fly Sarcophaga peregrina, a more distantly related species. To explain this unexpected finding, it is proposed that the Adh genes of the family Drosophilidae may not be orthologous to the Adh genes of the other two families, Tephritidae and Sarcophagidae.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1.) enzyme of Drosophila is a member of a family of proteins with very diverse functions known as short-chain dehydrogenases (~250 residues) and found from bacteria to mammals (Jornvall et al. 1995Citation ). The ADH enzymes from yeast, plants, and mammals are all grouped in the family of medium-chain dehydrogenases (~370 residues) and bear little, if any, sequence similarity to short-chain dehydrogenases (Jornvall, Persson, and Jeffery 1981Citation ; Danielsson et al. 1994Citation ). It is assumed that ADH in insects evolved independently of the more widespread medium-chain alcohol dehydrogenases.

From an evolutionary perspective, no other gene among the Diptera has been studied as extensively as the Adh gene of Drosophila. Duplications of a preexisting locus have occurred in the Adh region, often with subsequent functional differentiation (Powell 1997Citation ). The origin of the Adh gene in Drosophila may be partially explained by the observation that it is fairly similar to another Drosophila gene coding for the fat body protein P6 (Rat, Veuille, and Lepesant 1991Citation ), which has the features of the short-chain dehydrogenases. The proteins of this group, nevertheless, are too distantly related to Drosophila ADH to provide any information on how and when it acquired its catalytic activity. However, it has recently been suggested that Drosophila ADH coevolved with fleshy fruits of angiosperms and fermenting yeast (Ashburner 1998Citation ).

The acquisition of nucleotide sequence information from the Adh gene in a number of Drosophila species has increased substantially in recent years, allowing the interpretation of the genetic events which occurred over evolutionary time and the establishment of phylogenetic relationships between species and divergence times within this family (Sullivan, Atkinson, and Starmer 1990Citation ; Jeffs, Holmes, and Ashburner 1994Citation ; Russo, Takezaki, and Nei 1995Citation ). In addition, the question of whether the Drosophila ADH protein differentiation during evolution is associated with speciation through adaptation to new feeding niches or if it is an outcome of the time separation between species has been approached by Atrian et al. (1998)Citation , who analyzed the amino acid variability of 57 ADH sequences.

However, only one non-Drosophila Adh gene has been sequenced so far, that of the flesh fly Sarcophaga peregrina (Horio, Kubo, and Natori 1996Citation ), a higher dipteran quite divergent from Drosophila. We decided to extend the molecular study of ADH to two non-Drosophila fruit flies, the Mediterranean fruit fly (medfly) Ceratitis capitata, a major agricultural pest in tropical and subtropical regions, and the olive fly Bactrocera oleae, the major pest of olives. Both species belong to the family Tephritidae, which is estimated to have diverged from the family Drosophilidae between 80 and 100 MYA, an estimate based on immunological and molecular data (Beverley and Wilson 1984Citation ; Kwiatowski et al. 1994Citation ). In addition to being interesting in evolutionary studies, the fruit fly Adh genes are also of practical interest because they can be used as selectable markers in genetic engineering schemes for biological control (Robinson, Savakis, and Louis 1988Citation ).

Ceratitis capitata has two ADH isozymes, ADH-1 and ADH-2, which are expressed in different tissues (Gasperi et al. 1992Citation ) and are encoded by a pair of tightly linked genes (Malacrida et al. 1992Citation ). The two isozymes have been purified to homogeneity, and, although they also belong to the short-chain dehydrogenase family, they show no immunological cross-reactivity to Drosophila ADH (Gasperi et al. 1994Citation ). However, they cross-react to olive fly ADH protein (Gasperi et al. 1994Citation ), which has also been purified to homogeneity (Mazi et al. 1998bCitation ).

Here, we report the cloning and sequencing of cDNAs encoding the two medfly ADH isozymes and the tissue distribution of the corresponding mRNAs. We also report the sequence of an ADH cDNA of the olive fly B. oleae, which was cloned by functional complementation in yeast (Benos et al. 2000Citation ). We found that, contrary to expectation, the flesh fly ADH was more closely related to the tephritid than to the drosophilid ADH sequences. We hypothesize that the Drosophila Adh gene may not be orthologous with the other known Adh genes of higher diptera.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Flies
The Benakeion laboratory strain of the medfly C. capitata, raised at 22–25°C as previously described (Rina and Savakis 1991Citation ), was used for all experiments.

Nucleic Acid Preparation and cDNA Library Construction
Medfly genomic DNA and total RNA were prepared essentially as described previously (Holmes and Bonner 1973Citation ). Poly(A)+ RNA was separated from total RNA as described in Holmes and Bonner (1973)Citation using an oligo(dT) cellulose column from New England Biolabs. cDNA synthesis and library construction were done from poly(A)+ RNA from third-instar larvae using the lambdaZap-cDNA synthesis kit from Stratagene. Approximately 1 x 106 primary clones, more than 80% containing cDNA inserts, were recovered. All subsequent work was done with an amplified library.

Generation of an ADH-1–Specific cDNA Probe by Polymerase Chain Reaction
Medfly third-instar larva cDNA was synthesized from 20 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Stratagene) and oligo(dT) primer (Pharmacia) as described by the manufacturer (Stratagene). cDNA was amplified by the polymerase chain reaction (PCR) (30 cycles of 94°C for 1 min, 47°C for 1 min, and 72°C for 30 s). The three degenerate primers that were used (A, 5'-ta(tc)(tc)tNatgaa(tc)aa(tc)ga(tc)(tc)tNgc-3'; B, 5'-atNgc(tc)tcNgg(tc)tt(ag)tg-3'; C, 5'-taNgtNgg(tc)tccca(ag)taNac-3') were derived from reverse translation of the ADH-1 peptides (see Results).

Medfly cDNA Library Screening
Phages (2 x 105 PFU) were screened as described in Green and Struhl (1988)Citation , using 32P-labeled DNA as probe. The positive clones were isolated, and pBluescript phagemids containing the insert were excised in vivo using the R408 helper phage, as described in the ZAP-cDNATM synthesis kit manual from Stratagene.

Cloning of the 5' End of the ADH-1 cDNA
The 5' end of the ADH-1 cDNA was amplified directly from the lambdaZap cDNA library by PCR using an antisense primer specific for ADH-1 (5'-ACTTGCAGGCTTCATAAG-3') and a vector primer (the -20M13 universal primer). A ladder of fragment sizes was obtained. The longest fragment (approximately 230 bp) was cloned into Bluescript, and two clones were sequenced.

S1 Protection
A mixture of two oligonucleotides of different lengths, A1 (5'-CATATTTTATGCACTACTTTTTCGA-TTTGAACACTCACAGtctca-3') and A2 (5'-CTCAG-TCTCTATATATATTTACTTTCGTTCGCTAATGTGT-TTTTCTGCCTGtctca-3'), complementary to the untranslated region of ADH-1 and ADH-2, respectively, were used as probes for a quantitative analysis of the two transcripts according to the protocol described in Sambrook, Fritsch, and Maniatis (1989)Citation . Each of the two oligonucleotides contained a 5-nt-long tail at its 3' end (shown in lowercase) in order to distinguish the protected fragment from the probe. Total RNA (approximately 20 µg per reaction) was used in all experiments.

Cloning of an ADH cDNA from B. oleae by Complementation in Yeast
A cDNA library was constructed in the yeast expression vector pDB20 (Fikes et al. 1990Citation ) from RNA isolated from B. oleae adult flies using the cDNA synthesis reagents provided by Stratagene (Lambda ZAP) according to the kit's instructions. The ADH-deficient yeast strain MC892-1C (MAT-alpha, adh1, adh2, adh3, ura3-52, trp1, leu2, his3), kindly provided by Dr. M. von Ciriacy-Wantrup, was used for complementation. Complementation was performed by growing transformed yeast cells on minimal plates (2% glucose, 0.7% yeast nitrogen base without amino acids, and 2% agar) with the addition of the appropriate amino acids for 3 days, followed by replica plating on plates containing, additionally, 0.5 ppm antimycin A. The method for cloning ADH coding sequences by complementation in yeast is described in detail in Benos et al. (2000)Citation .

DNA and Protein Sequence Analysis
DNA and protein sequences were analyzed using the GCG programs in the Wisconsin Package, version 9.0, (Genetics Computer Group [GCG], Madison, Wis.). Multiple alignments, tree computation and bootstrap analysis were performed with the program CLUSTAL W (Thompson, Higgins, and Gibson 1994Citation ).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Generation of a Medfly ADH-1–Specific cDNA Probe and cDNA Cloning
The lack of immunological cross-reactivity observed between C. capitata and Drosophila melanogaster ADH proteins (Gasperi et al. 1994Citation ), along with the absence of cross-hybridization of D. melanogaster ADH probes to medfly sequences (unpublished data), suggested that the ADH isozymes of C. capitata may have diverged considerably from the Drosophila enzyme during evolution. Therefore, to clone medfly ADH, previously determined sequences of two ADH-1 peptide fragments (Gasperi et al. 1994Citation ) were used to design a set of oligonucleotide primers for PCR-amplification of ADH-1 sequences from medfly third-instar larva cDNA. The design of the primers was based on the observation that the first of the two medfly ADH-1 peptides showed similarity with the region between residues 25 and 47 of D. melanogaster ADH. The position of the second peptide was not known, as it did not show any similarity with Drosophila ADH, but we assumed that it would be located closer to the carboxyl terminus. Based on this assumption, three degenerate oligonucleotide primers derived by reverse translation were synthesized and used as primers in reverse transcription–PCR reactions: primer A, a "downstream" primer from the amino end of the first peptide, and primers B and C, "upstream" primers from the carboxyl end of the first peptide and from the second peptide, respectively. Primer A in combination with primer B or primer C gave amplification products of 66 bp and 700 bp, respectively. Cloning and sequencing of these amplified fragments showed that they corresponded to ADH-1 sequences: the 66-bp fragment contained the entire DNA sequence predicted from the 22-aa fragment, and the 700-bp fragment contained the two predicted amino acid sequences at its amino and carboxyl ends and an open reading frame with significant similarity to D. melanogaster ADH (data not shown).

The cloned 700-bp cDNA fragment was used to screen a cDNA library from third-instar larvae. More than 40 different positive clones were isolated, and six of them were sequenced. All six clones had an open reading frame (ORF) of 258 codons, a 58-bp 5' untranslated region (UTR), and a 143-bp 3' UTR. These clones could be divided into two sequence classes, each probably representing a polymorphic allele of the gene: three clones carried seven synonymous substitutions relative to the other three (data not shown). Surprisingly, the nucleotide sequence of these clones was only about 80% identical to the ADH-1 probe used for screening, showing many nonsynonymous codon differences. This suggested that the cDNA clones recovered probably corresponded to ADH-2 rather than ADH-1 cDNAs, a result not totally unexpected, since ADH-2 is the most abundant of the two isozymes.

Because of the low abundance of ADH-1 cDNA clones, the missing 5' end of ADH-1 was amplified by PCR from pooled DNA of the entire cDNA library using a combination of an ADH-1–specific primer and a primer corresponding to vector sequences. A full-length ADH-1 cDNA clone was recovered using this 5' end as probe and showed a 39-bp 5' untranslated sequence, an ORF encoding 257 amino acids, and a 3' UTR of 146 bp. The coding regions of the two genes were considerably divergent, with a total of 167 nucleotide differences (21.7%), of which 16% were synonymous substitutions and 5.7% resulted in amino acid replacements. The replacements accounted for 45 amino acid differences (17%) between the two protein sequences. Both protein sequences contained the "short-chain dehydrogenase" motif (PROSITE accession number PS00061; ADH_SHORT). The noncoding regions were considerably more divergent. It is notable, though, that the 3' UTRs of the two mRNAs were the same size (except for a 3-nt difference due to ADH-2 having one extra codon at the carboxy terminus). No significant similarities could be detected in the 5' UTRs.

In summary, we have shown that in the medfly, the two ADH isozymes are encoded by two similar genes, sharing approximately 83% amino acid sequence identity. The sequence similarity strongly supports the notion that these genes are products of a gene duplication. The relatively high degree of divergence between the two genes and the observation that most species of the family Tephritidae have two or three isoenzymes for ADH (Matioli et al. 1992Citation ) would support the view that this duplication is a rather early event in the radiation of Tephritidae.

Tissue Distribution of the Medfly ADH mRNAs
Previous studies have shown that the two ADH isozymes in the medfly have nonoverlapping tissue distributions. ADH-1 is found only in body muscles, and ADH-2 is in all other tissues in which ADH activity has been detected, mostly in the midgut, the fat body, and the ovary (Gasperi et al. 1992Citation ). Therefore, to confirm the identity of the two cDNA clones, the pattern of expression of the corresponding mRNAs was determined. Because the two mRNAs were very similar in length and in sequence, an S1 nuclease protection assay was employed to detect specific mRNAs, using probes specific for the divergent 5' UTRs of the two sequences. As shown in figure 1 , ADH-1 transcripts are present only in muscle, while ADH-2 is present in all other tissues examined, with the fat body showing the highest abundance and ovary the lowest. These data confirm the identity of the two ADH sequences. ADH-2 seems to be more similar to the Drosophila enzyme, considering its tissue distribution, while the unusual expression of Adh1 in the muscle raises a puzzle as to this isozyme function.



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Fig. 1.—Medfly tissue distribution of RNAs complementary to the two cDNA clones. A mixture of two antisense oligodeoxyribonucleotide probes, specific for ADH-1 and ADH-2, was used in the same S1 protection reaction. Equal amounts of RNA (approximately 20 µg per reaction) were used from all tissues. Muscle (M), fat body (FB), and gut (G) RNA were from third-instar larvae (6 days postoviposition); ovary mRNA (O) was from 4-day-old females. The probes are described in the text. The positions corresponding to the Adh-1 and the Adh-2 unprotected probes are indicated on the right. Arrowheads indicate the protected probes

 
Sequence of an ADH cDNA Clone from B. oleae
Fifteen olive fly cDNA clones that complemented ADH activity in yeast were end-sequenced and found to contain identical inserts. The complete sequence of one of these clones was determined (EMBL accession number AJ250007). It was 867 bp long and contains an ORF of 777 bp (starting at the first ATG), with 50 bp in the 5' UTR and 40 bp in the 3' UTR. The corresponding protein was predicted to be 258 amino acids long and had approximately 33%, 78%, and 84% overall amino acid identity with D. melanogaster ADH, medfly ADH-1, and medfly ADH-2, respectively. The sequence also contained the "short-chain dehydrogenase" motif (PROSITE accession number PS00061; ADH_SHORT). Based on phylogenetic analysis (fig. 2 ), the olive fly sequence seemed to be more closely related to the ADH-2 of the medfly.



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Fig. 2.—ADH sequence phylogeny. Top, Phylogenetic relationships between ADH and ADH-related protein sequences in Diptera. The human prostaglandin dehydrogenase, the closest to insect short-chain dehydrogenase, is included as the outgroup. Drosophila lebanonensis and Drosophila melanogaster are the most divergent of the available Drosophilid ADH sequences. The unrooted tree was constructed with the program CLUSTAL W using a distance matrix method. Branch lengths are proportional to amino acid differences. Values at nodes are percentage bootstrap values (1,000 replicates). The sequences are as follows: Homo sapiens prostaglandin dehydrogenase, PGDH_HUMAN; D. melanogaster ADH-related protein, ADHR_DROME; D. lebanonensis ADH-related protein, ADHR_DROLE; D. melanogaster ADH, ADH_DROME; D. lebanonensis ADH, ADH_DROLE; Sarcophaga peregrina ADH, ADH_SARPE; Bactrocera oleae ADH, ADH_BACOL; Ceratitis capitata ADH-1, ADH1_CERCA; Ceratitis capitata ADH-2, ADH2_CERCA. Bottom, Different topologies of the species and the sequence phylogenetic trees. The known phylogeny of Drosophila, Sarcophaga and the medfly are shown on the left; the protein phylogeny is shown on the right.

 
Relationships of Drosophila ADH, Sarcophaga ADH, and Related Proteins
Table 1 shows a measure of the phylogenetic distances between ADH and ADH-related protein sequences of selected higher Diptera, including ADH and ADHR sequences of Drosophila lebanonensis, the most distant Drosophila species relative to D. melanogaster. In terms of overall positional sequence identity, the three tephritid ADHs are very distantly related to the Drosophila ADHs. For example, medfly ADH-1 and olive fly ADH are 38% and 33% identical to Drosophila ADH, respectively. Indeed, the tephritid genes are as divergent from Drosophila Adh as they are from the Adh-related gene (Adhr), a gene that, most probably, does not encode an alcohol dehydrogenase. Adhr has been found immediately downstream of the Adh gene in species of the Sophophora subgenus of Drosophila; it has the same intron-exon structure as Adh, and it has been shown that in D. melanogaster these two genes are transcribed from the same promoter as a single dicistronic mRNA (Schaeffer and Aquadro 1987Citation ; Kreitman and Hudson 1991Citation ; Jeffs, Holmes, and Ashburner 1994Citation ; Brogna and Ashburner 1997Citation ). Drosophila ADH and ADHR are 38% identical in amino acid sequence; medfly ADH-1, for example, is 38% and 39% identical to Drosophila ADH and ADHR, respectively.


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Table 1 [bu792]Phylogenetic Distances Between ADH and ADH-Related Protein Sequences of Higher Diptera

 
Table 1 also shows an unexpected finding: the ADH of S. peregrina, the only other non-Drosophila ADH sequence in the database, is more closely related to the tephritid sequences, showing approximately 58% identity with medfly ADH-1, medfly ADH-2, and olive fly ADH, compared with 38% identity with D. melanogaster ADH. This result was unexpected because Ceratitis and Drosophila, members of the Acalyptrata subsection of Brachyceran Diptera, are more closely related to each other than to Sarcophaga, which belongs to the Calyptrata subsection.

This is also schematically represented in the phylogenetic tree of figure 2 (top), which is based on the same protein sequences. The human prostaglandin dehydrogenase gene was used to root this tree, as it was the closest noninsect "relative" to the short-chain dehydrogenases. The bootstrap analysis performed on these data was further supportive of this "paradoxical" evolution of the Adh gene in Diptera. In particular, three distinct clusters comprise this tree, all having bootstrap values of 1,000/1,000 (confidence 100%): one with the Adhr genes from the two most divergent Drosophila species, another with the Adh genes of the same Drosophila species, and a third with the Sarcophaga together with the tephritid genes. This conclusion was further supported by the pattern of indels in the aligned sequences. As shown in figure 3 , there are three internal indels in the aligned sequences, each involving one or two amino acids; they are located after positions 66, 168, and 193 of the D. melanogaster ADH sequence. The Sarcophaga and the tephritid ADHs share the same three indels, one of which is also in the ADH-related but not the ADH protein of Drosophila. This is additional evidence that the tephritid and Sarcophaga ADHs are more closely related to each other than to Drosophila ADH. The apparent lack of congruence between the tree based on the species and the tree based on protein phylogenies is illustrated in figure 2 (bottom).



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Fig. 3.—Alignment of the Bactrocera oleae and the two Ceratitis capitata ADH peptide sequences with representative Dipteran ADH and ADH-related sequences. Sequence names are as in figure 2 . Conserved positions are boxed in black. The three indels described in the text are in empty boxes

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
This paper extends our knowledge of the evolution of ADH in insects, which has previously been restricted to Drosophila species. Three nondrosophilid ADH sequences were considered, namely, those of C. capitata, B. oleae, and S. peregrina.

We cloned and sequenced cDNAs for the two ADH isozymes of the medfly C. capitata and for one ADH of the olive fly B. oleae. These represent the only known fruit fly Adh genes cloned outside of the family Drosophilidae. Because of the unexpectedly high degree of sequence divergence (<40% identity at the amino acid level between the known Drosophila sequences and medfly ADH), cloning of the medfly genes had to rely on partial sequencing of purified ADH-1 protein (Gasperi et al. 1994Citation ). For easier cloning of distantly related Adh genes, we have developed an efficient method based on complementation of ADH activity in yeast for the olive fly ADH. This method, which is presented elsewhere in detail (Benos et al. 2000Citation ), relies on the inability of yeast mutants lacking ADH activity to grow under anaerobic conditions, and it may be of more general use for cloning ADH from diverse sources.

Apart the evolutionary implication of the nondrosophilid ADH system, the availability of tephritid Adh genes clears the way for their use as selectable genetic markers in pest control schemes based on genetic engineering (Robinson, Savakis, and Louis 1988Citation ). In the olive fly, dramatic changes are observed in ADH allele frequencies when natural populations are introduced in the laboratory, and the ADH alleles involved differ in specific activity and enzymatic properties, suggesting that selection may be taking place (Mazi et al. 1998aCitation ). Cloning of olive fly ADH clears the way for investigating this interesting adaptive phenomenon.

Adh Duplications in Tephritids
The sequences and tissue distribution of the two cDNA classes from C. capitata confirm the existence of duplicated Adh genes in this species. It is known that the Adh locus has undergone multiple duplication events in insects. Russo, Takezaki, and Nei (1995)Citation proposed a scenario according to which as many as four independent duplications occurred in the last 180 Myr, resulting in the present status of the Adh region in drosophilid species.

Our data show that there has been at least one duplication event in tephritids, which resulted in medfly ADH-1 and ADH-2. These genes are tightly linked in the medfly genome (Malacrida et al. 1992Citation ) and have identical intron/exon structures (unpublished data). The observation that most tephritids have two ADH isozymes (Matioli et al. 1992Citation ), combined with the relatively high degree of sequence divergence between the two copies of the medfly ADH (17.5% amino acid substitutions), indicates that the duplication of the Adh gene in this family is old. The B. oleae ADH sequence is more closely related to that of medfly ADH-2; a second ADH isozyme is also electrophoretically detectable in the olive fly (unpublished data), although more work will be required to fully characterize the ADH system of this species.

Evolutionary Inferences from the ADH Sequences in Diptera
The finding that S. peregrina ADH is more closely related to the tephritid, rather than being equidistant to the Drosophila and tephritid sequences, is intriguing. The flesh fly S. peregrina is placed in the Calyptrata series of the division Schizophora (infraorder Cychlorrapha, suborder Brachycera, order Diptera), while the drosophilid and tephritid fruit flies belong to the Acalyptrata series of the Schizophora (Hennig 1958Citation ; Griffiths 1972Citation ). It would be expected, therefore, that the Sarcophaga ADH sequence is equally distant to Drosophila and to tephritid ADH sequences. In contrast, Sarcophaga ADH branches together with the tephritid sequences, while the Drosophila ADH sequences form a separate cluster. This conclusion is strongly supported by (1) bootstrap analysis of the distance-based phylogenetic tree and (2) the presence of three shared indels in the Sarcophaga and tephritid sequences. The Drosophila ADHs are actually as distant from the Sarcophaga/tephritid ADHs as from the ADH-related sequences, with the three branches forming a virtual trifurcation (fig. 2 ).

Two evolutionary scenarios may account for this unexpected finding; either the rate of evolution of ADH in the Drosophila lineage is (or has been) significantly faster than that in the other two lineages, or the Drosophila Adh genes are not orthologous to the tephritid and Sarcophaga genes. These two models are not, of course, mutually exclusive.

An acceleration in the rate of ADH evolution in drosophilids cannot be ruled out. If acceleration occurred, it would appear from figure 2 that it must have commenced or occurred in the ancestral Drosophila lineage. A temporal change in the rate of amino acid substitutions has been invoked to explain the apparently erratic evolution of glycero-3-phosphate dehydrogenase in higher Diptera (Kwiatowski et al. 1997Citation ). In this case, the rates of amino acid replacements appear to be slightly different between the two drosophilid taxa, Drosophila and Chymomyza, but higher when species of these two genera are compared with the medfly enzyme. Analysis of ADH sequences from diverse tephritid species will be required to test the possibility that Adh may have evolved at different rates in tephritids than in drosophilids.

However, being members of a multigene family, the tephritid/Sarcophaga and Drosophila Adh genes may be paralogous and therefore not connected to each other by the same phylogenetic tree as the corresponding lineages. It is possible that the ancient lineage that gave rise to the Calyptrata and the Acalyptrata was already endowed with two duplicated and already divergent genes and that each of the three lineages (Drosophilidae, Tephritidae, and Sarcophagidae) subsequently retained a different copy of these genes; under this scenario, the copy retained by tephritids would be the same as that used by the distantly related sarcophagids (fig. 4 ). This hypothesis is in agreement with the observation that Adh has undergone multiple duplication events during evolution. A related, "co-optation," scenario is that the ADH activity in both the Calyptrata and the tephritid lineages is associated with an orthologous gene (which has duplicated relatively recently, at least in the tephritid lineage), but this ortholog was lost in the drosophilid line in parallel with the de novo evolution of ADH activity from a different member of the preexisting multigene family of short-chain dehydrogenases. This scenario is also in agreement with the observation of emergence of new functions in duplicated members of the Adh family (Long and Langley 1993Citation ; Begun 1997Citation ). In either case, the Adh genes of, for example, Drosophila and the medfly would be paralogous, and their evolutionary distance would have no direct relation to that of the species lineages. No cross-hybridization could be detected between medfly Adh probes and Drosophila DNA or, reciprocally, between Drosophila Adh and medfly DNA (unpublished data). Moreover, the absence of regions of synteny between the two chromosomal Adh regions in the medfly and D. melanogaster may be an additional criterion for proposing a high evolutionary differentiation between the Adh systems of C. capitata and those of Drosophila (Malacrida et al. 1992Citation ). These data suggest that under the paralogy scenario, the ADH orthologs becoming pseudogenes may have diverged in nucleotide sequence to a degree that does not allow detection.



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Fig. 4.—A possible scenario of the evolution of the Adh gene in higher Diptera. A question mark indicates that the corresponding gene has not been identified, either because it has not been discovered yet or because it has been lost during the evolution of the lineage. Dashed arrows represent gene duplications. In this model, it is equally plausible that the first gene duplication generated the gray and black Adh genes, and the second duplication of one of these generated Adhr (white)

 

    Conclusions
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Analysis of the Adh and—possibly—Adhr genes from other Dipteran species and families and from different insect taxa, combined with the discovery of more members of the Adh family in the Drosophila genome and other genomes, should resolve the evolutionary history of this intriguing gene family. The co-optation hypothesis, if proven correct, would be of major interest, because it would provide a clear case of convergent evolution and emergence of new functions in a gene family. Since all duplicated sequences studied so far were proven to be due to tandem duplication events, the comparative study of genome organization of these loci from different insect species will be helpful in testing this hypothesis.


    Supplementary Material
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
The sequences reported in this paper have been deposited in EMBL/GenBank with accession numbers P48814, P48815, and AJ25000


    Acknowledgements
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Dr. T. Loukeris' advice and help and A. Babaratsas' expert technical assistance are acknowledged. The authors wish to thank Drs. E. Betran, S. Oehler, and F. C. Kafatos for critically reading an earlier version of the manuscript. This work was supported by European Union BIOTECH training grants to S.B. and G.G. and by a European Union research contract to C.S. S.B. and P.V.B. contributed equally to this work.


    Footnotes
 
Pierre Capy, Reviewing Editor

1 Present address: Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, Massachusetts. Back

2 Present address: Department of Genetics, Medical School, Washington University, St. Louis, Missouri. Back

3 Present address: Department of Animal Biology, University of Pavia, Pavia, Italy. Back

4 Keywords: Adh, ADH ADH evolution Ceratitis capitata, Bactrocera oleae, Sarcophaga peregrina. Back

5 Address for correspondence and reprints: Charalambos Savakis, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, P.O. Box 1527, Vasilika Vouton, Heraklion 71110, Greece. E-mail: savakis{at}imbb.forth.gr Back


    literature cited
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 Abstract
 Introduction
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 Discussion
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 Supplementary Material
 Acknowledgements
 literature cited
 

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Accepted for publication October 16, 2000.