Sequence Variation of Alcohol Dehydrogenase (Adh) Paralogs in Cactophilic Drosophila

Corresponding author: Luciano M. Matzkin, State University of New York, Stony Brook, NY 11794-5245., lmatzkin{at}life.bio.sunysb.edu (E-mail)

Communicating editor: S. W. SCHAEFFER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D. arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 ± 0.7 MY) and the age of the gene duplication (3.95 ± 0.45 MY).

THE strength of selection at a particular locus can be affected by changes in the environment experienced by its product (DYKHUIZEN and HARTL 1980 ; HARTL et al. 1985 ; DYKHUIZEN et al. 1987 ). Interspecific comparisons can identify differences in the evolutionary history of loci between species, but may not identify the environmental factors that changed the shape of the adaptive landscape. It is often possible to determine changes in the external environment experienced by species. An approach to this problem is to compare the evolutionary trajectories of recently duplicated loci, which have complete or partial nonoverlapping modes of expression.

Gene duplications underlie the diversification of genes and the origination of novel gene functions (OHNO 1970 ). Most models of gene evolution via duplication offer two possible fates for duplicated loci, either the creation of a gene with novel function or the formation of a pseudogene (OHNO 1970 ; OHTA 1988A , OHTA 1989 ). Other models have offered a third fate for recently duplicated loci. If the original locus had several modes of expression controlled by different regulatory sequences, after the duplication, mutations may occur in one or more of these regulatory sequences resulting in two loci having at least partially nonoverlapping modes of expression (HUGHES 1994 ; FORCE et al. 1999 ). At this point adaptive evolution can potentially occur since each locus will be released from the constraints of being expressed in all regions, effectively being placed in a new environment.

In Drosophila and other Diptera, there have been several independent duplications of the alcohol dehydrogenase (Adh) locus (GASPERI et al. 1992 ; RUSSO et al. 1995 ; GOULIELMOS et al. 2001 ). One such set of duplications in Drosophila occurs in the mulleri species complex. Within the mulleri species complex three Adh loci have been described (ATKINSON et al. 1988 ). An early duplication created a locus of abnormal function positioned in the 5' region of the Adh gene cluster. The exact function of this locus (Adh Finnegan) is unknown (BEGUN 1997 ). A later duplication resulted in Adh-2 and Adh-1, positioned 3 kb apart. ADH-1 activity is observed from egg until the 5-day-old larva stage (BATTERHAM et al. 1983 ). Just prior to pupal eclosion, both ADH-1 and ADH-2 activities are observed. In adults, only ADH-2 is expressed, except in females, where ADH-1 activity is limited to the ovaries.

The cactophilic mojavensis species cluster is within a set of 24 species that compose the mulleri species complex (WASSERMAN 1982 ). The mojavensis cluster is composed of D. navojoa, D. arizonae, and D. mojavensis, from basal to most derived (HEED 1982 ; RUIZ et al. 1990 ). Cactus hosts tend not to be shared across species: agria (Stenocereus gummosus) and organpipe (S. thurberi) are utilized by D. mojavensis, cina (S. alamosensis) and Opuntia spp. by D. arizonae, and Opuntia wilcoxii by D. navojoa (FELLOWS and HEED 1972 ; RUIZ and HEED 1988 ). Cactus host use does, however, vary with geographic location. Most importantly, the alcohol composition of the cactus rots varies with species (HEED 1978 ; VACEK 1979 ; FOGLEMAN 1982 ; KIRCHER 1982 ).

The D. melanogaster ADH allozyme polymorphism has become a textbook example of selection on a simple gene. The Fast/Slow (F/S) allozyme polymorphism shows parallel latitudinal clines in several continents, where the Fast allele is at its highest frequency in higher latitudes (OAKESHOTT et al. 1982 ). Population studies at the Adh sequence level have proposed that the pattern of variation observed at Adh is indicative of balancing selection (KREITMAN 1983 ). Centered around the replacement substitution producing the F/S polymorphism is an excess of silent polymorphisms that is consistent with models of balancing selection (HUDSON and KAPLAN 1988 ; HUDSON 1990 ). BERRY and KREITMAN 1993 observed that an indel (1) exhibited a stronger clinal pattern than that of the replacement polymorphism producing the F/S change. The 1 indel, located in the first intron of the larval transcript, has a significant effect on ADH activity (LAURIE and STAM 1994 ). Also, there is evidence of selection for the maintenance of pre-mRNA structure in Drosophila Adh (KIRBY et al. 1995 ), which could have potential effects on ADH activity. Hence the actual target of the proposed selection in D. melanogaster has not been unequivocally determined. In addition to D. melanogaster, population level sequence data have been collected in only a few species: D. pseudoobscura (PRAKASH 1977 ; SCHAEFFER and MILLER 1992A , SCHAEFFER and MILLER 1992B ), D. simulans (KLIMAN et al. 2000 ), D. willistoni (GRIFFITH and POWELL 1997 ), and D. americana americana (MCALLISTER and CHARLESWORTH 1999 ).

Alcohol dehydrogenase has been proposed to play an important role in the adaptation to alcohol environments (MERCOT et al. 1994 ). In Drosophila, a physiological and behavioral response to ethanol can be observed at both the larval and the adult stage (STARMER et al. 1977 ; GELFAND and MCDONALD 1980 ; GEER et al. 1990 ). There is a large amount of variation in hosts across Drosophila species, from flowers to crab excrement, and each particular host has its specific chemical makeup (POWELL 1997 ). Given what is known about the alcohol-dependent ecology of cactophilic Drosophila, the duplication and nonoverlapping modes of expression of Adh in D. mojavensis and D. arizonae, and the possible adaptive significance of Adh in Drosophila, it is of great value to examine the variation at Adh in these species. In this study we have focused on the allozyme and nucleotide variation of Adh paralogs of two cactophilic Drosophila sister species (D. mojavensis and D. arizonae) that utilize different cactus hosts.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isofemale line collections:
The D. mojavensis (MJBC) lines were collected in February 2001 from a geographic location 30 km south of La Paz, Baja California Sur, Mexico. Flies were aspirated from rotting sections of agria cactus and briefly placed in collecting tubes. Isofemale lines were set up in the field by placing a gravid female in an 8-dram vial containing standard banana-molasses medium. A similar technique was used in the collection of D. arizonae isofemale lines. D. arizonae isofemale lines were collected by Therese Markow in Tucson, Arizona (ARTU) and Guaymas, Sonora, Mexico (AR00) in 2000. The D. navojoa line is from the University of Arizona Drosophila species stock center (no. 15081-1374.0). While in the lab all lines were maintained in 8-dram vials containing standard banana-molasses media sprinkled with a few granules of live yeast. Lines were stored in a 25° incubator with a 14:10 light:dark cycle. Lines were transferred into new food vials every 3–4 weeks.

Allozyme survey:
Starch gel electrophoresis was used to estimate the level of ADH allozyme variation. A modified version of the BATTERHAM et al. 1983 protocol was used. Three one-fly samples per isofemale line were homogenized in 15 µl of Tris-boric acid buffer (41 mM Tris; 6 mM boric acid, pH 8.8) and transferred to filter paper. Samples were run at 4° through a 12% starch gel at 30 V/cm for 5 hr. A 1% agar overlay stain (100 mM Tris-HCl, pH 8.8; 260 mM 2-propanol; 0.75 mM NAD+; 0.61 mM methylthiazoletetrazolium; 0.03 mM phenazine methosulfate) was used to visualize ADH.

Sampling:
According to the allozyme variation observed in the D. mojavensis population, a constructed random sample (HUDSON et al. 1994 ) of 13 isofemale lines was PCR amplified and sequenced for both Adh-2 and Adh-1. The D. arizonae isofemale lines sampled varied slightly between Adh-2 (7 ARTU and 4 AR00 lines) and Adh-1 (6 ARTU and 7 AR00 lines). Overall there were 6 ARTU and 4 AR00 lines in common between the two samples that were sequenced for both paralogs. Additionally, one D. navojoa line was used to polarize the D. mojavensis and D. arizonae Adh sequence data for the purpose of performing a lineage-specific McDonald-Kreitman test.

PCR amplification and sequencing:
PCR amplification of CTAB genomic DNA preps (WINNEPENNINCKX et al. 1993 ) was done in a thermal cycler (MJ Research, Waltham, MA) and an Air-Thermo-Cycler (Idaho Technologies, Idaho Falls, ID) using GIBCO BRL (Carlsbad, CA) Taq DNA polymerase. Locus-specific primers were designed from the published D. mojavensis Adh-2/Adh-1 sequence (ATKINSON et al. 1988 ). Primers designed from the D. mojavensis Adh-1 sequence successfully amplified sequence from D. arizonae and D. navojoa. However, only a partial sequence (250 bp) was obtained for Adh-2 in D. navojoa using D. mojavensis primers. We used an inverse PCR technique explained by TRIGLIA et al. 1988 to obtain the complete D. navojoa Adh-2 sequence.

To examine the association between polymorphic sites between and within paralogs, we retrieved full haplotypes. Due to the fact that the Adh paralogs are autosomal loci, it was necessary to clone the Adh-2/Adh-1 gene cluster. This technique was used only for the D. mojavensis sequences. A 5.5-kb fragment containing Adh-2 and Adh-1 was amplified using the Expand Long Template PCR System (Roche, Mannheim, Germany). Fragments were then purified using QIAquick columns (QIAGEN, Valencia, CA) and cloned into a pCR 2.1 vector (TA cloning kit; Invitrogen, Carlsbad, CA). Colonies with inserts were picked and disrupted in 500 µl of ddH₂O. This solution was used as a template to PCR amplify Adh-2 and Adh-1 fragments individually. All PCR fragments were cleaned using the Prep-A-Gene kit (Bio-Rad, Hercules, CA) prior to sequencing.

Fragments were amplified and sequenced from three colonies per D. mojavensis individual to obtain the linkage phase and to correct for errors. The sequencing reactions were performed using the ABI Prism BigDye cycle sequencing kit v2.0, and reactions were run in an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The sequencing of the D. arizonae isofemale lines and the stock center D. navojoa line was done manually using the Sequenase kit v2.0 (United States Biochemical, Cleveland) and [³⁵S]dATP (Amersham, Buckinghamshire, England). All sequences are stored under GenBank accession nos. AY154827, AY154828, AY154829, AY154830, AY154831, AY154832, AY154833, AY154834, AY154835, AY154836, AY154837, AY154838, AY154839, AY154840, AY154841, AY154842, AY154843, AY154844, AY154845, AY154846, AY154847, AY154848, AY154849, AY154850, AY154851, AY154852, AY154853, AY154854, AY154855, AY154856, AY154857, AY154858, AY154859, AY154860, AY154861, AY154862, AY154863, AY154864, AY154865, AY154866, AY154867, AY154868, AY154869, AY154870, AY154871, AY154872, AY154873, AY154874, AY154875, AY154876, AY156524, and AY156525.

Data analysis:
Descriptive and statistical analysis of the sequence data was produced using SITES (HEY and WAKELEY 1997 ) and DnaSP version 3.53 (ROZAS and ROZAS 1999 ). The neighbor-joining gene tree using third base positions and bootstrapping was created using MEGA version 2.1 (KUMAR et al. 2001 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Allozyme variation:
Three individuals from each of the 41 D. mojavensis isofemale lines were used in the allozyme survey. Consistent with previous work (BATTERHAM et al. 1983 ) the ADH-2 enzyme had the strongest cathodal mobility. For ADH-2, 2 lines were homozygous for the Slow allele, 32 lines were homozygous for the Fast allele, and both alleles segregated in the 7 remaining lines. The weighted frequency of the ADH-2 Fast allele was 0.90; no allozyme variation was observed at ADH-1, which is in agreement with previous surveys (HEED 1978 ).

Sequence variation at alcohol dehydrogenase-2:
The gene structure in D. mojavensis and D. arizonae Adh-2 was similar to that observed in D. melanogaster Adh (KREITMAN 1983 ). In both species, the locus consists of a total of 880 bp, with two small introns of 55 bp (position 94–148) and 60 bp (position 554–613). D. mojavensis and D. arizonae Adh possess 2 fewer amino acid residues compared to the D. melanogaster locus. The inferred protein consists of 254 amino acid residues, which is the same as that observed in D. lebanonensis, the species for which a refined crystal structure of ADH is known (BENACH et al. 1998 , BENACH et al. 1999 ).

For D. mojavensis we sequenced a total of 12 Adh-2-F and 2 Adh-2-S alleles. The constructed random sample (CRS) consisted of 12 Fast alleles and 1 Slow allele; the variable sites are shown in Fig 1. All parameter estimates and tests were performed using this data set. The second Slow allele was omitted from the constructed random sample because there was evidence of a substantial gene conversion event between Adh-1 and Adh-2 (data not shown). There are a total of six replacement polymorphisms, where five separate the Fast and Slow alleles. Two of these replacement polymorphisms produce a charge change, which could be responsible for the Fast/Slow allozyme polymorphism: an arginine-to-serine change at position 84 and a histidine-to-tyrosine change at position 347. In an additional survey (L. M. MATZKIN and W. F. EANES, unpublished data) of sequence variation across D. mojavensis, we observed that only the replacement polymorphism at position 84 is unique to all Slow alleles. In the coding region there are 19 silent polymorphisms, plus a substantial level of variation segregating in the introns. Twenty-four segregating sites plus two small deletions are in the introns, and 20 segregating sites occur in the first intron alone.

View larger version (113K): In this window In a new window Download PPT slide   Figure 1. A list of inter- and intraspecific variation at Adh-2 in D. mojavensis and D. arizonae. The italicized nucleotide positions are sites located in the introns. Dagger indicates the D. mojavensis ADH-2 Slow allele. Shaded boxes indicate a replacement polymorphism within a species. Asterisk indicates replacement polymorphism producing a charge change. Dashes are sequence gaps produced by an insertion/deletion. Standard nomenclature was used to identify heterozygous sites: Y (C/T), W (A/T), M (A/C), and R (A/G). Double dagger indicates that D. arizonae line (ARTU 10) is not present in the Adh-1 data set.

The Adh-2 gene structure in D. arizonae was identical to D. mojavensis with the exception of a single-base deletion in both introns (Fig 1). Overall, we observed a greater number of segregating sites in D. arizonae. There were a total of 33 silent and 6 replacement polymorphisms, none of which produced a charge change (Fig 1). In 13 out of the 33 silent sites we found one or more individuals segregating for both bases, i.e., heterozygous sites. In such cases the alternative state was never a singleton. Similar to D. mojavensis there was a substantial amount of variation segregating in the introns, 38 sites, of which 30 occur in the first intron. Of the sites segregating in the first intron, 16 are shared with D. mojavensis (Fig 1). We found that the shared polymorphisms are due to gene conversion between the Adh-2 and Adh-1 intron 1, because the sequence of one of the Adh-2 intron haplotypes is identical to the Adh-1 intron.

The silent diversity () in D. arizonae was greater than that observed in D. mojavensis (Table 1). Given the nature of the sequence data in D. arizonae, an estimate of pairwise differences () can be performed only if we assume a random distribution of the variation across the alleles. A way to check for the validity of this assumption is by estimating pairwise differences from the seven D. arizonae individuals that contained zero or one heterozygous site. The estimated from these seven individuals ( = 0.05928) was similar to the overall estimate of (Table 1).

Sequence variation at alcohol dehydrogenase-1:
The gene structure observed at Adh-1 is similar to that of Adh-2 with the exception of a few small indels in the introns (Fig 2). As with ADH-2, 254 amino acid residues were in the inferred ADH-1 protein. For the D. mojavensis Adh-1 data set, we used the same population sample as in Adh-2. In D. mojavensis there were a total of 12 segregating sites; 10 occurred in the exons and none of those were replacement changes. There was very little variation in the introns. In D. arizonae, a total of 35 segregating sites were observed in the coding region, 31 silent and 4 replacement. Line ARTU 10 was not present in the Adh-1 analysis, unlike the D. arizonae Adh-2 data set. Additionally, lines AR00 2, 4, and 5 were included in the Adh-1 set but excluded for Adh-2. Due to the 16 changes that occur in intron 1 and the heterozygous nature of the data, no intron 1 data were collected for lines AR00 4, 6, and 14, because of difficulty in interpreting the autoradiographs. Once again, the overall level of variation in D. arizonae was greater than that observed in D. mojavensis (Table 1).

View larger version (51K): In this window In a new window Download PPT slide   Figure 2. A list of inter- and intraspecific variation at Adh-1 in D. mojanvensis and D. arizonae. The italicized nucleotide positions are sites located in the introns. Dagger indicates the D. mojaensis ADH-2 Slow allele. Shaded boxes indicate a replacement polymorphism within a species. Asterisk indicates replacement polymorphism producing a charge change. Dashes are sequence gaps produced by an insertion/deletion. Standard nomenclature was used to identify heterozygous sites: Y (C/T), W (A/T), K (T/G), S (C/G), and R (A/G). Double dagger indicates D. arizonae lines (AR00 4, 5, and 6) not present in the Adh-2 data set.

Interspecific and interparalog divergence:
Two different estimates of D. mojavensis/D. arizonae silent divergence were calculated, one for each paralog. Since nucleotide divergence K_s is based on pairwise differences (NEI 1987 ), for the comparison between species within paralog, we used only one D. arizonae line (ARTU 34). ARTU 34 was chosen for the analysis because it contained no heterozygous sites in Adh-1 and only one in Adh-2. POWELL 1997 suggested that the rate of silent substitution in Drosophila is between 1.0 and 2.7 x 10^-8 site changes per year with a mean of 1.9 x 10^-8 site changes per year; we used the mean rate of silent substitution to calculate divergence times. Using the locus divergence between Adh-1 (K_s = 0.065) and Adh-2 (K_s = 0.117) we obtained an estimate of time of divergence between D. mojavensis and D. arizonae of 1.7 and 3.1 million years (MY; for Adh-1 and Adh-2, respectively). Additionally, we calculated the silent divergence between D. navojoa and D. mojavensis (0.158 for Adh-1 and 0.133 for Adh-2) and D. arizonae (0.177 for Adh-1 and 0.155 for Adh-2). We obtained similar estimates for the divergence time between D. navojoa and D. mojavensis (3.5–4.1 MY) and D. navojoa and D. arizonae (4.1–4.6 MY). This method of estimating divergence ignores intraspecific variation; therefore it estimates only the divergence times of the genes, not the species (see DISCUSSION).

We also estimated the age of the duplication that gave rise to Adh-1 and Adh-2. The neighbor-joining tree in Fig 3 should be analogous to the estimated K_s values since the tree was constructed using third position sites only. As shown in Fig 3, D. mojavensis and D. arizonae share the same Adh duplication. Common ancestry of the Adh duplication is not evident for D. navojoa, but this may be largely due to the sequence homogenizing effect of gene conversion. The silent divergence between the D. mojavensis paralogs (0.133) resembles that of the D. arizonae paralogs (0.165). Therefore we can date the duplication event to 3.5–4.4 MYA, close to the time of the D. navojoa and D. mojavensis/D. arizonae divergence.

View larger version (21K): In this window In a new window Download PPT slide   Figure 3. Neighbor-joining tree of the Adh paralogs. Only third base positions were used to create the tree. The D. hydei (Adh-2) sequence was obtained from GenBank (accession no. X58694).

Linkage disequilibrium and recombination:
The level of linkage disequilibrium was determined only for D. mojavensis, since sequences in complete linkage phase were collected for D. mojavensis. Even though the paralogs were only 3 kb apart, there was little disequilibrium between the paralogs (Fig 4) as determined by the chi-square test using the Bonferroni correction for multiple comparisons. Markedly different levels of linkage disequilibrium were observed within the paralogs. There was a large amount of disequilibrium in Adh-2, mostly centered on the first intron. The cause of the disequilibrium in intron 1 is the segregation of two intron haplotypes that differ at 16 sites. The low-frequency (0.23) haplotype class in D. mojavensis was found to be segregating at a high frequency (0.82) in D. arizonae.

View larger version (60K): In this window In a new window Download PPT slide   Figure 4. Linkage disequilibrium in the Adh cluster of D. mojavensis. Linkage disequilibrium was determined using the chi-square test. The significance level was adjusted using the Bonferroni (B) correction for multiple comparisons. The darkened boxes represents exons, while the lines separating the boxes are introns. The dashed box reflects comparisons between the paralogs.

We determined the level of recombination using two methods: solving for the estimator C (HUDSON 1987 ) and solving for the less biased estimator (HEY and WAKELEY 1997 ). Both estimators of recombination are best presented as the ratio of the estimator over . This method provides an estimate of the rate of recombination (c) over that of the mutation rate (µ). Using C, the ratio is 0.173, while it was considerably greater (1.203) using the less biased estimator . The low c/µ ratio estimated from C was partly due to the intron 1 structure. Removing the effect of the intron 1 haplotype polymorphism greatly increased the c/µ ratios (1.072 and 2.700, estimated from C and , respectively).

Distribution of variation:
The pockets of linkage disequilibrium produced a heterogeneous distribution of variation (Fig 5). We examined the distribution of variation across the region using MCDONALD's (1996) DNA Slider program. There is a significant nonrandom distribution of segregating sites at Adh-2, both in D. mojavensis (G_mean = 4.416, P = 0.045) and in D. arizonae (G_mean = 5.080, P = 0.024). This heterogeneous pattern of variation was not observed at Adh-1, either in D. mojavensis (G_mean = 0.274, P = 0.962) or in D. arizonae (G_mean = 3.218, P = 0.096).

View larger version (18K): In this window In a new window Download PPT slide   Figure 5. Sliding-window analysis of inter- and intraspecific variation in D. mojavensis Adh-2 and Adh-1. The total (synonymous, nonsynonymous, and intron) level of variation () was determined for a sliding-window size of 100 bp and a step size of 25. The net nucleotide divergence (Da) was determined using one D. arizonae sequence (ARTU 34).

Frequency and test of independence analysis:
Both the TAJIMA 1989 and the FU and LI 1993 tests examine the frequency distribution of the variants. Significant negative values of the test statistic indicate a greater proportion of low-frequency variants, usually explained as being a result of an adaptive sweep or as a population expansion, while positive values indicate a greater proportion of high-frequency variants, possibly a result of balancing selection or population admixture. In D. mojavensis Adh-2, both Tajima's (D_Tajima = -1.549) and Fu and Li's (D_Fu&Li = -1.139) statistic were negative but not significantly. A similar result was observed at Adh-1 (D_Tajima = -1.007 and D_Fu&Li = -1.365). In D. arizonae Tajima's statistic was not significant for Adh-2 and Adh-1 (D_Tajima = 0.733 and D_Tajima = -1.023, respectively). The FU and LI 1993 test could not be performed for D. arizonae given the nature of the data.

Since polymorphism can eventually lead to fixation at both silent and replacement sites, fixed and polymorphic variation should be correlated. This can be addressed by implementing a MCDONALD and KREITMAN 1991 test. We examined this correlation in our data by using a G-test or Fisher's exact test when appropriate. In addition to analyzing the combined locus correlation (both D. mojavensis and D. arizonae), we also analyzed the lineage-specific correlation (see Table 2 and Table 3). An overall pooling of the fixed differences between two species in a McDonald/Kreitman (MK) test will tend to mask the pattern of evolution within each species lineage. Therefore, a way to implement an MK test is to partition the fixed differences into each species lineage. For the analysis of lineage-specific evolution, we used the D. navojoa sequence to polarize the data. As seen in Table 2 the correlation is present for Adh-2 at both the total locus and the species level. Conversely, this correlation does not seem to hold up when examining Adh-1 (Table 3). By doing the lineage-specific analysis we can observe that the significant deviation found at Adh-1 (G = 7.21, P = 0.0072) is due to D. mojavensis only. Two of the fixed replacement sites found in D. mojavensis are adjacent to each other, so it could be considered as either one or two mutational events. Overall this does not affect the result of the analysis if we assume it to be two mutational events; the deviation is significant (P = 0.015) as well if we assume it to be one event (P = 0.035).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Population size differences:
The overall level of variation in both D. mojavensis and D. arizonae differs in similar proportion in the Adh paralogs. D. arizonae has a level of gene diversity 1.5–2.5 times higher than that of D. mojavensis (Table 1). Our calculation of diversity from segregating sites (HUDSON 1990 ) estimates (WATTERSON 1975 ), which equals 4Nµ where N is the effective population size and µ is the neutral mutation rate. Assuming that the neutral mutation rates between D. mojavensis and D. arizonae are not different, this suggests a difference in population size between the species, D. arizonae being larger. Using the silent mutation rate (4.5 x 10^-9 per base pair per generation) calculated from D. melanogaster Adh (HEY and WAKELEY 1997 ) and the overall for Adh-1 and Adh-2 (0.0249 and 0.0469, for D. mojavensis and D. arizonae, respectively) we estimated the effective population sizes in D. mojavensis and D. arizonae to be 1.4 x 10⁶ and 2.6 x 10⁶, respectively. We can also compare the variation at Adh in these two species with what is known in other Drosophila species. The level of variation in both paralogs of D. arizonae resembles that found for D. pseudoobscura (MORIYAMA and POWELL 1996 ). There is a difference in the level of variation between the D. mojavensis paralogs. The variation found at Adh-1 is similar to what is observed in D. melanogaster, while the variation observed in Adh-2 resembles D. simulans (MORIYAMA and POWELL 1996 ). Given the significant MK test on Adh-1 (Table 3), the Adh-2 comparison is most adequate because hitchhiking can sweep out variation leading to a lower estimate of N.

Age of duplication and speciation:
In the original sequence characterization of the D. mojavensis Adh cluster, ATKINSON et al. 1988 concluded that the duplication event that produced Adh-1 and Adh-2 occurred about 17.9 MYA. Our estimate of the age of the duplication is 4.5-fold lower. There are two reasons for this large discrepancy between the estimates. First, sequencing errors in the initial inference of the sequence created an elevated level of divergence between the paralogs. The divergence between the paralogs (K_s = 0.197) in ATKINSON et al. 1988 was 1.5 times greater than that observed in this study. Second and most important, the rate of silent substitution used to estimate the age of the duplication was much slower than the rate used in this study. That study used the mammalian rate of silent substitution that was used by BODMER and ASHBURNER 1984 in a study of Drosophila Adh in the D. melanogaster species group. In this study we used the mean rate of silent substitution calculated from Drosophila (MORIYAMA and GOJOBORI 1992 ). Even if we estimate the age of the duplication using the slowest rate reported, 1.1 x 10^-8 sites/year, the age of the duplication estimated for D. mojavensis and D. arizonae (6.1 and 7.5 MY, respectively) is still about three times younger than the ATKINSON et al. 1988 estimate. This second, older calculation of the age of duplication using the slowest rate reported is in accordance with the estimate by RUSSO et al. 1995 of 6.5 ± 0.90 MY.

The phylogenetic relationships of the species in the D. mojavensis cluster are supported by previous work on the cytological evolution of these species (HEED 1982 ; RUIZ et al. 1990 ). In all studies, D. navojoa was found to be the most basal species of the species group, while D. mojavensis is the most derived (HEED 1982 ; RUIZ et al. 1990 ). Our estimate of the divergence between the D. mojavensis and D. arizonae Adh loci (1.7 and 3.1 MY for Adh-1 and Adh-2, respectively) was lower than that previously reported (RUSSO et al. 1995 ). To obtain an estimate of the divergence of the species, intraspecific variation must be included in our analysis. HUDSON et al. 1987 provide a set of equations in which the time of divergence T (in units of 2N generations) can be calculated taking into account intraspecific variation. We calculated the time of divergence (T) of D. mojavensis from D. arizonae to be 4.48N_e generations ago. Assuming that D. mojavensis goes through 6 generations in 1 year and using the above estimate of N_e we can estimate that the split between the species occurred 10⁶ years ago, more recent than our estimate using K_s. Using the HKA program (J. Hey) we performed 10,000 coalescent simulations based on observed levels of variation to estimate the 95% confidence intervals of T. We calculate the 95% confidence interval of the divergence time to be 0.19 x 10⁶ to 3.4 x 10⁶ years. This recent time of divergence supports many of the behavioral, physiological, and morphological studies on these species (MARKOW 1981 ; KREBS and MARKOW 1989 ; MARKOW and TOOLSON 1990 ; MARKOW 1991 ; ETGES 1992 ; STENNETT and ETGES 1997 ; KNOWLES and MARKOW 2001 ). Evidence on the reproductive behavior and physiology of D. mojavensis and D. arizonae suggests a possible rapid divergence propelled, at least in part, by sexually antagonistic coevolution (KNOWLES and MARKOW 2001 ).

We find that a gene conversion event has occurred between intron 1 of Adh-2 and Adh-1. Gene conversion will tend to homogenize the variation between the paralogs, causing an underestimation of the divergence between paralogs. In addition, gene conversion and hybridization will cause an underestimation of the sequence divergence. Species hybridization, followed by gene conversion between the paralogs will tend to have an effect on both paralogs, not just one intron; unless recombination and selection removed all the sequence belonging to one species, maintaining only the hybrid intron sequence, this scenario is highly unlikely. Therefore to avoid erroneous results we used only exons, more specifically third base positions, for constructing the gene tree (Fig 3).

Adaptive protein evolution at Adh-1:
Overall we found a significant MK test at Adh-1. The significance of the test was due to an excess in replacement fixations (Table 3). This pattern of variation has been previously described to be indicative of selection for advantageous amino acid mutations (MCDONALD and KREITMAN 1991 ; EANES et al. 1993 ). We were also interested in determining if this pattern of evolution is reflected in both the D. mojavensis and the D. arizonae lineages. It appears that the excess in replacement fixations is largely associated with the D. mojavensis lineage (Table 3).

Two out of the three amino acid differences that occur in the D. mojavensis lineage are near a functionally important region of the molecule. Residue Val-236 fixed in D. mojavensis is next to Asp-237, a residue involved in the noncovalent interaction at the dimer surface (BENACH et al. 1998 , BENACH et al. 1999 ). Residue Leu-61 is adjacent to residue Tyr-62, which comprises the coenzyme binding zone (BENACH et al. 1998 , BENACH et al. 1999 ). Much is known about the kinetics and functional importance of the ADH Fast/Slow allozyme polymorphisms in D. melanogaster (GELFAND and MCDONALD 1980 ; CHAMBERS 1988 ; GEER et al. 1990 ; FRERIKSEN et al. 1994 ), even though it is unclear if selection is specifically acting on the allozyme polymorphism itself or a linked site (BERRY and KREITMAN 1993 ). In D. melanogaster, the Fast/Slow polymorphism is due to a threonine-to-lysine substitution that occurs at residue 192. That residue is equivalent to Thr-191 in the D. lebanonensis ADH, for which the crystal structure is known (BENACH et al. 1998 , BENACH et al. 1999 ). Threonine 191 acts as a gate on the active site once the NAD-alcohol ternary complex is formed. The adaptive significance, if any, of the Fast allele in D. melanogaster is still not fully understood. What is known is that the Fast allele possesses higher protein levels (LAURIE and STAM 1988 ) and therefore exhibits a higher activity (FLETCHER et al. 1978 ; SAVAKIS et al. 1986 ), has different kinetic properties (WINBERG et al. 1985 ), and segregates at greater frequencies at higher latitudes (OAKESHOTT et al. 1982 ).

Without a detailed examination of the differences in the kinetic characteristics between the D. mojavensis and D. arizonae ADH-1 enzyme it is difficult to examine the possible adaptive significance of the three amino acid residues fixed between the two lineages. Other sources of evidence may shed some light on the possible selective forces responsible for the rapid fixation of amino acids in the D. mojavensis lineage. A cactus host shift occurred during the divergence of D. mojavensis from D. arizonae; this host shift might be responsible for creating the selective force (HEED 1982 ; RUIZ and HEED 1988 ; RUIZ et al. 1990 ). ADH-1 is expressed in the larval stage of D. mojavensis (BATTERHAM et al. 1983 ), and it is known that different cactus hosts exhibit different mixtures of alcohols (VACEK 1979 ; FOGLEMAN 1982 ; HEED 1982 ; KIRCHER 1982 ). The larval stage of the fly is intimately associated with its host. Since Adh-1 is expressed in the larvae, the host shift could have been responsible for the apparent adaptive evolution of Adh-1.

Maintenance of intron structure in Adh-2:
Unlike the pattern of adaptive protein evolution observed at Adh-1, Adh-2 does not seem to have undergone any recent episode of strong directional or balancing selection. One aspect of the variation at Adh-2 that is striking is the heterogeneous distribution of the polymorphisms, more specifically, the fact that there is notable variation in intron 1, which is due to the segregation of two distinct intron haplotypes in both species.

One of the Adh-2 intron 1 haplotypes is identical to the Adh-1 intron 1 [we refer to this haplotype as LA1 (like Adh-1) and the other as LA2]. Intron 1 is 55 bp long, but the intron haplotype is only 34 bp long (from position 108 to 141, see Fig 1). We propose that the LA1 haplotype originated from a gene conversion event between Adh-1 and Adh-2. The Adh-2 intron sequence was replaced by that of Adh-1. In D. mojavensis the LA1 haplotype is found at high frequency, while in D. arizonae it segregates at a low frequency. The Adh-1 intron 1 of D. mojavensis and the Adh-1 intron 1 of D. arizonae are almost identical (Fig 2). Gene conversion is a common phenomenon in duplicated genes in Drosophila [e.g., Amylase (HICKEY et al. 1991 ; POPADIC and ANDERSON 1995 ), Attacin (LAZZARO and CLARK 2001 ), and hsp70 (BETTENCOURT and FEDER 2002 )], as well as in other taxa [e.g., opsins in primates (BOISSINOT et al. 1998 ), MHC in red-winged black birds (EDWARDS et al. 1998 ), and HLA in humans (TAKAHATA and SATTA 1998 )]. Gene conversion has been generally viewed as a force slowing or even preventing the diversification of duplicated loci (WALSH 1987 ). Conversely, under certain circumstances gene conversions could aid in the adaptive evolution of paralogs by transferring mutation en masse (HANSEN et al. 2000 ). Such models of the possible effect of gene conversion do not place much emphasis on the transition stage (i.e., polymorphic). In this study we have observed gene conversion alleles segregating in Adh-2 in both D. mojavensis and D. arizonae.

There are three explanations for the existence of the same intron polymorphism in both species. The simplest of all possible explanations is that the LA1 haplotype independently originated in both species. This would encompass two independent and precisely identical gene conversion events, one in D. mojavensis and one in D. arizonae. Although simple, this seems highly improbable. Another explanation is that there was a gene conversion event between Adh-1 and Adh-2 in one species and a recent introgression introduced the LA1 haplotype into the other species. In lab conditions the two species can be forced to hybridize, but at very low frequency (WASSERMAN and KOEPFER 1977 ). Several character differences can affect the frequency of copulation in these species, such as behavior (MARKOW 1981 ) and cuticle hydrocarbon composition (MARKOW and TOOLSON 1990 ). As well as prezygotic mechanisms, postzygotic mechanisms such as a reduction in offspring viability and fertility of species hybrids have been observed (RUIZ et al. 1990 ). Introgression will produce a genome-wide effect; therefore evidence of this should be visible in several regions of the genome. If introgression were the source of the polymorphism, our data would suggest that only the intron 1 of Adh-2 was introgressed and nothing else, and this seems highly unlikely. As can be observed by the neighbor-joining tree (Fig 3), all D. mojavensis sequences within each paralog coalesce to one another. Therefore, there is no strong evidence of hybridization from the sequence data, and from the known behavioral data this seem unlikely. Therefore, an introgression event between the species might not be the most likely explanation for the intron 1 polymorphism in Adh-2. The apparent shared polymorphism observed in exon 2 (sites 430 and 460, see Fig 1) of Adh-2 is due to a second gene conversion between Adh-1 that occurred only in the D. mojavensis lineage. This will further be described in future work.

Third, the polymorphism in both species may extend from an ancient gene conversion that predates the divergence of D. mojavensis and D. arizonae and has been maintained in both species. Selection needs not be implied when explaining the sharing of a polymorphism between two species. Assuming the two alleles were equally common in the ancestral population the expected mean time loss of the shared polymorphism is 1.7N generations (CLARK 1997 ). The distribution of time to loss has a long tail; 95% of the time the losses occur by 3.8N generations and 99% of the time by 5.3N generations (CLARK 1997 ). Our data suggest that the divergence between D. mojavensis and D. arizonae occurred 4.48N generations ago; therefore it is not very likely that the shared polymorphism has been neutrally maintained.

Additionally, if the shared polymorphism is neutrally maintained, then we would expect that in each species a considerable amount of variation would be segregating in the two intron haplotypes. To examine this we have split our intron 1 data into three classes per species: LA2 haplotype, LA1 haplotype, and all Adh-1 haplotypes. The level of diversity () observed for the LA2 haplotype in D. mojavensis and D. arizonae was 0.025 and 0.097, respectively. The variation observed for the Adh-1 intron in D. mojavensis and D. arizonae was lower, 0.006 and 0, respectively. Additionally, the level of variation within the LA1 haplotype was also lower (0.019 and 0, for D. mojavensis and D. arizonae, respectively). Although variation has accumulated in the LA2 haplotype, variation has not greatly accumulated in the LA1 haplotype; this same pattern of low variation is also present in intron 1 of Adh-1. Therefore it is possible that selection might be preventing the accumulation of variation at the Adh-1 intron and the homologous LA1 haplotype. The difference in diversity between the LA1 and LA2 might be hard to reconcile with the proposed age of haplotypes. However, the possibility exists that the converted allele has simply had historically a smaller effective population size.

The pattern of linkage disequilibrium across intron 1 is striking given the observed levels of overall recombination apparent in the D. mojavensis Adh cluster. If selection is not involved, then we would expect that recombination would have broken down the linkage disequilibrium. Given the value of (0.0134/bp) estimated for D. mojavensis we can obtain an estimate c (9.6 x 10^-9), the recombination rate per base per generation. Using the D. mojavensis and D. arizonae divergence of 4.48N or 6.3 x 10⁶ generations and the fact that the intron haplotype is 34 bp, we can estimate that about two recombination events should occur in a 34-bp span of sequence since the species split. The estimate of two recombination events should be taken to be a maximum only. The number of sampled alleles and the fact that a recombination event between homologous alleles cannot be observed could be an explanation for the lack of recombinant introns in our sample. Although this is a possibility, we believe that the expected estimate of recombination does not agree with the levels and distribution of disequilibrium observed. This suggests that there might be selection to maintain the intron haplotype structure in D. mojavensis and D. arizonae. Similar patterns of disequilibrium in introns have been observed in other Drosophila.

Independent of D. mojavensis and D. arizonae, D. pseudoobscura also possesses a duplication of the Adh locus (SCHAEFFER and AQUADRO 1987 ). In D. pseudoobscura, two significant clusters of linkage disequilibrium were found, both occurring within introns (SCHAEFFER and MILLER 1993 ). The adult intron, intron 1, and intron 2 all form pre-mRNA structures, but the two major haplotypes segregating in intron 2 form highly stable pre-mRNA stem-loop structures (KIRBY et al. 1995 ). Pre-mRNA structures in Adh have also been suggested in several Drosophila species (STEPHAN and KIRBY 1993 ). The model for the maintenance of pre-mRNA structure is based on the selection for compensatory mutations that stabilize RNA structure (STEPHAN 1996 ). The function of pre-mRNA structures has recently been linked to the regulation of expression (ANTEZANA and KREITMAN 1999 ; CARLINI et al. 2001 ). Loci of low expression have low levels of codon bias and a more even distribution of G/A/T/C in the third position. Such low-expression loci tend to have more stable pre-mRNA structures (CARLINI et al. 2001 ). Highly stable pre-mRNA structures produce pauses in the translation of proteins, which may aid in protein expression and folding (NETZER and HARTL 1997 ; NIEPEL et al. 1999 ).

In our survey of Adh-2, no intron 1 recombinants were observed in either species. Evidence suggests that the structure of intron 1, especially in Adh-1 and the LA1 haplotype, has been independently maintained in both species. Both the LA2 and the LA1 haplotypes form pre-mRNA stem-loop structures that are more stable (calculated as the change in free energy) than all the potential recombinants between the two haplotypes (data not shown). Therefore, selection might be responsible for maintaining the intron haplotype structure of Adh in D. mojavensis and D. arizonae as it has been observed to occur in other Drosophila. The regulation of expression and translation seems the most likely role for the Adh pre-mRNA stem-loop structures. Further analysis of this latter point is needed.

Subfunctionalization of Adh paralogs:
This study shows that the Adh paralogs of D. mojavensis have traveled different evolutionary paths. It is of fundamental interest to understand why their evolutionary paths have diverged. Duplication followed by amino acid fixations has been traditionally placed as the major force in the evolution of novel gene functions (OHNO 1970 ; OHTA 1974 , OHTA 1987 , OHTA 1988A , OHTA 1988B ). These models assume that the duplicated locus is bombarded by mutations, most of them being deleterious, until a beneficial combination producing a novel function is created and fixed. These models do not take into account the role of gene expression in the evolution of paralogs. If the original locus had several modes of expression, following fixation, specialization of each paralog could occur if they limit their expression to that of a subset of the original locus (HUGHES 1994 ). This model has been further developed by FORCE et al. 1999 in which they propose that following the fixation of the paralogs, mutations will occur in one of the regulatory regions of a paralog that will silence expression. Assuming the original locus had several modes of expression, the end product of subfunctionalization will be the existence of paralogs with no or nearly no overlapping expression. Loci being expressed in different tissues or at different times during development will experience different environmental pressures, which may lead to the specialization and divergent evolution between the paralogs. The Adh system provides an especially good fit to the model, given the fact that it is known that the duplication event that produced Adh-1 and Adh-2 was not complete: the distal promoter for Adh-2 was lost during the duplication (ATKINSON et al. 1988 ).

The efficacy of natural selection on an enzyme locus is highly dependent on the environment in which the enzymes are expressed (DYKHUIZEN and HARTL 1980 ; HARTL et al. 1985 ; DYKHUIZEN et al. 1987 ). In D. mojavensis and D. arizonae the external and internal/cellular environments experienced by each paralog differ. According to metabolic flux analysis, the relative role of enzyme activity to overall pathway flux is quantified as the control coefficient (FELL 1997 ). In D. melanogaster, the control coefficient for ADH recorded in adults is 0.022 (MIDDLETON and KACSER 1983 ), while that measured in larvae is 1.0 (FRERIKSEN et al. 1991 ). This implies that changes that affect ADH enzyme activity will have a much greater effect on flux in the larval stage than in the adult stage. The differences in the modes of expression of Adh-1 and Adh-2 have provided for the possibility of natural selection to aid in their divergence.

The study of duplicated loci, most specifically recently duplicated loci, offers a very powerful system for the study of evolution at a locus. Paralogs with nonoverlapping modes of expression highlight how changes in the environment experienced by an enzyme have the potential to drastically alter its evolution. The combination of knowledge of the ecology of the study organism, population genetics studies of duplicated metabolic enzymes, and studies of enzyme function will aid in the study of the fundamental question of maintenance of natural variation.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY154827–AY154876, AY156524, and AY156525.

	ACKNOWLEDGMENTS

The authors thank Therese Markow for assistance in the field and for providing the D. arizonae isofemale lines and Brian Verrelli and Thomas Merritt for comments and suggestions on earlier versions of the manuscript. This research was supported by U.S. Public Health Service grant GM-45247 to W.F.E. and by National Science Foundation Predoctoral Fellowship and W. Burghardt Turner Fellowship to L.M.M. This is contribution no. 1103 from the Graduate Program in Ecology and Evolution, State University of New York at Stony Brook.

Manuscript received August 9, 2002; Accepted for publication October 4, 2002.

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