A comparison of the level of enzyme polymorphism in cosmopolitan Drosophila species between populations collected in distilleries and in their surroundings in Hungary

for the Adh locus in D. hydei in Hungary. The number of species was lower in distilleries than outside. The heterozygosity level in samples from distilleries was generally lower than in samples from outside. This result gives support to the hypothesis that the more diverse the environment the higher the level of polymorphism maintained.


Introduction
Genetic differentiation within a species is a common response to environmental heterogeneity. Some of the existing field studies indicate association between the level of polymorphism at several enzyme loci and the geographical variation of different environmen-tal factors (Nevo, 1978;Triantaphyllidis et al., 1980;Oakeshott et al., 1982;Singh et al., 1982;Van Delden, 1982;Oakeshott et al., 1983;Nevo et al., 1984).
Many authors have studied microdifferentiation of Drosophila populations living in wine cellars and in the surroundings (McKenzie and Parsons, 1974;Briscoe et al., 1975;McKenzie and McKenzie, 1978;Parsons, 1980;McKenzie and McKenzie, 1983). Their main interest was the gene frequency distribution at the Adh locus in populations from the 2 types of micro-habitats. It would also be interesting, however, to study the difference in the genetic diversity of the 2 kinds of populations. In the case of laboratory populations, several observations have revealed differences in the average frequency of heterozygotes when Drosophilids were kept in homogeneous and heterogeneous environments (Powell, 1971;McDonald and Ayala, 1974;Hale and Birley, 1983).
This study provides data for a comparison of the level of polymorphism at 4 enzyme loci among village populations of Drosophila melanogaster and D. hydei, and those living in distilleries. We have found that the average frequency of heterozygotes is higher in the village populations at the investigated loci.

Materials and Methods
Drosophilids were collected in 3 large regions of Hungary: the Central Tisza region (region I), the Bereg plain (region II) and the Sajo and Hernad valley (region 111). Signs on the map (Fig. 1) show the distilleries where collection took place. Enzyme polymorphism was determined from 13 samples with high individual counts of both D. melanogaster and D. hydei (full circles). In order to obtain field populations we used normal maizesucrose media as baits in the farmyards of the villages close to these distilleries. Similarly to the fermenting mash in the distilleries, this bait attracted the flies so we were able to collect them easily in the surroundings. A glass suction tube was used for the collection in both micro-habitats.

Statistical procedures
Standard errors of heterozygosity were calculated on a Commodore 64 computer by means of the Number Cruncher 1 programme.
As the proportion of heterozygotes was close to zero for most of the investigated loci, we used the angular transformation of frequency data when the t-tests were calculated. A paired t test was performed on a Commodore 64 computer using the Number Cruncher 1 programme.

Results
The common species in distilleries were D. melanogaster and D. hydei. Some individuals of other species also appeared, such as D. immigrans, D. funebris and D. busckii. The bait in the villages, however, attracted more species: besides the 2 common ones, we collected quite large samples of D. immigrans in each location and some samples of D. funebris and D. busckii in region I. Other species such as D. repleta., D. obscura and D. subobscura were scarce (Table 1).
The distribution of allele frequencies at the investigated loci in D. melanogaster populations collected in distilleries and in villages using baits is shown in Table Ila and lib, res-pectively. At the Adh locus, almost all the populations were polymorphic; however, the frequency of the slow allele was rather low. This is in good agreement with the European frequency gradient (Oakeshott et al., 1982). The populations investigated were less polymorphic at the Odh than at the Adh locus. For the Mdh and a-Amy loci, we found that the frequencies of alternative alleles were also rather low. As the a-amylase enzyme is enco-ded by a duplicated locus we did not calculate allele frequencies, thus only the phenotype frequencies are presented in the tables (Doane et al., 1975;Singh et al., 1982). At the a-Gpdh locus the average frequencies of the slow allele were 0.291 for the populations originating from distilleries and 0.265 for those collected in villages. On the basis of the results of a x 2 test we concluded that all the populations at all the investigated loci were in Hardy-Weinberg equilibrium.
Drosophila hydei was the other cosmopolitan species in our study. As opposed to D . melanogaster, this species did not occur in large masses either in distilleries or on bait.
The allele frequency values at all the investigated loci in D. hydei populations collected in distilleries and in villages by baits are presented in Table Illa and Illb, respectively. The Adh locus is known to be monomorphic in populations of D. hydei in the United States (Batterham et al., 1984). In some of the collecting sites, however, we found 2 different rare alleles at this locus. Figure 2 shows the new genotypes. The F allele was the most common, and the rare alleles showed either faster or slower migration. These rare alleles appeared only in a few populations, mostly in region I. At the Mdh locus 3 alleles, i.e. 6 genotypes, appeared in Hungarian populations. Allele S * was found only in populations collected on baits, and the frequency of allele F was slightly higher in these populations. The a-Gpdh locus was actually monomorphic with rare alleles appearing mainly in region II. Similarly to the Adh, the a Amy locus had 2 rare alleles (Doane et al., 1975) that were mainly found in populations of region 111.

Discussion
We compared the level of polymorphism in populations originating from distilleries to those collected in villages in the case of both species. Some important dataas a basis of comparisonare presented in Table IV for D. melanogaster populations. All 3 of the parameters -proportion of polymorphic populations (frequency of rare alleles > 0.01), average number of alleles (each investigated allele taken into account) and average heterozygosity -indicate a higher level of polymorphism in the field as compared with the distillery populations at 4 of the investigated loci. In D. melanogaster the highly polymorphic a!pdh locus was, however, an exception.
In the case of D. hydei populations, Table V shows the most basic data for comparison. The 3 examined parameters show the level of polymorphism to be higher in village populations for 3 of the investigated loci. The only exception was the highly polymorphic Mdh locus.
As the average frequencies of heterozygotes have rather high standard errors, we tested the statistical significance of differences between populations originating from the 2 habitats, villages versus distilleries. Results of the t-test are shown in Table Vi. The dif-ferences approached significance or were significant at all the investigated loci except a-Gpdh in D. melanogaster and Mdh in D. hydei; i.e., genic diversity appears higher in the villages as compared with the distilleries.
It can be concluded that field populations had a higher level of enzyme polymorphism in comparison with those living in distilleries. This tendency clearly appears at those enzyme loci with a low heterozygosity level. A possible explanation for the situation is that both species develop in villages in more diverse resources, in fermenting windfalls, in rotting vegetables, in rubbish, etc. In distilleries, however, Drosophilids grow in a more uniform environment, on mash with rather high alcohol concentrations. It is interesting, however, that the highly polymorphic loci (D. melanogaster: a-Gpdh, D. hydei: Mdh) do not show such a difference. Environments in nature are usually heterogeneous in time and space -the environment of the population has a grain structure. A fine grain would make polymorphism less likely to be achieved, or would reduce the stability of polymorphism already attained (Levins and Macartur, 1966). With coarseness of grain, however, the population may maintain some choice of genotypes over the types of conditions available (Levins and Macartur, 1966;Gillespie and Langley, 1974;Taylor, 1975). Our results support the hypothesis that the more diverse the environment, the higher the level of polymorphism that can be maintained (Powell, 1971;McDonald and Ayala, 1974;Nevo et al., 1984).