Isoenzymatic polymorphisms in urban populations of Drosophila willistoni Polimorffsmo isoenzfmatico en poblaciones urbanas de Drosophila willistoni

Natural populations of Drosophila willistoni collected both in urban sites and in the wild, as control, were analysed with respect to their isoenzyme polymorphisms in six loci of enzyme systems, through horizontal electrophoresis. The first pool of populations was collected and analysed during the years 1987 and 1988, and the other during 1992 and 1993, in comparable seasons. The level of enzymatic polymorphism first detected was smaller in comparison to that observed four years later, suggesting an increase of variability along the time elapsed since the first sampling of this species, in the city of Porto Alegre. Our findings are suggestive of an increasing adjustment of the species to this new environment.


INTRODUCTION
Marginal populations present a unique opportunity for evolutionary studies, since it is under such conditions that genetic adaptation to extreme environments could more frequently be found.In the Genus Drosophila, the genetics of marginal populations has been the matter of several classical studies (Da Cunha & Dobzhansky 1954, Da Cunha et al. 1959, Carson 1955, 1959, Dobzhansky 1965).
Drosophila willistoni is a wild, widespread species, native to hot and humid Neotropical forests, occurring from Florida and Mexico, that corresponds to the Northern range of its distribution, to Argentina, in the South (Spassky et al. 1971, Cordeiro & Winge 1995).Although classified as a "wild" species (Dobzhansky 1965, Carson 1965), D. willistoni seems to be capable of exploring man-altered environments (Dobzhansky 1965), due to its wide genetic variability expressed through several genetic markers until now surveyed.These include enzyme variability, documented by Borba & Napp (1986) for samples from southern latitudes.This potentiality was confirmed by the finding of this fly in samples from urban places in the city of Porto Alegre of southern Brazil, by Valente & Araujo (1986) and by Gofii et al. (1997Gofii et al. ( , 1998) ) in urban and suburban places of the city of Montevideo, in Uruguay.The present report is an attempt to describe the level of genetic variability of these urban populations of D. willistoni.

MATERIAL AND METHODS
Samples of natural populations of Drosophila willistoni were collected flying around banana baits, or as they emerged from rotten fruits of native and exotic plants, in parks and squares of Porto Alegre city (30° 1 0' S, 51 o 06' W), as well as in a wild control site, Eldorado do Sul (30° 05' S, 51 o 30' W) 40 km distant.
Fermented fruits colonised by preadult forms of Drosophila were carried to the laboratory and individually placed in tubes with culture medium (Marques et al. 1966) until the emergence of adult flies, according to Brncic & Valente (1978).The emerged flies were aspirated and classified by inspection of their distinctive characteristics of external genitalia.Isofemale lines of D. willistoni were immediately established, with one F1larva per female dissected, processed according to Ash burner ( 1967) and analysed with respect to its chromosomal polymorphism (Valente et al. 1993).Following this routine, through the analysis of the polytene chromosomes, we also discarded the possibility of misidentifications, since the sibling species D. paulistorum is sympatric to D. willistoni in almost all our samples.After their emergence, the flies were aged for a week, homogenized, and subjected to horizontal electrophoresis assays.
Expected heterozygosities per locus (Ayala et al. 1972) were compared to observed heterozygosities by the d test of Bailey (1974).
Two sample pools were analysed: the first collected between 1987 and 1988, and the other collected between 1992 and 1993, in comparable seasons.

RESULTS AND DISCUSSION
Table 1 corresponds to the data on the variation found in the Mdh locus analysed in D. willistoni populations.It can be observed that urban populations tended to become more polymorphic with time, as seen in the heterozygosis levels that appeared to increase from the first samples (1987)(1988) to the later ones (1992)(1993).This also seems to be true for the number of alleles detected in this enzymatic system.The Odh-2 locus (Table 2) clearly showed the same tendency, especially with regard to the comparison of the heterozygosis values, which in the 1987-1988 samples were similar but increased considerably in the urban populations, sampled in 1992 and 1993.On the contrary, the Odh-1 locus (Table 3 ), only analysed in the years 1992 and 1993, showed inverse tendencies in the number of alleles, in these two samples.In 1992, the urban populations showed more alleles than the wild one, contrary to what occurred in samples of 1993.In both samplings, however, the heterozygosis was higher in the urban populations than in the wild ones.
The a-Gpdh system, whose functional role in the wing vibration is well known for several winged insects and is also important to the dispersion of coloniser populations, had its variability diminished between the first and the second period of sampling, as can be seen in Table 4.This was true both for the heterozygosity measurement and for the number of alleles detected.Our interpretation for these findings is that in an initial phase, when the populations were exploiting the new urban environment, heterozygote flies had been favoured, and several alleles had been "tested" in face of the new challenges found.Later, when the populations were already established, the number of possible combinations dropped, a certain tendency to allele fixation emerging.For instance, among the samples of two contiguous and comparable places, as those of 0. B. Viana street (1988) and of M. Cardoso square (1992), only one of the alleles initially found showed the tendency of being fixed.
Table 5 shows data of the acid phosphatase (Acph) locus, in which we also observed a tendency for increasing variability between the two periods of sampling, but in this case this phenomenon seems also to occur in wild populations.Some urban populations, however, as those of Reden ao Park, that in the first period presented few alleles and low heterozigosity, doubled the number of alleles and showed high heterozygosity in the later samples.The same was verified in the samples from 0. B. Viana (1987)(1988) and from P. M. Cardoso (1992).

TABLE2
Allelic frequencies and heterozigosity in the Odh-2 locus of urban and wild populations of

Drosophila willistoni
Frecuencias alelicas y heterozigosidad en el locus Odh-2 de poblaciones urbanas y silvestres de   Finally, the Amy system (Table 6), analysed in samples collected in 1993, showed a higher number of alleles and heterozigosity in the urban population in contrast to the wild one.
The conclusions obtained from this analysis of all enzymatic loci comparing urban and wild is valid for the main proportion of the enzymatic systems evaluated.Considering the short generation period of this species, and the time elapsed between the two sampling periods ( 1987 I 1988-1992/1993 ), we suggest that the increase of genetic variability could be the consequence of the success of D. willistoni in becoming established in the new urban environment.The same populations were analysed with respect to their chromosomal polymorphism for paracentric inversions (Valente et al. 1993) and the results clearly showed a significant loss of structural chromosomic variability in urban populations.This finding support the idea of homoselection operating in marginal populations (Carson 1955(Carson , 1959)).According to this hypothesis, an increase of recombination allowed by the loss of inversions could generate higher allelic variability.This seems to be the case for our populations.We think that this is the most probable explanation for our findings, given our studies on both urban fly populations for more than fifteen years (Valente & Araujo 1985, Bonorino &  Valente 1989, Santos & Valente 1990, Valente et  al. 1989, 1993, Bonorino et al. 1993, Regner &  Valente 1993, Rohde & Valente 1996, 1997;  Valiati & Valente 1996, 1997)

TABLE 3
Allelic frequencies and heterozigosity in the Odh-1 locus of urban and wild populations of , and of wild ones (Valente & Araujo 1991, Saavedra et al. 1995).