Latitudinal clines for alcohol dehydrogenase allozymic variation and ethanol tolerance in Indian populations of Drosophila ananassae

Summary - Eight Indian geographical populations of D ananassae, collected along a 20°N latitudinal range, revealed significant clinal variation at the Adh (alcohol dehydrogenase) locus and Adh allelic frequency increased by about 1.5% with 1° latitude. Latitudinal increase of ethanol tolerance (1.8-3.7%) was observed in adults. Survival studies with adults showed that, in all cases, ethanol was used as a resource at low concentrations, while becoming a stress at higher concentrations. The resource/stress concentration threshold increased from 1.2 to 4% with latitude. Larval behaviour also exhibited an attraction/avoidance threshold, increasing from 1.6 to 4.4% ethanol with increasing latitude of origin. The parallel occurrence of latitudinal variation at the Adh locus and ethanol tolerance and utilisation in natural populations of D ananassae could be maintained by balancing the natural selection, which varies spatially along the northsouth axis of the Indian sub-continent. Drosophila ananassae / Adh polymorphism / ethanol utilisation / larval behaviour 1


INTRODUCTION
Colonising species populations offer the most suitable material for microevolutionary studies (Endler, 1977;. Eight Drosophila species are known as truly cosmopolitan while 21 Droso P hilia species have been designated as widespread (David and Tsacas, 1981). Studies on biogeography and evolutionary history, chromosomal and allozymic polymorphism, ecological, behavioural and quantitative traits were made in the colonising populations of D melanogaster but such studies are lacking for most of the successful colonising and widespread drosophilids (David and Tsacas, 1981; David and Capy, 1988). D ananassae constitutes one of the most successful colonising and domestic species of the Indian sub-continent and was first described by Doleschall (1958) from Indonesia. Chromosomal polymorphism has been extensively studied in Indian natural populations of D ananassae (Singh, 1984a,b;, but studies on characters such as enzyme polymorphism or physiological traits are totally lacking. To fill this gap, we have investigated Adh (alcohol dehydrogenase) polymorphism and ethanol tolerance in this species. D ananassae was found to exploit a variety of fermenting fruits in nature and larvae were observed physically immersed in fermented media. Since Adh is known to be involved in the utilisation and detoxification of exogenous alcohols, the present studies were made in order to analyse the extent of genic divergence at the Adh locus as well as ethanol tolerance in D ananassae populations from India.

MATERIALS AND METHODS
D ananassae, a member of the D melanogaster group in the Sophophora subgenus, is a successful colonising species throughout the Indian sub-continent. Isofemale lines were established from population samples of D ananassae from 8 Indian geographical sites (Rameswaram to Saharanpur; 9.17°N to 29.58°N, figure 1). Data on the number of isofemale lines, which were maintained for 5-6 generations in the laboratory, are given in table I. Homogenates of single individuals (one fly per isofemale line) were subjected to electrophoresis at 250 V and 25 mA at 4°C for 4 h. The gel slices were stained for the Adh gene-enzyme system by a standard staining procedure (Harris and Hopkinson, 1976). Genetic control of Adh banding patterns was interpreted from the segregation patterns of enzyme electromorphs of parents, F 1 and F 2 progeny of several single-pair matings.
The adult ethanol tolerance was assessed following the longevity test of Starmer et al (1977). In order to test ethanol utilisation, groups of 10 males or females, grown on killed yeast medium, were aged for 2 d on fresh food medium and then transferred to a set of 2 air-tight plastic vials which contained different ethanol concentrations (1-7%). All experiments were run in 5 replicates at 20°C and control experiments employed water in place of ethanol solution. Adult survivorship was monitored by daily observations of control and ethanol treatment experiments. The LT 50 values were calculated as the number of hours at which 50% of the flies had died and were estimated by linear interpolation. The ethanol resource utilisation values were represented by the ratio LT 50 ethanol/LT So control, ie if this ratio was > 1, ethanol was utilised as a resource, but if this value was < 1, it represented stress. The ethanol threshold concentration was obtained when LT 50 ethanol/LT SO control was equal to 1. The larval behaviour towards ethanol was analysed by following the method of Gelfand and McDonald (1983). The relative numbers of the larvae out of a total of 10 on the 2 sectors of agar Petri dishes (with and without ethanol) were noted after 20 min for each ethanol concentration. Five replicates were tested at each ethanol concentration at 20°C for each D ananassae population. The threshold values between attraction and avoidance after 20 min were then calculated.

Genetic basis of Adh polymorphism
The Adh enzyme in D ananassae revealed a single cathodal zone of activity. Segregating 2-banded patterns (of either faster or slower mobilities) and 4-banded patterns of Adh were observed in the individuals of D ananassae. Genetic crosses involving different 2-banded patterns resulted in 4-banded patterns in F 1 individuals, and 1:2:1 ratio of segregating 2-banded and 4-banded patterns in the F 2 progeny. Thus, Adh electrophoretic data of the parents and progeny of genetic crosses was found to be in agreement with a monogenic control of Adh patterns. The homozygous individuals exhibit a 2-banded pattern and the observed Adh electromorphs correspond to post-translational or conformational isozymes. The present observations correspond to what has been known for Adh for a long time in D melanogaster and other species. Ethanol utilisation by adults The D ananassae adults were analysed for their potential to utilise ethanol vapours in a closed system and the data from 8 geographical populations of D ananassae are given in figures 2 and 3. Adult longevity was found to increase in the range of 1 to 2% ethanol in south Indian populations while 1-4% ethanol revealed enhanced longevity in the north Indian populations (fig 2). The data revealed that the south

Adult ethanol tolerance
In 5 Indian populations of D ananassae that could utilise ethanol as a resource up to 1.5% longevities were compared at 1% ethanol and the data revealed interpopulational divergence (fig 4a). The toxic effects of ethanol concentrations were observed from mortality data on the 4th day of ethanol treatment of adults and LC 50 values revealed clinal variation from 1.8 to 3.7%, ie southern populations of D ananassae displayed significantly lower ethanol tolerance than the north Indian populations (fig 4b).
Larval behaviour The data on larval behaviour towards a range of concentrations of ethanol (1-6%) are represented in figure 5 and table II. The larval ethanol threshold values varied from 1.6% in the Rameswaram population to 4.4% in the Saharanpur population. The ranking order of populations is Saharanpur > Rohtak > Nagpur > Pune > Tirumala > Madras > Tiruchchirappalli > Rameswaram. The larval individuals of 8 populations of D ananassae revealed higher ethanol tolerance than those of adults but the pattern of clinal variation was found to be similar for both the adult and larval stages (table II). The ethanol indices in larval and adult individuals were found to vary latitudinally in all 8 populations of D ananassae (fig 5). The statistical correlations were found to be significantly higher among latitudinal variation versus larval and adult ethanol tolerance (table III). The Adh-F allelic frequency also revealed significant correlation with latitude. Thus, ethanol tolerance seems to be adaptively maintained by natural selection mechanisms.

DISCUSSION
The present data on clinal variation at the Adh locus in Indian populations of D ananassae further supported and validated the hypothesis that occurrence of latitudinal clines among geographical populations provides strong evidence of natural selection maintaining such clinal allozymic variation (Nagylaki, 1975;Oakeshott et al, 1982). The observed latitudinal variation in D ananassae concurred with other reports on the populations of D melanogaster, ie US populations (Marks et al, 1980;Van Delden, 1982); Australian populations (Oakeshott et al, 1982); and European and African populations (David et al, 1986). The observed data on D ananassae could be explained on the basis of the niche-width variation hypothesis, ie the amount of variation in a species was proportional to the niche-width. It has been argued that a species characterized by utilisation of diverse food resources and/or climatic adaptations should possess a significantly higher amount of genic divergence compared with narrow niche-width species (Parsons, 1983;Spiess, 1989).
The Indian geographical populations of D ananassae revealed significant genetic divergence in their potential to utilise ethanol. Adult longevity was found to increase significantly when ethanol increases from 1 to 2% for south Indian populations and from 1 to 4% for north Indian populations of D ananassae. The ethanol threshold values were found to vary clinally in the range of 1.2 to 4.0% in the case of adults and 1.6 to 4.4% for larvae in geographical populations of D ananassae from south to north localities. The LC 50 values revealed a clinal variation in the range of 1.8 to 3.7% ethanol, ie southern populations displayed lower ethanol tolerance than the northern populations. The ethanol tolerance threshold values in larval and adult individuals were found to vary latitudinally in different Indian populations of D ananassae. The present observations are in agreement with other reports on the evidence of action of natural selection at the Adh locus as well as for ethanol tolerance in some allopatric populations of D melanogaster (Hickey and Mclean, 1980). Thus, both these traits have adaptive significance and are maintained by natural selection mechanisms.