null Shamina

R. Parkash

Genetics Selection Evolution

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Original article
Latitudinal clines for alcohol
dehydrogenase allozymic variation
and ethanol tolerance
in Indian populations
of Drosophila ananassae
R Parkash Shamina
Maharshi Dayanand University, Department of Biosciences,
Rohtak, 124001, India
(Received 17 June 1993; accepted 18 November 1993)
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 AdhFallelic 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 north-
south axis of the Indian sub-continent.
Drosophila ananassae / Adh polymorphism / ethanol utilisation / larval behaviour 1
latitudinal cline
Résumé - Clines de latitude pour la variation allozymique de la déshydrogénase
alcoolique (Adh) et la tolérance à l’éthanol dans des populations indiennes de
Drosophila ananassae. Huit populations géographiques indiennes de D ananassae, récoltées
sur une étendue de 20 degrés de latitude nord, ont révélé une variation clinale significative
au locus Adh (déshydrogénase alcoolique), et une augmentation de la fréquence de l’allèle
AdhF d’environ 1,5 point de pourcentage par degré de latitude. Une augmentation de la
tolérance à l’éthanol avec la latitude (de 1,8 à 3,7%) a été observée chez les adultes.
* Correspondence and reprints: 446/23 (Near Park), DLF Colony, Rohtak, 124001,
Haryana, India.
Les études de survie sur adultes ont montré que, dans tous les cas, l’éthanol est utilisé
comme une ressource lorsqu’il est à de faibles concentrations, alors qu’il devient un
facteur de stress à des concentrations élevées. Le seuil entre ressource et stress se situait
entre 1,2 et 4% selon la latitude. Le comportement larvaire a aussi montré un seuil
d’attraction/évitement, augmentant de 1,6 à 4,4% d’éthanol avec l’augmentation de la
latitude d’origine. Le parallélisme observé entre les variations du locus Adh et la tolérance
à l’éthanol et son utilisation dans les populations naturelles de D ananassae en fonction de
la latitude pourrait être la conséquence d’une sélection naturelle équilibrante variant dans
l’espace le long de l’axe nord-sud du sous-continent indien.
Drosophila ananassae / polymorphisme de l’Adh / utilisation de l’éthanol / comporte-
ment larvaire / cline de latitude
INTRODUCTION
Colonising species populations offer the most suitable material for microevolution-
ary studies (Endler, 1977; 1986). Eight Drosophila species are known as truly cos-
mopolitan while 21 DrosoPhilia 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 exten-
sively studied in Indian natural populations of D ananassae (Singh, 1984a,b; 1989),
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 ex-
ploit 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, F1 and F2 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 LT50 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 LT50 ethanol/LTS 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 LT50 ethanol/LTSOcontrol 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.
RESULTS
Genetic basis of Adh polymorphism
The Adh enzyme in D ananassae revealed a single cathodal zone of activity. Segre-
gating 2-banded patterns (of either faster or slower mobilities) and 4-banded pat-
terns of Adh were observed in the individuals of D ananassae. Genetic crosses in-
volving different 2-banded patterns resulted in 4-banded patterns in F1 individuals,
and 1:2:1 ratio of segregating 2-banded and 4-banded patterns in the F2 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 homo-
zygous individuals exhibit a 2-banded pattern and the observed Adh electromorphs
correspond to post-translational or conformational isozymes. The present observa-
tions correspond to what has been known for Adh for a long time in D melanogaster
and other species.
Latitudinal Adh allozymic variation
The data on Adh allelic frequencies in 8 Indian populations are given in table I.
The Adh frequency increased significantly with increasing latitude (1.5% with 1°
latitude, r = 0.92). The Adh locus revealed significant interpopulation genotypic
heterogeneity (141.07) and allelic frequency heterogeneity (33.3) on the basis of
contingency chi-squared tests among the Indian populations. The data on Wright’s
fixation index (FST = 0.21) revealed significant genic divergence at the Adh locus in
Indian populations. Thus, the allelic frequency patterns at the Adh locus revealed
significant clinal variation (along the south-north axis) among Indian populations.
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
Indian population of Rameswaram had a longevity of 141 h compared with the
north Indian population of Saharanpur in which it was 175 h. However, the other
6 geographical populations revealed intermediate values (table II). The data on LT50
ethanol/LT50 control (which constitute the measure of resource versus stress) are
shown in figure 2. The adult ethanol threshold values were found to vary clinally
in the range of 1.2 to 4.0% among the 8 populations from south to north of the
Indian sub-continent (table II). Thus, ethanol concentration in the range of 3.4 to
4.0% served as a resource for north Indian populations while significantly lower
ethanol concentrations (1.2-2.8%) could be utilised by south Indian populations of
D ananassae.

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 LC50 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 LC50 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.
ACKNOWLEDGMENTS
Financial assistance from CSIR, New Delhi is gratefully acknowledged. We are grateful
to the reviewers for their helpful comments, JR David for his valuable guidance in the
present studies and M Weber for drawing the figures.
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Latitudinal clines for alcohol  dehydrogenase allozymic variation  and ethanol tolerance  in Indian populations  of Drosophila ananassae

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

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