Iran J Sci Technol Trans Sci

DOI 10.1007/s40995-017-0267-0

RESEARCH PAPER

Population Dynamics and Genetic Homogeneity in Natural

Populations of Drosophila melanogaster from Faisalabad, Pakistan
Muhammad Kashif Zahoor1 • Farah Batool1 • Shabab Nasir1 • Bilal Rasool1 •

Farhat Jabeen1 • Sarwat Zahoor2 • Humara Naz Majeed3

Received: 29 January 2015 / Accepted: 22 August 2015

Ó Shiraz University 2017

Abstract Drosophila melanogaster has been considered caused an increase in population number and vice versa.
an important model species to explore the patterns of Ten RAPD primers were used for PCR. Four primers were
diversity in natural populations. The current study was further selected on the basis of their efficiency and repro-
conducted to study population dynamics and genetic vari- ducibility. The number of amplification products varied
ability, gene flow and rate of migration in Faisalabad. The from 4 to 10 with an average of 7.0 bands per primer.
samples were collected from 17 different collection sites Mostly, all the samples showed monomorphic bands except
and cultured in the laboratory. The data for temperature for few samples from the south of the city. The allele and
and humidity were also recorded. It was observed that a genotype frequencies were measured at each RAPD loci.
decrease in temperature and increase in relative humidity Average observed heterozygosity (Ht 0.1886) and the
genetic variation (GST 0.129) showed that D. melanogaster
natural populations had low level of genetic diversity. The
Electronic supplementary material The online version of this dendrogram based on genetic similarities revealed three
article (doi:10.1007/s40995-017-0267-0) contains supplementary
material, which is available to authorized users. main groups of D. melanogaster populations. This pattern
of genetic homogeneity due to low genetic variability in
& Muhammad Kashif Zahoor local populations was attributed to the gene flow with no
[email protected] genetic drift in D. melanogaster populations in Faisalabad.
Farah Batool
[email protected] Keywords Drosophila melanogaster  Population
Shabab Nasir dynamics  Genetic variability  Homogeneity  RAPD-
[email protected] DNA markers
Bilal Rasool
[email protected]
Farhat Jabeen 1 Introduction
[email protected]
Sarwat Zahoor The fruit fly; Drosophila melanogaster, commonly known
[email protected] as vinegar fly, has been used in biological and medical
Humara Naz Majeed research for about 100 years because of many reasons, i.e.,
[email protected] the abundance of offspring, a rich catalog of characters, a
1
Department of Zoology, Government College University,
short generation time and the availability of accessible and
Faisalabad, Pakistan molecular tools (St Johnston 2002; Zahoor 2011). It is
2 established as a well-known model organism for genetics
Centre of Agricultural Biochemistry and Biotechnology
(CABB), University of Agriculture, Faisalabad, Pakistan and molecular studies around the world (Glinka et al. 2003;
3 Mateus and Sene 2003; Vaulin et al. 2007).
Department Pharmaceutical and Biological Chemistry,
School of Pharmacy, University College London, London, Drosophila melanogaster appeared in equatorial Africa
UK about 2–3 million years ago (MYA). The colonization in

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Asia and Europe occurred approximately and particularly in Southeast Asia are scarce (Vaulin et al.
10,000–15,000 years ago and then it migrated to America 2007).
and Australia (Nunes et al. 2008). Previously, it was The study on the genetic structure of natural populations
thought that D. melanogaster formed a single panmictic of D. melanogaster has not yet been conducted in Pakistan.
population. However, with the expansion of genomic Thus, the current study was designed to estimate the pop-
information of this species, genetic variations were ulation dynamics and to analyze the D. melanogaster nat-
observed in various geographical populations (Singh and ural populations to demonstrate the genetic variations, gene
Rhomberg 1987a, b; Schlötterer et al. 2006; Sella et al. flow and rate of migration from Faisalabad, Punjab, using
2009; Willi and Hoffmann, 2009). The level of variability random amplified polymorphic DNA (RAPD) markers.
showed its patterns of genetic diversity and evolutionary
history (Begun and Aquadro 1993; Sella et al. 2009; David
and Capy 1988). 2 Materials and Methods
The genetic diversity in natural populations could be
due to systematic process as mutation, natural selection, 2.1 Sampling of D. melanogaster and Collection
gene flow, inbreeding or a dispersive process as genetic of Environmental Data
drift (Falconer and Mackay 1996). Natural selection
caused by environmental factors is possible because Faisalabad stands between longitude 73°74 E and latitude
several authors have already reported that Drosophila is 30°31.5 N, with an elevation of 184 m (604 ft) above sea
sensitive to environmental changes such as temperature level. The city covers an area of approximately 1230 km2
and humidity (David and Capy 1988; Moraes and Sene (470 mi2). Seventeen different sampling sites were selected
2002; Mateus and Sene 2003; Ferreira and Tidon 2005; during the spring and summer seasons (March–August
Moraes and Sene 2007; Matilda et al. 2012). Drosophila 2012) from different areas of Faisalabad (Fig. 1). Sampling
colonizes an immense diversity of environments because was conducted by using glass bottles, jars and plastic
of its cosmopolitan nature (Ashburner and Bergman boxes. Different types of foods were used, i.e., melon,
2005). Many studies conducted on the effect of envi- guava, banana, grapes, apricot, apple, pomegranate,
ronment have shown genetic variations in natural pop- strawberry, plums or mango for trapping D. melanogaster.
ulations of Drosophila (Willi and Hoffmann 2009; A few pieces of fruit were placed in bottles that were then
Lachaise et al. 1988; Galtier et al. 2000; Willi and covered with muslin cloth with a few small holes to pro-
Hoffmann 2009). vide entry for flies (Fig. 2). The temperature and humidity
It is known that the pattern of DNA variation, poly- were measured by a hygrometer (for each collection day,
morphism and divergence are specific for individual D. noted separately and averaged) (Attaullah et al. 2015). The
melanogaster genes (Vaulin et al. 2007). Thus, to examine collected flies were shifted to the laboratory and carefully
the variations in the genome, several DNA-based tech- identified on the basis of morphological characteristics
niques are used to explain the patterns of genetic diversity using a standard microscope. The presence of sex combs
(Faith et al. 2004; Vaulin et al. 2007; Zahoor et al. 2013). and primary claspers in D. melanogaster was used as a
The RAPD-PCR is used to verify the existence of species confirmatory character as previously reported (Bock 1971).
population that has arisen either through genetic selection
under different environmental conditions or as a result of 2.2 DNA Extraction
genetic drift (Ayres et al. 2002; Faith et al. 2004; Jain et al.
2010). The collected samples of D. melanogaster were cultured in
Despite the popularity of this species in genetics, the the laboratory. For each population, multiple isofemale
global pattern of variations in natural populations has not lines were allowed to establish in the laboratory for a
yet been comprehensively described (Nunes et al. 2008). maximum of five generations. The flies collected for DNA
Mostly, studies on genetic variations focused on the Afri- extraction were transferred to 70% ethanol and stored at
can, American and European regions as representative -80 °C for molecular studies. For DNA extraction, a batch
examples of ancestral and derived populations. The few of 10–15 flies were homogenized in 400 ll of TNE buffer
studies in East Asian populations showed great variation in (Tris–NaCl–EDTA), and then 100 ll of 20 lg/ll of Pro-
morphological and physiological characters among D. teinase K and 40 ll of 20% sodium dodecyl sulfate (SDS)
melanogaster natural populations. However, a genetic were added. The homogenates were incubated at 55 °C for
homogeneity with regard to the common cosmopolitan 1 h, and 300 ll of 5 M NaCl was added and vortexed. The
inversions was reported from Southeast Asian populations mixture was centrifuged at 15,000 rpm for 10 min and the
(Glinka et al. 2005). However, overall the data on genetic supernatant was shifted to a separate Eppendorf tube. DNA
variation in natural populations of D. melanogaster in Asia was precipitated by adding 300–400 ll isopropanol or ice-

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Fig. 1 The collection sites for

Drosophila melanogaster from
Faisalabad. 1 Faisalabad
Airport, 2 Ayub Agriculture
Research Institute (AARI), 3
Sadhar, 4 Dijkot Road, 5 Canal
Road, 6 Peoples Colony, 7 Civil
lines, 8 Horticulture Garden, 9
FDA City, 10 Ghulam
Muhammad Abad (GM Abad),
11 Kalim Shaheed (Park), 12
Tariq Road, 13 University of
Agriculture Faisalabad (UAF),
14 Police lines, 15 Saman Abad,
16 Railway Station, 17
Government College University
Faisalabad (GCUF)

Fig. 2 Sampling of Drosophila melanogaster using fruits placed in University of Agriculture Faisalabad (UAF). c Bottle with fruit pieces
box/bottle. a Box containing guava and banana pieces placed on a placed inside Government College University Faisalabad (GCUF)
guava tree. b Bottle containing banana placed at Murva tree

cold 100% ethanol, kept at -21 °C for 1 h and then cen- (NH4)2SO4, MgCl2, dNTPs, 10-mer random primer and Taq
trifuged at 15,000 rpm for 10 min. The DNA pellet was DNA polymerase was optimized. The 10-base oligonu-
washed with chilled 70% ethanol, air-dried and re-sus- cleotide was obtained from Genelink Company (GL-C, D &
pended in 50 ll of sterile water (d3H2O). The DNA K-Series; Supplier: The Worldwide Scientific, Lahore) and
quantity was measured using UV spectrophotometer (U- used for the amplification of genomic DNA. Each PCR
2800, Hitachi). The optical density value (OD) of each reaction was carried out in a final volume of 25 ll containing
sample was calculated by measuring the absorbance at 260 approximately 100 ng of genomic DNA, 3 mM of MgCl2,
and 280 nm, respectively, and the concentration of total 20 pmol of primer, 2.5 ll buffer, 1.0 units of Taq DNA
genomic DNA was calculated accordingly. For the polymerase and 2.5 mM of each dNTPs. The PCR reaction
assessment of DNA quality, all the samples were run on was carried out using Personal Autorisierter Mastercycler of
1% agarose gel prepared in 0.59 TAE buffer and visual- Eppendorf, Germany. The PCR program comprised 35
ized under UV light (Farah 2012; Ashraf 2013; Zahoor cycles with initial denaturing of DNA at 95 °C for 5 min,
et al. 2013; Bibi et al. 2015). denaturation at 95 °C for 1 min, primer annealing at 37 °C
for 2 min, extension at 72 °C for 2 min, final extension at
2.3 RAPD-PCR Analysis 72 °C for 10 min and then holding at 4 °C until the tubes
were removed. The PCR products were analyzed on
For PCR of random amplified polymorphic DNA analysis, 1.5–1.8% agarose gel at 80 V for about 1 h (Farah 2012;
the concentration of genomic DNA, 109 PCR buffer with Ashraf 2013; Zahoor et al. 2013, Bibi et al. 2015).

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2.4 Statistical Analysis of Data

The fingerprints were examined under ultraviolet transil-

luminator and photographed using gel documentation sys-
tem (WEALTEC, Dolphin-DOC). The possibility of
contamination was checked using negative controls. The
negative control sample included all reaction materials
except template DNA. Amplified bands were then scored
by the presence (1) or absence (0) for all samples with all
primers, starting from the top of the lane to the bottom. The
ambiguous bands which could not be evidently discrimi-
nated were not counted. The RAPD markers were analyzed
using the method as described by Ayres et al. 2002. A
dendrogram was constructed using unweighted pair-group Fig. 4 Variation in relative humidity affecting the population of D.
mean analysis (UPGMA) (Nei 1978). Effective migration melanogaster. High relative humidity (March, July–August; C70%)
rates (Nm) were estimated based on inbreeding indices caused an increase in population number. A decrease in relative
(GST) (McDermott and McDonald 1993). Calculations humidity also caused a decrease in population (May and June;\60%)
were performed with the help of POPGENE version 1.32
software. caused a decrease in population. In March and April, the
temperature was low (±30 °C) and the population was
high, while with the rise in temperature in April, May and
3 Results June the population density started decreasing (C35 °C).
Then again in July and August when the temperature was
3.1 Population Dynamics with Reference decreasing (\35 °C), an increase was observed (Fig. 3).
to Environmental Conditions Figure 4 shows a variation in relative humidity affecting
the population of D. melanogaster. In March and April, the
The meteorological data for 6 months were recorded population size was higher, but as humidity was low in
simultaneously to check the environmental impact on May and June (\60%), the population also decreased. With
population dynamics of D. melanogaster (temperature, an increase in the humidity level during July and August
relative humidity). It was noted that low temperature (C70%), the population number of D. melanogaster
(March, April, August; Fig. 3) and high relative humidity increased. Thus, the present results showed that variation in
(March, August; Fig. 4) caused an increase in population temperature and humidity affected the population number
number. Moreover, higher temperature caused decrease in of D. melanogaster.
population. Subsequently, low relative humidity also
3.2 RAPD-PCR Analysis

Seventeen population samples of D. melanogaster were

analyzed by RAPD-PCR using ten oligonucleotide pri-
mers (Genelink; Supplementary Table). Four primers
were further selected which produced distinct, easily
detectable bands of variable intensities (Fig. 5; Table 1).
Indistinct bands produced by non-specific amplification
were ignored. The bands reproducible over repeated runs
with sufficient intensity to detect the presence or absence
with confidence were used for fingerprinting. The number
of amplification products produced per primer varied from
4 to 10 and ranged from 210 to 2500 bp size with an
average of 7.0 bands per primer (Table 1). The highest
number of loci (10) was obtained with primer C-04, while
Fig. 3 Variation of temperature affecting the population of D. the lowest number (1) was obtained with primer K-06
melanogaster. Lower temperature (March, April, August; ±30 °C) (data not shown). The level of 17 population samples
caused an increase in population number, whereas an increase in produced monomorphism with very few polymorphic loci.
temperature showed decrease in population (April, May and June;
C35 °C) The amplification of monomorphic loci depicts sharing of

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Fig. 5 Amplification profile of

Drosophila melanogaster
populations with primer K-09
(upper panel) and C-04 (lower
panel) by RAPD-PCR from
Faisalabad. Marker: 1 Kb ladder
A Government College
University Faisalabad (GCUF),
B Civil lines, C Tariq Road,
D horticulture garden,
E University of Agriculture
Faisalabad (UAF), F Peoples
Colony, G Canal Road,
H Railway Station, I Police
lines, J Kalim Shaheed (Park),
K Ghulam Muhammad Abad
(GM Abad), L FDA City,
M Dijkot Road, N Saman Abad,
O Ayub Agriculture Research
Institute (AARI), P Sadhar,
Q Faisalabad Airport

Table 1 Primers used in RAPD

Sr. no. Primers Nucleotide sequence Band size (bp) Fragments no.
analysis showing size and
number of amplified fragments 1 C-01 50 -TTCGAGCCAG-30 240–1500 9
obtained (no.) from specimens
of 17 Drosophila melanogaster 2 C-04 50 -CCGCATCTAC -30 200–2500 10
populations from Faisalabad 3 C-06 50 -GAACGGACTC-30 375–1200 7
4 K-09 50 -CCCTACCGAC-30 210–1300 4

common gene pool among different populations. Figure 5 Table 2 Summary of genetics data for D. melanogaster through
shows the amplification profile of all the samples with RAPD markers from Faisalabad
primers K-09 and C-04. The population samples from the Sr. no. Parameters Value
main city showed monomorphic bands compared to the
populations at the border of the city (Fig. 5; Lanes A–L 1 Total no. of loci 30
and Lanes M–Q). The percentage of polymorphism for 2 Average loci 7.0
each population ranged from 23.0 at the Government 3 GST (genetic variation) 0.129
College University site to 46.3 at the FDA city as shown 4 Nm (no. of migrants) 5.69
in Table 3. A low level of polymorphism, high level of 5 Total Ht (heterozygosity) 0.1886
similarity and low level of dissimilarity (data not shown) The genetic data analyzed through POPGENE showed high number
indicated very low level of divergence in D. melanogaster of migrants (Nm) (Nm C1.00 shows high migration rate) of D. me-
populations, suggesting a homogeneity pattern in D. lanogaster flies, which illustrates that the D. melanogaster popula-
tions exhibit gene flow among natural populations. In addition, the
melanogaster populations from Faisalabad. The genetic
low heterozygosity value (Ht) shows low level of dissimilarity; the
distance through Nei’s analysis shows a measure of low genetic variation (GST) value shows low level of variation among
divergence between species and populations of the same different populations
species. The gene diversity (heterozygosity) for D. mel-
anogaster populations from Faisalabad ranged from 0.121
to 0.254 (Table 3). The total average heterozygosity (Ht) 3.3 Cluster Analysis
was 0.1886. The overall genetic variation among 17
populations showed GST = 0.129 with a high rate of The UPGMA dendrogram based on the similarity matrix
migration (Nm = 5.69) (Table 2). of Nei’s genetic distances (Nei 1978) demonstrates three

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groups from Faisalabad populations (Fig. 6). One group city shows genetic interaction to GM Abad from the
(A) shows six populations and three subgroups, i.e., one north-west and Kalim Shaheed from the western area of
subgroup comprised Sadhar and Faisalabad Airport; one the city. In addition, Civil lines and Horticulture garden
subgroup of Ayub Agriculture Research Institute (AARI); area from the main city was showed relatedness to FDA
and one subgroup of Dijkot Road from the southern from the north site. The interaction between main city and
boundary of main Faisalabad City. All the collected the west-north sites shows that D. melanogaster has
samples (sites) showed a monomorphic banding pattern homogeneity pattern of genetic diversity. The group C
with each other, except three samples which were col- also shows some interesting patterns of genetic similarity,
lected from the boundary of the south-west of Faisalabad i.e., subgroup comprising UAF, Police lines and Railway
City. The RAPD primer (C-04) produced fragments that station from the main city was genetically more similar to
can clearly differentiate the populations from both main those of Saman Abad, the southern area of the city,
city and the southern area of Faisalabad (Fig. 5). Two respectively (Figs. 1, 5, 6; Table 3). The genetic simi-
populations of the third subgroup showed genetic simi- larity could be due to the transportation and the dispersal
larity to these subgroups in group A (Figs. 1, 6; Table 3). of D. melanogaster in these areas. The relatedness
Thus, the populations of the east and south-east of the between certain populations in Faisalabad populations and
city show genetic similarity (Group A). This relatedness the Nm (gene flow) value for an estimate of gene flow
among different populations shows that Drosophila pop- shows that the D. melanogaster populations exhibit gene
ulations are dispersing or migrating among these sites flow among natural populations. Moreover, a homogene-
along with a certain level of gene flow (Nm and GST; ity pattern was observed in many population samples.
Table 2). The group B also consists of six populations Thus, the monomorphic bands indicated low divergence
and two subgroups, i.e., Tariq Road area from the main between the sites and more gene flow among all the

Fig. 6 Dendrogram based on Nei’s genetic distances among and Canal Road from the eastern part to the southern part of the city.
Drosophila melanogaster populations from Faisalabad. The dendro- Group B consists of six populations and two subgroups, i.e., Tariq
gram demonstrates three groups from Faisalabad: Group A depicts six Road from the main city closely related to GM Abad from north-west
populations with three subgroups; subgroup comprising Sadhar and and Kalim Shaheed from the western area of the city. Civil lines and
Faisalabad Airport; one subgroup of Ayub Agriculture Research Horticulture Garden area from the main city related to FDA from the
Institute (AARI) and Dijkot Road from the southern boundary of main northern site. Group C comprising UAF, Police lines and Railway
Faisalabad City. Two populations of the third subgroup show Station from the main city shows genetic similarity to Saman Abad,
interesting genetic similarity pattern in group A from Peoples Colony the southern area of the city

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Table 3 Origin of 17 D.
S. no. Locality Polymorphism (%) Heterozygosity
melanogaster populations and
genetic variability indices 1 Faisalabad Airport 34.5 0.214
(polymorphism and gene
diversity) based on random 2 Ayub Agriculture Research Institute (AARI) 37.2 0.151
amplified polymorphic DNA 3 Sadhar 45.1 0.194
analysis 4 Dijkot Road 43.0 0.215
5 Canal Road 38.1 0.206
6 Peoples Colony 24.5 0.179
7 Civil Lines 27.2 0.197
8 Horticulture Garden 25.1 0.204
9 FDA City 46.3 0.179
10 Ghulam Muhammad Abad (GM Abad) 23.0 0.236
11 Kalim Shaheed (Park) 44.5 0.149
12 Tariq Road 27.2 0.144
13 University of Agriculture Faisalabad (UAF) 25.1 0.121
14 Police lines 24.5 0.213
15 Saman Abad 37.2 0.189
16 Railway Station 35.1 0.254
17 Government College University Faisalabad (GCUF) 23.0 0.162
N Individuals collected from each sampling site; percentage of polymorphism and heterozygosity revealed
through POPGENE software. Low level of polymorphism shows more genetic similarity which is con-
sistent with the heterozygosity values for each population of Drosophila melanogaster. Heterozygosity
value (Ht) approaching 1.00 shows maximum genetic dissimilarity among different populations, meaning
high genetic variations

populations in Faisalabad City (low polymorphism, However, D. melanogaster populations in Southeast Asia
Table 3). show genetic homogeneity which is maintained through
gene flow (Glinka et al. 2005). In parallel, an increase or
decrease in temperature causes a change in the metabolism
4 Discussion rate of Drosophila and ultimately causes a population
fluctuation. Subsequent studies with different insect popu-
Drosophila melanogaster has been considered to extend its lations showed an increase with high humidity levels and
range from tropical Africa to Asia 10,000–15,000 years lower temperature range (C25 °C) (Chernaki and Almeida
ago (Glinka et al. 2005). Due to the course of colonization 2001; Zahoor et al. 2003; Cooper et al. 2012). A combi-
events, the genetic composition of these flies has been nation of different features regarding a given environment
affected by both demographic and selective processes. The has also been recently observed in various studies (Dreyer
genetic diversity among populations can also arise sys- and Shingleton 2011; Rezaei 2012; Zahoor et al, 2013;
tematically due to exposure to different environmental Attaullah et al. 2015). Thus, keeping in mind the popula-
conditions. The differences in different populations of a tion dynamics, genetic variability and homogeneity pattern
particular species show divergence from one another in in D. melanogaster populations from different regions, the
their genetic composition (Glinka et al. 2003; Sella et al. current study was designed to estimate the genetic varia-
2009; Willi and Hoffmann 2009). It is reported that the tions, gene flow and rate of migration in D. melanogaster
genetic profile of natural populations typically varies from populations from Faisalabad. In the present study, it was
area to area. This variability may arise due to dispersal and observed that population of D. melanogaster increased
migration which could create a new population or changes with higher humidity and decreased with higher tempera-
in allele frequencies that result from chance mating in ture. The pattern of rise and fall of population showed a
small populations (genetic drift) (Primack and Hyesoon unique pattern which is true for other insect species (Za-
1989; Templeton 1991). hoor et al. 2003).
It has been reported that Asian populations of D. mel- The genetic distance through Nei’s analysis shows a
anogaster showed variation in both morphological and measure of divergence between species and populations of
physiological characters. These variations are said to be the same species (Zahoor et al. 2013; Bibi et al. 2015). The
maintained through genetic drift (David et al. 1976). heterozygosity for D. melanogaster populations from

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