Preliminary molecular variability among haplotypes of Saudi Arabian house sparrow <i>Passer domesticus</i>

doi:10.4236/oje.2011.13010

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Vol.1, No.3, 73-76 (2011)

http://dx.doi.org/10.4236/oje.2011.13010

Open Journal of Ecology

Preliminary molecular variability among haplotypes of

Saudi Arabian house sparrow Passer domesticus

Sayed A. M. Amer1,2*, Metwally M. Montaser1,3, Mohammed Shobrak1

1Faculty of Science, Taif University, Taif, Kingdom of Saudi Arabia; *Corresponding Author: [email protected]

2Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt;

3Department of Zoology, Faculty of Science, Al-Azhar Uni ver sity, Cairo, Egypt.

Received 22 September 2011; revised 20 October 2011; accepted 30 October 2011.

ABSTRACT

Genetic variability of Passer domesticus from

different localities of Saudi Arabia was investi-

gated. Six hundred and fifty nine nucleotides

were sequenced from the mitochondrial cyto-

chrome b gene. There was a slight difference

among the studied haplotypes and most substi-

tutions were synonymous. In some haplotypes

at the west of Arabian Peninsula (mountainous

habitat), two transitions only were non-synony-

mous. The data were used to construct the rela-

tionship of the Arabian house sparrow to its con

specific taxa from Europe and America. Neigh-

bor-joining (NJ), maximum-parsimony (MP) and

maximum-likelihood (ML) analytical methods

were used. The three methods showed cluster-

ing of the Arabian haplotypes in one group and

their sister relationship with the haplotypes fr-

om Netherlands and America. A spanish haplo-

type came basal to both groups. Collecting

more samples and more DNA data could be of

necessary to clearly address the genetic vari-

ability of this rapidly propagated species in Ara-

bia.

Keyw ords: Saudi Arabia; House Sparrow;

Mitochondrial DNA; Genetic Variability

1. INTRODUCTION

The house sparrow, Passer domesticus is distributed

in Europe, Nor t h Af ri ca and p a rts of Asia such as Middle

East, Indian subcontinent and a narrowing band from

northern Asia toward the Pacific coast. Its large-scale

spread includes North and South America, South Africa,

Australia and New Zealand [1-3].

The genetic diversity among the avian fauna is found

to be low, probab ly due largely to the greater mobility of

birds, resulting in higher levels of gene flow [4]. There

have been several studies of the genetics of house spar-

rows on species and population level [5-11]. Moreover,

the relationships among their congeneric species and to

other finches (Passeridae and New World sparrows) have

been tackled molecularly, for the first time, by Allende et

al. [12]. Recently, the genetic relationships among pas-

serines and other related families were studied using

mitochondrial and nuclear genome’s data [13]. Dor and

Lotem [14] have studied the parentage of the house

sparrows on a molecular basis.

Quantifying and characterizing avian genetic variabil-

ity should be a priority in conservation to evaluate the

effect of recent drastic population changes, to preserve

present-day diversity and, eventually, to provide guide-

lines for future conservation plans [15]. Despite its close

proximity to man, relatively little is known about the

connectivity between house sparrow populations. Do the

various sites that has been occupied by the species form

a continuous population, or are we dealing with a more

meta population type of population structure? These

Conservation information should take into account kn-

owledge on genetic population structure. Therefore the

aim of the present study was to collect samples of the

house sparrow from around the Kingdom of Saudi Ara-

bia in order to sequence an orthologous gene from each

of these samples. Such data could aid to understand the

relatedness of these haplotypes and to get an overall pic-

ture of the genetic variability of this passerine bird pro-

viding guidelines for its conservation.

2. MATERIALS AND METHODS

We collected 17 samples of P. domesticus from the

wild of 6 localities from Saudi Arabia (Arar, Qassim,

Tabuk, Al-Madina, Taif and Jazan). See Figure 1 that

explains the exact localities. Blood and tissue samples

have been numbered and labeled immediately in the lab

and preserved in –80˚C for further molecular studies.

S. A. M. Amer et al. / Open Journal of Ecology 1 (2011) 73-7 6

Figure 1. Map of the Arabian Peninsula showing the Saudi

Arabian localities from which the studied samples have been

collected.

DNA was extracted from 0.5 ml blood samples with QI-

AGEN spin-column kits according to the manufacturer ’s

instruction. Extracted DNA was spectrophotmetrically

quantified at 260/280 nm and was used for polymerase

chain reaction (PCR).

PCR was performed in 50 µl total volume of reaction

buffer containing 0.2 mM dNTPs, 1.5 mM MgCl2, 2 µl of

DNA solution and 0.25U of DNA Taq-polymerase (Invi-

trogen). 0.2 µM of each of the L14841 5’-AAAAAG-

CTTCCATCCAACATCTCAGCATGATGAAA-3’ and H

15767 5’-ATGAAGGGATGTTCTACTGGTTG-3’ as de-

tailed by Edwards et al. [16] were also added. The reac-

tion mixture was put into a 0.2 ml thin-walled PCR tube

and amplification was performed in PXE 0.5 thermal

cycler (Thermo Electron Corporation Co.) with the fol-

lowing profile: 94˚C for 5min followed by 30 cycles of

94˚C for 1min, 58˚C for 1min and 72˚C for 1min. A final

strand elongation at 72˚C was done for an additional

7min.

The resultant solutions were electrophoresed on a 1.5%

agarose gel in TAE (40 mM Tris, 40 mM acetic acid and

1 mM ethylenediamine-tetra acetic acid) and the gels

were stained with ethidium bromide. 100 bp DNA Lad-

der (Biolabs) was used as a marker for the molecular

weight size. The PCR products were then purified from

gel with the use of spin column according to the Kit

Manual.

Sequencing reactions were performed in a MJ Re-

search PTC-225 Peltier Thermal Cycler using a ABI

PRISM. BigDyeTM Terminator Cycle Sequencing Kits

with AmpliTaq-DNA polymerase (FS enzyme) (Applied

Biosystems) following the protocols supplied by the ma-

nufacturer were used. A single-pass sequencing was per-

formed on each template using the last mentioned PCR-

primers. The fluorescent-labeled fragments were purified

from the unincorporated terminators with an ethanol pre-

cipitation protocol. The samples were resuspended in

distilled water and subjected to electrophoresis in an ABI

3730xl sequencer (Applied Biosystems).

Nucleotide sequences of the mitochondrial cytb gene

(659 bp) were aligned with the same fragment for other

haplotypes from the DDBJ database (1 from USA, 1

from Netherlands, 1 from Spain, 1 P. flaveolus and 1

from P. luteu s). One additional outgrou p taxan (Petronia

petron ia) was included in the alignment in order to root

the tree. The alignment was carried out by using the

DNASIS 3.5 (Hitachi) and MacClade 4.03 (Sinauer As-

sociates, Inc.) with manual adjustments. We conducted

the tree analyses by neighbor-joining (NJ), maximum-

parsimony (MP) and maximum-likelihood (ML) meth-

ods. These analyses were done in PAUP* 4.0b10 [17] by

heuristic searches with the TBR branch swapping, 10

random taxon additions and 1000 bootstrap replications

for each method. Juckes-Cantor distance model was used

to construct a neighbor-joining tree [18].

3. RESULTS AND DIS CUSSION

Unambiguous 659 sites from cytb gene for 17 samples

of the house sparrow P. domesticus were sequenced in

this study. These data were deposited in DDBJ/EMBL

GenBank database with their accession numbers (AB-

671331-AB671347). The sequences were aligned and wer e

used for an al ysis. Thes e da ta show ed b ase fr equ encies of A

= 29.3%, C = 35.1%, G = 12.8% and T = 22.8%. O f th e s e

nucleotides, 558 were constant and 101 were variables.

Sixty seven of the variable sites were parsimony-unin-

formative and 34 were informative under parsimony cri-

terion. The estimated uncorrected pair wise distance

(Table 1) was zero among the different Saudi Arabian

haplotypes except those from Jazan, which showed very

low distance to other Arabian haplotypes (D = 0.002).

Jazan and other Arabian individuals showed close dis-

tance to both American and Netherlands haplotypes. A

Spanish haplotype was equally distant from other Ara-

bians, Netherlands and American individuals (D = 0.006)

and more distant from Jazan haplotype (D = 0.008). Fig-

ure 2 depicts an NJ tree that has been constructed using

the aligned sequences and Neighbor-joining algorithm

[18] with Juckes-Cantor. Similar topology was obtained

by maximum-parsimony under similar conditions. An

optimal ML tree was also found with similar topology

and a negative log likelihood of 1369.479. The three

analytical methods (NJ, MP and ML) showed the ho-

mogeneity of the house sparrows globally (bootstrap =

100, 100, 99, respectively) and clustering of the Arabian

S. A. M. Amer et al. / Open Journal of Ecology 1 (2011) 73-7 6

7575

Ta bl e 1. Uncorrected pair wise distances determined between

haplotypes from different localities. “Others” refer to all Saudi

Arabian haplotypes except those from Jazan since they showed

zero distance when they compared.

Other localities Jazan USA Netherlands

Other localities -

Jazan 0.002 -

USA 0.003 0.005 -

Netherlands 0.003 0.005 0.000 -

Spain 0.006 0.008 0.006 0.006

Jazan

Taif

Qassim

Tabuk

Al-Madina

USA [AY030117]

Netherlands [AY495393]

Spain [AF230906]

P. flaveolus [L77904]

P. luteus [AY495394]

Petronia petronia [AF230914]

0.005 substitutions/site

Arar

96, 92

100, 100, 99

88, 77, 64

63, 72, 63

69, 72, 73House s

arro

Saudi Arabia

Figure 2. Neighbor-joining tree constructed from 659 bp of

cytb gene among the different studied haplotypes. The boot-

strap values are showed at nodes for neighbor-joining, maxi-

mum-parsimony and maximum-likelihood methods, respec-

tively, when they are more than 50%.

house sparrow (from different localities) in one group

with the American and Netherlands sparrows (bootstrap -

ping = 69, 72 and 73, respectively). The Spain sample

came basal to both groups. This relationship was trust-

able because of the reasonably strong statistical support.

One cannot discriminate any intra-population variation

for this bird in Saudi Arabia.

Nucleotide substitutions are generally considered in

terms of transitions and transversions. The sequenced fr-

agment of cytb gene showed 24 substitutions among the

different haplotypes of which two were transversions.

Among these polymorphic changes, 22 were in the third

position, and 2 were in the first and second po sitions an d

therefore, 2 of these su bstitutions were non-synonymous

and the 22 were synonymous. One of the non synony-

mous changes was found in only one haplotype from Al-

Madina and involved a substitution of phenylalanine

with Sereine at T191 ➝ C191. The position of this ami-

no acid in the frame of cytb gene is at 64. The second

substitution was found in different haplotypes inhabiting

Qassim, Tabuk, Jazan and Taif and showed a transition

for the amino acid number 116 from valine to isoleucine

at A346 ➝ G346 (Figure 3). It is notable that the non

Figure 3. The aligned translated amino acids of the sequenced

fragment of cytb gene for different haplotypes. The underlined

letters are those amino acids with polymorphism. Note that this

alignment is for the portion exhibited polymorphism (from 59

to 116).

synonymous mutations were found in the samples inhab-

iting the localities at the vicinity of west Saudi Arabia,

which is characterized by mountainous habitat. These

habitats are acquired abundance of vegetations and cha-

racterized by different climate (temperature, rainfall and

humidity). Further study is necessary in which more

statistical packages for more samples and data should be

used to relate these chang es to ecology.

Due to their ability to fly, birds can move over large

geographical scales, and their populations are therefore

often spatially more homogeneous than in some other

taxonomic groups [4,19,20] . The genetic variabili ty wit hin

house sparrow populations in Saudi Arabia were in the

same category as previously estimated for other areas in

the world [9,11,21]. Genetic variability within its popu-

lations was very limited probably due to the connection

between them [1]. We found a weak differenttiation am-

ong some of the population s, mainly in tho se at the west-

ern edge of the Kingdom, but other areas showed no

significant popu lation difference (Table 1). A ccordingly,

we could not differentiate any type of clusters of indi-

viduals within the collected data. Exten siv e homogeneity

of populations was most likely due to populations are

better connected. We, therefore, concluded that Saudi

Arabian house sparrow need s collection of more samples

and DNA data and manipulates these data by up to data

statistical programs to address its genetic diversity.

4. ACKNOWLEDGEMENTS

This work has been done under the financial support of Taif Univer-

S. A. M. Amer et al. / Open Journal of Ecology 1 (2011) 73-7 6

sity of Kingdom of Saudi Arabia (Project number 1/432/1066).

OPEN ACCESS

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