Molecular classification of Pakistani collared dove through DNAbarcoding

Ali Raza Awan • Emma Umar • Muhammad Zia ul Haq •

Sehrish Firyal

Received: 15 February 2013 / Accepted: 14 September 2013 / Published online: 26 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Pakistan is bestowed by a diversified array of

wild bird species including collared doves of which the

taxonomy has been least studied and reported. DNA bar-

coding is a geno-taxonomic tool that has been used for

characterization of bird species using mitochondrial cyto-

chrome c oxidase I gene (COI). This study aimed to

identify taxonomic order of Pakistani collared dove using

DNA barcoding. Purposely herein, we present a phyloge-

netic analysis of Pakistani collared dove based on 650 base

pairs of COI gene sequences. Analysis of phylogenetic tree

revealed that Pakistani collared dove shared a common

clade with Eurasian collared dove (Streptopelia decaocto)

and African collared dove (Streptopelia roseogrisea) which

indicated a super-species group in Streptopelia genus. This

is the first report of molecular classification of Pakistani

collared dove using DNA barcoding.

Keywords: Dove � Pakistan � Phylogenetic �Molecular � DNA barcoding � Cytochrome c oxidase I

(COI)

Introduction

Pakistan is bestowed by a diversified array of bird spe-

cies including collared doves. The Pakistani collared

dove has been least studied especially no genetic

data is yet available. It is a medium-sized bird having a

body drab brown in color with white patches in the outer

tail feathers, narrow half black ring edged with white

around the back of the neck and reddish pink legs and

feet.

To comprehend natural biodiversity and its interaction

with the human society, identification of species has been

considered as an imperative task. Phenotypic plasticity

and genetic variability in species may lead to incorrect

taxon recognition. Sometimes morphologically cryptic

taxa common in many groups are overlooked by this

approach [1]. In biological studies accurate species iden-

tification is very important. Biological properties of

mitochondrial (mt) DNA make it suitable marker for

studying molecular biodiversity. Being maternally inher-

ited, usually it does not undergo recombination, and can

be said to present itself as a whole unchanging genome

and it evolves in a neutral pattern. The evolutionary rate

of mtDNA behaves like a clock, which is in the absence

of mutations spreading through natural selection, accu-

mulates slight differences in time. Knowing mt diver-

gence level, divergence time can also be estimated [2, 3].

mt genes are among the major sources of data for evo-

lutionary studies in birds. Indeed, complete mt genomes

are actively been used to unveil the phylogenetic rela-

tionships among major orders, whereas single genes

especially Cytochrome c oxidase I (COI) are considered

standard for species delimitation and identification (DNA

barcoding) [4, 5]. DNA barcoding using COI gene has

been proved to be an efficient method for species iden-

tification of birds and other animals [5–10].

Hence the present study aimed the taxonomic identifi-

cation of Pakistani collared dove using COI gene poly-

morphism (DNA barcoding) and its phylogeny.

A. R. Awan (&) � E. Umar � M. Zia ul Haq � S. Firyal

Institute of Biochemistry & Biotechnology, University of

Veterinary and Animal Sciences, Civil Lines, Outfall Road,

Lahore, Pakistan

e-mail: arawan77@uvas.edu.pk

S. Firyal

e-mail: sehrishfiryal@uvas.edu.pk

123

Mol Biol Rep (2013) 40:6329–6331

DOI 10.1007/s11033-013-2747-4

Materials and methods

To investigate the phylogenetic classification of Pakistani

collared dove, blood samples (50 lL) of ten Pakistani col-

lared dove were collected from different regions of Lahore

city of Pakistan. The samples were randomly named from

PKD1 to PKD10. Genomic DNA was extracted from each

blood sample using standard phenol–chloroform extraction

method [11]. Reported primers (BirdF1: TTCTCCAACC

ACAAAGACATTGGCAC BirdR1: ACGTGGGAGATAA

TTCCAAATCCTG) were used for amplification of 650 base

pairs (bp) of COI gene sequences [5]. Purified amplicons

were sequenced with both forward and reverse primers using

BigDye terminator cycle sequencing kit (Applied Biosys-

tems, USA) on ABI 3130XL Genetic Analyzer. The eluci-

dated sequences were submitted to GenBank for public

record. Multiple sequence alignments were performed with

NCBI BLAST freeware (http://blast.ncbi.nlm.nih.gov/Blast.

cgi). Using Fast Minimum Evolution algorithm of NCBI

BLAST a phylogenetic tree was constructed [12] in which

the COI gene sequences of the Pakistani collared dove was

compared with all available sequences of doves to

investigate the taxonomy and phylogeny of the Pakistani

collared dove.

Results and discussion

In this study, the COI gene of Pakistani collared dove was

sequenced. The gene sequences were submitted to NCBI

GenBank (Accession No. KC182062 to KC182071). The

homology analysis of the COI gene sequences and the

reference sequence of closest species; the Eurasian collared

dove (Streptopelia decaocto; accession no. HQ168040)

revealed 5 single nucleotide polymorphisms (SNPs) in the

COI gene sequences at different sites. The observed poly-

morphisms were C511T in PKD 1 and PKD 9, G473A in

PKD2, A226G in PKD7 and T448C in PKD 10. Using Fast

Minimum Evolution algorithm, a phylogenetic tree was

constructed in which the COI gene sequences of the

Pakistani collared dove was compared with all available

sequences of doves. All samples of Pakistani collared dove

shared a single clade and found to be the same species that

is S. decaocto). Sequences of the Collared dove showed a

Fig. 1 The phylogeny based on the Cytochrome c oxidase I gene sequences of doves indicating the phylogenetic and molecular classification of

the Pakistani collared dove

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very close relationship with Streptopelia roseogrisea

(African collared dove) (Fig. 1). Furthermore, the phylo-

genetic tree provided evidence in favor of several classical

theories viz; (i) Eurasian doves are close relative to African

collared dove, (ii) The Mourning collared dove(Streptop-

elia decipiens) is not a close relative of the North American

Mourning Dove, (Zenaida macroura) rather it is compar-

atively a close relative to Eurasian-, African- and Paki-

stani-collared doves, (iii) The Slender-billed Cuckoo-Dove

and Brown Cuckoo-Dove belong to same species although

some authorities consider it disputed [13].

Mutations have strong influence on mtDNA diversity

patterns in birds. There is a strong relationship between

species maximal longevity and mt mutation rate which is in

agreement with the mt theory of ageing [14]. The mt

sequences along with multiple nuclear genes can also be

used to distinguish between speciation arising from high

regional selective sweeps [15]. The simplest test of species

identification by DNA barcode is the variation of COI gene

sequences between two species. The second test is whether

the variation of COI gene within species is much less than

those among species. Previously, the COI gene has been

successfully used to identify most of the bird species [5].

Furthermore, Phylogenetics can explore the range of

genetic divergences among taxa [16] and mtDNA

sequences have been used successfully to estimate phylo-

genetic relationships among bird taxa, and to study the

population genetics and molecular evolution [17]. The

polymorphism in COI gene of the Pakistani collared dove

showed that this species is under the process of evolution.

As the doves and pigeons have been domesticated and

found to be the most diversified species in different geo-

graphic regions so it can be the reason of rapid evolution

[18]. Distinct geography of Pakistan might have induced

the local doves for their independent evolution. In our

study, the alignment of the COI gene sequences of Paki-

stani dove samples showed lesser intra-species polymor-

phism than that of inter-species. The finding indicates the

taxonomic status of Pakistani collared dove as an inde-

pendent species or at least as a sister species of Eurasian-

and African-collareds doves. ‘‘Recent molecular systematic

studies using mtDNA sequence revealed that traditionally

accepted species-level taxa often conceal genetically

divergent sub-specific lineages found to represent new

species upon close taxonomic scrutiny, suggesting that

Intraspecific mtDNA variation is considered useful in

species discovery’’ [19]. This is the first report of genetic

characterization and molecular classification of Pakistani

collared dove using COI gene polymorphism; the DNA

barcoding.

References

1. Knowlton N (1993) Sibling species in the sea. A Rev Ecol Syst

24:189–216

2. Galtier N, Nabholz B, Glemin S, Hurst GD (2009) Mitochondrial

DNA as a marker of molecular diversity: a reappraisal. Mol Ecol

18(22):4541–4550

3. Gissi C, Iannelli F, Pesole G (2008) Evolution of the mitochon-

drial genome of Metazoa as exemplified by comparison of con-

generic species. Heredity 101(4):301–320

4. Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S,

Escalante AA (2011) Evolution of modern birds revealed by

mitogenomics: timing the radiation and origin of major orders.

Mol Biol Evol 28(6):1927–1942

5. Hebert PD, Stoeckle MY, Zemlak TS, Francis CM (2004) Iden-

tification of Birds through DNA Barcodes. PLoS Biol 2(10):e312

6. Kerr KC, Stoeckle MY, Dove CJ, Weigt LA, Francis CM, Hebert

PD (2007) Comprehensive DNA barcode coverage of North

American birds. Mol Ecol Notes 7(4):535–543

7. Remigio EA, Hebert PD (2003) Testing the utility of partial COI

sequences for phylogenetic estimates of gastropod relationships.

Mol Phylogenet Evol 29(3):641–647

8. Greenstone MH, Rowley DL, Heimbach U, Lundgren JG,

Pfannenstiel RS, Rehner SA (2005) Barcoding generalist preda-

tors by polymerase chain reaction: carabids and spiders. Mol Ecol

14(10):3247–3266

9. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005)

DNA barcoding Australia’s fish species. Philos Trans R Soc Lond

360(1462):1847–1857

10. Smith MA, Fisher BL, Hebert PD (2005) DNA barcoding for effective

biodiversity assessment of a hyperdiverse arthropod group: the ants of

Madagascar. Philos Trans R Soc Lond 360(1462):1825–1834

11. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory

manual III. Cold Spring Laboratory Press, Cold Spring Harbour

12. Desper R, Gascuel O (2004) Theoretical foundation of the bal-

anced minimum evolution method of phylogenetic inference and

its relationship to weighted least-squares tree fitting. Mol Biol

Evol 21(3):587–598

13. David G, Barnes E, Cox J (2001) Pigeons and doves: a guide to

the pigeons and doves of the world. Pica Press, East Sussex

14. Nabholz B, Glemin S, Galtier N (2009) The erratic mitochondrial

clock: variations of mutation rate, not population size, affect

mtDNA diversity across birds and mammals. BMC Evol Biol 9:54

15. Baker AJ, Tavares ES, Elbourne RF (2009) Countering criticisms

of single mitochondrial DNA gene barcoding in birds. Mol ecol

resour 9(1):257–268

16. Gibb GC, Penny D (2010) Two aspects along the continuum of

pigeon evolution: a South-Pacific radiation and the relationship of

pigeons within Neoaves. Mol Phylogenet Evol 56(2):698–706

17. Kan XZ, Yang JK, Li XF, Chen L, Lei ZP, Wang M, Qian CJ,

Gao H, Yang ZY (2010) Phylogeny of major lineages of galli-

form birds (Aves: Galliformes) based on complete mitochondrial

genomes. Genet Mol Res 9(3):1625–1633

18. Stringham SA, Mulroy EE, Xing J, Record D, Guernsey MW,

Aldenhoven JT, Osborne EJ, Shapiro MD (2012) Divergence,

convergence, and the ancestry of feral populations in the

domestic rock pigeon. Curr Biol 22(4):302–308

19. Mila B, Tavares ES, Munoz Saldana A, Karubian J, Smith TB,

Baker AJ (2012) A trans-Amazonian screening of mtDNA reveals

deep intraspecific divergence in forest birds and suggests a vast

underestimation of species diversity. PLoS One 7(7):e40541

Mol Biol Rep (2013) 40:6329–6331 6331

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