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Molecular Classification of Pakistani Wild Rose-Ringed Parakeet through Novel Cytochrome B Gene Polymorphism




Molecular Classification of Pakistani Wild Rose-Ringed Parakeet through Novel Cytochrome B Gene Polymorphism

Ali Raza Awan*, Sehrish Firyal, Muhammad Tayyab, Lala Rukh, M. Zia ul Haq, Shagufta Saeed and Muhammad Wasim

Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Civil Lines, Outfall Road, Lahore, Pakistan


Rose-ringed Parakeet (Psittacula krameri) is sub-divided in 4 sub-species (P. krameri krameri, P. krameri parvirostris, P. krameri manillensis and P. krameri borealis). Identification of the sub-species is an intricate chore. This study aimed to genetically identify and classify the indigenous wild Rose-ringed Parakeet of Pakistan using Cytochrome b (Cytb) gene polymorphism. Mitochondrial DNA of 24 unrelated Pakistani wild Ring-rose Parakeets was isolated and utilized for amplification and DNA sequencing of Cytb gene. The Phylogenetic analysis of the Cytb gene indicated that the Pakistani wild Rose ringed Parakeet was mono-phyletically claded with P. k. manillensis with a sequence similarity of 99.37%. Comparative analysis indicated 4 Single Nucleotide Polymorphism (SNPs) sites in the Pakistani wild Rose-ringed parakeet’s Cytb gene which tags the peculiarity to the Pakistani wild Rose-ringed parakeet. This is the first report that lays bare the molecular classification of Pakistani wild Rose-ringed Parakeet at sub-specie level using novel Cytb gene polymorphism.

Article Information

Received 05 May 2016

Revised 31 August 2016

Accepted 06 September 2016

Available online 05 January 2017

Authors’ Contributions

ARA, SF conceived and designed the study, executed the experimental work, analyzed the data, and wrote the article. LR and MZH helped in sampling of birds. MT helped in data analysis. SS and MW helped in preparation of manuscript.

Key words

Parakeet, Parrot, Wildlife, Molecular classification, Cytochrome b.

* Corresponding author:

0030-9923/2017/0001-0283 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan





Pakistan is a habitat to hundreds of avian species with unique bio-diversity (Awan et al., 2013). Among the birds, parrots are considered unique cage-birds due to their bright colors, intelligence, social and affectionate nature and ability to imitate human voices. They attracted the focus of scientists due to their unique features i.e. vocal communication, brain evolution, nesting behavior, life-history trait evolution, global patterns of species richness, and evolution of mitochondrial control region duplications. Three hundred and seventy two species of parrots belong to order Psittaciformes and many of these species are the members of family Psittacoidae (Iwaniuk et al., 2004).

An indigenous parakeet species of Pakistan is phenotypically similitude to Rose-ringed Parakeet (Psittacula krameri). The local name of this parakeet is “Kathy” and this parakeet is least documented. The rose-ringed parakeets (P. krameri) are sub-divided into 4 sub-species; P. k. krameri, P. k. parvirostris, P. k. manillensis and P. k. borealis (Clements, 2007). Mead et al. (1993) differentiated the four sub-species on the basis of phenotypic characteristics including wing and tail length, body weight and differences in upper and lower mandible. Most phylogenetic studies (Astuti et al., 2006; Schweizer et al., 2009; Wright et al., 2008) have encompassed only the parrot genera giving a broad perspective of evolutionary trends (Kundu et al., 2012). Identification of wild rose-ringed parakeet sub-species is a complex chore because these sub-species share several common features and adaptive radiation wields strong influence on evolution of the parakeet species and sub-species along geographic coordinates. The taxonomic delimitation of taxa has to be carefully reviewed before any conclusion drawn with respect to bio-geographical history or population dynamics (Ribas et al., 2007). Dearth of dependable method for identification of wild rose-ringed parakeet at sub-species level led us to analyze Pakistani wild rose-ringed parakeet using Cytb gene polymorphism. Cytb gene analysis have become important tool in population genetics and in determination of phylogenetic relationship especially among the closely related avian species and sub-species (Allan and Max, 2010). In the present study, we have classified the indigenous rose-ringed parakeet of Pakistan at sub-species level using novel Cytb gene polymorphism.


Materials and Methods


To explore the polymorphism in Cytb gene of Pakistani wild rose-ringed Parakeets; blood samples from 24 unrelated parakeets with typical phenotypic features were selected from different regions of Pakistan. The samples were named from PKPrC1 to PKPrC24. Standard organic method was used for isolation of the genomic DNA. Forward (5’-TCCTCCGCACTATCAATCCT-3’) and reverse (5’-ATGCAAATAGGAAATACCATTC-3’) primers were used for the amplification of Cytb genes of Pakistani wild rose-ringed parakeets using PCR. DNA sequencing of the Cytb genes was done using big dye terminator cycle sequencing kit and ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, USA). Sequence data was edited manually using Chromas Ver. 1.45, ( and BioEdit (Hall, 1999). Sequence homology analysis was performed using DNA sequences of Cytb gene of the Pakistani wild rose-ringed parakeets and the reported Cytb genes of Psittacula species available at NCBI Genbank; http://www.ncbi.nlm.nih. gov/genbank/ (Table I). Molecular Evolutionary Genetics Analysis (MEGA, V 5.0,) was used to estimate the evolutionary distances between sequences by computing the nucleotide differences between each pair of sequences. Phylogenetic tree was constructed using TreeView software. The rooted tree was formed by placing a root in the middle of the longest edge (Desper and Gascuel, 2004).


Table I.- The sequences utilized for the construction of phylogenetic tree.

Species Accession No. Species Accession No.
Brotogeris pyrrhopterus FJ652864 Pionus sordidus ponsi EF517634
Chalcopsitta scintillate AB177955 Pionussordidus corallines EF517628
Eclectus roratus AB177965 Pionus sordidu smindoensis EF517629
Eclectus roratus roratus AY220101 Pionus tumultuo susseniloides EF517615
Lorius garrulous AB177951 Psittacula calthorpae GQ996512
Psittacula alexandri AB177958 Psittacula columboides AY220108
Psittacula cyanocephala AY220109 Psittacula echo AY220113
Pionus chalcopterus EF517621 Psittacula eupatria AY220115
Pionus cyanescens EF517619 Psittacula finschii GQ996510
Psittacula longicauda longicauda GQ996509 Pseudeos fuscata AB177964
Pionus maximiliani lacerus EF517626 Psittacula krameri AY220117
Pionus maximilianisiy EF517624 Psittacula krameri KC876659
Pionus menstruus AY669403 Psittacula krameri KC876665
Pionus menstruus AY286207 Psittacula krameri manillensis GQ996517
Pionus menstruus EF517604 Psittacula krameri parvirostris GQ996497
Pionus menstruus reichenowi EF517613 Psittacula roseate AY220107
Pionus menstruus rubrigularis EF517606 Pionus tumultuosus EF517614
Pionus sordidus antelius EF517632 Tanygnathussumatranus AB177962
Pionus senilis U89179 Vini australis AF346339





The comparative analysis of Cytb gene sequence of Pakistani wild rose-ringed parakeet with reported Cytb gene sequences indicated 4 novel SNPs sites in Pakistani wild rose-ringed parakeet (Fig. 1). The four SNPs were the substitutions of G to A at 270, T to C at 273, T to C at 276 and C to T at 663 positions in Cytb gene sequence of Pakistani wild rose-ringed parakeet (Fig. 1). All nucleotide substitutions were found at third base position of the codons and were not found changing any amino acids. The same pattern of same-sense substitutions were previously reported in Neotropical parrots (Tavares et al., 2006).

Psittacula krameri manillensis was found to be the closest homologue of Pakistani wild rose-ringed parakeet according to the sequence similarity analysis which is 99.37%.

Genetic distances were calculated for determination of relationship of Pakistani wild rose-ringed parakeet with other parrot species (Table II). Data demonstrated that, greater the genetic distance, more distinct will be the species. A value of 0.015 indicated closely related homologues e.g. Pakistani wild rose-ringed parakeet and P. k. manillensis share 99.37% identity. Similarly, genetic distance of 0.088 between Pakistani wild rose-ringed parakeet and P. eupatria corresponds to the sequence identity of 92.74% (Table II). Whereas a higher value (0.101) indicated that Pakistani wild rose-ringed parakeet was more genetically distinct from P. alexandri as these two share 91.1% sequence identity.




This study describes the classification of indigenous Rose-ringed parakeet of Pakistan at sub-specie level using Cytb gene polymorphism. A wider relationship between evolution of morphology and distribution exists within Psittacula genus and make it highly speciose (Kundu et al., 2012). These spontaneous variations in Psittacula morphology signify the identification of the Psittaculla species and sub-species through molecular tools. In the genomic era, phenotypic characters of species cannot represent the true depiction of the taxonomy as classical identification approaches are not sufficient to elucidate the genetic bio-diversity and arrangement of hierarchical taxonomic and evolutionary tree (Ijaz et al., 2017). Mitochondrial genes are the molecular instruments used for genetic characterization and taxonomic investigations of avian families, species, sub-species and populations. Among the mitochondrial genes Cytb gene is extensively used for species and sub-species identification and has been considered one of the most useful genes for evolutionary studies. It has been extensively employed to address the diversity questions as it contains both rapidly and slowly evolving codon positions (Saif et al., 2012).

Cytb gene fragments of 799 base pairs (bp) from 24 unrelated Pakistani wild rose-ringed parakeet were amplified, sequenced and submitted to National Center of Biotechnology Information (NCBI) GenBank under the accession No. KC876642 to KC876665. The sequence similarity analysis shows the similarity of Pakistani Rose ringed parakeet with P. k. parvirostris (98.37%), P. k. borealis (98.2%), P. echo (96.37%), P. k. krameri (96.12%), Psittacula columboides and Psittacula eupatria (92.74%), Psittacula alexandri (91.99%), Psittacula longicauda (91.61%), Psittacula calthorpae (91.49%) and 90.99% with Psittacula roseata.

Further, phylogenetic analysis demonstrated that Indian P. k. manillensis was found to be the contiguous taxa of Pakistani wild rose-ringed parakeet, whereas Ethiopian P. k. parvirostris, Indo-Pak P. k. borealis, Mauritiusal P. echo and African P. k. krameri were found more distinct from the Pakistani wild rose-ringed parakeet (Fig. 2). P. k. parvirostris and P. echo were revealed polyphyletic with respect to other members of P. krameri, whereas, Kundu et al., (2012) reported the monophyletic existence of various Psittacula species.


Table II.- Genetic distances between the various parrot species.

Psittacula krameri parakeets< (KC876642)

Psittacula alexandri fasciata (GQ996507.1) 0.101                      
Psittacula himalayana (AY220102.1) 0.097 0.096                    
Psittacula alexandri (AY220105.1) 0.101 0.000 0.096                  
Psittacula alexandri (AY220106.1) 0.101 0.000 0.096 0.000                
Psittacula cyanocephala< (AY220109.1) 0.091 0.054 0.062 0.054 0.054              
Psittacula cyanocephala cyanocephala (GQ996508.1) 0.096 0.058 0.066 0.058 0.058 0.004            
Psittacula eupatria (AY220115.1) 0.087 0.104 0.101 0.104 0.104 0.099 0.09          
Psittacula eupatria magnirostris (GQ996496.1) 0.092 0.066 0.083 0.066 0.066 0.078 0.07 0.07        
Psittacula krameri manillensis (AY220110.1) 0.015 0.091 0.097 0.091 0.091 0.091 0.09 0.08 0.08      
Psittacula krameri manillensis (AY220111.1) 0.019 0.096 0.102 0.096 0.096 0.096 0.10 0.08 0.08 0.04    
Psittacula krameri borealis (AY220116.1) 0.019 0.096 0.101 0.096 0.096 0.096 0.10 0.07 0.08 0.04 0.07  
Psittacula krameri parvirostris (GQ996497.1) 0.015 0.092 0.097 0.092 0.092 0.092 0.09 0.07 0.08 0.07 0.01



On the basis of sequence homology of Cytb gene, the Pakistani wild rose-ringed parakeet can be classified as sub-species of P. k. manillensis but the deviation in Cytb gene sequence of Pakistani wild rose-ringed parakeets is pointing towards independent evolution of this species as an ecocline in Pakistan. The geographical heterogeneity exerts evolutionary pressure to a specie to adopt the status of an ecocline to that extent which evolves it into sub-species (Groombridge et al., 2004). For example mandible size in the African rose-ringed parakeets (P. k. krameri) increases westwards across its geographical range in Africa and mandible color of Asian rose-ringed parakeets changes from red (P. k. borealis) to black (P. k. manillensis) in longitudinal coordinates of South-Asia (Forshaw, 2006). The same pattern of adaptive radiation is observed in Pakistani wild rose-ringed parakeet which has been evolved independently in the indigenous environment.




This is the first report of classification of wild rose-ringed parakeet of Pakistan at sub-species level using novel Cytb gene polymorphism and this study indicated that the Pakistani wild rose ringed parakeet is mono-phyletically claded with P. k. manillensis having a sequence similarity of 99.37%.


Statement of conflict of interest

Authors have declared no conflict of interest.




Allan, G.J. and Max, T.L., 2010. Molecular genetic techniques and markers for ecological research. Nature Education Knowledge, 3: 2.

Astuti, D., Azuma, N., Suzuki, H. and Higashi, S., 2006. Phylogenetic relationships within parrots (Psittacidae) inferred from mitochondrial Cytochrome-B gene sequences. Zool. Sci., 23: 191-198.

Awan, A.R., Umar, E., Zia Ul Haq, M. and Firyal, S., 2013. Molecular classification of Pakistani wild collared dove through DNA barcoding. Mol. Biol. Rep., 40: 6329-6331.

Clements, J.F., 2007. The clements checklist of the birds of the world. Christopher Helm, London.

Desper, R. and Gascuel, O., 2004. Theoretical foundation of the balanced minimum evolution method of phylogenetic inference and its relationship to weighted least-squares tree fitting. Mol. Biol. Evol., 21: 587-598.

Forshaw, J.M., 2006. Parrots of the world; an identification guide. Princeton University Press, Princeton, New Jersey.

Groombridge, J.J., Jones, C.G., Nichols, R.A., Carlton, M. and Bruford, M.W., 2004. Molecular phylogeny and morphological change in the Psittacula parakeets. Mol. Phylogen. Evolut., 31: 96-108.

Hall, T.A., 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/Nt. In: Nucleic acids symposium series, pp. 95-98.

Ijaz, U., Tahir, M.T., Majeed, K.A., Iqbal, S., Huma, I., Firyal, S., Ahmad, I., Chohan, S. and Khan, A.R., 2017. Molecular characterization and phylogeny of Panthera pardus (Common Leopard) in Pakistan. Pakistan. J. Zool., 49: 65-69.

Iwaniuk, A.N., Dean, K.M. and Nelson, J.E., 2004. Interspecific allometry of the brain and brain regions in parrots (Psittaciformes): Comparisons with other birds and primates brain. Behav. Evolut., 65: 40-59.

Kundu, S., Jones, C.G., Prys-Jones, R.P. and Groombridge, J.J., 2012. The evolution of the Indian ocean parrots (Psittaciformes): Extinction, adaptive radiation and eustacy. Mol. phylogen. Evolut., 62: 296-305.

Mead, C.J., Clark, J.A. and Peach, W.J., 1993. Report on bird ringing in Britain and Ireland 1992. Ring. Migr., 14: 152-200.

Ribas, C.C., Tavares, E.S., Ysoshihara, C. and Miyaki, C.Y., 2007. Phylogeny and biogeography of yellow-headed and blue-fronted parrots (Amazona ochrocephala and Amazona aestiva) with special reference to the South American taxa. Ibis, 149: 564-574.

Saif, R., Babar, M.E., Awan, A.R., Nadeem, A., Hashmi, A.S. and Hussain, T., 2012. DNA fingerprinting of Pakistani wild buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers. Mol. Biol. Rep., 39: 851-856.

Schweizer, M., Seehausen, O., Guntert, M. and Hertwig, S.T., 2009. The evolutionary diversification of parrots supports a taxon pulse model with multiple trans-oceanic dispersal events and local radiations. Mol. phylogen. Evolut., 54: 984-994.

Tavares, E.S., Baker, A.J., Pereira, S.L. and Miyaki, C.Y., 2006. Phylogenetic relationships and historical biogeography of neotropical parrots (Psittaciformes: Psittacidae: Arini) inferred from mitochondrial and nuclear DNA sequences. System. Biol., 55: 454-470.

Wright, T.,F., Schirtzinger, E.E., Matsumoto, T., Eberhard, J.R., Graves, G.R., Sanchez, J.J., Capelli, S., Müller, H., Scharpegge, J., Chambers, J.K. and Fleischer, R.C., 2008. A multilocus molecular phylogeny of the parrots (Psittaciformes): support for a gondwanan origin during the cretaceous. Mol. Biol. Evolut., 25: 2141-2156.

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Pakistan Journal of Zoology


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