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Mitochondrial DNA Diversity in Francolinus Pondicerianus Interpositus (Grey Francolin, Galliformes) from Pakistan IMRAN KHALIQ1,2 , M

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Mitochondrial DNA Diversity in Francolinus Pondicerianus Interpositus (Grey Francolin, Galliformes) from Pakistan IMRAN KHALIQ1,2 , M Hereditas 148: 70–76 (2011) Mitochondrial DNA diversity in Francolinus pondicerianus interpositus (grey francolin, Galliformes) from Pakistan IMRAN KHALIQ 1,2 , M. TERESA TEJEDOR3 , LUIS V. MONTEAGUDO 3 , MARIA RIAZ 2 and ALEEM A. KHAN 2 1 Department of Biological Sciences, Government College Dera Ghazi Khan, Pakistan 2 Institute of Pure and Applied Biology, Bahaudin Zakariya University, Multan, Pakistan 3 Department of Anatomy, Embryology and Genetics, Faculty of Veterinary Sciences, Universidad de Zaragoza, Zaragoza, Spain Khaliq, I., Tejedor, M. T., Monteagudo, L. V., Riaz, M. and Khan, A. A. 2011 . Mitochondrial DNA diversity in Francolinus pondicerianus interpositus (grey francolin, Galliformes) from Pakistan. – Hereditas 148 : 70–76. Lund, Sweden. eISSN 1601-5223. Received 22 November 2010. Accepted 15 March 2011. Francolinus pondicerianus interpositus (grey francolin, Galliformes) is the only francolin present in the Suleiman Range (central Pakistan), one of the poorest and least developed areas in Pakistan. As a game bird, the francolin is an important income source for the region, but no demographic data are available. Therefore, the aim of this work was to study the polymorphism pattern of the Control Region gene (mitochondrial DNA, mtDNA), in order to obtain some initial information about genetic diversity, possible structure and demographic dynamics in this population. In 29 individuals captured in four sampling areas in the western and the eastern Suleiman Range, we detected nine polymorphic sites in a 511 bp fragment of the mtDNA Control Region gene, resulting in seven haplotypes. Haplotype (h ϭ 0.818 Ϯ 0.032) and nucleotide diversity ( π % ϭ 0.308 Ϯ 0.210) values suggested a large popula- Φ ϭ ϭ tion size and a low divergence among the haplotypes. AMOVA ( ST 0.005; P 0.352) did not detect any signifi cant differences among the western and eastern populations; therefore, specimens of both sampled areas could be considered as drawn from a single population. The observed distribution of pairwise mismatches was bimodal, revealing signifi cant departure from a growing–decreasing population model (P ϭ 0.030); these results would point to a demographic equilibrium. Tribal control of hunting might provide an explanation for this situation, but future overhunting would threaten the survival of this population. Luis V. Monteagudo, Department of Anatomy, Embryology and Genetics, Faculty of Veterinary Sciences, Universidad de Zaragoza, Miguel Servet 177, ES-50013 Zaragoza, Spain . E-mail: [email protected] Francolinus pondicerianus (Gmelin 1789) (Galliformes) (D EL HOYO et al. 1994), although the global population is one of the most common game birds in the Indostan size is unknown. Asian area. Its distribution range spreads along a large Only the subspecies F. p. interpositus occurs in the proportion of Pakistan: the lower hills of Makran and Suleiman Range ( ALI and RIPLEY 1983; ROBERTS 1992). Lasbela, Sindh, and the Punjab including the Salt Range, The Suleiman Range (central Pakistan) lies between a large proportion of the Thar Desert and the Suleiman 28 ° 05 ′ N to 31 ° 80 ′ N and between 67 ° 20 ′ to 71 ° 55 ′ E Range ( ALI and RIPLEY 1983; ROBERTS 1992). (Fig. 1). The average elevation is about 1500 m, even Three F. pondicerianus subspecies are recognised though the hills in the extreme north reach 3441 m. The ( CLEMENTS et al. 2007). Francolinus p. pondicerianus lives climate of the region is exceedingly dry in these hills in in southern India and SriLanka and is characterized by summer as well as in winter. This ecoregion is made up of showing the centre of throat ochraceous. Francolinus. p. gravel and scree slopes with widely scattered isolated tufts interpositus is found in northwestern India and Pakistan of bunch grasses, thorny hassock-shaped clumps of plants and it shows paler colours, with the centre of throat such as Onobrychis and Acantholimon spp. Forest cover is creamy-white. Francolinus. p. mecranensis lives in arid sparse and highly concentrated in gullies. As it refers to southeastern Iran and south Pakistan; its plumage is the biodiversity features, it is predominantly Palearctic in origin palest, generally greyer and less chestnut than the other but does contain some Indo-Malay affi nities (WWF 2008). subspecies . This subspecies mostly occurs along permanent water According to ROBERTS (1992), populations of F. pondic- sources during late summer and fall ( ROBERTS 1992). It erianus are on the verge of decline in Asia because of the roosts at night and rests during the day, primarily on the loss of food, excessive predation, hunting and habitat ground in dense cover (tamarisk thickets, reeds grass clumps destruction and extended use of guns for hunting. How- and cultivated crops) and it lives singly or in scattered pairs ever, F. pondicerianus is common in most parts of its range and parties of 3-5 individuals ( MAHMOOD et al. 2010). © 2011 The Authors. This is an Open Access article. DOI: 10.1111/j.1601-5223.2010.02206.x Hereditas 148 (2011) Mitochondrial DNA diversity in the grey francolin 71 The aim of this study is to describe the polymorphism pattern of the CR in F. pondicerianus interpositus for the fi rst time, in order to get some initial information about genetic diversity, possible structure and demographic dynamics for the Suleiman Range population. The data obtained could be the basis for further decisions about management and protection of this population. MATERIAL AND METHODS Biological sampling Fig. 1. The Sulaiman Range (SR) position in Pakistan. Samples were collected in the four areas indicated. Feathers were obtained post-mortem from twenty nine Francolinus pondicerianus interpositus individuals shot The Suleiman Range is a Provincially Administered Tribal by hunters at four different locations in the Suleiman Area (PATA), i.e. under the control of different Balouch Range: Khalchas (69 ° 33 ’ N, 29 ° 23 ’ E; n ϭ 12), Higlon tribes. The whole area is one of the poorest and least devel- (70 ° 01 ’ N, 30 ° 30 ’ E; n ϭ 4), Berg (69 ° 80 ’ N, 30 ° 25 ’ E; oped in Pakistan. The main source of income in the area is n ϭ 11) and Bait Suwai (30 ° 45 ’ N, 70 ° 50 ’ E; n ϭ 2) in either through rearing goats or through cultivation depend- the 2008 winter hunting season (Fig. 1). We grouped ing upon the seasonal rainfall. However, due to the lack of these locations, located in both sides of the Suleiman income and due to the increasing population in some areas Range, as western side (Kalchas) and eastern side (Berg, of Suleiman Range, people have started exploiting the for- Hinglon and Bait Suwai) populations. merly undisturbed wildlife of the area. A single grey franco- We clipped eight to ten feathers from each bird by lin can be sold for 5 – 10 US dollars in the market. Therefore, means of canine nail clippers. As the birds are dead when hunting is becoming a very attractive and easy option for the feathers are collected, we can take a number of feathers locals. In recent years this trend has increased ( KHALIQ suitable to carry out several analyses in both Pakistan and unpubl.), but the possible impacts on population genetics Spain. We cleaned the birds ’ skin with 10% Povidone- and demography have not yet been evaluated. iodine surgical scrubs before clipping for preventing even- In some parts of Pakistan, only a small amount of popu- tual contamination. We air dried feather snips prior to lation data is available about F. pondicerianus . MAHMOOD placing them in individual sterile marked plastic bags. All et al. (2010) estimated an averaged population density of the work with the birds complied with all Spanish and 0.47 Ϯ 0.09 individuals ha Ϫ1 and a total size of 3327 birds Pakistani laws. in the Lehri Nature Park (Punjab, Pakistan), but no infor- mation is available about the Suleiman Range. DNA extraction Mitochondrial DNA (mtDNA) is relatively easy, rapid For each individual, we cut off 2 mm fragments from the and inexpensive to sequence and research on rapidly proximal tip of two feathers. The fragments were immersed evolving loci provides suffi cient variation to draw infer- in 200 μ l of a 5% Chelex 100 resin solution in pH 8 TE ences on the structure of populations ( BROWN et al. 1982, buffer (10 mM TrisHCl, 0.1 mM EDTA) containing C LAYTON 1984, SACCONE et al. 1991). The Control Region Proteinase K (1.2 mg ml –1 ) and 2% sodium dodecyl gene (CR, also called hypervariable region or D-loop) sulfate. After overnight incubation at 55 ° C, samples were often evolves faster than the rest of the mtDNA ( BAKER vortexed and incubated for 10 min at 100 ° C. They were and MARSHALL 1997) and it appears to be highly variable vortexed again and incubated for 15 min at the same in birds ( WENINK et al. 1994). In birds, several studies temperature before centrifugation at 11 000 g for three showed that the CR is not the fastest evolving locus in min. The supernatant was transferred to a fresh tube and mtDNA and that this trend is genus specifi c ( ZINK and conserved at – 20 ° C. This supernatant was included at B LACKWELL 1998; ZINK et al. 1998; LEE et al. 2001; 35 v/v % (volume/volume percentage) in the fi nal PCR R UOKONEN and KVIST 2002); several mtDNA coding loci, reaction mix. Neither RNAse treatment nor DNA precipi- such as cytochrome b and ND2 (mitochondrially encoded tation are necessary. NADH dehydrogenase 2) evolve as fast as the CR. How- ever, analysis of polymorphism in the CR has proved to be Polymerase chain reaction and sequencing useful in preliminary studies on genetic variation and structure and phylogeography in birds ( MERIL Ä et al. 1997; We designed polymerase chain reaction (PCR) primers G ODOY et al.
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