MOLECULAR PHYLOGENY AND DIVERSITY ANALYSIS OF BOVIDAE (BOSELAPHUS TRAGOCAMELUS, ANTILOPE CERVICAPRA) AND CERVIDAE (AXIS AXIS, AXIS PORCINUS) IN

BY

GHULAM ABBAS 2011-VA-748

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

MOLECULAR BIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF VETERINARY & ANIMAL SCIENCES,

2016

The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore.

We, the supervisory committee, certify that the contents and form of the thesis, submitted by Mr. Ghulam Abbas, Regd. No. 2011-VA-748 have been found satisfactory and recommend that it be processed for the evaluation by the External Examiner(s) for the award of the degree.

Supervisor: ______Dr. Asif Nadeem

Member: ______Prof. Dr. Masroor Ellahi Babar

Member: ______Dr. Muhammad Tayyab

DEDICATED To My Dear Parents

i

ACKNOWLEDGEMENTS

All praises to “ALLAH”, the Almighty, Most Gracious, the most Merciful and the Sustainer of the worlds, Who created the jinn and humankind only for His worship and raised among the inhabitants of Makkah the Holy Prophet “Muhammad” (peace be upon him.) from among themselves, who recited to them His revelations and purified them, and taught them the Book (Holy Quran) and the Wisdom.

Foremost thanks to Almighty Allah Who blessed me with good health and valued teachers to accomplish my Ph. D studies. I feel great pleasure in expressing my heartiest gratitude and deep sense of obligation to my supervisor Dr. Asif Nadeemand to the distinguished member of my supervisory committeeProf. Dr. Masroor Ellahi Babar (Tamgha-e-Imtiaz) for their able guidance, keen interest, skilled advice, constructive criticism, constant encouragement, valuable suggestions and kind supervision throughout the course of my study and research work. In addition, I owe my thanks to my committee memberDr. Muhammad Tayyabfor his suggestions.

I need to offer my heartiest thanks to my friends, research fellows and colleagues at the University especially to Dr. Mohammad Sajid Tahir, Dr. Tanveer Hussain, Dr. Mohammad Haseeb Saeed and Mr. Muhammad Aqeel for their support and fruitful suggestions duringmy research. I would like to offer my special thanks to Mr. Ghulam Abbas Pathan and all those who helped me during the sample collection

In the end, I could not envisage the endeavors that my parents made for successful completion of my higher education. They sacrificed all available resources in their lives for making me able to get higher education that ultimately lead me to successful completion of the degree. No acknowledgement could ever adequately express my obligation to my affectionate parents for leading their children into intellectual pursuits. “May Allah rest their souls in piece” (Aa’meen). I also extend my heartiest compliments to my wife, kids and sister whose love, prayers and support made it possible to reach this position.

Ghulam Abbas Haral ii

TABLE OF CONTENTS

DEDICATION…………………………………………………………i ACKNOWLEDGEMENTS……………………………………………ii TABLE OF CONTENTS………………………………………………iii LIST OF TABLES……………………………………………………..iv LIST OF FIGURES……………………………………………………v

SR. NO. CHAPTERS PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 8

3 MATERIALS AND METHODS 24

4 RESULTS 45

5 DISCUSSION 106

6 SUMMARY 115

7 LITERATURE CITED 116

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LIST OF TABLES

TABLE TITLE PAGE NO. NO. 3.1a Sampling details of Boselaphus tragocamelus 30 3.1b Sampling details of Antilope cervicapra 31 3.1c Sampling details of Axis axis 32 3.1d Sampling details of Axis porcinus 33 3.2 List of primer sequences 39 3.3 Recipe for each multiplex of primers 40 3.4 PCR reaction composition for sequencing products 42 3.5 PCR reaction conditions for sequencing products 42 4.1 Polymorphisms identified in cytochrome-b gene of Boselaphus 46 tragocamelus 4.2 Polymorphisms identified in cytochrome-c gene of Boselaphus 50 tragocamelus 4.3 Polymorphisms identified in d-loop region of Boselaphus tragocamelus 54 4.4 Cytochrome-b, cytochrome-c and d-loop region based evolutionary 59 analyses of Boselaphus tragocamelus 4.5 Polymorphisms identified in cytochrome-b gene of Antilope cervicapra 62 4.6 Polymorphisms identified in cytochrome-c gene of Antilope cervicapra 65 4.7 Polymorphisms identified in mitochondrial d-loop region of Antilope 68 cervicapra 4.8 Cytochrome-b, cytochrome-c and d-loop region (collectively) based 72 evolutionary analyses of Antilope cervicapra 4.9 Polymorphisms identified in cytochrome-b gene of Axis axis 74 4.10 Polymorphisms identified in cytochrome-c gene of Axis axis 77 4.11 Polymorphisms identified in mitochondrial d-loop of Axis axis 79 4.12 Cytochrome-b, cytochrome-c and d-loop region based evolutionary 83 analyses 4.13 Polymorphisms identified in cytochrome-b gene of Axis porcinus 84 4.14 Polymorphisms identified in cytochrome-c gene of Axis porcinus 87 4.15 Polymorphisms identified in mitochondrial d-loop region of Axis porcinus 90 4.16 Cytochrome-b, cytochrome-c and d-loop region (collectively) based 94 Evolutionary analyses of Axis porcinus

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LIST OF FIGURES

FIG. TITLE PAGE NO. NO. 3.1 Map of Pakistan showing selected areas for sampling of Boselaphus tragocamelus 26 3.2 Map of Pakistan showing selected areas for sampling of Antilope cervicapra 27 3.3 Map of Pakistan showing selected areas for sampling of Axis axis 28 3.4 Map of Pakistan showing selected areas for sampling of Axis porcinus 29 Photograph of Boselapus tragocamelus Antilope cervicapra (Bovidae), Axis axis 3.5 34 and Axis porcinus (Cervidae) Multidimensional scaling plot for cytochrome -b gene of Boselaphus 4.1 47 tragocamelusof various regional origins Cytochrome-b gene based genetic variation plot of Boselaphus tragocamelus of 4.2 48 various regional origins Phylogenetic Neighbor Joining tree (circular) of cytochrome-b gene of Boselaphus 4.3 49 tragocamelus Multidimensional scaling plot of mitochondrial genomic gene, cytochrome-c for 4.4 51 Boselaphus tragocamelus 4.5 Cytochrome-c based genetic variation plot of Boselaphus tragocamelus 52 4.6 Phylogenetic tree (circular) of cytochrome-c gene of Boselaphus tragocamelus 53 Multidimensional scaling plot of mitochondrial genomic d-loop region for 4.7 Boselaphus tragocamelus 55 4.8 D-loop sequence based Genetic variation plot of Boselaphus tragocamelus 56 4.9 Phylogenetic tree (circular) of d-loop region of Boselaphus tragocamelus 57 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.10 58 cytochrome-c and d-loop region for Boselaphus tragocamelus Cytochrome-b, cytochrome-c and d-loop region based genetic variation plot of 4.11 Boselaphus tragocamelus 59 Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.12 61 of Boselaphus tragocamelus. 4.13 cytochrome-b based multidimensional scaling plot of Antilope cervicapra 63 4.14 Cytochrome-b based genetic variation plot of Antilope cervicapra 63 4.15 Phylogenetic tree (circular) of cytochrome-b gene of Antilope cervicapra. 64 4.16 Multidimensional scaling plot of cytochrome-c gene for Antilope cervicapra 66 4.17 Cytochrome-c based genetic variation plot of Antilope cervicapra 66 4.18 Phylogenetic tree (circular) of cytochrome-c gene of Antilope cervicapra 67 4.19 Multidimensional scaling plot of d-loop region for Antilope cervicapra 69 4.20 d-loop region based genetic variation plot of Antilope cervicapra 69 4.21 Phylogenetic tree (circular) of d-loop region of Antilope cervicapra 70 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.22 cytochrome-c and d-loop region (collectively) for Antilope cervicapra 71 v

Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic 71 4.23 variation plot of Antilope cervicapra Phylogenetic tree (rectangular) of Cytochrome-b, cytochrome-c and d-loop region 4.24 73 (collectively) of Antilope cervicapra 4.25 Multidimensional scaling plot of mitochondrial cytochrome-b gene for Axis axis. 75 4.26 Cytochrome-b based genetic variation plot of Axis axis 75 4.27 Phylogenetic tree (circular) of cytochrome-b region of Axis axis 76 4.28 Multidimensional scaling plot of cytochrome-c gene for Axis axis 77 4.29 Cytochrome-c based genetic variation plot of Axis axis 78 4.30 Phylogenetic tree (circular) of cytochrome-c region of Axis axis 78 4.31 Multidimensional scaling plot of mitochondrial d-loop region for Axis axis 80 4.32 D-loop region based genetic variation plot of Axis axis 80 4.33 Phylogenetic tree (circular) of d-loop region of Axis axis 81 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.34 82 cytochrome-c and d-loop region (collectively) for Axis axis Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic 4.35 variation plot of Axis axis 82 Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.36 (collectively) of Axis axis 83 4.37 Multidimensional scaling plot of mitochondrial cytochrome-b for Axis porcinus 85 4.38 Cytochrome-b based genetic variation plot of Axis porcinus 85 4.39 Phylogenetic tree (circular) of cytochrome-b region of Axis porcinus 86 4.40 Multidimensional scaling plot for Axis porcinus based on cytochrome-c 88 4.41 Cytochrome-c based genetic variation plot of Axis porcinus 88 4.42 Phylogenetic tree (circular) of cytochrome-c region of Axis porcinus 89 4.43 Multidimensional scaling plot of d-loop region for Axis porcinus 91 4.44 D-loop region based genetic variation plot of Axis porcinus 91 4.45 Phylogenetic tree (circular) of d-loop region of Axis porcinus 92 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.46 93 cytochrome-c and d-loop region for Axis porcinus Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic 4.47 93 variation plot of Axis porcinus Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.48 95 (collectively) of Axis porcinus Phylogenetic tree (radiation) of cytochrome-b gene of selected four species 4.49 96 (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus). Phylogenetic tree (circular) of cytochrome-b gene (collectively) of selected four 4.50 species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis 97 porcinus) Phylogenetic tree (radiation) of cytochrome-c region (collectively) of selected four 4.51 species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis 98 porcinus) Phylogenetic tree (circular) of cytochrome-c region of selected four species 4.52 99 (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus)

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Phylogenetic tree (ractangular) of d-loop region of selected four species 4.53 100 (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus) Phylogenetic tree (circular) of d-loop region of selected four species (Boselaphus 4.54 101 tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus) Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.55 cytochrome-c and d-loop region for Bovidae (Boselaphus tragocamelus and 102 Antelope cervicapra). Cytochrome-b, cytochrome-c and d-loop region based Genetic variation plot of 4.56 102 Bovidae (Boselaphus tragocamelus and Antelope cervicapra) Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.57 103 cytochrome-c and d-loop for Cervidae (Axis axis and Axis porcinus) Cytochrome-b, cytochrome-c and d-loop region based genetic variation plot of 4.58 103 Cervidae (Axis axis and Axis porcinus) Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.59 104 cytochrome-c and d-loop region (collectively) for selected four species. Cytochrome-b, cytochrome-c and d-loop region based variation plot of selected 4.60 104 four species Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.61 105 of Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus

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CHAPTER 1 INTRODUTION

Vertebrates are an endlessly fascinating group of animals dominating the planet earth.

They are important not only because we are familiar with them, but also because we, humans, are also vertebrates. Among vertebrates, are unique group of animals. They have mammary glands and are found in diverse environmental conditions and are able to use wide range of resources on the earth ranging from north to south poles and from mountain peaks to depth of oceans. This group comprises 1135 genera, 136 families and 26 orders (Wilson and

Reeder, 1993).

Bovidae and Cervidae are families of class Mammalia. Bovidae represent ungulates with unbranched horns. This distinguishes Bovidae from other cud-chewing artiodactyls especially family Cervidae. Fifty five percent of modern ungulates belong to this family. Bovidae has 50 genera and 143 different species. Most of these species are wild and only sheep, goat and three bovine species are domesticated (Hassanin et al. 2012). Cervidae (deer family), comprises 40 species. It is largest family of Ruminantia. It is widely distributed and has ample population.

Cervidae along with Moschidae, Giraffidae and Antilocapridae is grouped into superfamily,

Cervoidea. Some of the worth mentioning features of Cervidae are, they shed their antlers annually and they do not have gall bladder. On the basis of different features like morphology of limbs, chromosomal makeup of their somatic cells and behavior and ecology, this family is subdivided two main groups. One of them is subfamily cervinae i.e. plesiometacarpal, that is further divided into two tribes Cervini and Muntiacini. Second group called telemetacarpal is divided into subfamilies Odocoileinae and Hydropotinae.

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INTRODUCTION

Having 40 species of deer, family Cervidae has highest number of species after Bovidae.

Antlers are one of unique characteristics of this family. As compared with horns, that always remain attached to the skull, antlers are caste off every year (Wilson and Reeder, 1993).

Cervidae is very diverse regarding its size, habitat and behavior. Some of the synapomorphies like presence of antlers in the males are used to group closely related cervids. According to

Grubb (2005), sixteen genera of family Cervidae are grouped in four sub families. These sub families are Cervinae, Muntiacinae, Hydropotinae, and Odocoileinae. Sub family Cervinae is further divided into four genera. One, Dama is found in Eurasia and have two species of fallow deer. Second, Axis has four Asian species including Hog deer (Axis porcinus) and (Axis axis). Third, Elaphurus davidianus (Père David’s deer) is found in and fourth, Cervus is widely distributed genus having nine species found in Asia. Among those Cervus elaphus, is found all over northern hemisphere (Gilbert et al. 2006).

The largest of the Asian antelopes, the (Boselaphus tragocamelus) is a bovid that was first time defined in 1766 by Pallas. The body of female Nilgai is yellow-brown in color. In males, yellow-brown color steadily changes into blue-grey when they approach the mature age.

Their nape and back have erectable mane. A "hair pennant" is visible in the middle of the underside of the neck. White patterns are visible in the form of cheek spots, edges of the lips along with those, they have throat bib also. Underside of body has a thin white stripe which expands in size as it proceeds towards backside. Legs are slim that provide sustenance to stocky body. The body slopes downwards from front towards back. The head is long and slim. Horns of males Blue bull (Boselaphus tragocamelus) are 20 to 25 cm long. Horns are straight and tilted forward slightly. Favorite habitat of Nilgai is woodlands area and grassy steppe. Abodes of

Nilgai in Pakistan are pure desert areas of Cholistan and Thar in Punjab and Sind respectively.

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INTRODUCTION

Nowadays, this Antilope is naturally found in narrow strip of land along Indian border in the eastern part of Pakistan. They are not found in herds in this area. Presence of Nilgai in Changa

Manga Plantation, near Lahore, has been reported also. According to Sind Wildlife Department,

220 Nilgai were found in Tharparkar district in 1999 (Maan and Chaudhry 2001).

Blackbuck (Antilope cervicapra) has morphologically different male and female animals.

Male and female animals have white spots on their lower sides. They have white circle around the eye and also have white chin. Females and their babies are yellow in color and most of them do not have horns. Body is thin and slim. Males have horns and those grow before color is changed. Black buck have lateral size of 35 to73 cm. Though breeding can occur all over the year, but March to May and August to October are top breeding periods. show diurnal habit when season is comparatively cool and remain active sporadically during whole day time. But when weather is hot they graze only early morning and late afternoon and shelter from the sun during remaining time. can run as fast as 80 km/hrs. In contrast to hog deer, black bucks were social and lived in groups.

Blackbucks originally belong to . They were found everywhere in this area from plains up to mountains. Nowadays, human activities have destructed their habitat so they are found only in isolated places. In our country of Pakistan, blackbucks are found in

Cholistan and Thar area of Punjab and Sind along eastern border areas. At present, there is no specific area for their living in Pakistan. Now they are found as captive in different places like

Kirthar and Lal Sohanra National Parks. Once common in the desert belt where agriculture existed, the antelope species is now extinct in Sindh. However, a large population of the species exits in Khairpur’s Mehrano reserve, a private farm supported by the Sindh wildlife department.

Some other places where black buck are found in Pakistan are and different wildlife centers.

3

INTRODUCTION

Some people also have limited number of black bucks in their private forms. In 2008; more than

1500 Black Bucks were found in different breeding centers of Punjab and Sindh in Pakistan

(Mallon 2008).

The Spotted Deer or Chital (Axis axis) is only Cervid within genus Axis. Three other species that were part of this genus are now grouped in on the basis of their genetic evidence. Axis axis is also known as chital deer, spotted deer or axis deer. Axis Axis is very common in Indian subcontinent. They live in herds. Antlers of spotted deer are lyre-shaped and way of howling is primitive as compared to other species of deer. Spotted deer is very common in forests of , Sri Lanka, Nepal, , Bhutan, but it has very small population in

Pakistan (Duckworth et al. 2008).

Hog deer (Axis porcinus) is member of the family Cervidae. It is found in grasslands along the rivers. With slight variation in preference regarding habitat, hog deer prefer to live in flood plain grass lands, coastal grasslands, grasslands dominated by blady grass (Imperata cylindrica) and alluvial floodplain, lightly wooded and also mountain areas in India, Nepal,

Thailand, China, Vietnam and Bangladesh (www.wildlifeofpakistan.com dated 26-04-2015).

Biological diversity means variation in life forms like different types of plants, animals and microorganisms and diverse ecosystems where they live in. Change and destruction of ecosystem result in reduction of biodiversity of plants and animals in that area. It is common opinion of different Scientists that now a day’s world flora and fauna is becoming extinct faster than ever in the history. This loss will have far reaching negative impact on lives of human. So conservation of biodiversity is a major concern all over the world, today. It is crucial to safeguard remains of our biodiversity resources from the increasingly consumptive lifestyle and

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INTRODUCTION escalating urbanization overtaking countries and their wild areas. Unplanned human growth and settlements throughout the world are causing enormous pressure on wildlife resources.

We have some information about 1.5 million species but still a lot of them are to be defined. Increasing population and more consumption has resulted in loss of natural resources of earth at rate of 5% per decade (Seabury et al. 2004). This loss to biological wealth of earth is catastrophic and it has aesthetic, economic and ecological importance for us. This shocking damage of species and their abodes has awakened conservationists and other environmental activists all over the world to make efforts for conservation of this wealth around the globe by sharing knowledge and resources.

Comprising approximately 800000 km2, Pakistan is 7th largest country in Asia and could be divided into six bio-geographical regions (Shackleton 1997). Having diverse ecology in its wide spreading latitudinal and immensely varied altitudinal range, Pakistan comprises very important and remarkable ecological region of the world. This ecologically diverse region ranges from the mangrove forests bordering Arabian Sea to the snow- capped mountain tops at the amalgam of Western , Hindukush and Karakorum. The habitats of these regions provide sustenance to different of species of plants, mammals, , , amphibians, fishes and invertebrates. Collectively, they constitute biodiversity of Pakistan (Sheikh and Molur,

2004).

Pakistan has opulent number of species but population of most of wild animal species is declining in Pakistan because of illegal hunting and decreasing natural habitats.

Traditionally, Pakistanis are fond of hunting and development of modern weapons of hunting has further made it easy. All large mammals have declined with respect to their number and

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INTRODUCTION distribution. Some of the major causes of decreasing animal biodiversity in Pakistan are rapidly increasing human population, weak governance and poverty.

Population of a lot of mammal species has decreased during last two centuries because of human activities and the uncontrolled use of natural resources. Under the influence of human activities in present times, a lot of animal species have become extinct or they are in the danger of extinction. This extinction of animal species not only has ecological but also economic and social loss for human being. Along with aforementioned implications, wild life is also source of food and earnings for local people. So, management of wildlife is necessary for maintenance of genetic diversity and species survival. Small population size due to excessive hunting results in loss of genetic diversity that is required for acclimatization of a population in changing environmental conditions (Thevenon et al. 2004).

Different planning and actions are oriented towards restoration of such species that had plentiful populations in the past. One of the most common actions in this regard is trans-location of individuals from viable populations. This causes loss of genetic diversity. So, present day attention is focused to avoid or reverse the loss of genetic diversity in such populations or to establish populations with high genetically diversity in former areas (Leberg 1990).

It is consensus among the conservation geneticists that genetic diversity within and among populations is necessary for survival of different populations. The main reason of genetic depletion is when there is no genetic flow in populations of small effective size. Recent advancement in molecular biology techniques has helped to get some realistic data regarding structure and dynamics of population genetics. Some issues regarding practical utilization of this data are needed to be resolved. Reduced genetic diversity within (sub) populations and increased

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INTRODUCTION genetic diversity between different populations are two most important factors affecting the population structures (Hartl et al. 2003).

Management of wild animal resources without taking in consideration their population genetic structure usually leads to sudden reduction of genetic diversity of that animal population

(Markov et al. 2015). So, present day biological studies are needed to be focused on conservation of genetic diversity. Knowledge of genetic diversity helps in sustainable utilization of aforesaid animal resources and then maintenance of biological stability of species. Thus the assessment of the genetic diversity is needed for maintenance of sustainable hunting and conservation and improvement of genetic resources of animals of a specific population (Markov et al. 2015). For phylogenetic studies and species identification of different animals, Cytochrome-b along with other mitochondrial DNA markers has been used frequently in the recent times (Hsieh et al.

2001).

In this study different mitochondrial DNA regions were sequenced to identify the polymorphisms in two species of each of bovidae (Boselaphus tragocamelus and Antilope cervicapra) and cervidae (Axis axis and Axis porcinus) families and their phylogenetic relationships with other animals were constructed.

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CHAPTER 2 REVIEW OF LITRATURE

Because of having hair, three middle ear bones and mammary glands, mammals are endothermic amniotes and so are different from reptiles and birds. Brain of mammals controls temperature and the circulatory system. They have four-chambered heart. Mammals are biggest animals on earth. They are also one of the animals having high intelligence. They live on earth, sea and air also. Mammals are known to everyone on earth and are very important part of ecosystem. Improbable numbers of the mammalian species revealed astounding evolutionary pattern and mode of adaptation and has its own behavior and attitude on the earth. Mammals are the most important biotic component of the forest ecosystem (Webb and Bartlein, 1992).

Mammals are source of meat protein, milk protein, collagen, leather, bones and sinew for various tools from the time man was started hunting of these animals. Mammals are also used as means of transportation and for varying heavy articles and also pulling different thing like carts.

They are also considered as companion of man. So it may be said that domestication resulted in human civilization. With the human civilization, some of the mammals were domesticated for the production of meat and milk. With the experience regarding the production of different valuable product man can came to know that bovine, porcine, ovine and caprine meaning cattle, swine, sheep and goat species were important. Different types of domestic animals were later on selectively bred for their better traits in coming generations regarding production of different valuable products.

In Pakistan, Hog deer are limited to forests along the rivers, grasslands and especially in those areas having dense grass with scattered plants of Saccharum spontaneum, Saccharum munja and Tamarix dioica. Hog deer are native to the Indus eco-region and its habitat exists up

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REVIEW OF LITERATURE to Attock. Indus river forests reserves in Sind have good number of its population. The animal is breeding in captivity at the Khirthar National Park. Chashma Barrage Wildlife Sanctuary, Head

Islam/Chak Kotora Game Reserve, Lal Suhanra National Park , Wildlife Sanctuary of Taunsa Barrage and Rasool Wildlife Sanctuary are important habitats of Hog deer (Arshad et al. 2012).

Deer evolved some omnivores not having antlers that later on evolved into large herbivores having branched antlers. On the basis of paleontological and morphological information about deer family, Dremomeriinae group in Miocine period evolved into many subgroups like Dromomericinae and Lagomericinae (extinct). These branches further evolved into Odocoileinae and Muntiacinae (muntjaks) and they are believed to be ancestors of subfamily

Cervinae. Hydropotes genus of water deer intoseparate family Hydropotinae was made on the basis of some of the primitive features of these animals like absence of antlers. So, Hydropotes genus is believed to be most primitive genus of Cervidae family. Set of chromosomes of water deer was considered original primitive on the basis of reestablishment of the karyotype evolution.

Cervidae family comprises 90 species of the deer. These are found in tropical and sub- tropical zones of the world. Body weight of deer lies in between 30 to 300 kg. The average weight of smallest species (northern pudú) is 10 kg and of the largest species (the moose) is 431 kg. Such species are supple with solid bodies having long and powerful legs that are fit for rough terrains of woodlands. Deer can jump and swim excellently. They are ruminants, they chew cud and their stomach is four-chambered. They have teeth that are evolved for vegetation feeding.

They do not have upper incisors like other ruminants.

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REVIEW OF LITERATURE

Miller et al. (1992) established that deer were reason of damaging 98 threatened or endangered plant species. Enser (2002) stated that plant New blazing star was protected by fence in Rhode Island part of Block Island. American ginseng populations are being ruin utterly by deer in the central Appalachians. A research conducted by McGraw and Furedi (2005) established that seeds and fruits of many plant species are eaten by White-tailed deer. Majority of those seeds is able to germinate after excretion by deer. Williams and Ward (2006) during another research in Connecticut established that fifty seven species of plants were able to germinate from pellets of deer. Thirty two of these fifty seven species were exotic and few of these like autumn olive, wine raspberry, and multiflora rose were too much invasive. Authors concluded that this dense population was creating negative and long lasting effects on the forests.

Deer, grazing in such forests, have preferences for seedling of specific species. As a result species diversity is reduced at such an extreme level that mono cultures are created. So, hundreds of years are required to restore the forests to their original diversity. In this way deer are able to change the ecology of forest by altering the course of development of forest vegetation.

Sometime Deer have harmful effects on forest by damaging plant.

According to Mills (2007), keeping in view the role of genetic polymorphism in the survival and changing aspects of wildlife populations, four populations of red deer were studied in Brittany in order to assess the level of their genetic variability and genetic structure of populations. It was studied whether deer living in isolated forests were genetically penurious as compared to individuals living in large and unbroken forests in other areas of Western Europe.

They searched the effects of genetic isolation, due to shattering of habitat along with management actions taken to confine spreading of deer into smaller isolated forests. By taking

Chambord as reference, they were able to judge indigenous position of Brittany populations in

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REVIEW OF LITERATURE their study. At last, as a practical consequence of the conservation genetic work, their aim was to judge that either genetic makeup of Brittany’s populations permitted uncovering illegal activities like of hunted samples so that hunting quota could be fulfilled.

Hajji et al. (2007) stated that Killarney population has levels of polymorphism similar to those present in threatened red deer populations in Tunisia. Records of history regarding the

Killarney population show a bottleneck (Ryan et al. 2001). This happened before red deer were protected by Irish Wildlife Act of 1976. He further stated that on the basis of data available from

18th century onwards, it may be said that population of red deer remained small even after last population of wolfs was eliminated. So, no evidence regarding increase in population of red deer was available after elimination of the natural predator. Isolation of the remaining population in

Ireland, along with the non availability of data, might be causes of low levels of genetic diversity.

Ecosystem is prime one which gives benefits to humans by natural ways. It is no doubt that role of mammals in ecosystem is of utmost importance because they are involved in provision of some of the important services like dispersal of seeds, regulation of population of insects their role as indicator of health of ecosystem. Nonetheless, macroecological and macroevolutionary processes that are key stone for present and past patterns of biodiversity are only starting point to be explored worldwide. As far as crisis of worldwide extinction is concerned, it is also important to comprehend these processes so that this ecosystem related knowledge could be used to avoid future biodiversity and ecosystem services losses. Unluckily, different efforts made to have knowledge about biodiversity of mammals have been hindered due to unavailability of data. Integrated approaches based upon information regarding current species distributions, ecologies and evolutionary histories make it possible to understand this

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REVIEW OF LITERATURE biodiversity. On the basis of the above mentioned factors and investigation conducted on the basis of this data, we are able to explore evolution of biodiversity of mammals along with their past and present ecology and this may further help in hypothesizing future of the mammals.

The importance to maintain molecular biodiversity in animals is strongly advocated by

Food and Agriculture Organization. Identification of mammal species is essential for conservation of native genetic pools which is primarily to fill the unanticipated pure breeding demands in the future.

Pakistan is situated at the Western end of South Asian sub-continent and its fauna and flora are blend of indomalayan, Palearctics and Ethiopian regions and is rich in biodiversity.

More than two-third of the country is arid or semi-arid. Mammal’s fauna in Pakistan is diversified but not investigated yet. Among these, deer populations are the common mammals belonging to different families. However, a large population of the black buck species exists in

Khairpur’s Mehrano reserve, a private farm supported by the Sindh wildlife department. Some other places where black buck are found in Pakistan are zoos and different wildlife centers. Some people also have limited number of black bucks in their private forms. More than 1500 Black

Bucks were found in different breeding centers of Punjab and Sindh in Pakistan (Mallon 2008).

In the past, fossil records were also used for the deciphering of evolutionary track of organisms. Limitation of this approach is that fossils records can provide estimate of age on the basis of radioactive dating (Severin, 2006). In addition, biochemical and immunological methods were also used to characterize the lineage pattern. These methods use antibodies against soluble proteins that are species-specific. Therefore, these methods can only be used for such materials that contain extractable proteins. Another drawback of these methods is that only those species

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REVIEW OF LITERATURE can be identified for which antibodies are available. In addition, blood plasma and serum proteins and blood groups were also used for characterization (Severin, 2006).

Tamate et al. (1995 & 1998), Tamate and Tsuchiya (1995), Nagata et al. (1995) established that two distinct groups of sub-species among in their phylogenetic trees were the same as suggested by phylogenetic investigations. One of those was south western sika deer including C.n. mageshimae, C.n. pulchellus, C.n. keramae and C.n. Nippon and the second was Hokkaidu Honshu sika deer, including C.n. centralis and C.n. yesoensis. They were believed of having morphological features other than those of other sika deer within Japan. So they were able to be included as part of mainland sika deer (C. n. hortulorum) as suggested by Imaizumi

(1970).

According to Imaizumi (1970), Corbet (1978), Matsumoto et al. (1984) and Endo and

Gill (1996), there were conflicts about the taxonomy of the Tsushima population. Geist and

Sunderam (1992) stated that Artiodactyla’s comparative morphometry was not always important taxonomically. The reason was that morphological features of such animals may change significantly with conditions of the environment. Sika deer shows remarkable diversity regarding their body weight in north and south islands in Japan. Largest species being C. n. yesoensis, compared with smallest is C. n. yakushimae that have weight less than half of the formerly mentioned. So, in spite of the fact that subspecies of sika deer have remarkable differences regarding size of body and morphology, taxonomy of these subspecies has always been controversial. As it has already been described that there is no differences between Honshu centralis and Hokkaidu yesoensisas as far as mitochondrial DNA, 12S rRNA and cytochrome b sequences is concerned. Also sequences of both genes in the Tsushimacentralis are also clearly different from other two sub-species as stated by Nagata et al. (1995). Tamate and Tsuchiya

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REVIEW OF LITERATURE

(1995) while working on restriction site mitochondrial polymorphisms stated that genetic differences exist between Hokkaido island yesoensis, Honshu mainland centralis, Tsushima

Island centralis and yakushima of Yakushima Island. Restriction site pattern of Honshu centralis was regarded closer to yesoens than the observed patterns. These sub species were different from

Tsushima centralis and yakushimae. Tamate et al. (1998) described two phylogenetic groups of

Japanese sika deer on the basis of sequences of cytochrome b. Their names are northern and southern Hokkaido-Honshu and Kyushu groups respectively. So, molecular data validates previous classifications of sub-species of sika deer based on morphological features.

The distribution of C. elaphus since the last ice age generally appears continuous across

Europe and Asia, however, a study of conditions during past glaciations suggests otherwise.

Cervus species are thought to have originated in Asia and appeared in Europe with both coronate

(crown-like) and acoronate forms during the Gunz, Cromerian, Mindel, and Holsteinianinter glacials (Dolan, 1988), a time span from about 0.6 to 0.2 million years ago. Separation between the Asian and European forms was possibly provided by periods of glaciations (Webb and

Bartlein, 1992) that occurred about every 100,000 years. Records show that during the Riss or

Salian glacial period, from about 0.2 to 0.15 Mya, most of northern Europe, western Siberia, and northern Asia was covered with ice, steppe tundra, and desert, while southern Europe and southern Asia were dry steppe and grasslands (Adams and Faure, 1997). Connecting Europe and

Asia was a bridge of semi-desert or desert that would have restricted any movement of deer between the two regions. Although a warming phase took place during the Eemian interglacial

(0.13– 0.115 Mya), it was not until the last 10,000 years that Europe and Asia were reconnected with vegetation (Adams and Faure, 1997) that would have supported the exchange of deer.

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REVIEW OF LITERATURE

In addition to the traditional morphological and behavioral traits, molecular markers recently have been used to support taxonomic distinctions. Comparisons of allozymes and mitochondrial DNA (mtDNA) from European red deer and North American wapiti clearly indicate separation between the two groups (Dratch and Gyllensten, 1985; Dratch, 1986; Cronin,

1993; Polziehn et al. 1998). Although few protein variants were found to discriminate red deer from wapiti, the control region of mtDNA sequences region of red deer indicated the loss of a large repeat and about 5% sequence divergence from wapiti (Polziehn et al. 1998). The loss/gain of repeats in the mtDNA control region appears to be consistent among each Cervus species, and due to an absence of variation within individuals, the repeats themselves have been used for phylogenetic comparisons (Cook et al. 1999). Sampling from the ends of a cline, however, cannot address the possibility of a gradient in characters across the range of a species (Karl and

Bowen, 1999) or the randomness at which molecular markers are lost from a population (Avise,

1998). Discrete distributions of the molecular study based data would support the possibility of a species distinction if the distribution coincided with the ranges of wapiti or red deer. Currently, there is no molecular information available from most Asian subspecies to determine if a gradient of characters exists between Europe and North America.

Most imperious molecular technique used for the detection of genetic variation at DNA level opened the new dimensions of reserach. These include DNA hybridization, repetitive DNA sequences like microsatellites and DNA sequencing of both nuclear and mitochondrial DNA

(Carmela et al. 2000). It has been known since 1985 that mitochondrial DNA is a helpful marker for phylogeoraphy research and molecular biologist are continuously identifying different species on the basis of unique mitochondrial loci analysis. In comparison to the mitochondrial loci proved to be much sensitive indicators for mammalians species identifications. Genome

15

REVIEW OF LITERATURE analysis has been used by biologists for characterization of different genes and as molecular clocks (Bromham, 2003), and can even be used where no fossils records are available. As a molecular clock, it has a potential to define the time of divergence between closely related species and finally depict the molecular distance among the species.

In isolated populations which have low polymorphism, inbreeding and reduced fitness and, genetic structure is most important regarding species management. It has been proven that isolation has visible impact on their genetic diversity and also results in reduced mean number of alleles and heterozygosity (Webley et al. 2007). The consequences of impact of bottleneck have been reported in detail and historical records that is also matching, most of the times, with molecular technique based information (Broders 1999, Williams et al. 2002, Webley et al. 2004).

MtDNA has become most accepted marker for analysis of molecular based diversity for last three decades. It has been used because it is technically easy to use, it was thought to be clonal, near-neutral and clocklike nature of substitution rate. These facts may question the importance of mitochondrial DNA as evidence of recent speciation and population history.

Ballard and Rand (2005), due to its clonal, neutral, and clock like nature, mitochondrial

DNA has proved an ideal onlooker of population and species history. Maternally inherited genes of mitochondrial genome are involved in ATP metabolism and protein biosynthesis. This is the reason that mtDNA genetic diversity may have role in expression of several traits of individuals like growth rate and feed efficiency. According to Rokas et al. (2003) genome level methodologies have played an important role in the extrapolation of evolutionary relationships between different individuals. Tiny size of genome, rapid substitution rate and maternal inheritance are some of the distinguishing qualities of mitochondrial genome, contrary to nuclear genome, that make mitochondrial genome most favorite marker for study of phylogenetics and

16

REVIEW OF LITERATURE population genetics like investigation of gene flow, hybridization and introgression (Moore

1995; Wang et al. 2008).

A lot of phylogenetics of mammals are analyzed by using twelve peptide coding region of the mtDNA in the Artiodactyla but Cervidae (Cao et al. 1994, Ursing et al. 2000, Arnason et al. 2002). Another study regarding phylogeny analysis was conducted using cytochrome b (Cytb) gene, in different orders of eutherian animals including Cervidae and Bovidae, but they were not able to differentiate between Cervidae and Bovidae families (Irwin et al. 1991). A phylogeny analysis was performed by Honeycutt et al. (1995) by using Cytb & cytochrome c oxidase subunit II (COII) genes in different species of mammals, including Cervidae and Bovidae, and different topologies were observed between Cyt-b and COII genes. According to Irwin et al.

(1991), on the basis of study of Cytb gene, Cervidae and Bovidae were not differentiated. On the other hand, phylogenetic tree constructed on the basis of COII gene, clearly differentiated

Cervidae and Bovidae, Phylogenetic analysis based upon a single gene was considered untrustworthy as far as topology is concerned, particularly in situations when larger classifications between species, such as interorder or interfamily, was under consideration. The reason was different evolutionary rates between different mitochondrial genes. So, phylogenetic analysis of Cervidae and Bovidae using more mitochondrial genes was needed.

Wada et al. (2007) described whole mitochondrial genome nucleotide sequences of

Japanese Sika deer (Cervusnippon), Yeso (C. n. yesoensis), Honshu (C. n. centralis) and

Yakushima Sika deer (C. n. yakushimae).Their genomes sizes were 16543, 16663 and 16440 base pairs, respectively. Several tandem repeats observed in D-loop of mitochondrial genome of

Japanese Sika deer were different, ranging from 4 to 10 repeats each. About ten repeats were observed in Honshu Sika deer samples collected from Okutama city, Tokyo. This was unique

17

REVIEW OF LITERATURE number of repeats in Japanese Sika deer. Based upon this study, it was observed that complete nucleotide sequence similarity of DNA (mitochondrial), fluctuated from 94.1% to 96.7% while similarity of amino-acid sequence was ranged from 98.5% to 99.4%. A phylogenetic tree based on 13 peptide coding genes was constructed on the basis of nucleotide sequences and amino-acid sequence of thirteen peptide coding genes presented a topology of Cervidae, Bovidae and

Equidae with high bootstrap values. Cyt-b is mostly used in phylogenetic analyses of mammals.

Main issue is the difficulty in assessing the conservation status of the deer species, Boselaphus tragocamelus and Antilope cervicapra, which is partially caused by disagreements on whether

Antilope cervicapra should be divided into two species, more commonly known as red deer and wapiti (Bryant and Maser, 1982). Contributing to the current classification of Boselaphus tragocamelus, its geographic distribution and phenotypic cline that extend from Europe, across

Asia, and over to North America (Geist, 1987). Groves and Grubb (1987) categorized

Boselaphus tragocamelus sub-species into three contiguous groups. In contrast, Whitehead

(1972) and Cockerill (1984) maintained that a distinction between European red deer and

American wapiti corresponded to the geographic barriers presented by the Gobi Desert and the

Himalayan Mountains, and that the differences between the two deer groups were sufficient to raise the status of wapiti to Boselaphus tragocamelus.

Chikuni et al. (1995) and Matthee et al. (2001) observations based upon the modern molecular genetics studies, recommend close relation of Cervidae with Bovidae then with

Moschidae. But Giraffidae family appears more distant from Cervidae and closer to

Antilocapridae. On the hand, Chikuni et al. (1995) and Matthee et al. (2001) based upon larger number of mammals and using different genes, observed that Giraffidae and Cervidae were joined as stated by Gatesy and Arctander (2000), Beintema et al. (2003) and Hassanin and

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REVIEW OF LITERATURE

Douzery (1999). Investigations by Buntjer et al. (1998) and Lee et al. (2002) by using microsatellite DNA and by Potapov et al. (1997) and Comincini et al. (1996) by using RAPD–

PCR showed that mtDNA genomes of different individuals of Odocoileinae sub-family reveal more dissimilarity from each other as compared to the genomes of species belonging to subfamily Cervinae. Kraus and Miyamoto, (1991) stated that Phylogenetic relationships in

Cervidae family, that was deduced on the basis of study of nucleotide sequences of ribosomal

RNA genes of mitochondria and amino acid sequences, showed the origin of this family from single parents. Though, information regarding phylogenetic relationships between subfamilies and genera, those fall in divergent orders within the family, is somewhat controversial.

Information based upon control mtDNA advocate origin of subfamilies Cervinae and

Odocoileinae from same ancestors but study of Cytochrome-b gene of mitochondria didn’t upkeep theory based on origin of telemetacarpal deer from common ancestors (Douzery and

Randi (1997). On the basis of information available till recent times, Odocoileinae comprises two clades, one of those include genera, Alces of moose, Hydropotes of water deer, and

Capreolus of roe deer. Second clade includes, genera Odocoileus of American deer, Mazama of mazama, and Rangifer of reindeer (Ref). Genus Hydropotesis a close relative of Capreolus and is also included Odocoileinae. Randi et al. (1998) showed lack of antlers in the water deer is not due to original primitive state but result of secondary reduction. Later on reported phylogenetic trees by Douzery and Randi (1997) were obtained on the basis of different genes like three mtDNA and four nuclear ones and those didn’t differ from the phylogenetic trees as constructed by Randi et al. (1998). But investigation by Douzery and Randi (1997 and Randi et al. (1998) analysed the same genes and placed the musk deer (Moschus) in Bovidae family.

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REVIEW OF LITERATURE

Population of most of mammalian species has decreased during last two hundred years due to different kind human activities. These kerfuffle by humans resulted in destruction and loss of habitat due to unmanageable utilization of natural resources. So, it was needed to restore those populations that were abundant in the past. This goal was tried to be achieved by trans-locating individuals of these populations to some suitable habitats. Another issue is the low genetic diversity appeared as a result of these actions. So, currently efforts regarding restoration of such animals were based upon reversing the loss of genetic diversity in these populations in the areas that were formerly occupied by these populations (Leberg 1990). It was not possible during translocation programmes, initiated in the past, to establish link between genetic variability, stock source and number of founders. It was observed after such restoration efforts that most of such populations had to pass through genetic bottlenecks, founder effects or had to suffer extended periods of small population sizes. Managers of such programs are worried about negative impacts of low genetic diversity on the fitness and viability such populations in future

(Allendorf and Leary, 1986).

Approximately after every 700 bp in Bostaurus and every 300 bp in Bosindicus cattle a single nucleotide polymorphism (SNP) takes place (The Bovine Genome Sequencing and

Analysis Consortium, 2009). SNP is a single nucleotide variation at a particular location of the

DNA in the genome of any individual. And this polymorphism is more than 1% of the population (Brookes, 1999). Almost more than two million single nucleotide polymorphism

(SNPs) have been found in the genes of the cattle which would provide infinite DNA variation advancing the discovery of causal mutations for quantitative traits (Sellner et al., 2007). By making utilization of next-generation sequencing, Van Tassel et al. (2008) reported as more as

23000 SNPs in the SNP collection of bovine populations. This study was based on sixty six

20

REVIEW OF LITERATURE cattle from different lines of Holstein, other dairy breeds, and beef breeds that are most common, such as Angus, Red Angus, Charolaise, Gelbvieh, Hereford, Limousine and Simmental (Hayes et al. 2009). These SNPs, along with other reported variations, were used to assemble arrays with up to fifty thousand SNPs and were applied for genomic selection (VanRaden et al., 2009).

With the help of high throughput technologies, genotyping of large amount of SNPs from the whole genome has become possible (Seide, 2010). Development of bovine SNP chips is the main example of the high throughput technology. Bovine SNP50 Genotyping Bead Chip from the company Illumina is the most commonly used for cattle (Illumina Inc., San Diego, CA, USA). It contains 54690 evenly spread out SNP based probes that span all the genome of bovine (Illumina

Inc., San Diego, CA, USA). It was assessed that, in all genome of cattle, more than thirty million

SNPs would be needed to attain optimal marker coverage Goddard and Hayes (2009). In addition, Hawken et al. (2004) reported 17,344 SNPs in coding-region of bovine through the analysis of expressed sequence tags (ESTs). Sequencing of whole human genome facilitated the positional cloning of genes that were responsible for different diseases (Lander et al. 2001).

More than 1500 genes were documented but success was inadequate for infrequent diseases developed due to genetic mutations. Study of complex diseases needed approach based on whole-genome association by using thousands of genetic markers and samples. On the basis of fact that these genetic markers were already identified and validated, so the project of

“HapMap” was initiated in order to confine genetic variation and providing haplotype information (Sachidanandam et al. 2001). The second stage of this project has created more than

3.1 million SNPs even though the real number is in the region of 10 million (Altshuler et al.

2005). By combining the technologies developments, enlightened genomic deviation in databases was utilized by different companies to create low-priced and high turnout genotyping

21

REVIEW OF LITERATURE products which are appropriate for study of linkage and association of whole genome. These technologies were used in well- planned studies to meet the criteria regarding establishment of positive replication report (Chanock et al. 2007). For studies covering the whole genome, maximum SNPs are required for the developments of technologies were assay call and error rates concordance with presented data should be reported.

Genotyping technologies important in genetic research are Illumina Infinium Beadchips,

Affymetrix GeneChip; Perlegen and Invader. The systems for appropriate findings and to approaches the candidate gene or linkage analysis are Affymetrix Gene Chip, Molecular

Inversion Probes, Goldengate by Illumina and Infinium assays, Invader, Sequenom Mass Array;

SNP stream, SNPlex, Centaurus and Taqman assays.

Perlegen Method of genotyping has established genotyping assay on the basis of

Affymetrix designs and chips. So, Perlegen is able to use not only perfect match or mismatch probes analogous to Affymetrix but also the strips that probe through the SNP location in forward as well reverse strand orientations. Interrogating the position of SNP, it moves in 25 bp probes by 4 bp between two positions (Hinds et al. 2004). A chain of long-range PCR reactions is involved to prepare the target DNA that amplifies the loci having the SNPs from each sample.

The biotin-ddUTP/dUTP and terminal deoxynucleodityl transferase are used to label the PCR products. Patil et al. (2001), confocal scanner of high resolution is applied to identify the hybridization. The large number of interrogated SNPs might be possible by means of Affymetrix

GeneChip but Perlegen has monitored 220 k SNPs because of target preparation approach based upon PCR and high pass rate (Kooner et al. 2008).

Cytochrome b and D-loop parts of mitochondria are potent markers that can be used for characterization of different genetic resources (Goldstein & Pollock, 1997). According to Babar

22

REVIEW OF LITERATURE et al. (2015), as for as diversity between species is concerned, mtDNA show high and distinguishing mutations rate, but on the other hand those are very rare within species,

Cytochrome b and D-loop parts of mitochondria have their role as markers in describing milk production and solving forensic cases relating to conflicts of parentage.

Statement of the Problem

The main objectives of this research were:

1) To assess the polymorphisms in mitochondrial Cytochrome-b gene in bovidae

(Boselaphus tragocamelus and Antilope cervicapra) and cervidae (Axis axis and Axis

porcinus) in Pakistan.

2) To identify the polymorphisms in mitochondrial Cytochrome-c gene in bovidae

(Boselaphus tragocamelus and Antilope cervicapra) and cervidae (Axis axis and Axis

porcinus) in Pakistan.

3) To study polymorphisms in mitochondrial D-loop region in bovidae (Boselaphus

tragocamelus and Antilope cervicapra) and cervidae (Axis axis and Axis porcinus) in

Pakistan.

4) To analyse diversity and construct mitochondrial Cytochrome-b, Cytochrome-c and D-

loop based phylogenetic trees of bovidae (Boselaphus tragocamelus and Antilope

cervicapra) and cervidae (Axis axis and Axis porcinus) of each with other animals.

23

CHAPTER 3 MATERIALS AND METHODS

Present investigation was planned to perform molecular phylogeny and diversity analysis of Pakistani bovidae and cervidae families using mitochondrial DNA markers. Research was conducted at Molecular Biology and Genomic Laboratory, Institute of Biochemistry and

Biotechnology, University of Veterinary and Animal Sciences, Lahore.

Selection of animals:

Four species of animals, two each from Bovidae and Cervidae family were selected for this study. A total of hundred (Twenty five from each selected species of the Bovidae and

Cervidae) were selected for sampling. The selection was purely based upon phenotypic characteristics. Sampling was done from natural habitats, zoos, parks, wildlife parks and captive breeding centers at different places of Pakistan. Distances among sampling area reflect that animals are unrelated

Collection of fecal samples:

Approximately 20 g of fecal samples were collected from above the mentioned natural habitats, parks, zoos and captive breeding centres using disposable plastic gloves and stored in

95% ethanol using a polypropylene bottle at room temperature. Name and code of each animal was marked on each sample. Fecal sampling was done for animals in captive breeding where they were kept individually. Blood sampling was done for animals living in groups to avoid ambiguity.

24

MATERIALS AND METHODS

Collection of blood samples:

Blood sample from each animal was collected aseptically into EDTA (Ethylenediamine tetra acetic acid) coated tube. In order to prevent coagulation of blood, tubes were inverted up and down multiple times so that EDTA could mix completely with blood. Blood samples were shifted to ice immediately in field and then transferred to the laboratory. During sampling few things were considered, from one place only single sample was taken, sampling was conducted in intervals after every fresh arrival at and finally more samples were collected from individual captive breeding centres. So animals were totally unrelated (for record: for atleast three generations).

Storage of DNA samples:

When reached in the laboratory, list of all collected samples of each species was made.

Collected samples were temporarily stored at -20oC before the DNA extraction.

25

MATERIALS AND METHODS

Figure 3.1: Map of Pakistan showing selected areas for sampling of Boselaphus tragocamelus

(Blue bull).

The fig was modified from http://www.ezilon.com (20-4-2015).

26

MATERIALS AND METHODS

Figure 3.2: Map of Pakistan showing selected areas for sampling of Antilope Cervicapra (Black buck).

The fig was modified from http://www.ezilon.com (20-4-2015).

27

MATERIALS AND METHODS

Figure 3.3: Map of Pakistan showing selected areas for sampling of Axis axis (Spotted deer).

The fig was modified from http://www.ezilon.com (20-4-2015).

28

MATERIALS AND METHODS

Figure 3.4: Map of Pakistan showing selected areas for sampling of Axis porcinus (Hog Deer).

The fig was modified from http://www.ezilon.com (20-4-2015).

29

MATERIALS AND METHODS

Table 3.1: Sampling details of samples of all Four Animals used in this study (Year Span= March’2012 to October’2014) a) Boselaphus tragocamelusa

Sr. Number No Samples Source Google Coordinates of . Samples 1 BTa5 BT17 29°24'8.7"N 71°40'54.5"E 2

2 BT7, BT23 Bahria Town Lahore 31°18'51.5"N 74°12'11.7"E 2 BT10, BT19, 3 Bahria Town Rawalpindi 33°29'45.2"N 73°6'20.3"E 3 BT20 4 BT8 Basti Bahadurpur Multan 30°15'27.9"N 71°29'48.2"E 1

5 BT2 Kasur 31°5'19.3"N 73°57'44.7"E 1

6 BT4 Charagh Abad, T T Sing 31°20'6.3"N 72°46'2.4"E 1 Gatwala Wildlife Breeding 7 BT15 BT22 31°28'42.7"N 73°12'36.7"E 2 Centre, Faisalabad 8 BT16 Lahore , Lahore 31°22'53.9"N 74°12'41.6"E 1

9 BT18 , Lahore 31°33'22.7"N 74°19'34.0"E 1 Lal Suhanra National Park 10 BT9, BT11 29°19'1.4"N 71°54'16.4"E 2 Bahawalpur Lohi Bher Wildlife Park 11 BT14, BT21 33°57'49.5"N 73°11'93.1"E 2 Rawalpindi 12 BT24 Indo-Pak border, Bahawalnagar 29°59'57.1"N 73°15'31.8"E 1

13 BT25 BT3 Peerowal Khanewal 30°20'22.7"N 72°2'2.4"E 2

14 BT1 BT6 Head Balloke Raavi River 31°11'25.9"N 73°52'32.6"E 2

15 BT13 Vehari Wildlife Park Vehari 30°2'14.7"N 72°21'2.6"E 1 Wildlife Park Kamalia, T T 16 BT12 30°42'52.9"N 72°40'25.9"E 1 Sing TOTAL 25

30

MATERIALS AND METHODS b) Antilope cervicaprab

Sr. Number No Samples Source Google coordinates of . Samples 1 ACb18,AC19 Bahawalpur Zoo, Bahawalpur 29°24'8.7"N 71°40'54.5"E 2

2 AC13, AC21 Bahria Town Lahore 31°18'51.5"N 74°12'11.7"E 2

3 AC20, AC12 Bahria Town Rawalpindi 33°29'45.2"N 73°6'20.3"E 2

4 AC16 Basti Bahadurpur Multan 30°15'27.9"N 71°29'48.2"E 1

5 AC9 Changa Manga, Kasur 31°5'19.3"N 73°57'44.7"E 1

6 AC24 Charagh Abad, T T Sing 31°20'6.3"N 72°46'2.4"E 1

7 AC2 Peerowal Khanewal 30°20'22.7"N 72°2'2.4"E 1

8 AC25 Khangur Ghotki, Sind 27°52'10.8"N 69°25'2.5"E 1

9 AC6 AC10 Kirthar National Park, Sind 25°41'27.8"N 67°31'23.4"E 2

10 AC3 Lahore safari park 31°22'53.9"N 74°12'41.6"E 1

11 AC5 Lahore Zoo, Lahore 31°33'22.7"N 74°19'34.0"E 1

12 AC15, AC11 Lal Suhanra National Park 29°19'1.4"N 71°54'16.4"E 2 Lohi Bher Wildlife Park 13 AC4 33°57'49.5"N 73°11'93.1"E 1 Rawalpindi Mir of Khairpurs Mehrano 14 AC17 27o16’47.1"N 68o40’32.6"E 1 reserve, Sind 26o47’37.6"N 67o 15 AC7 New Jatoi, Nawab Shah, Sind " 1 59’28.0 E 16 AC1 AC23 Rajoa Saadat, 31o38’35.5"N 72o58’26.7"E 2

17 AC22, AC14 Head Balloke Raavi River 31°11'25.9"N 73°52'32.6"E 2 Wildlife Farms Raiwind Road 18 AC8 31°23'5.5"N 74°14'8.5"E 1 Lahore TOTAL 25

31

MATERIALS AND METHODS c) Axis axisc

Sr. Number No Samples Source Google coordinates of . Samples 1 AAc1, AA10 Bahawalpur Zoo, Bahawalpur 29°24'8.7"N 71°40'54.5"E 2

2 AA6, AA7 Bahria Town Lahore 31°18'51.5"N 74°12'11.7"E 2

3 AA4, AA5 Bahria Town Rawalpindi 33°29'45.2"N 73°6'20.3"E 2

4 AA11, AA12 Basti Bahadurpur Multan 30°15'27.9"N 71°29'48.2"E 2

5 AA22 Shabbir Abad, 31°23'29.9"N 72°24'33.1"E 1

6 AA14, AA15 , Karachi 24°52'32.9"N 67°1'22.7"E 2

7 AA16, AA17 Lahore safari park, Lahore 31°22'53.9"N 74°12'41.6"E 2

8 AA19, AA18 Lahore Zoo, Lahore 31°33'22.7"N 74°19'34.0"E 2 Lohi Bher Wildlife Park 9 AA20, AA2 33°57'49.5"N 73°11'93.1"E 2 Rawalpindi 10 AA13 , Jhnag 31°23'14.9"N 72°27'50.7"E 1

11 AA3 Peerowal Khanewal 30°20'22.7"N 72°2'2.4"E 1 AA23, 12 Rana Resort Head Balloke 31°11'25.9"N 73°52'32.6"E 3 AA24, AA25 13 AA8, AA21 Wildlife farms Lahore 31°23'5.5"N 74°14'8.5"E 2

14 AA9 Wildlife Park Vehari 30°2'14.7"N 72°21'2.6"E 1

TOTAL 25

32

MATERIALS AND METHODS d) Axis porcinusd

Sr. Number No Samples Source Google coordinates of . Samples 1 APd24, AP4 Bahawalpur Zoo, Bahawalpur 29°24'8.7"N 71°40'54.5"E 2

2 AP22, AP5 Bahria Town Lahore 31°18'51.5"N 74°12'11.7"E 2

3 AP10, AP12 Bahria Town Rawalpindi 33°29'45.2"N 73°6'20.3"E 2

4 AP16 Basti Bahadurpur Multan 30°15'27.9"N 71°29'48.2"E 1

5 AP21 Shabbir Abad, Jhang 31°23'29.9"N 72°24'33.1"E 1

6 AP19 Changa Manga, Kasur 31°5'19.3"N 73°57'44.7"E 1 Gatwala Wildlife Breeding 7 AP3, AP8 31°28'42.7"N 73°12'36.7"E 2 Centre, Faisalabad 8 AP9, AP11 Gulshan-e-Iqbal Park, Lahore 31°30'46.2"N 74°17'31.2"E 2

9 AP17, AP20 Lahore safari park, Lahore 31°22'53.9"N 74°12'41.6"E 2

10 AP23, AP2 Lahore Zoo, Lahore 31°33'22.7"N 74°19'34.0"E 2 Lohi Bher Wildlife Park 11 AP6, AP7 33°57'49.5"N 73°11'93.1"E 2 Rawalpindi 12 AP13, AP15 Peerowal Khanewal 30°20'22.7"N 72°2'2.4"E 2

13 AP18, AP25 Head Balloke Raavi River 31°11'25.9"N 73°52'32.6"E 2

14 AP14 Wildlife Park Vehari 30°2'14.7"N 72°21'2.6"E 1 Taunsa Barrage Wildlife 15 AP1 30°30'46.4"N 70°50'58.9"E 1 Sanctuary TOTAL 25

33

MATERIALS AND METHODS

Figure 3.5: Photograph of Boselapus tragocamelus Antilope cervicapra (Bovidae), Axis axis and

Axis porcinus (Cervidae)

34

MATERIALS AND METHODS

Extraction of DNA from Fecal samples:

Fecal DNA was extracted following Zhang et al. (2006) using the reagents and protocol are given below;

1. 1–1.5 g of feces material was weighed into a 15 mL centrifuge tube.

2. The material was vortexed and washed using 5 mL ethanol, then centrifuged (4000 × g, 2

min) to pellet the fecal particle and the supernatant was discarded. The washing step was

repeated once using 5 mL TE (10 mM Tris, 1 mM EDTA, pH 8).

3. Three mL TNE (10 m mol/L Tris-Cl, 0.5% SDS, 1 m mol/L CaCl2) and 50 μL Proteinase

K (20 mg/mL) were added to the centrifuge tube, and the whole was incubated at 55◦C

for 1–2 hour(s).

4. The lysate was centrifuged (4000 × g, 1 min) to pellet the fecal particle.

5. The supernatant was then transferred into a new 15 mL centrifuge tube containing 3 g

potato starch (Sigma).

6. The tube was continuously vortexed for 1 min to suspend the starch completely, and the

suspension was incubated for 1 min at room temperature. The starch tube was centrifuged

(8000 × g, 3 min) to pellet the starch.

7. Then 600 μL supernatant was pipetted into a new 2 mL centrifuge tube, to which 150 μL

NaCl solution (3.5 mol/L) and 250 μL CTAB solution (0.7 M NaCl, 10% cetyl trimethyl-

ammonium bromide, CTAB) were added, followed by incubation at 70◦C for 10 min.

8. The mixture was extracted twice using an equal volume of phenol: chloroform: isoamyl

alcohol (25:24:1), the supernatant was then transferred to a new tube.

35

MATERIALS AND METHODS

9. An equal volume of binding buffer (4 M guanidine hydrochloride, 1 M potassium acetate,

pH 5.5) was added to the tube, mixed gently, and applied to a spin column (from EZNA

Cycle-pure kit, Omega; loading Ultra free-MC 30000 filter membrane, Millipore)

10. Then loaded in a 2 mL microcentrifuge tube and centrifuged at 6000 × g for 30 s.

11. Subsequently, the filter membrane was washed twice by centrifuge (8000 × g, 1 min)

using 750 μL 75% ethanol.

12. The DNA was eluted with 200 μL TE, and 50 μg/mL RNase was added.

Extraction of DNA from blood samples:

DNA was extracted from blood samples by inorganic method (Sambrook and Russel,

2001) for PCR amplification. The detailed stepwise methodology adopted for DNA extraction is described below:

13. One mL of T.E. lysis buffer (Tris HCl 10mM, EDTA 0.2mM) was added to 0.5 mL

of blood and was mixed by vertex for 3-5 minutes.

14. The tubes were centrifuged at 3500 rpm for 5 min at 4o C.

15. Supernatant was discarded and the pellet was obtained.

16. Then pellet was suspended in 200 l T.A.E. (Tris acetic acid EDTA) buffer

17. Steps # 2 & 3 were repeated, until pellet became light pink.

18. Supernatant was discarded after last washing with lysis buffer. 25L of 10% SDS

(Sodium dodecyl sulfate) , 10g proteinase K (50 L of 10µg/uL conc.) and 5 mL

BufferA1 was added to the pellet, shacked and incubated overnight in water bath at

37oC for complete digestion of cellular proteins.

36

MATERIALS AND METHODS

19. 500 L of PCI (phenol chloroform isoamyle alcohol) was added in the digested

samples and contents were emulsified by inversion.

20. These samples were centrifuged at 3715 × g (4150 rpm for 15 minutes).

21. After centrifuge, three layers appeared. Aqueous layer containing DNA appeared on

upper side while on lower side appeared layer containing phenol and proteins.

Middle whitish layer was of proteins. Upper layer containing DNA was transferred

to 1.5 mL fresh Eppendorf tube.

22. Equal volume of Isopropanol was added and the tubes were inverted gently till

DNA threads were visible and tubes were kept at room temperature for 10 minutes.

23. The contents were centrifuged at 3715 × g (4150 rpm for 5 min) at 0o C and

supernatant was discarded carefully.

24. DNA pellet was washed with 200 L of 70% ethanol for 2-4 hours on shaking stand

and then centrifuged at 3715 × g (4150 rpm for 5 min) at 0o C.

25. 70% ethanol was discarded carefully saving the DNA pellet.

26. Washing with 200 L of 100% ethanol was done for 2-4 hrs on shaking stand.

27. The DNA pallet was air dried at 37o C in an incubator or at room temperature until

the smell of ethanol ends.

28. 500-1000 μL low T.E. buffer (Tris HCl 10mM, EDTA 0.2mM) was added

according to amount of DNA pellet. At this step DNA was dissolved in the low T.E.

buffer by gentle shaking.

29. Tubes were placed in a shaking water bath at 70°C for 30 minutes to inactivate

nucleases.

37

MATERIALS AND METHODS

DNA Quantification:

DNA quantification of the samples was done using a Nanodrop (Thermoscientific,

Wilmington USA). All samples were brought at same concentration level i.e. 50 ng/uL of low

T.E. buffer.

Primers Designing and Synthesis:

Reference sequences of complete mitochondrial genome including d loop, cytochrome b and Cytochrome c regions for Axis axis (Accession No NC_020680), Axis porcinus (Accession

No. JN632600), Boselaphus tragocamelus (Accession No.NC_020614) and Antilope cervicapra

(Accession No.NC_012098) were taken from NCBI (www.ncbi.nlm.nih.gov). Sequences were aligned by the Molecular Evolutionary Genetics Analysis (MEGA) software to design primers from loci of interest. Close homology among the sequences of each loci allowed designing of a common pair of suitable primers for each locus, hence three pairs of primers were designed for complete amplification of three loci. Moreover, two additional forward primers were also designed for the purpose of sequencing of cytochrome c. All primers were designed using the primer blast of NCBI (www.ncbi.nlm.nih.gov) and synthesized from Genelink, USA.

38

MATERIALS AND METHODS

Table 3.2: List of primer sequences.

Primer Locus PRIMER SEQUENCE (5’ TO 3’) Product TA Name size (bp) (°C)

CytoB-F Cytochrome-b GTCATTCAACTACAAGAACACTA 1289 510C

CytoB-R Cytochrome-b TAAATAGAACTTCAGCTTTGGG 510C

CytoC- Cytochrome-c GCTTCAATCTACTTCTCCCG 1761 530C

F1

CytoC-R Cytochrome-c GTGGTTATGATGTTGGCTTG 530C

CytoC- Cytochrome-c CACCTRGCAGGYGTCTC 1242 530C

F2

CytoC- Cytochrome-c GATCACCTGCYATAATATGAGC 650 530C

F3

DLF D- Loop AGCCTCACTATCAACACCCA 1020 540C

DLR D- Loop CACATAGGTTTGGTCCCAGC 540C

Optimization of PCR conditions:

Primers were optimized for their annealing temperatures for successful Polymerase

Chain Reaction (PCR). Initially, all the primers were amplified by a temperature gradient PCR in which a range of annealing temperature (500 C- 600 C) was used in the Bio-Rad Thermocycler.

The temperatures at which primers of different markers showed best results were observed and selected. The subsequent PCRs were carried out at optimized annealing temperatures.

39

MATERIALS AND METHODS

DNA Amplification:

PCR was performed using Bio-Rad Thermocycler. All DNA samples were amplified using locus specific primers. Each pair of primers was used in amplification of each DNA sample of selected species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus). Amplification reactions were carried out at temperatures, specific for each primer given in Table-3.2. Amplification was performed with a final reaction volume of 25 µL, which included forward and reverse primers, dNTPs (Deoxy Nucleoside Tri-Phosphates), Taq buffer A,

DNA Taq Polymerase (Roche Diagnostics, Basel, Switzerland), double distilled Deionized water and DNA. Amplification of DNA samples was done for each pair of primer at a time. A master mixture for amplification reaction, having all the components, was prepared fresh before every

PCR reaction.

Table 3.3: Recipe for each multiplex of Primers

No. Reagents Quantity Concentration

1 DNA (Sample) 2 L 50ng / L

2 PCR Buffer 3L 10X

3 dNTPs 3L 2.5mM / L

4 Forward Primer 1L 10 mM / L

5 Reverse Primer 1L 10mM / L

6 Taq Polymerase 1 L 5U / L

7 Deionized Water 14L NIL

Total reaction volume = 25 L

PCR was carried out in four steps. First step was initial denaturing at 95oC for 2 minutes.

Second step comprised of 30 cycles each for 30 seconds at 95oC for denaturation, 30 seconds at

40

MATERIALS AND METHODS

51oC-54oC (as optimized per locus) for primer annealing and 2 min at 72o C for extension. Third step was final extension for 10 min at 72o C and Fourth step was maintaining the final temperature at 4oC until the collection of PCR products from Thermocycler.

PCR products along with 1 kb ladder were run on 1.2 % agarose gel at 100 volts for 35 minutes to visualize the bands of amplified products. Amplification bands of all samples were visualized and compared with standard ladder in UV light chamber and pictures were saved in gel documentation system.

Gel Electrophoresis:

To detect the PCR product by gel electrophoresis, DNA grade Agarose, 1X TAE Buffer

(pH-8.0), Ethedium bromide, Gel loading dye (Bromophenol blue), Molecular weight marker,

Gel electrophoresis equipment, UV- Transilluminator, Gel documentation system were used.

Precipitation of amplicons:

Amplicons were run on 1.2 % agarose gel followed by precipitation.

Protocol used is given below;

30. Full volume of amplicon and 100 ul 80% ethanol were poured in 1-5 mL tube.

31. Mixture was placed in dark for 1 hour.

32. This incubated mixture was centrifuged 2300 x g (5000 rpm for 15 minutes)

33. Supernatant was discarded and waited till pellet was dried.

34. Dried pellets were then dissolved in nuclease free water.

41

MATERIALS AND METHODS

Sequencing the PCR products:

Precipitated PCR products were sequenced using dye labelled dideoxy terminatore sequencing using ABI Genetic Analyzer 3130 XL (Applied Biosystem Inc., Foster city, CA,

USA) with standard protocols. Following reagents were used;

Table 3.4: PCR reaction composition for sequencing products

No. Reagents Volume

1 DNA 2 L (50ng/uL)

2 Big dye sequencing mix 3L

3 Primer 3L

4 5X buffer 1L

5 Total 9L

Table 3.5: PCR reaction conditions for sequencing products

No. Steps Temperature Time

1 Initial denaturation 95oC 1 min

2 Denaturation 96oC 30 sec

3 Annealing 90oC 15 sec

4 Extension 60oC 4 min

5 Repeat step 2-4 for 35 cycles

6 Final Extention 60o C 5 min

Multiple amplifications (30X) from the original samples were done to minimize the possibility of identifying PCR-induced mutations.

42

MATERIALS AND METHODS

Precipitation of sequencing products:

Ethanol was used for precipitation sequencing products. 40 L of 75% ethanol was added to each 10 L reaction to final concentration of 60%. The reaction mixtures were mixed using vortex and then were left for room temperature for 20 minutes. Then, these were centrifuged at

16000 x g (14000 rpm for 20 minutes) at 4oC. The supernatant was discarded and pellets were washed with 100 L of 70% ethanol. Then pellets were dried. After this pellets were dissolved in

15 L of deionized water before loading in ABI Genetic Analyzer 3130 XL according to manufacturer’s instructions given in technical manual. After each run samples were analyzed using ABI PRISM sequencing analysis software version 3.7.

Analysis of the Sequences:

Alignment of the sequences was done using NCBI. Alignments of sequences were done with the help of Blast 2 sequences and Clustal W (Thompson et al. 1994). The Single Nucleotide

Polymorphisms (SNPs) were detected in sequences. Maximum Parsimony Tree and Bayesian

Phylogenetic tree was prepared by using MEGA 6 (Tamura et al. 2013). Statistical analysis was done by using Bioconductor in R (Gentleman et al. 1999) that helped in determining the status of our selected individuals regarding their evolutionary history as well as marker significance by neutralizing the effect of environment.

Sequence alignment and phylogenetic analysis:

The mitochondrial d-loop, cytochrome b and cytochrome c region sequences of selected species of was aligned with reference sequences using the ClustalW (Thompson et al. 1994) multiple alignment program of MEGA 6 (Tamura et al. 2013), which automatically applied the gap and slide alignments. The data were then converted to FASTA formats, and sites at which

43

MATERIALS AND METHODS the primary homology was observed, manually excluded for future analysis. The mitochondrial d-loop, cytochrome b and cytochrome c gene sequences (separately and combined) were subjected to three types of phylogenetic analysis using the MEGA 6.0 software package (Tamura et al. 2013) the distance based Neighbor-Joining (NJ) (Saitou and Nei, 1987) and minimum evolution (ME) (Rzhetsky and Nei, 1992) methods using the Kimura-2-Parmeter distance measurement, and an equally weighted maximum parsimony (MP) analysis (Fitch 1971), was performed using the maximum likelihood (ML) algorithm.

Work Place

Molecular Biology and Genomic Laboratory, Institute of Biochemistry and

Biotechnology, University of Veterinary and Animal Sciences, Lahore.

44

CHAPTER 4 RESULTS

Phylogeny and Diversity Analysis

1. Boselaphus tragocamelus 1.1 Mitochondrialcytochrome-b gene based analysis 1.2 Mitochondrial cytochrome-c gene based analysis 1.3 Mitochondrial d-loop region based analysis 1.4 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis 2. Antilope cervicapra 2.1 Mitochondrialcytochrome-b gene based analysis 2.2 Mitochondrial cytochrome-c gene based analysis 2.3 Mitochondrial d-loop region based analysis 2.4 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis 3. Axis axis 3.1 Mitochondrialcytochrome-b gene based analysis 3.2 Mitochondrial cytochrome-c gene based analysis 3.3 Mitochondrial d-loop region based analysis 3.4 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis 4. Axis porcinus 4.1 Mitochondrialcytochrome-b gene based analysis 4.2 Mitochondrial cytochrome-c gene based analysis 4.3 Mitochondrial d-loop region based analysis 4.4 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis 5. Boselaphus tragocamelus &Antilope Cervicapra and Axis Axis & Axis Porcinus 5.1 Mitochondrial cytochrome-b gene based analysis 5.2 Mitochondrial cytochrome-c gene based analysis 5.3 Mitochondrial d-loop based analysis 5.4 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis of Boselaphus tragocamelus & Antelope cervicapra (Bovidae) 5.5 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis of Axis axis & Axis porcinus (Cervidae) 5.6 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis of Boselaphus tragocamelus & Antelope cervicapra (Bovidae) and Axis axis & Axis porcinus (Cervidae)

45

RESULTS

1. Phylogeny and Diversity Analysis of Boselaphus tragocamelus

1.1 Phylogenetic and diversity analysis of Boselaphus tragocamelus using cytochrome-b Gene

1.1.1 Identification of polymorphisms in Cytochrome-b gene of Boselaphus tragocamelus

Polymorphisms were identified in Cytochrome-b gene of Boselaphus tragocamelus as given in Table 4.1. Allele frequency was calculated. As no heterozygosity was observed so genetypic frequency was found same as allele frequency.

Table 4.1: Polymorphisms identified in cytochrome-b gene of Boselaphus tragocamelus

Change in Nucleotide Allele Frequency No. Base Positiona (Wild to Mutant) A B 1 14180 A→C 0 1b

2 15286 C→T 0 1 b

3 15302 T→G 0 1 b

4 14293 C→T 0.92 0.08

5 14635 C→T 0.64 0.36

6 15121 T→C 0.92 0.08

7 15134 C→T 0.92 0.08

8 15135 T→C 0.92 0.08

9 15138 G→A 0.92 0.08

10 15154 C→T 0.64 0.36

11 15179 G→A 0.80 0.20

12 15252 A→G 0.84 0.16

13 15290 T→C 0.60 0.40 a. with reference to NC_020614 b. all samples were monomorphic

46

RESULTS

1.1.2 MDS Analysis of Variation

Multidimensional scaling (MDS) plot was generated by using ‘R’. Plot was figured out by using 1st and 2nd dimensional transformations showing symmetrical variation of genetic distance values in MDS plot. Which was further used to represent the genetic differentiation between population within a two dimensional plot. In order to cluster the data, suitable transformation was done with an eigen value implicitly and calculated into its linear combinations. During MD scaling two eigen values were computed and scaled to get two linear combinations of variable data. Linear combination in 1st dimension was obtained by using

1steigen value while 2nd dimension transformation vector was computed with the help of 2nd value. In this way, ‘n-1’ eigen values were achieved to get ‘n-1’ number of data shapes/transformations (Cox and Cox, 2001). After obtaining data in two new shapes, they were clustered based on small difference between them. In total, 25 sequences were analyzed to get evolutionary distance matrix. That matrix was then utilized to plot MDS as shown in Figure 4.1

Figure 4.1: Multidimensional scaling plot of mitochondrial genomic region, cytochrome-b for Boselaphus tragocamelus.

47

RESULTS

1.1.3 Pair Wise Evolutionary Distance

Computational model (BH87) was used to generate distance profile for Cytochrome-b sequence of Boselaphus tragocamelus. Genetic variation plot was created by computational model for Boselaphus tragocamelus of various regional origins. Horizontal axis contains breeds while genetic distance values are taken at vertical axis. Figure 4.2 was generated by using the genetic distance dataset of mitochondrial genomic region of Cytochrome-b.

Figure 4.2: Cytochrome-b based Genetic variation plot of Boselaphus tragocamelus

1.1.4 Phylogenetic Tree of Boselaphus tragocamelus using Cytochrome-b gene

The Neighbor joining phylogenetic tree was constructed by using maximum likelihood method implemented in a desktop application named as MEGA 6. Boselaphus tragocamelus was the target species. DNA sequences of Cytochrome-b gene of Boselaphus tragocamelus were were processed for the phylogenetic analyses. Target species was encircled in the figure 4.3.

48

RESULTS

Figure 4.3: Phylogenetic Neighbor joining tree (circular) of Cytochrome-b gene of Boselaphus tragocamelus

49

RESULTS

1.2 Phylogenetic and diversity analysis of Boselaphus tragocamelus using cytochrome-c Gene

1.2.1 Identification of polymorphisms in Cytochrome-c gene of Boselaphus tragocamelus

Polymorphisms were identified in Cytochrome-c gene of Boselaphus tragocamelus as given in Table 4.2. No heterozygosity was observed. Allele frequency “1” represent that all samples were monomorphic.

Table 4.2: Polymorphisms identified in cytochrome-c gene of Boselaphus tragocamelus.

Change in Nucleotide Allele Frequency No. Base Positiona (Wild to Mutant) A B 1 5497 T→ A 0 1

2 6260 G→ A 0 1

3 6395 G→ A 0 1

4 6668 G→ A 0 1

5 6707 C→ T 0 1

6 5469 A→G 0.80 0.20

7 5472 C→T 0.80 0.20

8 5493 A→G 0.80 0.20

9 5496 G→A 0.80 0.20

10 5867 C→T 0.64 0.36

11 5871 A→G 0.64 0.36

12 5937 T→C 0.64 0.36

13 6260 G→A 0.88 0.12

14 6395 G→A 0.88 0.12

15 6668 G→A 0.88 0.12

16 6818 C→T 0.68 0.32 a. with reference to NC_020614

50

RESULTS

1.2.2 MDS Analysis of Variation

Multidimensional scaling (MDS) plotwas generated by using ‘R’ as described earlier in this chapter. Plot was figured out by using 1st and 2nd dimensional transformations showing symmetrical variation of genetic distance values in MDS plot. Figure was generated by targeting the genetic distance profiles of mitochondrial genomic region, cytochrome-c as shown in Figure

4.4

Figure 4.4: Multidimensional scaling plot of mitochondrial genomic gene, Cytochrome-c for

Boselaphus tragocamelus.

51

RESULTS

1.2.3 Pair Wise Evolutionary Distance

Computational model (BH87) was used to generate distance profile for Cytochrome-c sequence of Boselaphus tragocamelus. Genetic variation plot was created by computational model for Boselaphus tragocamelus of various regional origins. Horizontal axis contains breeds while genetic distance values were taken at vertical axis. Figure was generated by using the genetic distance dataset of mitochondrial Cytochrome-c.

Figure 4.5: Cytochrome-c based genetic variation plot of Boselaphus tragocamelus

1.2.4 Phylogenetic Tree of Boselaphus tragocamelus using Cytochrome-c gene

Phylogenetic tree was constructed by using maximum likelihood method implemented in a desktop application as described ealier. Target species was encircled in the figure 4.6.

52

RESULTS

Figure 4.6: Phylogenetic tree (circular) of cytochrome-c gene of Boselaphus tragocamelus

53

RESULTS

1.3 Phylogenetic and diversity analysis of Boselaphus tragocamelus using Mitochondrial d-loop region

1.3.1 Identification of polymorphisms in d-loop region of Boselaphus tragocamelus

Polymorphisms were identified in mitochondrial d-loop region of Boselaphus tragocamelus as given in Table 4.3. No heterozygosty was observed.

Table 4.3: Polymorphisms identified in d-loop region of Boselaphus tragocamelus

No. Base Positiona Change in Nucleotide Allele Frequency (Wild to Mutant) A B 1 15684 T→ C 0 1 2 15834 C→ T 0 1 3 16284 C→ G 0 1 4 15556 G→A 0.68 0.32 5 15606 T→C 0.76 0.24 6 15647 A→G 0.88 0.12 7 15662 T→C 0.88 0.12 8 15663 C→T 0.88 0.12 9 15685 C→T 0.64 0.36 10 15705 T→C 0.88 0.12 11 15713 G→A 0.88 0.12 12 15835 T→C 0.64 0.36 13 16085 T→C 0.88 0.12 14 16241 A→G 0.80 0.20 15 16283 A→T 0.80 0.20 16 16285 C→G 0.64 0.36 17 16288 A→G 0.80 0.20

a. with reference to NC_020614

54

RESULTS

1.3.2 MDS Analysis of Variation

Multidimensional scaling (MDS) plot was generated and figured out by using 1st and 2nd dimensional transformations. Figure was generated by targeting the genetic distance profiles of mitochondrial genomic d-loop region as shown in Figure 4.7

Figure 4.7: Multidimensional scaling plot of mitochondrial genomic d-loop region for Boselaphus tragocamelus.

1.3.3 Pair Wise Evolutionary Distance

Computational model (BH87) was used to generate distance profile for d-loop region sequence of Boselaphus tragocamelus. Horizontal axis contains breeds while genetic distance values are taken at vertical axis (Figure 4.8).

55

RESULTS

Figure 4.8: d-loop sequence based genetic variation plot of Boselaphus tragocamelus

1.3.4 Phylogenetic Tree of Boselaphus tragocamelus using d-loop region

Phylogenetic tree was constructed by using maximum likelihood method implemented

(MEGA 6). DNA sequences of d-loop region of animals under study were processed for the phylogenetic analyses (figure 4.9).

56

RESULTS

Figure 4.9: Phylogenetic tree (circular) of d-loop region of Boselaphus tragocamelus

57

RESULTS

1.4 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis

1.4.1 MDS Analysis of Variation

Sequences of three genes (cytochrome-b, cytochrome-c and d-loop) were combined and multidimensional scaling plot was generated. Plot was figured out by using 1st and 2nd dimensional transformations showing symmetrical variation of genetic distance values in MDS plot.

Figure 4.10: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop region for Boselaphus tragocamelus.

1.4.2 Pair Wise Evolutionary Distance

Computational model (BH87) was used to generate distance profile for cytochrome-b, cytochrome-c and d-loop region (collectively) sequence of Boselaphus tragocamelus. Genetic variation plot was created by computational model for Boselaphus tragocamelus of various regional origins. Horizontal axis contains breeds while genetic distance values are taken at

58

RESULTS vertical axis. Figure was generated by using the genetic distance dataset of mitochondrial genomic region namely cytochrome-b, cytochrome-c and d-loop region (collectively).

Figure 4.11: Cytochrome-b, cytochrome-c and d-loop region based genetic variation plot of Boselaphus tragocamelus

1.4.3 Estimates of Evolutionary Divergence between Sequences

[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 [ 1] [ 2] 0.00365 [ 3] 0.00169 0.00253 [ 4] 0.00478 0.00281 0.00365 [ 5] 0.00309 0.00169 0.00197 0.00337 [ 6] 0.00197 0.00281 0.00084 0.00393 0.00225 [ 7] 0.00112 0.00478 0.00281 0.0059 0.00422 0.00309 [ 8] 0.0045 0.00309 0.00393 0.0059 0.00309 0.00365 0.00393 [ 9] 0.00422 0.0045 0.00253 0.00562 0.00393 0.00281 0.00422 0.00478 [10] 0.00393 0.0059 0.00506 0.00534 0.00478 0.00534 0.00393 0.00618 0.00253 [11] 0.00309 0.00169 0.00197 0.00337 0 0.00225 0.00422 0.00309 0.00393 0.00478 [12] 0.00253 0.00393 0.00141 0.00506 0.00337 0.00112 0.00197 0.00309 0.00281 0.00534 0.00337 [13] 0.0045 0.00422 0.00337 0.00197 0.00309 0.00365 0.00393 0.00393 0.00422 0.0045 0.00309 0.00309 [14] 0.00478 0.00393 0.00422 0.00675 0.00337 0.00337 0.00478 0.00365 0.00169 0.00365 0.00337 0.00337 0.00534 [15] 0.00365 0.00056 0.00309 0.00337 0.00225 0.00281 0.00422 0.00309 0.00506 0.00646 0.00225 0.00393 0.00478 0.00393 [16] 0.00253 0.00393 0.00141 0.00506 0.00337 0.00169 0.00365 0.00253 0.00393 0.00646 0.00337 0.00225 0.00478 0.00506 0.00393 [17] 0.00141 0.00225 0.00309 0.00337 0.00393 0.00281 0.00253 0.00478 0.00506 0.00422 0.00393 0.00393 0.00478 0.00562 0.00225 0.00393 [18] 0.00253 0.00112 0.00141 0.00393 0.00056 0.00169 0.00365 0.00253 0.00337 0.00534 0.00056 0.00281 0.00365 0.00281 0.00169 0.00281 0.00337 [19] 0.0045 0.00084 0.00337 0.00197 0.00141 0.00365 0.00562 0.00393 0.00534 0.00506 0.00141 0.00478 0.00393 0.00478 0.00141 0.00478 0.00309 0.00197 [20] 0.00197 0.00225 0.00028 0.00337 0.00169 0.00056 0.00309 0.00365 0.00225 0.00478 0.00169 0.00169 0.00309 0.00393 0.00281 0.00169 0.00281 0.00112 0.00309 [21] 0.00365 0.00393 0.00253 0.00112 0.00225 0.00281 0.00478 0.00534 0.0045 0.00478 0.00225 0.00393 0.00141 0.00562 0.0045 0.00393 0.0045 0.00281 0.00309 0.00225 [22] 0.00365 0.00281 0.00253 0.0045 0.00112 0.00337 0.00309 0.00253 0.00393 0.00478 0.00112 0.00225 0.00253 0.00337 0.00337 0.00393 0.00506 0.00169 0.00253 0.00281 0.00337 [23] 0.00309 0.00337 0.00197 0.0045 0.00337 0.00112 0.00253 0.00253 0.00281 0.00478 0.00337 0.00056 0.00253 0.00337 0.00337 0.00281 0.00337 0.00281 0.00422 0.00169 0.00393 0.00281 [24] 0.00112 0.00422 0.00225 0.00365 0.00253 0.00309 0.00225 0.00562 0.00478 0.00281 0.00253 0.00365 0.00393 0.0059 0.00478 0.00365 0.00253 0.00309 0.00337 0.00253 0.00253 0.00309 0.00422 [25] 0.16554 0.16582 0.1647 0.16554 0.1661 0.1647 0.16554 0.16695 0.1661 0.16835 0.1661 0.16498 0.16554 0.16695 0.16526 0.16554 0.16582 0.16554 0.16667 0.1647 0.16526 0.16639 0.16526 0.16667 [26] 0.00225 0.00309 0.00169 0.00422 0.00253 0.00141 0.00225 0.00281 0.00253 0.0045 0.00253 0.00141 0.00281 0.00309 0.00309 0.00253 0.00309 0.00197 0.00393 0.00141 0.00309 0.00253 0.00141 0.00337 0.1647 Table 4.4: Cytochrome-b, cytochrome-c and d-loop region based evolutionary analyses of Boselaphus tragocamelus.

59

RESULTS

1.4.4: Phylogenetic Tree of Boselaphus tragocamelus using Cytochrome-b, cytochrome-c and d-loop region (combined sequence)

Phylogenetic tree was constructed by using maximum likelihood method as described earlier. DNA sequences of Cytochrome-b, cytochrome-c and d-loop region (combined sequence) of animals under study were routed for phylogenetic analyses (figure 4.12).

60

RESULTS

Figure 4.12: Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region (combined sequence) of Boselaphus tragocamelus.

61

RESULTS

2. Phylogeny and Diversity Analysis of Antilope cervicapra

2.1 Phylogenetic and diversity analysis of Antilope cervicapra using cytochrome-b Gene

2.1.1 Identification of polymorphisms in Cytochrome-b gene of Antilope cervicapra

Polymorphisms were identified in Cytochrome-b gene of Antilope cervicapra. Detail is given in Table 4.5.

Table 4.5: Polymorphisms identified in cytochrome-b gene of Antilope cervicapra.

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 14174 C→A 0 1 2 14177 A→C 0 1 3 14256 G→A 0 1 4 14544 A→G 0 1 5 15220 G→A 0 1 6 15293 G→A 0 1 7 13650 G→A 0.56 0.44 8 13657 C→T 0.52 0.48 9 13660 A→G 0.80 0.20 10 13664 A→G 0.56 0.44 11 14532 T→C 0.76 0.24 12 14710 A→G 0.80 0.20 13 14735 T→C 0.80 0.20

a. with reference to NC_012098

62

RESULTS

2.1.2 MDS Analysis of Variation

Figure 4.13: cytochrome-b based multidimensional scaling plot of Antilope cervicapra

2.1.3 Pair Wise Evolutionary Distance

Figure 4.14: Cytochrome-b based genetic variation plot of Antilope cervicapra

63

RESULTS

2.1.4 Phylogenetic Tree of Antilope cervicapra using Cytochrome-b gene

Figure 4.15: Phylogenetic tree (circular) of cytochrome-b gene of Antilope cervicapra.

64

RESULTS

2.2 Phylogenetic and diversity analysis of Antilope cervicapra using cytochrome-c Gene

2.2.1 Identification of polymorphisms in Cytochrome-c gene of Antilope cervicapra

Polymorphisms were identified in Cytochrome-c gene of Antilope cervicapra. Detail is given in Table 4.6.

Table 4.6: Polymorphisms identified in cytochrome-c gene of Antilope cervicapra

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 5890 C→T 0 1 2 6577 A→G 0 1 3 5420 A→G 0.96 0.04 4 5459 G→A 0.92 0.08 5 5510 G→A 0.96 0.04 6 5536 C→T 0.88 0.12 7 5579 C→T 0.96 0.04 8 5616 T→C 0.92 0.08 9 5665 C→T 0.84 0.16 10 5811 T→C 0.96 0.04 11 5990 A→G 0.96 0.04 12 6032 G→A 0.80 0.20 a. with reference to NC_012098

65

RESULTS

2.2.2 MDS Analysis of Variation

Figure 4.16: Multidimensional scaling plot of cytochrome-c gene for Antilope cervicapra

2.2.3 Pair Wise Evolutionary Distance

Figure 4.17: Cytochrome-c based genetic variation plot of Antilope cervicapra

66

RESULTS

2.2.4 Phylogenetic Tree of Antilope cervicapra using Cytochrome-c gene

Phylogenetic tree was constructed by using maximum likelihood method as described earlier.

Figure 4.18: Phylogenetic tree (circular) of cytochrome-c gene of Antilope cervicapra.

67

RESULTS

2.3 Phylogenetic and diversity analysis of Antilope cervicapra using d-loop region

2.3.1 Identification of polymorphisms in d-loop region of Antilope cervicapra

Polymorphisms were identified and detail is given in Table 4.7.

Table 4.7: Polymorphisms identified in mitochondrial d-loop region of Antilope cervicapra.

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 15578 A→G 0 1 2 15591 G→A 0 1 3 15644 A→G 0 1 4 15676 A→G 0 1 5 15681 G→A 0 1 6 15699 G→A 0 1 7 15725 A→G 0 1 8 15762 A→G 0 1 9 15781 T→C 0 1 10 15876 A→G 0 1 11 15446 T→C 0.84 0.16 12 15455 A→G 0.84 0.16 13 15473 A→G 0.72 0.28 14 16386 C→T 0.88 0.12 15 16387 A→G 0.92 0.08 16 16397 T→C 0.88 0.12 a. with reference to NC_012098

68

RESULTS

2.3.2 MDS Analysis of Variation

Figure 4.19: Multidimensional scaling plot of d-loop region for Antilope cervicapra

2.3.3 Pair Wise Evolutionary Distance

Figure 4.20: D-loop region based genetic variation plot of Antilope cervicapra.

69

RESULTS

2.3.4 Phylogenetic Tree of Antilope cervicapra using D-loop region

Figure 4.21: Phylogenetic tree (circular) of d-loop region of Antilope cervicapra

70

RESULTS

2.4 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysisof Antilope cervicapra.

2.4.1 MDS Analysis of Variation

Figure 4.22: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop region (collectively) for Antilope cervicapra.

2.4.2 Pair Wise Evolutionary Distance

Figure 4.23: Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic variation plot of Antilope cervicapra.

71

RESULTS

2.4.3 Estimates of Evolutionary Divergence between Sequences

[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 [ 1] [ 2] 0.00309 [ 3] 0.00169 0.00309 [ 4] 0.00197 0.00338 0.00141 [ 5] 0.00056 0.00253 0.00113 0.00141 [ 6] 0.00169 0.00253 0.00225 0.00197 0.00113 [ 7] 0.00281 0.00141 0.00281 0.00309 0.00225 0.00281 [ 8] 0.00197 0.00338 0.00141 0.00281 0.00141 0.00253 0.00309 [ 9] 0.00084 0.00225 0.00141 0.00113 0.00028 0.00084 0.00197 0.00169 [10] 0.00197 0.00281 0.00253 0.00281 0.00141 0.00197 0.00309 0.00281 0.00169 [11] 0.00281 0.00141 0.00169 0.00197 0.00225 0.00281 0.00113 0.00309 0.00197 0.00309 [12] 0.00197 0.00338 0.00028 0.00169 0.00141 0.00253 0.00309 0.00169 0.00169 0.00281 0.00197 [13] 0.00253 0.00113 0.00253 0.00281 0.00197 0.00253 0.00084 0.00281 0.00169 0.00281 0.00084 0.00281 [14] 0.00197 0.00281 0.00141 0.00281 0.00141 0.00197 0.00309 0.00169 0.00169 0.00225 0.00309 0.00169 0.00281 [15] 0.00169 0.00366 0.00225 0.00253 0.00113 0.00225 0.00338 0.00084 0.00141 0.00253 0.00338 0.00253 0.00309 0.00253 [16] 0.00141 0.00281 0.00197 0.00169 0.00084 0.00141 0.00253 0.00225 0.00056 0.00225 0.00253 0.00225 0.00225 0.00225 0.00197 [17] 0.00113 0.00253 0.00056 0.00197 0.00056 0.00169 0.00225 0.00084 0.00084 0.00197 0.00225 0.00084 0.00197 0.00084 0.00169 0.00141 [18] 0.00113 0.00197 0.00169 0.00141 0.00056 0.00056 0.00225 0.00197 0.00028 0.00141 0.00225 0.00197 0.00197 0.00141 0.00169 0.00084 0.00113 [19] 0.00084 0.00281 0.00141 0.00169 0.00028 0.00141 0.00253 0.00169 0.00056 0.00169 0.00253 0.00169 0.00225 0.00169 0.00141 0.00113 0.00084 0.00084 [20] 0.00141 0.00338 0.00197 0.00225 0.00084 0.00197 0.00309 0.00056 0.00113 0.00225 0.00309 0.00225 0.00281 0.00225 0.00028 0.00169 0.00141 0.00141 0.00113 [21] 0.00281 0.00141 0.00281 0.00309 0.00225 0.00281 0.00113 0.00309 0.00197 0.00309 0.00113 0.00309 0.00084 0.00309 0.00338 0.00253 0.00225 0.00225 0.00253 0.00309 [22] 0.00197 0.00281 0.00141 0.00281 0.00141 0.00197 0.00309 0.00169 0.00169 0.00225 0.00309 0.00169 0.00281 0.00113 0.00253 0.00225 0.00084 0.00141 0.00169 0.00225 0.00309 [23] 0.00113 0.00253 0.00169 0.00141 0.00056 0.00113 0.00225 0.00197 0.00028 0.00197 0.00225 0.00197 0.00197 0.00197 0.00169 0.00084 0.00113 0.00056 0.00084 0.00141 0.00225 0.00197 [24] 0.00141 0.00225 0.00197 0.00225 0.00084 0.00141 0.00253 0.00225 0.00113 0.00056 0.00253 0.00225 0.00225 0.00169 0.00197 0.00169 0.00141 0.00084 0.00113 0.00169 0.00253 0.00169 0.00141 [25] 0.01125 0.01322 0.01181 0.0121 0.01069 0.01181 0.01294 0.0121 0.01097 0.0121 0.01294 0.0121 0.01266 0.0121 0.01181 0.01153 0.01125 0.01125 0.01097 0.01153 0.01294 0.0121 0.01125 0.01153 [26] 0.16793 0.16934 0.16793 0.16878 0.16737 0.16821 0.16962 0.16765 0.16765 0.1685 0.16962 0.16821 0.16934 0.16793 0.16793 0.16821 0.16737 0.16765 0.16765 0.16765 0.16934 0.16793 0.16793 0.16793 0.1654 Table 4.8 Cytochrome-b, cytochrome-c and d-loop region (collectively) based evolutionary analyses of Antilope cervicapra

2.4.4 Phylogenetic Tree of Antilope cervicapra using Cytochrome-b, cytochrome-c and d- loop region (collectively)

Phylogenetic tree was constructed by using maximum likelihood method implemented in a desktop application named as MEGA 6. DNA sequences of Cytochrome-b, cytochrome-c and d-loop region (collectively) were processed for the phylogenetic analyses (figure 4.24).

72

RESULTS

Figure 4.24: Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region (collectively) of Antilope cervicapra.

73

RESULTS

3 Phylogeny and Diversity Analysis of Axis axis

3.1 Phylogenetic and diversity analysis of Axis axis using cytochrome-b Gene

3.1.1 Identification of polymorphisms in Cytochrome-b gene of Axis axis

Polymorphisms were identified in Cytochrome-b gene of Axis Axis. Detail is given in

Table 4.9.

Table 4.9: Polymorphisms identified in Cytochrome-b gene of Axis Axis

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 14221 A→G 0 1 2 14598 C→T 0 1 3 15099 A→G 0 1 4 15199 A→G 0 1 5 15219 T→C 0 1 6 15249 T→C 0 1 7 14221 A→G 0.80 0.20 8 15299 A→G 0.56 0.44 9 15372 G→A 0.92 0.08 10 15439 T→C 0.92 0.08 11 15449 T→C 0.56 0.44

a. with reference to NC_020680

74

RESULTS

3.1.2 MDS Analysis of Variation

Figure 4.25: Multidimensional scaling plot of mitochondrial cytochrome-b gene for Axis axis.

3.1.3 Pair Wise Evolutionary Distance

Figure 4.26: Cytochrome-b based genetic variation plot of Axis axis

75

RESULTS

3.1.4 Phylogenetic Tree of Axis Axis using Cytochrome-b gene

Figure 4.27: Phylogenetic tree (circular) of cytochrome-b region of Axis axis

76

RESULTS

3.2 Phylogenetic and diversity analysis of Axis axis using cytochrome-c Gene

3.2.1 Identification of polymorphisms in Cytochrome-c gene of Axis axis

Table 4.10: Polymorphisms identified in cytochrome-c gene of Axis axis.

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 5414 T→A 0 1 2 5441 G→A 0 1 3 6684 T→C 0 1 4 5414 T→A 0.84 0.16 5 5441 G→A 0.84 0.16 a. with reference to NC_020680

3.2.2 MDS Analysis of Variation

Figure 4.28: Multidimensional scaling plot of Cytochrome-c gene for Axis axis

77

RESULTS

3.2.3 Pair Wise Evolutionary Distance

Figure 4.29: Cytochrome-c based genetic variation plot of Axis axis

3.2.4 Phylogenetic Tree of Axis axis using Cytochrome-c gene

Figure 4.30: Phylogenetic tree (circular) of cytochrome-c region of Axis axis

78

RESULTS

3.3 Phylogenetic and diversity analysis of Axis axis using Mitochondrial d- loop region

3.3.1 Identification of polymorphisms in D-loop region of Axis axis

Table 4.11: Polymorphisms identified in mitochondrial d-loop of Axis axis

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 15647 C→T 0 1 2 15673 C→T 0 1 3 15676 C→T 0 1 4 15679 T→C 0 1 5 15723 G→A 0 1 6 15763 T→C 0 1 7 15472 T→A 0.92 0.08 8 15610 A→G 0.92 0.08 9 15701 A→G 0.84 0.16 10 15783 C→T 0.92 0.08 11 15843 C→T 0.84 0.16 12 15934 C→T 0.84 0.16 13 15997 T→C 0.92 0.08 14 15997 C→T 0.92 0.08 15 16141 G→A 0.92 0.08 16 16264 T→C 0.80 0.20

a. with reference to NC_020680

79

RESULTS

3.3.2 MDS Analysis of Variation

Figure 4.31: Multidimensional scaling plot of mitochondrial d-loop region for Axis axis

3.3.3 Pair Wise Evolutionary Distance

Figure 4.32: D-loop region based genetic variation plot of Axis axis

80

RESULTS

3.3.4 Phylogenetic Tree of Axis axis

Figure 4.33: Phylogenetic tree (circular) of d-loop region of Axis axis

81

RESULTS

3.4 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes collectively) based analysisof Axis axis.

3.4.1 MDS Analysis of Variation

Figure 4.34: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop region (collectively) for Axis axis

3.4.2 Pair Wise Evolutionary Distance

Figure 4.35: Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic variation plot of Axis axis

82

RESULTS

3.4.3 Estimates of Evolutionary Divergence between Sequences

[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 [ 1] [ 2] 0.00368 [ 3] 0.02546 0.02348 [ 4] 0.00141 0.00339 0.02518 [ 5] 0.00085 0.00283 0.02461 0.00057 [ 6] 0.00057 0.00311 0.02489 0.00085 0.00028 [ 7] 0.00141 0.00283 0.02461 0.00113 0.00057 0.00085 [ 8] 0.00481 0.00566 0.02687 0.00396 0.00396 0.00424 0.00396 [ 9] 0.0116 0.01414 0.02178 0.01245 0.01188 0.01216 0.01188 0.01103 [10] 0.00339 0.00028 0.02376 0.00311 0.00255 0.00283 0.00311 0.00594 0.01443 [11] 0.02518 0.02376 0.00028 0.02489 0.02433 0.02461 0.02489 0.02716 0.02207 0.02348 [12] 0.00113 0.00368 0.02546 0.00028 0.00085 0.00057 0.00141 0.00424 0.01273 0.00339 0.02518 [13] 0.00509 0.00594 0.02716 0.00424 0.00424 0.00453 0.00368 0.00028 0.01132 0.00622 0.02744 0.00453 [14] 0.00113 0.00255 0.02433 0.00085 0.00028 0.00057 0.00028 0.00368 0.0116 0.00283 0.02461 0.00113 0.00396 [15] 0.00057 0.00311 0.02489 0.00141 0.00085 0.00113 0.00085 0.00424 0.01103 0.00339 0.02518 0.0017 0.00453 0.00057 [16] 0.00396 0.00651 0.02772 0.00424 0.00424 0.00453 0.00481 0.00085 0.01075 0.00622 0.02744 0.00453 0.00113 0.00453 0.00396 [17] 0.01216 0.01358 0.02122 0.01188 0.01132 0.0116 0.01132 0.01047 0.00057 0.01386 0.0215 0.01216 0.01075 0.01103 0.0116 0.01132 [18] 0.0017 0.00311 0.02489 0.00028 0.00085 0.00113 0.00085 0.00368 0.01216 0.00339 0.02518 0.00057 0.00396 0.00057 0.00113 0.00453 0.0116 [19] 0.00113 0.00255 0.02433 0.00085 0.00028 0.00057 0.00028 0.00368 0.0116 0.00283 0.02461 0.00113 0.00396 0 0.00057 0.00453 0.01103 0.00057 [20] 0.00339 0.00368 0.02546 0.00368 0.00311 0.00283 0.00368 0.00707 0.01499 0.00339 0.02518 0.00339 0.00736 0.00339 0.00396 0.00736 0.01443 0.00396 0.00339 [21] 0.0611 0.06167 0.0826 0.06082 0.06025 0.06054 0.06082 0.06365 0.07072 0.06139 0.08232 0.0611 0.06393 0.06054 0.0611 0.06393 0.07016 0.0611 0.06054 0.06025 Table 4.12 Cytochrome-b, cytochrome-c and d-loop region based evolutionary analyses.

3.4.4: Phylogenetic Tree of Axis axis using Cytochrome-b, cytochrome-c and d-loop region

Figure 4.36 Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region (collectively) of Axis axis.

83

RESULTS

4 Phylogeny and Diversity Analysis of Axis porcinus

4.1 Analysis of Axis porcinus using Cytochrome-b Gene

4.1.1 Identification of polymorphisms in Cytochrome-b gene of Axis porcinus

Table 4.13: Polymorphisms identified in cytochrome-b gene of Axis porcinus.

No. Base Positiona Change in Nucleotide Allele Frequencies (Wild to Mutant) A B 1 14297 A→G 0 1 2 14522 G→A 0 1 3 14733 A→G 0 1 4 14871 T→C 0 1 5 15295 G→A 0 1 6 14297 G→A 0.60 0.40 7 14402 A→G 0.72 0.28 8 14532 C→T 0.84 0.16 9 14875 T→C 0.60 0.40 10 15143 A→G 0.60 0.40 11 15149 G→A 0.92 0.08 12 15207 T→C 0.92 0.08 13 15232 C→T 0.76 0.24 a. with reference to JN632600

84

RESULTS

4.1.2 MDS Analysis of Variation

Figure 4.37: Multidimensional scaling plot of mitochondrial cytochrome-b for Axis porcinus

4.1.3 Pair Wise Evolutionary Distance

Figure 4.38: Cytochrome-b based genetic variation plot of Axis porcinus

85

RESULTS

4.1.4 Phylogenetic Tree of Axis porcimus using Cytochrome-b gene

Figure 4.39: Phylogenetic tree (circular) of cytochrome-b region of Axis porcinus

86

RESULTS

4.2 Phylogenetic and diversity analysis of Axis porcinus based on Cytochrome-c Gene

4.2.1 Identification of polymorphisms in Cytochrome-c gene of Axis porcinus

Table 4.14: Polymorphisms identified in cytochrome-c gene of Axis porcinus.

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 6547 C→T 0 1 2 6781 T→C 0 1 3 5475 A→G 0.80 0.20 4 5501 A→G 0.84 0.16 5 5628 G→A 0.80 0.20 6 5804 G→A 0.84 0.16 7 5808 C→A 0.72 0.28 8 5858 C→T 0.80 0.2 9 5940 G→T 0.80 0.2 10 5965 A→G 0.92 0.08 11 6426 T→C 0.72 0.28 12 6585 G→A 0.92 0.08 a. with reference to JN632600

87

RESULTS

4.2.2 MDS Analysis of Variation

Figure 4.40: Multidimensional scaling plot for Axis porcinus based on cytochrome-c

4.2.3 Pair Wise Evolutionary Distance

Figure 4.41: Cytochrome-c based genetic variation plot of Axis porcinus

88

RESULTS

4.2.4 Phylogenetic Tree of Axis porcimus using Cytochrome-c gene

Figure 4.42: Phylogenetic tree (circular) of cytochrome-c region of Axis porcinus

89

RESULTS

4.3 Phylogenetic and diversity analysis of Axis porcinus using Mitochondrial d-loop region

4.3.1 Identification of polymorphisms in d-loop region of Axis porcinus

Table 4.15: Polymorphisms identified in mitochondrial d-loop region of Axis porcinus.

Change in Nucleotide Allele Frequencies No. Base Positiona (Wild to Mutant) A B 1 15547 T→C 0 1

2 15582 T→C 0 1

3 15639 A→G 0 1

4 15697 G→A 0 1

5 15731 A→G 0 1

6 15747 T→G 0 1

7 15852 C→T 0 1

8 15597 G→A 0.80 0.20

9 15737 A→G 0.92 0.08

10 15859 G→A 0.84 0.16

11 15891 C→T 0.96 0.04

12 15919 T→C 0.80 0.2

13 15995 C→G 0.84 0.16

14 16059 C→T 0.92 0.08

15 16106 C→T 0.92 0.08

16 15189 T→C 0.84 0.16

17 16243 C→T 0.84 0.16

18 16283 A→G 0.96 0.04

19 16314 A→G 0.84 0.16 a. with reference to JN632600

90

RESULTS

4.3.2 MDS Analysis of Variation

Figure 4.43: Multidimensional scaling plot of d-loop region for Axis porcinus

4.3.3 Pair Wise Evolutionary Distance

Figure 4.44: D-loop region based genetic variation plot of Axis porcinus

91

RESULTS

4.3.4 Phylogenetic Tree of Axis porcinus using Cytochrome-c gene

Figure 4.45: Phylogenetic tree (circular) of d-loop region of Axis porcinus

92

RESULTS

4.4 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes collectively) based analysis of Axis porcinus

4.4.1 MDS Analysis of Variation

Figure 4.46: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop region for Axis porcinus.

4.4.2 Pair Wise Evolutionary Distance

Figure 4.47: Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic variation plot of Axis porcinus.

93

RESULTS

4.4.3 Estimates of Evolutionary Divergence between Sequences

[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 [ 1] [ 2] 0.0017 [ 3] 0.00254 0.00424 [ 4] 0.00367 0.00311 0.00396 [ 5] 0.00311 0.0048 0.00283 0.00452 [ 6] 0.00254 0.00198 0.00452 0.00339 0.00509 [ 7] 0.00198 0.00028 0.00396 0.00283 0.00452 0.0017 [ 8] 0.00141 0.00311 0.00113 0.00452 0.00396 0.00339 0.00283 [ 9] 0.00254 0.00198 0.00452 0.00113 0.00509 0.00226 0.0017 0.00339 [10] 0.00113 0.00283 0.00367 0.0048 0.00198 0.00367 0.00311 0.00254 0.00367 [11] 0.00198 0.00367 0.0017 0.00226 0.00226 0.00396 0.00339 0.00283 0.00283 0.00311 [12] 0.00311 0.00254 0.00339 0.0017 0.00283 0.00283 0.00226 0.00396 0.00283 0.00311 0.00283 [13] 0.00113 0.00283 0.00254 0.0048 0.00424 0.00254 0.00311 0.00141 0.00367 0.00226 0.00311 0.00424 [14] 0.00028 0.00198 0.00226 0.00339 0.00283 0.00226 0.0017 0.00113 0.00226 0.00141 0.0017 0.00283 0.00141 [15] 0.00113 0.0017 0.00367 0.0048 0.00311 0.00367 0.00198 0.00254 0.00367 0.00113 0.00311 0.00424 0.00226 0.00141 [16] 0.00311 0.00254 0.0017 0.00226 0.00339 0.00283 0.00226 0.00283 0.00283 0.00424 0.00226 0.0017 0.00424 0.00283 0.00424 [17] 0.00254 0.00424 0.0017 0.00226 0.00339 0.00452 0.00396 0.00226 0.00339 0.00367 0.00113 0.00283 0.00254 0.00226 0.00367 0.00339 [18] 0.00424 0.00367 0.00396 0.00226 0.00226 0.00396 0.00339 0.00509 0.00283 0.00424 0.00339 0.0017 0.00537 0.00396 0.00537 0.00226 0.00452 [19] 0.00254 0.00424 0.0017 0.00339 0.00226 0.00452 0.00396 0.00226 0.00452 0.00254 0.00226 0.00283 0.00367 0.00226 0.00254 0.00226 0.00226 0.00452 [20] 0.00226 0.0017 0.0048 0.00254 0.00537 0.00141 0.00198 0.00367 0.00141 0.00339 0.00311 0.00311 0.00339 0.00254 0.00339 0.00311 0.00367 0.00424 0.0048 [21] 0.00057 0.00226 0.00311 0.00424 0.00254 0.00311 0.00254 0.00198 0.00311 0.00057 0.00254 0.00254 0.0017 0.00085 0.0017 0.00367 0.00311 0.00367 0.00311 0.00283 [22] 0.00311 0.00254 0.00283 0.00113 0.00339 0.00283 0.00226 0.00396 0.0017 0.00424 0.00113 0.0017 0.00424 0.00283 0.00424 0.00113 0.00226 0.00226 0.00339 0.00198 0.00367 [23] 0.0599 0.06103 0.06075 0.06188 0.06132 0.06132 0.06075 0.06075 0.06132 0.06103 0.06019 0.06132 0.06103 0.05962 0.06103 0.06075 0.06132 0.06188 0.06132 0.0616 0.06047 0.06075 [24] 0.00678 0.00791 0.00763 0.00819 0.00819 0.00819 0.00763 0.00763 0.00819 0.00791 0.00706 0.00763 0.00791 0.0065 0.00791 0.00763 0.00763 0.00876 0.00763 0.00848 0.00735 0.00763 0.06019

Table 4.16 Cytochrome-b, cytochrome-c and d-loop region (collectively) based Evolutionary analyses of Axis porcinus

94

RESULTS

4.4.4: Phylogenetic Tree of Axis porcinus using Cytochrome-b, cytochrome-c and d-loop region

Figure 4.48 Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region (collectively) of Axis porcinus.

95

RESULTS

5 Phylogeny and Diversity Analysis of Boselaphus tragocamelus & Antilope Cervicapra (Bovidae) and Axis Axis & Axis Porcinus (Cervidae)

5.1 Mitochondrial cytochrome-b Gene based analysis

Figure 4.49: Phylogenetic tree (radiation) of cytochrome-b gene of selected four species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).

96

RESULTS

Figure 4.50: Phylogenetic tree (circular) of cytochrome-b gene (collectively) of selected four species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).

97

RESULTS

5.2 Mitochondrial cytochrome-c Gene based analysis

Figure 4.51: Phylogenetic tree (radiation) of cytochrome-c region (collectively) of selected four species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).

98

RESULTS

Figure 4.52: Phylogenetic tree (circular) of cytochrome-c region of selected four species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).

99

RESULTS

5.3 Mitochondrial d-loop region based analysis

Figure 4.53 Phylogenetic tree (ractangular) of d-loop region of selected four species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).

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RESULTS

Figure 4.54: Phylogenetic tree (circular) of d-loop region of selected four species (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).

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RESULTS

5.4 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis of Boselaphus tragocamelus & Antelope cervicapra (Bovidae)

5.4.1 MDS Analysis of Variation

Figure 4.55: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) for Bovidae (Boselaphus tragocamelus and Antelope cervicapra).

5.4.2 Pair Wise Evolutionary Distance

Figure 4.56: Cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based Genetic variation plot of Bovidae (Boselaphus tragocamelus and Antelope cervicapra)

102

RESULTS

5.5 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis of Axis axis & Axis porcinus (Cervidae)

5.5.1 MDS Analysis of Variation

Figure 4.57: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop for Cervidae (Axis axis and Axis porcinus)

5.5.2 Pair Wise Evolutionary Distance

Figure 4.58: Cytochrome-b, cytochrome-c and d-loop region based genetic variation plot of Cervidae (Axis axis and Axis porcinus)

103

RESULTS

5.6 Mitochondrialcytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis of Boselaphus tragocamelus & Antelope cervicapra (Bovidae) and Axis axis & Axis porcinus (Cervidae)

5.6.1 MDS Analysis of Variation

Figure 4.59: Multidimensional scaling plot of mitochondrial genomic cytochrome-b, cytochrome-c and d-loop region (collectively) for all four species.

5.6.2 Pair Wise Evolutionary Distance

Figure 4.60: Cytochrome-b, cytochrome-c and d-loop region based variation plot of selected four species.

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RESULTS

5.6.3 Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based phylogenetic analysis

Figure 4.61: Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region of Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus.

105

CHAPTER 5 DISCUSSION

Pakistan is faced with many threats of environment degradation for species ecosystem.

Diverse climatic and ecological conditions and wide latitudinal and altitudinal geographical spread make Pakistan favorite habitat for large variety of mammals. Among the mammals, different number of both domestic and wild animals belonging to bovidae and cervidae are very common in Pakistan. But present time invention of modern tools of land cultivation, illegal hunting and rapid urbanization led to devastating decrease in wild animals. Currently, only limited patches at different locations of Pakistan have small populations of bovidae (Boselaphus tragocamelus, Antilope cervicapra) and cervidae (Axis axis, Axis porcinus).So these species are on their way towards possible scantiness especially from Indo-Pak region.

This study was planned to investigate the Molecular phylogeny and genetic diversity of bovidae (Boselaphus tragocamelus, Antilope cervicapra) and cervidae (Axis axis, Axis porcinus) families in Pakistan on the basis of mitochondrial cytochrome b, cytochrome c and d-loop regions. Aim of this study was to genetically differentiate bovidae and cervidae. It is implicit that mitochondrial genes evolved according to neutral model of evolution (Slatkin, 1995). These genes have been used as tools to resolve issues regarding taxonomic levels (Zardoya et al. 1998).

The study is first ever effort regarding molecular phylogeny and diversity analysis of bovidae (Boselaphus tragocamelus, Antilope cervicapra) and cervidae (Axis axis, Axis porcinus) in Pakistan. Cytochrome-b gene can be employed to study both systematic deep phylogeny and divergences in the recent times (Kirchman et al. 2000). D-loop is high polymorphic region of mitochondria and sequences in this region evolve rapidly. Effectiveness of d-loop region for taxonomic level study of phylogeny has already proved in vertebrates. So it is an excellent tool 106

DISCUSSION for study of genetic variation within and among the closely related organisms (Wu et al. 2010).

Similarly Cytochrome-c gene is widely used for identification of species on the basis of molecular data and for establishment of relationship between closely related species (Struder-

Kypke and Lynn 2010). Several molecular studies on cervidae have been contributed considerably to resolve evolutionary relationships among deer species based on amino acid, mitochondrial DNA and nuclear DNA sequences (Miyamoto et al. 1990; Cronin et al. 1996;

Douzery and Randi, 1997; Randi et al. 2001; Pitra et al. 2004 and Gilbert et al. 2006).

Genome conservation plays important role in survival of the species and the ecosystem.

For the purpose of genetic information for Boselaphus tragocamelus, Antilope Cervicapra, Axis

Axis and Axis Porcinus species in Pakistan, Mitochondrial cytochrome-b, cytochrome-c and d- loop region have been used to determine genetic diversity and genetic structure which can be exploited in deriving information on phylogenetic relationships (Feral et al. 2006; Mwacharo et a1, 2006; Giovambattista et a1, 2001), DNA sequence based techniques are useful for assessing genetic variability within and among populations (Haig, 1998). The conservation of wild endangered species is dependent on maintenance of genetic variations (Crozier, 1992; Lynch and

Milligan, 1994) which is an indication of variation in genomes.

According to previous reports, The Cervidae is composed of four subfamilies: Cervinae,

Muntiacinae, Odocoileinae and Hydropotinae (Eisenberg, 1981). There are two groups within

Cervidae, that is plesiometacarpalia (Cervinae and Muntiacinae) and telemetacarpalia

(Odocoileinae and Hydropotinae) by condition of the lateral metacarpals. Any variation in the given pattern was explained by Hassanin at al. (2012) with following four assumptions: The first two hypotheses propose that the samples were extracted from individuals of the same species, because of either species misidentification (human error) or imperfect taxonomy (species

107

DISCUSSION synonymy). The third hypothesis implies that the mtDNA genome of one species was transferred into the other by inter-specific hybridization, a process known as mtDNA introgression. The fourth hypothesis suggests a recent speciation event. Our results illustrate normal distribution patterns of all four species but over all tendency was observed towards homozygosity.

To assess the level of genetic variation, an 1139 bp fragment in the Cytochrome-b gene of the mitochondrial DNA from 25 deer individuals was PCR-amplified and sequenced and analysed. A total of thirteen variable sites were observed in Cytochrome-b gene of Boselaphus tragocamelus. Out of these, three variations were found monomorphic for mutant allele.

Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 10 transitions and three transversions. Allele frequency of all variations was calculated and very low frequency of mutant allele was observed. As no heterozygous individuals were found so allelic frequency and genotypic frequency was same. The average heterozygosity values for endangered and non-endangered populations are often lower and higher values, respectively (Frankham et al. 2002). So, our results illustrate distribution of specie inclined more towards endangered, which is alarming and demands immediate measures for its conservation. In some other related studies as well, overall genotypic frequency has been found tendency to be homozygous as Nielsen et al. (2008), Qureshi et al. (2004) and Dellicour et al.

(2011).

Nucleotide sequence of mitochondrial Cytochrome-c gene (1544 bp fragment) from

Boselaphus tragocamelus individuals was amplified, sequenced and analysed. A total of sixteen variable sites were observed. Out of these, five variations were monomorphic for mutant allele.

Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 15 transitions and one transversion. Allele frequency of all variations was

108

DISCUSSION calculated and low mutant allele frequency was observed. No heterozygous individuals were found.

Polymorphisms in d-loop region (749 bp) of Boselaphus tragocamelus were identified.

Out of seventeen variable sites, three variations were found monomorphic for mutant allele.

Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 15 transitions and two transversions. Allele frequency of all variations was calculated and very low frequency of mutant allele was observed. As no heterozygous individuals were found so allelic frequency and genotypic frequency was same.

In Antilope cervicapra, 1139 bp fragment sequenced of mitochondrial Cytochrome-b gene was analysed. A total of thirteen variable sites were observed. Out of these, six variations were found monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 11 transitions and two transversions. Low frequency of mutant allele and no heterozygous individuals was observed.

In the Cytochrome-c gene of the mitochondrial DNA, 1544 bp fragment from Antilope cervicapra individuals was analysed. A total of twelve variable sites were observed. Out of these, three variations were found monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. There were 12 transitions but no transversion was observed. Allele frequency of all variations was calculated and very low frequency of mutant allele was observed. As no heterozygous individuals were found so allelic frequency and genotypic frequency was same.

Mitochondrial d-loop region (997 bp fragment) of Antilope cervicapra individuals was sequenced and analysed. A total of sixteen variable sites were observed. Out of these, ten variations were monomorphic for mutant allele. Remaining animals were also homozygous both

109

DISCUSSION for wild and mutant allele. The variable sites were comprised of 16 transitions and no transversion. No heterozygous individuals were found.

A total of eleven variable sites were observed in Cytochrome-b gene of Axis Axis. Six variations were monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. Transition substitution was found on all sites. All individuals were homozygous. Allele frequency of mutant allele was found low.

Cytochrome-c gene (1544 bp fragment) of Axis Axis was analysed and five variable sites were observed. Out of these, three variations were monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of three transitions and two transversions. Allele frequency of all variations was calculated and low mutant allele frequency was observed. No heterozygous individuals were found.

Polymorphisms in d-loop region of Axis Axis were identified. Out of sixteen variable sites, six variations were found monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 15 transitions and one transversion. Allele frequency of all variations was calculated and very low frequency of mutant allele was observed. As no heterozygous individuals were found so allelic frequency and genotypic frequency was same.

In Axis porcinus, 1139 bp fragment sequenced of mitochondrial Cytochrome-b gene was analysed. A total of thirteen variable sites were observed. Out of these, five variations were found monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 13 transitions. Low frequency of mutant allele and no heterozygous individuals was observed.

110

DISCUSSION

In the Cytochrome-c gene of the mitochondrial DNA, 1526 bp fragment from Axis porcinus individuals was analysed. A total of twelve variable sites were observed. Out of these, two variations were found monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. Eleven transitions and one transversion were observed. Allele frequency of all variations was calculated and very low frequency of mutant allele was observed. As no heterozygous individuals were found so allelic frequency and genotypic frequency was same.

Mitochondrial D-loop region of Axis porcinus (688 bp fragment) individuals was sequenced and analysed. A total of nineteen variable sites were observed. Out of these, seven variations were monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 18 transitions and one transversion. No heterozygous individuals were found.

Multidimensional scaling (MDS) plot of mitochondrial cytochrome-b, cytochrome-c and d-loop region for Boselaphus tragocamelus, Antilope Cervicapra, Axis Axis and Axis Porcinus was generated. Single gene and three genes combined sequences based MDS plot was generated for i) each species of deer, ii) Boselaphus tragocamelus & Antelope cervicapra (Bovidae), iii)

Axis axis & Axis porcinus (Cervidae)and iv) Boselaphus tragocamelus & Antelope cervicapra

(Bovidae) and Axis axis & Axis porcinus (Cervidae).

Cytochrome-b, cytochrome-c and d-loop based MDS plot for Boselaphus tragocamelus showed that samples are clustered. The greater clustering of the samples indicates that they contain lower within-population genetic variability. Same pattern was observed when combined sequence of three genes based plot was constructed. The degree of sample clustering shown on

111

DISCUSSION the MDS plot for Antilope cervicapra based on single gene and combined sequence is also informative. The clustered samples pattern showed less genetic variability.

Overlap was found in the overall genetic profile of the Axis axis as identified by analysis.

This result indicates that there is high genetic similarity. The greater clustering of the Axis porcinus samples also indicates lower genetic variability. Comparison of the combined sequence based genetic profiles of the Boselaphus tragocamelus and Antelope cervicapra found that the amount of genetic variation contained in each population is similar in each population but both species of Bovidae were significantly different. Differences were found between genetic variations among Cervidae. Overall significant genetic differences between species were found which showed that each species of deer were more tightly clustered around the range of genetic variation.

Pairwise evolutionary distance also showed that populations were significantly different from each other. Genetic variability and phylogenetic relationship within and between analysed groups was evaluated based on polymorphic loci from Bovidae and cervidae. The level of heterozygosity was relatively comparable in all evaluated groups of animals. The highest homozygosity was observed in all selected species of Bovidae and cervidae and differences were very low.

Cytochrome-b, cytochrome-c and d-loop based phylogenetic analysis of the gene sequences revealed that each species individuals comprise a clade that is clearly distinct from the clade comprised of other species of deer selected for this study. The neighbour-joining tree created from mitochondrial genes based on data set from cervidae and bovidae showed clear differentiation between Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis

112

DISCUSSION porcinus. Finding of this study indicated that these four species of deer have significant genetic variations that differentiate them from each other.

DNA sequence conservation occurs in Boselaphus tragocamelus (Pepin et al. 1995).

Gallagher et al. (1998) concluded that B.tragocamelus was karyotypically derived with conditions unique to species of Bovinae; some derived chromosomal conditions common to

Boselaphus tragocamelus, the African buffalo (Syncerus caffer), and some Tragelaphini could be convergent. Gene sequences for interleukin 2 (Das et al. 2006), Toll like receptor 3 (Dhara et al.

2007), and prion protein gene (Seabury et al. 2004) suggest that Boselaphus tragocamelus shares a common lineage with water buffalo (B. bubalis) and kudu (Tragelaphus) more than with domestic cattle.

In this study, we found evidence for a significant reduction in genetic diversity of the members of bovidae and cervidae from different regions of Pakistan. Both allelic richness and observed heterozygosity were lower in these populations compared to the larger populations from other regions previously reported by (Frantz et al. 2006, 2008; Hmwe et al. 2006; Kuehn et al. 2004; Martı´nez et al. 2002; Pe´rez-Espona et al. 2008; Zachos et al. 2007). It is likely that this reduction in genetic diversity is the direct result of a combination of geographic isolation and small population sizes in the recent past. Similar observations have also been reported by

Dellicour et al. in 2011.

Genetic variation is a base material for animal survival, which acts as the genetic source for prediction of future in conservation that leads to the conservation of animals, to analyze the status of conservation. Molecular markers plays initial guide for evaluation of the genetic variation (Allendorf et al. 2010). Therefore, the information on this level of genetic variation is prerequisite for designing effective strategy in future effective practices for conservation

113

DISCUSSION

(Crandall et al. 2000). However further genomic investigations should be carried out at larger scale.

Overall, low level of generic diversity revealed with higher level of genetic differentiation among different populations. The genetic analysis showed the generic variation at species level. Combined effects of field and molecular techniques standardize the conditions for genomic amplification and analysis for status of population structure in bovidae and cervidae species. The mitochondrial genome sequence will directly facilitate conservation, population, management, and genetic epidemiology research in bovidae and cervidae while also enabling conservation genomics in the endangered species of deer.

114

CHAPTER 6 SUMMARY

Many species of mammals have declined within the past two centuries due to human caused disturbances and the unsustainable use of natural resources. Molecular methods have an important role in phylogeny and diversity analysis. The present study was designed for diversity analysis of Boselaphus tragocamelus & Antelope cervicapra (Bovidae) and Axis axis & Axis porcinus (Cervidae) family in Pakistan. A total of 25 samples from each of the four species were collected from different parks, zoos and natural habitats. DNA was extracted, PCR primers were designed and cytochrome-b, cytochrome-c gene and d-loop regions were amplified by PCR.

PCR products were sequenced bi-directionally by Big DyeTM Terminator. Bioinformatics tools,

Blast 2 sequences, Clustal-W, MEGA-6, Bioconductor in “R” were applied for analysis. The clustering of the samples indicates that each species contains less within-population genetic variability. Same pattern was observed when sequence of three genes was combined and MDS plot was constructed. Phylogenetic analysis of the gene sequences revealed that each species comprised a clade that is clearly distinct from the clade comprised of other species of deer selected for this study. Finding of this study indicated that these species of deer have significant genetic variations among-species that differentiate them from each other. This is the first report from our region. The information of selected species of deer is prerequisite for designing effective strategy in future conservation practices. However further genomic investigations should be carried out at larger scale.

115

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