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Habitat Distribution, Diversity and Systematics of Mus Spp

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Habitat Distribution, Diversity and Systematics of Mus Spp HABITAT DISTRIBUTION, DIVERSITY AND SYSTEMATICS OF MUS SPP. IN POTHWAR, PAKISTAN SHAHNAZ BIBI 03-arid-783 Department of Zoology/Biology Faculty of Sciences Pir Mehr Ali Shah Arid AgricultureUniversity, Rawalpindi Pakistan 2017 2 HABITAT DISTRIBUTION, DIVERSITY AND SYSTEMATICS OF MUS SPP. IN POTHWAR, PAKISTAN by SHAHNAZ BIBI (03-arid-783) A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Zoology Department of Zoology/Biology Faculty of Sciences Pir Mehr Ali Shah Arid AgricultureUniversity, Rawalpindi Pakistan 2017 3 CERTIFICATION I hereby undertake that this research is an original and no part of this thesis fall under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences. Name:Shahnaz Bibi Signature: ______________________ Registration No: 03-arid-783 Date: __________________________ Certified that the contents and form of thesis entitled “Habitat Distribution, Diversity and Systematics of Mus spp. in Pothwar, Pakistan” submitted by Ms. Shahnaz Bibi have been found satisfactory for the requirement of degree. Supervisor:________________________ (Dr. M. Sajid Nadeem) Member: __________________________ (Prof. Dr. Mirza Azhar Beg) Member:___________________________ (Dr. Ghazala Kaukab Raja) Chairperson: ________________ Dean:____________________ 4 Director, Advanced Studies: _________________________ To my parents, because I owe it all to them. Many Thanks! 5 CONTENTS LIST OF FIGURES 10 LIST OF PLATES 13 LIST OF ABBREVIATIONS 14 ACKNOWLEDGMENTS 14 ABSTRACT 16 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 05 2.1 MOLECULAR STUDIES ON MUS 06 2.2 MORPHOMETRIC AND CRANIAL STUDIES 09 2.3 PELAGE COLOUR VARIATIONS IN MUS 12 2.4 HABITAT DISTRIBUTION OF MUS 13 3 MATERIALS AND METHODS 16 3.1 STUDY AREA 16 3.2 HABITAT STUDY AND SAMPLE COLLECTION 18 3.3 MOLECULAR ANALYSIS 19 3.3.1 Extraction of DNA 19 3.3.2 PCR Amplification of Cytochrome b 22 3.3.3 Gel Electrophoresis of PCR Products 23 6 3.3.4 Clean Up of PCR Products 23 3.3.5 Sequencing of Cytochrome b 23 3.3.6 Population Expansion, Mismatch Analysis and Divergence 24 Time Estimation 3.4 MORPHOMETRIC AND CRANIAL ANALYSIS 29 3.5 PELAGE COLOUR ANALYSIS 31 3.6 STATISTICAL ANALYSIS 31 4 RESULTS AND DISCUSSION 34 4.1 MOLECULAR ANALYSIS 34 4.1.1 Mismatch Distribution 35 4.1.2 Pak-India Centre of Diversity and Expansion Events 36 4.1.3 Population Expansion 44 4.2 MORPHOMETRIC AND CRANIAL ANALYSIS 46 4.2.1 Morphometric Variations at Species Level 51 4.2.1.1 Morphometric Variations Among M. musculus Sub-spp. 52 4.2.1.2 Morphometric Variations Among M. m. castaneus Sub- 54 lineages 4.2.2 Cranial Variations 56 4.3 PELAGE COLOUR VARIATIONS 68 7 4.4 HABITAT DISTRIBUTION 74 4.4.1 Relative Abundance of The Collected Taxa 74 4.4.2 Species Richness 75 4.4.3 Geographical Segregation 77 CONCLUSION AND RECOMMENDATIONS 85 SUMMARY 87 LITERATURE CITED 89 LIST OF TABLES Table No. Page 3.1. Sampling localities, habitat type, sample size (N) and 20 geographical coordinates of 145 mice specimens analyzed in this study 3.2. List of Mitochondrial DNA primers used in this study 25 3.3. List of cytochrome b and mtDNA control region sequences 28 along with accession numbers and localities used as reference in this study 4.4. Genetic diversity indices of four M. m. castaneus Cyt b 41 lineages. Sample size (N), number of haplotypes (H), number of polymorphic sites (S), haplotype diversity (Hd), nucleotide diversity (Pi), mean number of pairwise differences among sequences (K), Tajima’s D, and FU’s Fs values of neutrality tests from mismatch distribution analysis 8 4.5. Genetic diversity indices of M. m. castaneus mitochondrial 42 control region data. Sample size (N), number of haplotypes (H), number of polymorphic sites (S), haplotype diversity (Hd), nucleotide diversity (Pi), mean number of pairwise differences among sequences (K), Tajima’s D (with its P value), and FU’s FS values of neutrality tests from mismatch distribution analysis 4.6. Estimation of population expansion time (years before present) 43 for all M. m. castaneus sublineages based on Cyt b under two substitution rates 4.7. Comparison of body weight (BW), head and body length 53 (HBL), tail length (TV), hind foot length (HFL) and ear length (EL) between M. cookii, M. musculs and M. terricolor. Body weight is in g all other measurements are in mm 4.8. Comparison of body weight (BW), head and body length 55 (HBL), tail length (TV), hind foot length (HFL) and ear length (EL) between three sub-species of M. musculs. Body weight is in g, all other measurements are in mm 4.9. Morphometric measurements of four Cyto b lineages of mice 57 of M. musculus castaneus (CAS). Statistics given are no of specimens (N), mean (X), standard deviation (SD), and coefficient of variation (CV). Morphometric variants included are body wt (BW), head and body length (HBL), tail vertebrae length (TV), hind foot length (HF) and ear length (E). Body weight is in g and the linear measurements are in mm 9 4.10. Percentage count of the number of specimens correctly 58 classified to the respective sub-lineage on the basis of Discriminant Function Analysis (DFA) of morphological variables 4.11. Standardized Canonical Discriminant Function Coefficients of 58 morphological variables for M. m. castaneus sub-lineages 4.13. Summery statistics of cranial and dental variables of M. 60 Cookii, M. musculs and M. terricolor 4.14. Summary statistics of cranial and dental variables of three sub- 62 species of M. musculus 4.15. Summary statistics of cranial and dental variables of four sub- 63 lineages of M. m. castaneus 4.16. Percentage count of the number of specimens correctly 64 classified to the respective sub-lineage on the basis of Discriminant Function Analysis (DFA) of cranial variables 4.17. Standardized Canonical Discriminant Function Coefficients of 64 cranial variables for M. m. castaneus sub-lineages 4.18. Munsell colour chart values for four Cytb sub-lineages of mice 70 of M. musculus castaneus (CAS). 10 4.19. Percentage count of the number of specimens correctly 71 classified to the respective sub-lineage on the basis of Discriminant Function Analysis (DFA) of coat colour variable 4.20 Standardized Canonical Discriminant Function Coefficients of 71 coat colour variables for M. m. castaneus sub-lineages LIST OF FIGURES Figure No. Page 3.1. Map of study area with sampling localities 17 4.1 NJ tree for all Cyt b sequences done during the study 40 depicting Mus species and sub-species recovered from the study area 4.2. ML tree for Cyt b sequences from M. m. castaneus 47 referred to as CAS1, CAS2, CAS3, and CAS4 along with M. m. domesticus used as outgroup 4.3. Median-Joining Network depicting four cyto b lineages 48 of M. m. castaneus 4.4. MJ Network for concatenated sequences (Cyt b and 49 control region) from individuals for which sequences from both genes were available. 11 4.5. Mismatch distribution for all M. m. castaneus sub 50 lineages (a) CAS1, (b) CAS2, (c) CAS3 a, (d) CAS3 b and (e) CAS4 4.6 Results of Discriminant Function Analysis (1st two 59 functions) of 5 morphological variables 4.7 Results of Discriminant Function Analysis (1st two 65 functions) of cranial variables 4.8 Results of Discriminant Function Analysis (1st two 72 functions) of coat colour variables 4.10 The proportion of mice specimens taken from different 76 habitat types in study area 4.11 The proportion of different Mus spp. collected from the 76 study area 4.12 The proportion of different sub-spp. of M. musculus 78 collected from the study area 4.13 The proportion of different lineages of M. m. castaneus 78 collected from the study area 4.14 The proportion of occurrence of three Mus spp. in each 79 habitat type 12 4.15 The proportion of occurrence of three subspp. of M. 79 musculus in each habitat type 4.16 The proportion of occurrence of different lineages of M. 80 m. castaneus in each habitat type 4.17 The proportion of occurrence of three Mus species in 82 different districts of Pothwar 4.18 The proportion of occurrence of three sub-spp. of M. 82 musculus in different districts of Pothwar 4.19 The proportion of occurrence of different CAS lineages 83 in each district in study area 13 LIST OF PLATES Plate No. Page 3.1. Cytochrome b gene amplified from different Mus specimens. 26 M is ladder (easy ladder100-2000bp). 3.2 Mitochondrial DNA control region amplified from different 27 Mus specimens. M is ladder (easy ladder100-2000bp). 4.1 Coat colour variations in M. m. castaneus. (A) Golden brown, 69 (B) Yellowish brown back and golden legs, (C) Pure dark grey with white feet, (D) Dark brown. 14 LIST OF ABBREVIATIONS PCR Polymerase Chain Reaction Dbndd1 Dysbindin (dystrobrevin binding protein 1 Tcf Transcription factor 25 (basic helix-loop-helix) Afg3l1 ATPase family gene 3 like-1 (yeast) mg Milligram Cyt b Cytochrome b mm Millimeter µl Microliter scnDNA Single-copy nuclear DNA mtDNA CR Mitochondrial DNA control region CAS Mus musculus castaneus MAFFT Multiple alignment using fast fourier transform τ Tau MEGA Molecular evolutionary genetics analysis Myr Million years DNAsp DNA sequence polymorphism HBL Head and body length TL Tail length EL Ear length ACKNOWLEDGMENTS 15 All thanks to Allah Almighty, the source of knowledge and wisdom endowed to mankind, who gave me courage to complete this thesis. Countless salutations are upon the Holy Prophet Muhammad (PBUH), who is forever a symbol of direction and torch of guidance for humanity as whole. I would like to extend thanks to the many people, in many countries, who so generously contributed to the work presented in this thesis.
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