COMPARATIVE PHYLOGEOGRAPHY OF SCHIZOTHORACINAE FISH IN NORTHERN PAKISTAN AND WESTERN CHINA MUHAMMAD FIAZ KHAN DEPARTMENT OF ZOOLOGY HAZARA UNIVERSITY, MANSEHRA PAKISTAN 2016 HAZARA UNIVERSITY, MANSEHRA DEPARTMENT OF ZOOLOGY COMPARATIVE PHYLOGEOGRAPHY OF SCHIZOTHORACINAE FISH IN NORTHERN PAKISTAN AND WESTERN CHINA BY MUHAMMAD FIAZ KHAN This research study has been conducted as partial fulfillment of the requirement for the Degree of Doctor of Philosophy in Zoology, Hazara University Mansehra, Pakistan August 22, 2016 COMPARATIVE PHYLOGEOGRAPHY OF SCHIZOTHORACINAE FISH IN NORTHERN PAKISTAN AND WESTERN CHINA SUBMITTED BY: MUHAMMAD FIAZ KHAN (PhD scholar) SUPERVISOR: Dr. Muhammad Nasir Khan Khattak Assistant Professor Department of Zoology Hazara University, Mansehra CO-SUPERVISOR: Prof. Chen Yifeng Institute of Hydrobiology Chinese Academy of Sciences Wuhan, China DEPARTMENT OF ZOOLOGY HAZARA UNIVERSITY, MANSEHRA PAKISTAN, 2016 DEDICATION THIS WORK IS DEDICATED TO MY PARENTS AND MY SUPERVISOR Table of Contents Chapter 1 1 INTRODUCTION 1 1.1 Study Area 1 1.2 Introduction to Qinghai-Tibetan China 3 1.3 Classifications of Fishes 3 1.4 Pakistan freshwater resources 4 1.5 Fishes of Pakistan 5 1.6 Fishes of China 6 1.7 Taxonomy of genus schizothorax 6 1.8 Origin of Schizothoracinae Fishes 8 1.9 Pleistocene glaciations 8 1.10 Phylogeographic predictions based on glaciations history 10 1.11 Role of Phylogeography in evolution 12 1.12 Rationale of the study 13 1.13 The objectives of the current study was 13 Chapter 2 14 REVIEW OF LITERATURE 14 2.1 Phylogeography 14 2.2 Genetic markers used in phylogeography 16 2.2.1 Mitochondrial genes (mtDNA) 16 2.2.2 Characteristics of Mitochondrial DNA 16 i 2.2.3 Cytochrome B Gene 19 2.2.4 Control region (D-Loop) 20 2.3 Importance of genetic material in fish identification 22 2.4 Disadvantages of mitochondrial material 23 2.5 Phylogeography of Schizothoracines fishes 23 2.6 Worldwide Distribution of Schizothorax 23 2.7 Role of glaciations history in Phylogeography 26 Chapter 3 29 MATERIALS AND METHODS 29 3.1. Chemicals used during experiment 29 3.2. List of solution used during experiment 29 3.3. Sampling sites 30 3.4 Collected species 31 3.5 Sample collection and preservation 31 3.6 Samples labeling 32 3.7 Fixing of whole specimens 34 3.8 Fish Identification 34 3.9 DNA extraction protocol 35 3.10 Gel Electrophoresis 36 3.11 Primers designing 37 3.12 Laboratory procedures 40 3.13 PCR Procedure 41 3.14 PCR mixture for 60ul 42 ii 3.15 Sequencing 43 3.16 Blast searches 43 3.17 Data Analysis 43 3.17.1 Gene identification and genome analyses 43 3.17.2 Phylogenetic Analysis 44 Chapter 4 46 RESULTS 46 4.1 Genome organization and composition 48 4.2 Overlapping regions and intergentic spacer 49 4.3 OL Region 50 4.4 Protein-Coding Genes 50 4.5 Comparative gene arrangement comparison of S. esocinus, S. plagiostomus and S. labiatus 51 4.6 Base composition in Schizothoracine fishes 61 4.6.1 Base composition in Schizothorax esocinus 61 4.6.2 Base composition in Schizothorax plagiostomus 64 4.6.3 Base composition in Schizothorax labiatus 67 4.7 Frequency of amino acid in protein coding gene 70 4.7.1 Frequency of amino acid in protein coding gene of S. esocinus 70 4.7.2 Frequency of amino acid in protein coding gene of S. plagiostomus 73 4.7.3 Frequency of amino acid in protein coding gene of S. labiatus 76 4.8 Codon usage in protein coding genes of schizothoracine 79 4.8.1 Codon usage in protein coding genes of S. esocinus 80 iii 4.8.2 Codon usage in protein coding genes of S. plagiostomus 84 4.8.3 Codon usage in protein coding genes of S. labiatus 86 4.9 Transfer RNA Genes 89 4.10 AT skew and GC skews value 93 4.11 Noncoding Sequences in Schizothoracinae 100 4.12 Phylogenetic of complete genomes 103 4.13 Comparative study tRNAs in genomes Schizothoracines species 108 4.14. Comparative of protein coding genes 109 4.15 Stop and start codon in protein coding genes 110 4.16 Comparative study of amino acid 111 4.17 Comparative size of other genes 112 4.18 Phylogenetic Analysis 113 4.19 Haplotype diversity in Schizothoracine fishes 113 4.20 Phylogenetic analysis of Cytb 116 4.21 Phylogenetic of Dloop region 118 4.22 Phylogenetic analysis of Cytb and Dloop 120 4.23 Divergence time of Schizothoracinae fishes in Pakistan and Tibet 122 Chapter 5 124 DISCUSSION 124 5.1 Genomic organization 124 5.2 Protein coding genes 125 5.3 Non-coding region 127 5.4 Transfer and Ribosomal RNA Genes 129 iv 5.5 Phylogeography 132 5.6 Divergence time of Schizothoracines fishes 136 CONCLUSIONS 139 RECOMMENDATIONS 140 Chapter 6 141 REFERENCES 141 APPENDICES 167 Appendix i: Best model test of Cytb. 167 Appendix ii: Best model test of Dloop. 170 Appendix iii: Best model test of combined data (Cytb and Dloop). 174 v List of Figures Fig 1: Map of Pakistan, showing major river system (Google, 2015). 2 Fig. 2: Cytochrome b protein (Google, 2015) 20 Fig. 3: Mitochondrial D loop showing different regions (Google, 2015) 21 Fig 4: Different steps and condition for PCR. 41 Fig. 5: DNA extraction bands of complete genome on gel electrophoresis. 46 Fig. 6: PCR results of complete genome on gel electrophoresis. 46 Fig. 7: PCR bands of Cytb gene on gel electrophoresis 47 Fig. 8: PCR bands of Dloop gene on gel electrophoresis 47 Fig. 9: Graphical chart of complete mitochondrial genome of Schizothorax esocinus. 54 Fig. 10: Graphical chart of complete mitochondrial genome of Schizothorax plagiostomus. 57 Fig. 11: Graphical chart of complete mitochondrial genome of Schizothorax labiatus. 60 Fig. 12: Percentile of amino acid in mitochondrial genome Schizothorax esocinus. 72 Fig. 13: Percentile of amino acid in mitochondrial genome Schizothorax plagiostomus. 75 Fig. 14: Percentile of amino acid in mitochondrial genome of Schizothorax. Plagiostomus. Error! Bookmark not defined. Figure 15: Percentile of amino acid in mitochondrial genome of Schizothorax. labiatus. 78 Fig. 16: Comparative study of amino acid in protein coding gene of Schizothoracines. 79 vi Fig. 17: The secondary structure of the 22 tRNA genes encoded by Schizothoracine mtDNA represented in cloverleaf form. Standard base pairings (G-C and A-T) are indicated by colons (.). 91 Fig. 18: Sequences of the control region from the S. esocinus mitochondrial genome. 101 Fig. 19: Sequences of the control region from the S. plagiostomus mitochondrial genome. 101 Fig. 20. Sequences of the control region from the S. labiatus mitochondrial genome. 102 Fig. 21. The evolutionary history was inferred using the Neighbor-Joining method. 105 Fig. 22. Neighbor joining phylogenetic tree of complete mitochondrial genomes of S. plagiostomus with closely related species. 106 Fig. 23. The evolutionary history was inferred using the Neighbor-Joining method. 107 Fig. 24: Phylogenetic tree showing the relationship of Schizothoracines fishes on the basis of Cytb gene. Each branch showing Bayesian posterior probability (PP) ≥ 0.95 %. 117 Fig. 25: Phylogenetic tree showing the relationship of Schizothoracines fishes on the basis of Dloop. Each branch showing Bayesian posterior probability (PP) ≥ 0.95 %. 119 Fig. 26: Phylogenetic tree showing the relationship of Schizothoracines fishes on the basis of combined mtDNA (Cytb, Dloop) gene. 121 Fig. 27: Ultra metric ML tree of Schizothoracine fishes based on the NPRS transformation using Cytb data. 123 vii List of Tables Table: 1. Details of specimens collected from different locations 33 Table 2: PCR and sequencing primers designed from the complete mitochondrial genome 38 Table 3: PCR and sequencing primers used for amplification of Cytb and Dloop. 40 Table 4. Characteristics of genes in the mitochondrial genome of Schizothorax esocinus 52 Table. 5: Characteristics of the mitochondrial genome of S. Plagiostomus 55 Table 6. Characteristics of the mitochondrial genome of S. Labiatus 58 Table 7: The base composition in different regions of mitochondrial genome of Schizothorax esocinus. 62 Table 8: The base composition in different regions of mitochondrial genome of Schizothorax plagiostomus. 65 Table 9: The base composition in different regions of mitochondrial genome of Schizothorax labiatus. 68 Table 10: Frequency of amino acid in protein coding gene in Schizothorax esocinus are given in percent. 71 Table 11: Frequency of amino acid in protein coding gene in schizothorax plagiostomus are given in percent. 74 Table 12: Frequency of amino acid in protein coding gene in Schizothorax labiatus. 77 Table 13: Amino acid, codon number, frequency and codon usage in protein coding gene of S. esocinus. 81 viii Table 14: Amino acid, codon number, frequency and codon usage in protein coding gene of S. plagiostomus. 84 Table 15: Amino acid, codon number, frequency and codon usage in protein coding gene of S. labiatus. 87 Table 16: Table shows the AT skew and GC skews value of all genes in mitochondrial genome of S. esocinus. 94 Table 17: Table shows the AT skew and GC skews value of all genes in mitochondrial genome of S. plagiostomus. 96 Table 18: Table shows the AT skew and GC skews value of all genes in mitochondrial genome of S. labiatus. 98 Table 19: Comparative size (bp) of transfer RNA in S. Plagiostomus (bp), S. esocinus (bp) and S. labiatus (bp) 108 Table 20: Comparative size (bp) of protein coding gene in S. Plagiostomus (bp), S. esocinus (bp) and S. labiatus (bp). 109 Table 21: Start and stop codon in protein coding gene of S. Plagiostomus (bp), S. esocinus (bp) and S. labiatus (bp). 110 Table 22: Comparative number of amino acid in protein coding gene of S.