Phylogenetic relationships in Apocynaceae based on both nuclear and plastid molecular datasets A Dissertation Submitted in the Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in PLANT SCIENCES BY NAZIA NAZAR DEPARTMENT OF PLANT SCIENCES, FACULTY OF BIOLOGICAL SCIENCES, QUAID-I-AZAM UNIVERSITY ISLAMABAD, PAKISTAN 2012 Phylogenetic relationships in Apocynaceae based on both nuclear and plastid molecular datasets Doctor of Philosophy in Plant Sciences BY NAZIA NAZAR DEPARTMENT OF PLANT SCIENCES, FACULTY OF BIOLOGICAL SCIENCES, QUAID-I-AZAM UNIVERSITY ISLAMABAD, PAKISTAN 2012 Dedicated To My Parents Table of Contents Index of figures i Index of tables ii Acknowledgements iii List of abbreviations iv Abstract v Chapter One — General Introduction 1 1.1 Distribution of Apocynaceae 1 1.1.1 Asclepiadoideae 1 1.1.2 Secamonoideae 2 1.1.3 Periplocoideae 2 1.1.4 Apocynoideae and Rauvolfioideae 3 1.2 Economic importance of Apocynaceae 3 1.3 Apocynaceae ─ since Brown’s treatise 4 1.4 An overview of subfamilies in Apocynaceae sensu lato 7 1.4.1 Rauvolfioideae 7 1.4.2 Apocynoideae 10 1.4.3 Periplocoideae 13 1.4.4 Secamonoideae 15 1.4.5 Asclepiadoideae 17 1.5 The use of molecular data in cladistic analysis 20 1.6 Utility of plastid and nuclear regions for phylogenetic reconstruction in plants 21 1.7 Nuclear gene (Phytochrome A) 22 Chaper two — Materials and Methods 25 2.1 Taxon sampling 25 2.2 DNA extraction 25 2.3 Polymerase chain reaction (PCR) 34 2.3.1 trnL-F intron-spacer region 37 2.3.2 Nuclear gene PHYA 37 2.3.3 Promoter region of atpB gene 37 2.4 Purification of amplified products 37 2.5 Sequencing 38 2.6 Data analysis 38 2.6.1 Parsimony analysis 38 2.6.2 Bayesian analyses 38 Chapter three — Results 55 3.1 DNA extraction and PCR 55 3.2 Purification and sequencing of amplified products 55 3.3 Phylogenetic analysis based on plastid trnL-F intron region sequences 55 3.4 Phylogenetic analysis of Apocynaceae by using nuclear region (PHYA) 63 3.5 Combined phylogenetic analyses (PHYA and trnL-F) of Apocynaceae 64 3.6 Phylogenetic analysis based on promoter region of atpB gene promoter 79 Chapter four — Discussion 82 4.1 Incongruence 82 4.2 Asclepiadoideae 83 4.2.1 Fockeeae 83 4.2.2 Ceropegieae 83 4.2.3 Marsdenieae 87 4.2.4 Asclepiadeae 88 4.2.5 Asclepiadeae – ACT group 88 4.2.6 Asclepiadeae ─ MOG group 88 4.3 Secamonoideae 91 4.4 Periplocoideae 92 4.5 Apocynoideae 93 4.5.1 APSA clade 93 4.5.2 Nerieae 96 4.5.3 Malouetieae 96 4.5.4 Odontadenieae 97 4.5.5 Mesechiteae 97 4.5.6 Echiteae 98 4.5.7 Apocyneae 98 4.5.8 Baisseeae 98 4.6 Rauvolfioideae 99 4.6.1 Alstonieae and Aspidospermeae 100 4.6.2 Alyxieae 100 4.6.3 Vinceae 100 4.6.4 Plumerieae 103 4.6.5 Tabernaemontaneae 103 4.6.6 Melodineae 104 4.6.7 Carisseae 104 4.7 Phytochrome A 104 4.8 Conclusion 105 Chapter five — References 106 Appendix One 128 QIAquick PCR purification kit protocol 128 Appendix two 129 Cycle sequencing 129 Purification of cycle sequencing product 129 Index of figures Figure 1.1 A summary of the relationships among five subfamilies and twenty two tribes of Apocynaceae..................................................................................................24 Figure 2.1 Map showing the areas of plant collection in Pakistan..............................32 Figure 2.2 Map showing different countries of the world, from where different taxa of Apocynaceae were collected and stored in herbarium or as living collection in The Royal Botanic Gardens, Kew, London.........................................................................33 Figure 2.3 Showing locations of primers used to amplify different regions of plastid and nuclear genomes....................................................................................................35 Figure 3.1 Extraction of genomic DNA and amplified products of different plastid and nuclear regions.......................................................................................................56 Figure 3.2 One of the most parsimonious trees for Apocynaceae based on the sequences of plastid trnL-F region...............................................................................58 Figure 3.3 The Bayesian tree resulting from the analysis of the trnL-F sequences for Apocynaceae................................................................................................................60 Figure 3.4 Parsimony analysis of only PHYA sequences from different taxa of Apocynaceae................................................................................................................65 Figure 3.5 Bayesian tree based on only PHYA sequences...........................................67 Figure 3.6 Parsimony analysis of trnL-F sequences of taxa present in combined analyses........................................................................................................................69 Figure 3.7 Bayesian analysis of trnL-F sequences of taxa present in combined analyses....................................................................................................................... 71 Figure 3.8 One of the most parsimonious trees for Apocynaceae based on sequences of the combined dataset (PHYA and trnL-F)................................................................73 Figure 3.9 Bayesian analysis of Apocynaceae by using combined datasets (PHYA and trnL-F)..........................................................................................................................75 Figure 3.10 Parsimony tree generated by using molecular combined data set (trnL-F, atpB and PHYA)...........................................................................................................80 Figure 3.11 Majority rule consensus tree based on the Bayesian analysis of the combined molecular datasets (trnL-F, atpB and PHYA)..............................................81 i Index of tables Table 2.1 A list of the samples from Pakistan and The Royal Botanic Gardens Kew, London with vouchers information and place of collection.........................................26 Table 2.2 Sequences and sources of different primers used in this study...................36 Table 2.3 A list of taxa with GenBank accession numbers used in trnL-F and PHYA analyses........................................................................................................................39 Table 4.1 Current phylogenetic positions of taxa in different groups of Asclepiadoideae............................................................................................................84 Table 4.2 Phylogenetic position of taxa in different groups of Apocynoideae in comparison with previous studies................................................................................94 Table 4.3 Comparative overview of phylogenetic position of Rauvolfioideae’s taxa within present phylogenetic analyses and previous classifications............................101 ii Acknowledgment In the name of Allah, the Most Gracious and the Most Merciful Alhamdulillah, all praises to Allah for the strengths and His blessing in completing this thesis. First and foremost I wish to thank my supervisor Dr. Tariq Mahmood (Department of Plant Sciences) for his guidance, suggestion and encouragement throughout the Ph.D study. I wish to express my heartfelt appreciation and deep sense of devotion to Prof. Mark W. Chase (Royal Botanic Gardens, Kew, London) who was a permanent source of encouragement and guidance for completion of my research work in Jodrell laboratory. His scholarly ideas beautified the scientific nature of this research work. He always directed to enlighten the ways of life as well. I would also like to pay my cordial thanks to James J Clarkson and David Goyder (from Royal Botanic Gardens Kew, London) for their continuous cooperation, munificent guidance, moral inputs, support, unconditional help and valuable advices they offered for completion of my research. Their continuous encouragement and concern are highly appreciated. It gives me great pleasure to express my gratitude to Higher Education Commission of Pakistan, for providing Indigenous and IRSIP scholarships. I would like to express my gratitude to the Dean, Faculty of Biological Sciences, Prof. Dr. Asghari Bano and also to the faculty members of Department of Plant Sciences, Dr. Mushtaq Ahmad and Dr. Mir Ajab Khan for their support and help. I would also like to thanks late Dr. Muhammad Arshad (Cholistan Institute of Desert Studies, The Islamia University of Bahawalpur) for providing plant specimens from Pakistan. My acknowledgement also goes to all the technicians and office staffs of Department of Plant Sciences for their co-operations. My special thanks to my friends and fellows Sobia Kanwal, Ishrat Naveed, Faiza Munir, Shazia Rehman and all junior students of the Lab. I am deeply indebted to my parents for their support, both emotional and financial, over the years. Last, but certainly not least, I would like to give special thanks to Muhammad Sarfraz, who is a great listener and has always been at my side during the highs and lows of this project. Nazia Nazar iii List of Abbreviations ACT Asclepiadinae, Cynanchinae and Tylophorinae AIDS Acquired Immunodeficiency Syndrome APG Angiosperm Phylogeny Group APSA Apocynoideae, Periplocoideae, Secamonoideae and Asclepiadoideae bp Base Pair BP Bootstrap BSA Bovine serum albumin CI Consistency Index CsCl-EtBr Cesium Chloride Ethidium Bromide CTAB Cetyl Trimethyl Ammonium Bromide EDTA Ethylene diamine Tetra Acetic Acid ITS Internal Transcribed