Investigation into the Use of Molecular Methods to Distinguish between Species of Caladenia Subgenus Calonema Chutima Kongjaroon A thesis submitted in a fulfillment of the requirements for the degree of Doctor of Philosophy School of Engineering and Sciences Faculty of Health, Engineering and Science Victoria University Melbourne Australia 2012 Thesis abstract The phylogeny of typical spider orchid (Caladenia subgenus Calonema) is investigated for the first time. The analyses were performed using 17 RAPD and 10 ISSR primers on 30 taxa representing the three spider orchid groups (the dilatata, patersonii and reticulata groups) yielding 135 RAPD and 63 ISSR polymorphic markers. The average number of polymorphic markers produced from 17 RAPD and 10 ISSR primers were 5.12 and 4.48, respectively. 76 RAPD markers and 38 ISSR markers were polymorphic within spider orchid species. The highest number of amplified DNA fragments were produced from OPE15 (8.77 fragments) and UBC 842 (6.71 fragments) while OPF04 (2.93 fragments) and UBC 825 (3.02 fragments) gave the smallest number of amplification products. The average Dice genetic similarity of pairs of individuals within a species ranged from 0.772 to 0.939 based on RAPD and from 0.770 to 0.976 for ISSR data. The 117 spider orchid individuals analysed by RAPD, ISSR and combined data using cluster analysis were classified into three groups that correlate with those based on morphological analysis: reticulata, patersonii and dilatata. The phylogenetic analysis based on three data sets, presented relationships at the intraspecific level that were well supported by bootstrapping. However, the interspecific relationships within the groups remained unclear. Principal component analysis of RAPD and ISSR data also conducted indicating closed relationships between the reticulata and patersonii groups. High correlation (r=0.90) was detected between RAPD and ISSR markers by mean of Mantel test. ii Phylogenetic relationships within the dilatata group were also investigated using ITS and the trnT (UGU) and trnL (UAA) 5’exon spacer sequences. The ITS and the trnT- L had the approximate sizes of 750 bp and 700 bp, respectively. The G+C content of ITS sequences varied from 41.67 to 42.12% which is higher than the G+C content of the trnT-L (25.80 to 26.26%). The aligned data matrix of the ITS region included a total of 671 sites including 544 constant characters, 102 parsimony uninformative characters and 25 parsimony informative characters. The aligned data matrix of the non-coding region of the trnT-L included a total of 677 sites that consisting of 617 constant characters, 49 parsimony uninformative characters and 11 parsimony informative characters. The strict consensus tree inferred from both ITS and the trnT-L sequences and combined data had poor resolution of the dilatata grouping as indicated by unresolved polytomies that were supported with relatively low bootstrap value. The ITS phylogeny well resolved the separation of C. venusta, C. cardiochila, C. tessellata and C. patersonii from the dilatata spider orchid. The trnT-L phylogenetic relationships provided sufficient information to resolve the relationships between C. flaccida, C. latifolia and L. menziesii and typical spider orchids. The phylogenetic tree constructed from combined data tended to have the feature of ITS phylogenetic tree, as ITS sequences had more polymorphisms. The trnT-L sequence of C. verrucosa also showed specific sequences that might be useful in the identification of this species. The use of molecular characters to assess the phylogenetic relationships among the species of Caladenia subgenus Calonema does not address the position of spider iii orchids within their belonging group. This evidence indicates that taxonomic re- examination of the Caladenia subgenus Calonema is needed. iv Student Declaration “I, Chutima Kongjaroon, declare that the PhD thesis entitled Investigation into the Use of Molecular Methods to Distinguish between Species of Caladenia Subgenus Calonema is no more than 100,000 words in length including quotes and exclusive of tables, figures, appendices, bibliography, references and footnotes. This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma. Except where otherwise indicated, this thesis is my work”. Chutima Kongjaroon December, 2011 v Acknowledgements I would like to acknowledge the assistance of my principal supervisor Dr. Randall Robinson, my co-supervisors Dr. Swati Baindur Hudson, Wendy Probert and Neville Walsh. As for my supervisors, their assistance has been most invaluable to my research. Without their assistance my work would have been impossible to finalize. I am sincerely grateful to my supervisors. I greatly appreciate the assistance of Professor Mark Burkman and Sita Ariati School of Botany, The University of Melbourne and Dr. Mark Dobrowolski, Department of Primary Industries for their valuable suggestion with regards to the statistical analysis of phenetic analysis. I express my sincere appreciation to Gary Backhouse, Michele Duncan, Andrew Pitchard and Jeffrey Jeanes for providing necessary information and valuable discussion. I would like to express my gratitude to the following people who provided me with plant materials for this study: Gary Backhouse, Fiona Coates, Michele Duncan, C. Gordes, F. Gordes, Glen Johnson, Mick Kelgan, Andrew Pitchard, Gail Pollard, Paul Scannell Gidja Walker and Nevill Walsh. I give my special thanks to Gary Backhouse, Malcolm Thomas and Dick Thomson for photographs of orchids. vi I have also appreciated the warm welcome as well as valuable discussion from members of Royal Botanic Gardens, Melbourne and Australian Orchid Society: Elizabeth James, Rob Cross and Dick Thomson. I would like to thank my lab mates and technical staff: Dr. Joshuan Jonhson, Dr. Bogden Zisu, Dr. Panuwat Suppakul, Dr, Thanut Amatayakul, Belinda Davis, Kathryn Lauder, Umi Purwandari, Gurdeep Singh, Nayana Bandara, Osaana Donkor, Ajith Bandara, Dale Tomlinson, Nikola Popovik, Charmaine DiQuattro, Irawati Prasatya, Stacey Lloyd, Min Thi Nguyen, Joseph Pelle, and Michael Rogerson. Special thank goes to Dr. Sarah Fraser for her warm help. I would like to express my sincere thankfulness to Mr. Wiboon Chulerttiyawong, former Minister Counselor (Education) and Mr. Phanu Sungkhavon, Minister Counselor (Education) as well as Ms. Somjit Wattanatassi, Secretary Officer, at the Royal Thai Embrassy, Canberra and Ms. Rujirat Duangjino, Ms. Somkid Ratanatanyong at the Office of the Civil Service Commission, Bangkok, Thailand for their support and contributions. I am indebted to the following import organizations and contributors of my study, the Royal Thai Government and Maejo University. This research was supported in part with funds from the Australian Orchid Foundation and the Australian Biological Resources Study. vii The support of monks, ubasoks, and ubasikaes as well as the members of the Dhammakaya International Society of Australia Inc. who have been supporting me physically and spiritually, especially Jessada Suttinon and Atorn Nillapong. I would like to express my most sincere thanks to the following people: Venerables (Phra) Chalard Yimsoomboon, Ronrawee Rawiphapho, Sakcsith Siripaththo and the Detphrakoune family who have been very helpful to me during my stay in Australia. Without their support, I will proprably encounter enormous difficulties. Finally, and most importantly, to my dearest parents, I would like to give my heart- felt thanks for the great start they gave me in life, for teaching me a sense of prospective, for their emotional and spiritual support, not only during my study life but during my entire life. More than any statement, my wholehearted gratitude goes to them. viii Table of Contents Title i Abstract ii Student Declaration v Acknowledgements vi Table of Contents ix List of Figures xiii List of Table xvii Chapter 1:Introduction 1 1.1 Genus Caladenia 2 1.2 Plant description 2 1.2.1 Floral characteristic 6 1.3 Ecology 7 1.4 Taxonomy of genus Caladenia 9 1.4.1 Subtribe Caladeniineae 9 1.4.2 Genus Caladenia – early classification systems 11 1.4.3 Genus Caladenia classification in 20th century 13 1.5 Spider orchids in Victoria 19 1.6 Spider orchid classification status 39 Chapter 2:Background 41 2.1 Plant taxonomy 41 2.2 Plant genomes 44 2.3 Types of markers 45 2.3.1 Morphological and anatomical and physiological characters 46 2.3.2 Molecular characters 46 2.3.2.1 Type of DNA markers 48 2.4 Uses of molecular markers in plant research 53 2.5 Constructing a phylogenetic tree with molecular markers 54 2.5.1 Phylogenetic trees 54 2.5.2 Methods for building phylogenetic trees 56 2.5.3 Phylogenetic tree construction methods used in this study 56 2.5.3.1 Making trees using UPGMA distance-based method 57 2.5.3.2 Trees generated by maximum parsimony character- 58 based methods 2.5.4 Evaluation tree using randomizing tests and bootstrapping 59 2.6 The used of molecular marker in orchid phylogenetic relationships 62 2.7 Current status of molecular based classification in spider orchids 67 Chapter 3:Genetic Relatedness among Caladenia Species Using RAPD 68 Markers 3.1 Introduction 68 3.2 Material and methods 70 3.2.1 Plant materials 70 3.2.2 DNA extraction 79 3.2.3 Amplification of RAPD markers 80 3.2.4 Data analysis 81 ix 3.3 Results 83 3.3.1 RAPD marker profile 83 3.3.1.1 Number of monomorphic and polymorphic bands 83 produced within spider orchid species and over the whole sample