A molecular systematic study of the African endemic cycads by PHILIP ROUSSEAU Dissertation submitted in fulfilment of the requirments for the degree MAGISTER SCIENTIAE in BOTANY in the FACULTY OF SCIENCE DEPARTMENT OF BOTANY AND PLANT BIOTECHNOLOGY at the UNIVERSITY OF JOHANNESBURG SUPERVISOR: PROF. M. VAN DER BANK CO-SUPERVISOR: DR. P.J. VORSTER FEBRUARY 2012 Declaration I declare that this dissertation has been composed by me and the work contained within, unless otherwise stated, is my own. Philip Rousseau February 2012. II Table of contents Abstract p. V Foreword p. VI Acknowledgements p. VII List of abbreviations p. VIII Chapter 1. Introduction and aims 1.1. General introduction p. 1 1.2. Taxonomy p. 3 1.3. Distribution p. 5 1.4. Conservation status p. 6 1.5. Intra-generic concepts p. 9 1.5.1. Stangeria p. 9 1.5.2. Encephalartos p. 10 1.6. Aims and hypotheses p. 12 Chapter 2. Material and methods 2.1 Taxon sampling p. 29 2.2 DNA extraction p. 29 2.3 Polymerase chain reaction (PCR) p. 29 2.4 DNA sequencing p. 30 2.5 Sequence alignment and analysis of molecular data p. 30 2.5.1. Phylogenetic analysis p. 31 2.5.2. DNA barcoding analysis p. 32 Chapter 3. Phylogenetic analyses: Results and discussion 3.1. Molecular evolution p. 46 3.2. Combined plastid analysis p. 46 3.3. Nuclear ITS analysis p. 47 3.4. Combined molecular analyses p. 47 3.4.1. Clade A: lineage 1 p. 47 3.4.2. Clade B p. 50 Clade B: lineage 2 p. 50 Clade B: lineage 3 p. 51 Clade B: lineage 4 p. 52 Clade B: lineage 5 p. 53 Lineage 5: sub-lineage A p. 53 Lineage 5: sub-lineage B p. 54 3.4.3. Clade C p. 55 Clade C: sub-clade I: lineage 6 p. 55 III Lineage 6: sub-lineage A p. 56 Lineage 6: sub-lineage B p. 56 Clade C: sub-clade II: lineage 7 p. 57 Lineage 7: sub-lineage A p. 57 Lineage 7: sub-lineage B p. 58 Clade C: sub-clade III: lineage 8 p. 59 Clade C: sub-clade III polytomy p. 60 Encephalartos ferox p. 61 Clade C: sub-clade III: lineage 9 p. 61 Clade C: sub-clade III: lineage 10 p. 62 Encephalartos hirsutus and E. inopinus p. 63 3.5. Conclusions p. 63 Chapter 4. DNA Barcoding: Results and discussion 4.1. General introduction p. 100 4.2. Results p. 101 4.2.1. Sequencing success rates p. 101 4.2.2. Genetic variation p. 102 4.2.3. Species resolution and species concepts p. 103 4.3. Discussion p. 103 4.3.1. Success rates p. 103 4.3.2. Genetic variation p. 104 4.3.3. Species resolution p. 106 4.4. Conclusions p. 108 Chapter 5. General conclusions p. 137 Chapter 6. References p. 139 Appendix 1. Complete collection list p. 155 Appendix 2. Canadian Center for DNA Barcoding protocols p. 163 IV ABSTRACT Africa's cycads (66 species and 2 subspecies in two endemic genera: Encephalartos and Stangeria) are extremely endangered with four species extinct in the wild and 80% threatened (CR, EN, or VU) with all included in CITES Appendix 1. Although South Africa has some of the world’s strictest cycad legislation, these plants are still under threat from illegal collection for horticulture and medicine especially where they are seized in an unidentifiable condition. Currently developed legislation demands accurate identification for permit issue. Ex situ conservation of genetic and locality based diversity is paramount. Furthermore, taxonomically many species of unknown origin are difficult to identify especially when diagnostic characters are absent. Species delimitation and numbers are uncertain with field observations often contradicting current understanding. DNA barcoding can assist with all the above-mentioned scenarios. In the current study all proposed DNA barcoding regions (matK, rbcLa, psbA-trnH, and nrITS) along with several additional regions were tested on ~350 samples from which a phylogeny of 63 of the 65 Encephalartos species was also constructed. Results show general good amplification and sequencing success of proposed barcoding regions, although a shift to specialist primers was made in several cases. Genetic variation however was extremely low as is resolution at species level, even when multi-locus barcodes were employed. Results obtained from the phylogenetic analyses show an increase in resolution at both species and higher levels compared to previous work and as such several new groupings are delimitated. Each species grouping is characterised by shared, derived morphological, ecological, and geographic characters and when compared to previous phylogenetic studies are supported to some extent. The current study provides the first step towards a much-needed monograph and revision of the entire genus Encephalartos. V FOREWORD ThIs dissertation is presented in six chapters. Chapter 1 focuses on the general importance of the world’s cycads with emphasis on the study group. Key areas are the taxonomy, distribution, and conservation with the aims and hypothesis stated. Importantly all species authorities are listed in Table 1.1 while generic authorities are in Table 1.2. Chapter 2 outlines the general methods used in the study. Note that all samples from the New York Botanical Garden were not submitted to GenBank, as they are the property of Dr. Damon Little. Chapter 3 presents results and a discussion of the phylogenetic relationships within the genus Encephalartos. Chapter 4 presents results of the DNA barcoding study, a discussion thereof with concluding remarks. Chapter 5 presents a general conclusion on all aspects of the study including hypotheses and aims as set out in Chapter 1. Chapter 6 contains all literature referred to in the preceeding chapters. Lastly, Appendix 1 contains a complete collection list and Appendix 2 contains all protocols as performed at the Canadian Centre for DNA Barcoding. All photographs, figure and tables, unless otherwise indicated, were done by P. Rousseau. VI ACKNOWLEDGEMENTS This work would not have been possible without the financial assistance of the following insitutions: National Research Fund of South Africa (NRF), Royal Society UK, University of Johannesburg, the National Science Foundation (EF-0629890), the Alfred P. Sloan Foundation at the Cullman Program for Molecular Systematics at the New York Botanical Garden. This project was also partly funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (2008-OGI-ICI-03). Firstly, I would like to acknowledge Prof. Braam van Wyk who promted me into this project, your wisdom and vision has once more humbled me. For samples of these rare and difficult taxa, a special thanks goes to the Pretoria National Botanical Garden, the University of Johannesburg, the University of Pretoria and especially Susan Myburgh, the Lowveld National Botanical Garden and especially Karin van der Walt, Andre Cilliers and Robert Rousseau for access to their prized collections, Adolf Fanfoni for his multiple contributions to the project, and for the very rare samples donated by Dr. Xander de Kock and Prof. Nat Grobbelaar. Also for the various photographs of some of the rarest plants in the world a special thanks to the members of the cycad forum and Art Vogel and Anders Lindström. A special word of thanks is in order for Prof. Grobbelaar whose opinion was consulted and whose reference works were of undeniable value. To Prof. Dennis Stevenson, Dr. Roy Osborne and Prof. John Donaldson, whom I have had the privilege of communicating with on occasion, thanks for the advice and the motivation. This work would most certainly not be possible without the gracious help of Dr. Damon Little whose appreciation of the difficulties in doing molecular work on cycads was a welcome justification for the troubles endured and overcame with his help. At the University of Johannesburg numerous individuals have made significant contributions to the project, in particular I would like to thank Mr. Thinus Fourie for getting me started and all the administrative assistance, Mrs. Helen Long for administrative help, and Dr. Olivier Maurin for assistance with the numerous tasks demanded by the project. Also to my life long friend Wikus van Wyk for the help with the data manipulation in excel, automating hours of manual labour. To my supervisor Prof. Michelle van der Bank, thank you for your patience and commitment even though there were easier more rewarding projects, thank you for indulging a student his passion. Also for the numerous opportunities you have entrusted to me, you have contributed so much to the scientist I am today. Lastly to my co-supervisor Dr. Piet Vorster, a giant in the field, thank you so much for your genuine concern, open and friendly sharing of your wealth of knowledge and shared excitement surrounding these wonderful plants. Finally this work is dedicated to my father Robert Rousseau whose love of these plants has shaped so much of my life. Ek het oor die twee jaar so baie van Pa in my leer sien, dankie vir wat Pa-hulle in my gekweek het. VII LIST OF ABBREVIATIONS The following abbreviations are used throughout the text: ACDB: African Centre for DNA Barcoding BLAST: Basic Sequence Alignment Search Tool BOLD: Barcoding of Life Data Systems BP: Bootstrap Percentages CBOL: Consortium for the Barcode of Life CCDB: Canadian Centre for DNA Barcoding CI: Consistency Index CITES: Convention on International Trade in Endangered Species CR: Critically Endangered CTAB: Hexadecyltrimethylammonium Bromide CWOFI: Cycad World of Innovation DD: Data Deficient ddH20: Double Distilled Water DNA: Deoxyribonucleic Acid DNTP: Deoxyribonucleotide DRC: Democratic Republic of the Congo EF: Experimental Farm (UP) EN: Endangered EW: Extinct in the Wild IBOL: International Barcode of