An Assessment of the Genetic Diversity and Origin of the Invasive Weed

AN ASSESSMENT OF THE GENETIC DIVERSITY AND ORIGIN OF THE INVASIVE WEED CHROMOLAENA ODORATA (L.) KING AND ROBINSON IN SOUTH AFRICA A thesis submitted in fulfilment of the requirements for the degree of MASTER OF SCIENCE of RHODES UNIVERSITY by INGE VON SENGER July 2002 Abstract Chromolaena odorata (L.) King and Robinson is a n alien invasive weed to most of the Old World tropical regions of the earth, including South Africa where it is morphologically distinct from most other C. odorata plants examined from both its native and invasive range. It is thought that these morphological differences are related to difficulties encountered in successful establishment of biological control agents on the South African population of C. odorata. It has been postulated that the source population of the South African population will harbour potential biocontrol agents that will be suited to successful establishment on the South African plants. Several morphological, cytological and isozyme studies have been attempted to identify the source population of the South African population, but these have failed to identify the origin of the South African population. In this dissertation two PCR-based methods were attempted, in an investigation into whether the morphological differences and difficulties in establishment of biocontrol agents have a genetic basis. The two techniques attempted were: Inter Simple Sequence Repeat (ISSR) amplification, and DNA sequencing. Results could not be obtained using the ISSR method, and the reason for this was not discovered despite extensive trials. The internal transcribed spacer region and the external transcribed spacer region sequences were obtained from five samples, and compared. It was found that the ETS region gave more phylogenetic signal at the intraspecific level than the ITS region. However, due to difficulties in amplification of the external transcribed spacer region, work here focussed on obtaining Internal Transcribed Spacer sequences for 61 samples. Each of the samples sequenced had a unique ITS sequence, displaying a high level of intraspecific genetic diversity. The degree of this diversity is discussed with reference to the possible influences of polyploidy and concerted evolution on genetic structure. The ITS data indicated that some of the physical traits used to define ‘morphotypes’ of C. odorata were not correlated to genotype. From discussion and comparison of morphological character distributions and the ITS-based phylogeography it is suggested that the geographical origin of the South African population is Greater Antilelan, rather than from the continents of North and South America, which is where the Australasian, West African and Mauritian infestations are suggested to have originated. ii Table of Contents ABSTRACT ii TABLE OF CONTENTS iii LIST OF FIGURES vii LIST OF TABLES ix LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS xi CHAPTER 1 - CHROMOLAENA ODORATA AS AN INVASIVE ALIEN WEED 1 The Approaching Biological-Extinction Event 1 Weeds in the Extinction Crisis 1 The Effect of Weeds on Ecology 2 Weeds Collapse Mutualisms 4 Hybridisation of Weeds 5 Polyploidy 6 Control of Weeds 8 Biocontrol 9 History of Biological Control of Weeds 10 Biocontrol in South Africa 10 Measures for the Control of Chromolaena odorata 12 Native Distribution of Chromolaena odorata 14 Morphology of Chromolaena odorata 15 Morphological Variation 16 Ecological Tolerance of Chromolaena odorata 18 Review of the Invasive Character of Chromoalena odorata 19 Harmful Properties of Chromolaena odorata 23 Benefits of Chromolaena odorata 25 History of C. odorata's spread 26 Australasia 26 Africa 28 Clues to the Origin of South Africa's C. odorata 29 Motivation for DNA-Based Study 33 AIMS OF THIS STUDY 35 CHAPTER 2 – DNA FINGERPRINTING OF CHROMOLAENA ODORATA 36 PCR-BASED MOLECULAR MARKERS 36 Random Amplified Polymorphic DNA PCR 36 Advantages of RAPDs 38 Disadvantages of RAPDs 38 Studies in Plants 40 Amplified Fragment Length Polymorphisms 41 Advantages 42 Disadvantages 42 Studies in Plants 42 Variable Number Tandem Repeats 43 VNTRs in Plants 44 Inter Simple Sequence Repeats 45 Advantages 46 iii Disadvantages 47 Studies in Plants 47 MATERIALS AND METHODS 51 Gel Visualisation 51 Comparisons of DNA Extraction Protocols 52 CTAB-Based Extraction Protocols 52 Commercial DNA Extraction Kit 53 Quantification of DNA Extract 54 Initial PCR Thermal Cycling Conditions 54 PCR Reagents 54 Primers 55 Primer Screening 55 Primer Volumes 55 Primer Degradation 55 Optimisation of Magnesium Concentration 55 DNA Polymerase Concentration 55 dNTPs 56 Additives 56 Template Concentration 56 Tissue Preservation 56 Fresh Leaf Tissue 56 Saturated NaCl /CTAB Buffer 57 Test for Inhibitory Compounds 57 Non - C. odorata Samples 57 Ammonium Acetate Precipitation 57 Thermal Cycling Profiles 58 Thermal Cyclers 58 RESULTS AND DISCUSSION 59 Gel Visualisation 59 Comparisons of DNA Extraction Protocols 59 CTAB-Based Extraction Protocols 59 UV Absorbance Values 60 Commercial DNA Extraction Kit 60 Observations from Multiple Extractions 60 PCR Reagents 61 Primers 61 Primer Screening 61 Primer Volumes 61 Control Against Primer Degradation 61 Optimisation of Magnesium Concentration 63 DNA Polymerase 63 Additives 63 Template Concentration 63 Tissue Preservation 64 Fresh Leaf Tissue 64 Saturated NaCl/CTAB Buffer 64 Test for Inhibitory Compounds 64 Thermal Cycling Profiles 64 Thermal Cyclers 65 Molecular Weight of Occasionally-Produced ISSR Bands 65 SUMMARY AND FUTURE RECOMMENDATIONS 66 CHAPTER 3 - SEQUENCING 67 INTRODUCTION 67 Intraspecific Molecular Variation in Plants 67 iv Intergenic Spacer Region 72 External Transcribed Spacer Regions 72 Internal Transcribed Spacer Regions 73 Use of ITS in Asteraceae 73 Potential for ITS in Intraspecific and Phylogeographic Use 74 MATERIALS AND METHODS 77 Sampling Strategy 77 DNA Isolation, PCR Amplification, and Sequencing 77 Sequence Alignment and Phylogenetic Reconstructions 81 Choice of Analytical Methods 82 Maximum Parsimony 84 Neighbour Joining Distance Analysis with Jukes Cantor Correction 84 Statistical Parsimony 84 Distance (define according to Arlequin) 85 RESULTS AND DISCUSSION 86 Comparison of ETS and ITS Phylogeny Reconstructions 86 Discussion of Relative Usefulness of ETS and ITS in Intraspecific 87 Studies Phylogenetic Analysis of ITS Sequence Data 90 Maximum Parsimony Analysis 90 Statistical Parsimony Analysis 93 Neighbour Joining Distance Analysis 93 Pairwise Differences Analysis 93 Phylogeography of C. odorata 100 Australia and Other Regions 100 Phylogeography of C. odorata in the Americas 102 Phylogeography of South African C. odorata 104 Significance of Differences in Flower Colour and Root System 111 Distribution of Morphotypes 112 Possibility of Finding Consensus Between Previous and Current Findings 113 Significance of Genetic Diversity of the South African C. odorata population. 117 Possible Roles of Polyploidy and Concerted Evolution in Genetic Diversity of C. 118 odorata FINAL CONCLUSIONS 121 LITERATURE CITED 123 APPENDICES 142 Appendix 1 Alternative Names for Chromolanena odorata 142 Appendix 2 Silver Staining Protocol 143 Appendix 3 Modified CTAB Plant DNA Extraction Protocol 144 Appendix 4 ISSR Protocol Variations 145 Appendix 5 Ammonium Acetate Precipitation Protocol 154 Appendix 6 Sequence Alignments 155 ETS Sequence Alignment 155 ITS Sequence Alignment 157 Appendix 7 Jukes Cantor Distance Matrix 170 v List of Figures Figure 1.1 Map showing spread and distribution of Chromolaena odorata in South Africa. Figure 2.1 ISSR PCR. A single primer targeting a (CA)n repeat is used to amplify genomic sequence flanked by two inversely oriented (CA)n elements (from Ziętkiewicz et al., 1994). Figure 2.2: Initial successful ISSR PCR amplification. Numbers refer to primers [each with two samples: Mexico10 (M) and SA_Dbn13 (D)]. Vertical arrow shows gel direction. Figure 2.3 1% Agarose gel with ethidium bromide showing ISSR PCR amplification (with background smears). Molecular markers on the right hand side indicate that the ISSR bands are between 1000 and 300bp long. Sample names are: M (molecular markers); V (Venezue2); B (Brazil_5); J (Jamiaca8); Me (Mexico10); U (USA_Fl12); S (SA_Dbn13); M (molecular markers). Figure 3.1 The nuclear ribosomal 18S to 26S repeat unit, showing positions of the ETS, and ITS-1 & ITS-2. According to Baldwin (1993) and Baldwin and Markos (1998). Gene sizes are not shown to scale. (ITS = Internal Transcribed Spacer, ETS = External Transcribed Spacer, NTS = Non- Transcribed Spacer, IGS = Intergenic Spacer) Figure 3.2 Unrooted ETS and ITS maximum parsimony trees with bootstrap values Figure 3.3 Unrooted Neighbor Joining Jukes Cantor distance trees, with bootstrap values > 50%. (ITS did not have bootstrap values >50%) Figure 3.4 Strict consensus parsimony tree for two most parsimonius trees of 673 steps. CI = 0.932; RI = 0.881 Colours indicate morphotypes: Blue = South African, Red = Floridean, Green = Venezuelan, Black = other/unknown Figure 3.5 TCS Minimum spanning network for complete ITS data set. Empty circles indicate missing haplotypes, and rectangles indicate probable outgroups. Colours indicate morphotypes: Blue = South African, Red = Floridean, Green = Venezuelan, Black = other/unknown Figure 3.6 Rooted Jukes Cantor Neighbour Joining distance tree. Numbers behind brackets indicate bootstrap values for bracketed taxa. Figure 3.7 The same Jukes Cantor Neighbour Joining distance trees as Figure 3.6, but showing only the outgroup closest to the ingroup, to allow better resolution of

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