Taxonomic delimitation and the evolutionary history of the Australasian Lautusoid group of Senecio (Asteraceae) ________________________________________________________________ A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Evolutionary Biology of the University of Canterbury by Chia-Sin (Jasmine) Liew University of Canterbury 2016 ____________________________________________________________ Table of Contents ACKNOWLEDGEMENTS…………………………………………………………….. 1 ABSTRACT……………………………………………………………………………… 2 CHAPTER 1: Introduction……………………………………………………………… 4 1.1. Thesis overview 4 1.2. Background and aims of this study 4 1.2.1. The Lautusoid group of Senecio 5 1.2.2. The delimitation and evolution of the Lautusoid group (Chapter 2) 8 1.2.3. Resolving the Senecio glaucophyllus complex (Chapter 3) 9 1.2.4. Testing the infraspecific delimitation of Senecio “pseudoglaucophyllus” (Chapters 4 & 5) 10 1.2.5. Aims of this thesis 11 1.3. Taxonomic delimitation 11 1.3.1. Species delimitation 11 1.3.2. Supra-specific and infraspecific taxonomic delimitation 12 1.3.3. Taxonomic delimitation in the presence of hybrids 14 1.3.4. Species delimitation in the presence of morphologically cryptic or complex species 16 CHAPTER 2: The delimitation and evolutionary history of the Australasian Lautusoid group of Senecio (Asteraceae; Senecioneae)……………………………….. 19 2.1. ABSTRACT 19 2.2. INTRODUCTION 19 2.3. MATERIALS & METHODS 24 2.3.1. Taxon sampling 24 2.3.2. DNA extraction, PCR amplification and sequencing 25 2.3.3. Recombination detection 26 2.3.4. Phylogeny reconstruction 26 2.3.5. Testing topological hypotheses 27 2.4. RESULTS 32 2.4.1. Phylogenetic analyses of the nuclear regions 32 2.4.2. Phylogenetic analyses of the plastid regions 33 2.4.3. Australasian Senecio lineages 34 2.4.4. Topological test for the monophyly of the Lautusoid group in the plastid phylogeny 36 2.5. DISCUSSION 36 2.5.1. An explanation for phylogenetic incongruence between plastid and nuclear Senecio phylogenies 36 2.5.2. The taxonomic delimitation of the Lautusoid group 39 2.5.3. Morphological differences between the Lautusoid group and other Australasian Senecio lineages 40 2.5.4. At the periphery of the Lautusoid group: hybrids with other Australasian Senecio lineages 43 2.5.5. Evolutionary relationships among Lautusoid species 46 2.6. CONCLUSION 48 CHAPTER 3: Hiding in plain sight: cryptic species in the Senecio glaucophyllus complex………………………………………………………………………………….... 49 3.1. ABSTRACT 49 3.2. INTRODUCTION 49 3.3. MATERIALS AND METHODS 52 3.3.1. Sampling and morphometric data collection 52 3.3.2. Morphometric data analysis 54 3.3.2.1. PCoA analyses 54 3.3.2.2. Random Forest analysis 55 3.3.3. Molecular phylogenetic analyses 56 3.4. RESULTS 57 3.4.1. PCoA analyses 57 3.4.2. Random Forest analysis 59 3.4.2.1. Predictive power of the morphological data 59 3.4.2.2. Diagnostic characters 60 3.4.3. Molecular phylogenetic analyses 62 3.5. DISCUSSION 64 3.5.1. Distinguishing Senecio aff. glaucophyllus from S. glaucophyllus subsp. glaucophyllus sensu Ornduff 64 3.5.1.1. Morphological differences 64 3.5.1.2. Molecular phylogenetic differences 66 3.5.2. Taxonomic realignment of Senecio aff. glaucophyllus and S. glaucophyllus sensu Ornduff 67 3.5.3. Geography, ecology, and conservation 69 3.6. CONCLUSION 70 CHAPTER 4: Patterns of morphological diversity in Senecio “pseudoglaucophyllus”…………………………………………………………………... 73 4.1. ABSTRACT 73 4.2. INTRODUCTION 73 4.2.1. The taxonomic history of Senecio “pseudoglaucophyllus” 74 4.2.2. The infraspecific groups of Senecio “pseudoglaucophyllus” 75 4.2.3. Problems with the infraspecific taxonomic delimitation of Senecio “pseudoglaucophyllus” 77 4.3. AIMS 77 4.4. MATERIALS AND METHODS 78 4.4.1. A phenetic approach to testing morphology-based taxonomical hypotheses 78 4.4.2. Morphometric data collection 79 4.4.3. Morphometric data analysis 82 4.4.3.1. Multivariate analyses 82 4.4.3.2. Univariate analyses 83 4.4.3.3. Geographical patterns in the morphometric data set 84 4.5. RESULTS 84 4.5.1. Multivariate analyses 84 4.5.1.1. PCoA and NMDS analyses 84 4.5.1.2. Cluster analysis 89 4.5.1.3. ANOSIM 93 4.5.2. Univariate analysis 93 4.5.3. Correlation between geographic distance and morphological dissimilarity 95 4.6. DISCUSSION 97 4.6.1. Patterns of morphometric variation in Senecio “pseudoglaucophyllus” 97 4.6.2. Morphological delimitation and diversity of the infraspecific groups of Senecio “pseudoglaucophyllus” 98 4.6.3. The delimitation and morphological affinities of the two Marlborough morphotypes 100 4.6.4. Geographical patterns in the morphological data 102 4.6.5. The infraspecific classification of Senecio “pseudoglaucophyllus” 103 4.7. CONCLUSIONS 105 CHAPTER 5: An integrative approach to revisiting the infraspecific classification of Senecio “pseudoglaucophyllus”……………………………………………………… 117 5.1. ABSTRACT 117 5.2. INTRODUCTION 117 5.3. AIMS 120 5.4. MATERIALS AND METHODS 120 5.4.1. Sampling and DNA extraction 120 5.4.2. ITS sequencing and phylogeny construction 123 5.4.3. AFLP analysis 124 5.4.3.1. Restriction 124 5.4.3.2. Linker ligation 124 5.4.3.3. Pre-selective amplification 125 5.4.3.4. Selective amplification 126 5.4.4. Genotyping and scoring of AFLP fragments 126 5.4.5. AFLP data analyses 129 5.4.5.1. Bayesian inference of genetic structure 129 5.4.5.2. Multivariate analyses 130 5.4.5.3. Testing for isolation by distance 130 5.4.5.4. Analysis of molecular variance (AMOVA) 130 5.5. RESULTS 131 5.5.1. ITS data 131 5.5.2. AFLP data 131 5.5.3. AFLP analyses 134 5.5.3.1. Bayesian clustering using STRUCTURE 134 5.5.3.1.1. Number of genetic clusters (K) 134 5.5.3.1.2. Genetic structure of Senecio “pseudoglaucophyllus” 135 5.5.3.2. Geographical structure of AFLP data 137 5.5.3.3. Multivariate analyses 139 5.5.3.3.1. PcoA 139 5.5.3.3.2. AMOVA 140 5.6. DISCUSSION 141 5.6.1. Patterns of genetic diversity in Senecio “pseudoglaucophyllus” 141 5.6.2. A comparison of morphological and genetic patterns of diversity 143 5.6.3. Conclusion and taxonomic implications 145 CHAPTER 6: Summary and conclusions………………………………………………. 147 6.1. Overview 147 6.2. The delimitation and evolution of the Lautusoid group of Senecio (Chapter 2) 149 6.3. Resolving the Senecio glaucophyllus complex (Chapter 3) 151 6.4. Testing the infraspecific delimitation of Senecio “pseudoglaucophyllus” (Chapters 4 & 5) 152 REFERENCES…………………………………………………………………………... 154 APPENDIX……………………………………………………………………………….. 186 ACKNOWLEDGEMENTS To Pieter and Peter, my supervisors, thank you for guiding me through my study and this thesis. Without your patience and dedication, I would not have been able to do it. To the curators and managers of AD, AK, CANU, CHR, BRI, MEL, PERTH, and WELT, thank you for approving my herbarium loans and giving me permission to sample tissue materials. A special shout out to all the kind souls at the Allan Herbarium (CHR): Mary, Debby, Sue, Charles, Kerry, Rob, and especially Ines, who endured so many requests from me and without whom my research would not have progressed. To numerous people from different institutions who have been lending me a hand with collecting samples: Shannel Courtney, Nicholas Head, John Barkla, Jeremy Rolfe, Daniel Kimber, Hugh Wilson, Mike Thorsen, Mike Harding, Alice Shanks, Miles and Gillian Giller, Di Carter, Trevor Partridge and Peter Johnson, thank you so much. I would not have been able to carry out my research if it was not for your help. Thanks to Maggie, Craig, Claire, Nicole, Gavin, Nicki, Penny and Thomas at the UC School of Biological Sciences, who helped me in various ways during my study. Thanks to Tim, who kindly contributed his R code and detailed instructions of how to draw pie charts on a map and being a friend throughout my study. Special thanks to Rachel, Ilina, Natalie, Michael, Stephanie, Jacob, Jasper, Sarah, Stephen, Denise, Ayla, and Valentin, thank you for being my lab and office buddies. Your support and encouragement in times of need are what kept me going and are what I missed the most since leaving New Zealand. Also thanks to Tammy and Marie, who provided much needed guidance, assurance and help from time to time. I am absolutely grateful and can not thank Mom and Dad enough, who are monumental in my journey of doing a PhD. Thanks for getting me home, keeping me alive and meeting all my needs while I wrestled with my thesis. Your unconditional love and support is what kept me motivated and inspired in life. Also thanks to my brothers, I have come a long way under your love and support. Thanks for providing me a place to stay and feed me when needed throughout my thesis writing. Derek, your patience and love keep me forward when it is hard. Thanks for all the treats and love you lots. Funding for this research project was provided by a University of Canterbury Doctoral Scholarship and the School of Biological Sciences Strategic Investment Fund to Pieter Pelser. 1 ABSTRACT Taxonomic delimitation can be a challenging task for systematists, because of the dynamic and complex evolutionary processes that shape patterns of biodiversity. Yet, it is an essential aspect of biology, because it defines units of evolutionary significance, which form the basis for studying all aspects of biodiversity. In this thesis, I studied the taxonomic delimitation and evolutionary history of the Australasian Lautusoid group of Senecio at the infrageneric, species, and infraspecific level. Members of the Lautusoid group are morphologically very diverse and occupy a wide array of habitats. Moreover, the Lautusoid group has a large diversity of chromosome profiles compared to other Australasian Senecio, which indicates the possible occurrence of hybridization in its evolutionary history.