Faculty of Science and Engineering Department of Environment and Agriculture Function, Ecology and Evolution of Seed Size in Hakea (Proteaceae) Sh-Hoob Mohamed El-Ahmir This thesis is presented for the degree of Doctor of Philosophy of Curtin University December 2015 Declaration To the best of my knowledge and belief this thesis contains no material previously published by any other person except where due acknowledgment has been made. This thesis contains no material that has been accepted for the award of any other degree or diploma in any university. Date ……14/12/2015……………. ii Abstract Southwestern Australia is one of the global biodiversity hotspots for its richness in endemic plants including grevilleas and hakeas. The genus Hakea (Proteaceae) has an evolutionary history of c.18 million years, and displays exceptional high functional diversity with adaptation to nutrient impoverished soil, granivory, droughts and recurrent fires. Seeds play a critical role in the life cycle of a plant. Research into function, ecology and evolution of seed size will provide critical insights into evolution, adaptation and diversification of flora in Southwestern Australia subject to nutrient impoverished and water deficient soil. In this thesis, I first assembled a time-based phylogeny for Hakea (Proteaceae) with 82 species, reconstructed ancestral state for six functional traits and determined their evolutionary trajectories in response to the advent or increasing presence of fire, seasonality, aridity, nectar-feeding birds and (in)vertebrate herbivores/granivores in Chapter 2. In Chapter 3, the interaction network between seed size and fecundity, postfire regeneration strategy, fruit size, plant height and serotiny (canopy seed storage) among 82 species of Hakea was investigated using Structural Equation Modelling (SEM) and correlated trait evolution analysis. From this analysis suggested that the ancestral Hakea arose c. 18 million years ago and was broad-leaved, non-spinescent and insect- pollinated, with medium-sized, serotinous fruits and resprouted after fire. The results also showed the causal correlations between seed size and fruit size (strong) and fecundity (weak) in Hakea, and between fecundity and postfire regeneration strategy (strong), but not between seed size and regeneration strategy. Overall, evolutionary histories have had most control over seed size variation among Hakea species. In two glasshouse experiments, I explored the relationship between capacity of tolerating cotyledon damage and seed size by removing part of cotyledons in Hakea species with different seed size in Chapter 4, assessed whether the critical nutrients in cotyledon can be compensated by external nutrients, and whether seed mass is consistently associated with a seedling’s iii ability to cope with cotyledon damage in Chapter 5. I found that small-seeded Hakea species may gain competitive advantages over larger those with seeds due to earlier germination, faster seedling emergence, rapid true leaf emergence and quicker transfer nutrient to early seedling’s growth. Removal of the cotyledons but addition of a balanced nutrient solution failed to restore complete growth of any experimental Hakea species, but the root: shoot ratio was maintained, as was the extension of roots for the four species with smallest seeds. The cotyledons provide the essential nutrients, N, P and K, to support early growth of Hakea seedlings but other nutritional roles of the cotyledons are also implicated. Hakea species with small seeds can only tolerate damage to the cotyledons better than large seeds when they have ready access to soil nutrients. iv Acknowledgements This thesis took a lot of time to be completed and was possible thanks to the support of many people. I am really grateful to my supervisors, Dr Tianhua He and Prof Byron Lamont, for their expertise, patience and encouragement, constructive comments and suggestions. Tianhua particularly taught me a great deal about research and helped throughout the project, from glasshouse to laboratory, to paper and thesis production. Co-supervisor Prof Mark Gibberd is thanked for his additional support and assistance. Special thanks to Dr. Philip Groom who supervised me in the preparation of the proposal at the beginning of this study. Dr Sim Lin Lim is thanked for his valuable assistance in Hakea DNA sequence and phylogenetic analysis. Thanks are also due to the staff and students of the Department of Environment and Agriculture of Curtin University and Plant Ecology and Evolution Group for assistance and use of facilities. For technical and administrative support, thanks to Charles Lacoste, William Parkinson, and Aimee Tournay. I also acknowledge financial support from Libyan Government, Australian Research Council, Office of Research and Development and Graduate Research School at Curtin University. Last but not least, I have to thank my family and friends, who believed in me and to those who have contributed to this work who are not mentioned. v Table of contents Declaration ........................................................................................................... ii Abstract ............................................................................................................... iii Acknowledgements .............................................................................................. v Table of contents ................................................................................................. vi List of figures .................................................................................................... viii List of tables ......................................................................................................... x Supplementary .................................................................................................... xi Appendicces........................................................................................................ xii List of abbreviation ........................................................................................... xiv Chapter 1: Ecology of seed size, and ecology and adaptation of Hakea: A general introduction .......................................................................................................... 1 1.1. Background ................................................................................................ 1 1.1.1. Natural variation of seed size in plants ..................................................... 1 1.1.2. The relationship between seed size and cotyledon size ............................. 4 1.2. An overview of Australian genus Hakea ..................................................... 6 1.2.1. Mediterranean-climate ecosystems and Southwest Australia as a biodiversity hotspot ........................................................................................... 7 1.2.2. Southwestern Australia as the diversity centre for Hakea ......................... 9 1.2.3. Morphology and function attributes in Hakea (stems, leaves, florescence, flowers, fruit, Seed) ......................................................................................... 14 1.3. Evolutionary history and phylogeny of Hakea ........................................... 15 1.3.1. Fire strategy ........................................................................................... 16 1.3.2. Variation of seed size in Hakea .............................................................. 17 1.3.3. Serotinous fruits..................................................................................... 19 1.3.4. Pollination ............................................................................................. 20 1.3.5. Type, size and functions of cotyledons in Hakea .................................... 21 1.3.6. The evolution and adaptation of Hakea .................................................. 23 1.4. Objectives and organization of this thesis ................................................. 28 1.5. References ................................................................................................ 32 Chapter 2: Evolution of functional traits and effects on diversification in Hakea ............................................................................................................................ 57 2.1. Abstract .................................................................................................... 57 2.2. Introduction .............................................................................................. 58 2.2.1. Consideration of theory and concepts ..................................................... 58 2.3. Materials and Methods.............................................................................. 61 2.3.1. Phylogenetic reconstruction ................................................................... 61 2.3.2. Trait data ............................................................................................... 61 2.3.3. Trait reconstruction through the phylogeny ............................................ 62 2.3.4. Speciation and trait proliferation rates .................................................... 63 2.4. Results ...................................................................................................... 64 2.4.1. Hakea time-based phylogeny ................................................................. 64 2.4.2. Evolutionary trajectories for two leaf attributes ...................................... 64 vi 2. 4.3. Evolutionary