The evolutionary significance of parthenogenesis and sexual reproduction in the Australian spiny leaf insect, Extatosoma tiaratum Yasaman Alavi Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy October 2016 School of BioSciences Faculty of Science The University of Melbourne i ii Abstract The costs and benefits of sexual reproduction has long been a subject of debate in biology. The paradox arises from the fact that theoretically, sex is associated with many costs, yet it is the most prevalent mode of reproduction in the tree of life. Facultative parthenogenetic systems, in which females can reproduce both sexually, and in the absence of sperm, parthenogenetically, provide suitable systems to compare costs and benefits of reproductive modes, while minimizing confounding effect that are not directly related to reproductive modes. In this thesis, I used the Australian Phasmatid, Extatosoma tiaratum, to investigate the evolutionary significance of facultative parthenogenesis, and compare fitness consequences of sex and parthenogenesis. The evolutionary significance of facultative parthenogenesis is unknown but male or sperm limitations are potential factors. I investigate male mating frequency and variation in ejaculate size and quality in E. tiaratum. I show that most, but not all, males are able to mate multiply, but ejaculate size decreases with increased number of matings. In addition, ejaculate size increased with increasing time interval between matings, suggesting that E. tiaratum males require time to replenish ejaculate reserves. These findings suggest male sperm limitation may be an important factor influencing the evolution of parthenogenesis in this system. iii The cytological mechanism of parthenogenesis determines the genetic diversity and heterozygosity levels of the offspring and is thus an important component of the comparison between reproductive modes. Using microsatellite markers that I developed for E. tiaratum, I investigated the cytological mechanism of parthenogenesis in this species. I demonstrated that the most likely mechanism of parthenogenesis in E. tiaratum is automixis with terminal fusion, resulting in substantial loss of heterozygosity in the first generation parthenogenetic offspring. Based on these results I predicted parthenogenesis to be associated with fitness reduction in E. tiaratum. I then investigated the fitness consequences of sex and parthenogenesis in E. tiaratum, in terms of offspring immunity and reproductive success. I show that parthenogenesis is associated with fitness reduction both in terms of immune function and reproductive success. Females derived from sexually conceived mothers have higher immune response compared to females derived from parthenogenetically conceived mothers. A female’s reproductive success is substantially higher if she is conceived sexually and produces sexual offspring. In fact, the cost of parthenogenesis on reproductive success is high enough to eliminate the two-fold cost of sex in E. tiaratum. Nevertheless, the cost of parthenogenesis can be compensated in the next generation, if parthenogenetically females produce daughters sexually. The results of this thesis offer a new perspective in understanding the evolution and maintenance of facultative parthenogenesis in E. tiaratum, and suggest that females iv enjoy the benefit of parthenogenesis allowing them to reproduce in the absence of sufficient or preferred sperm, while mediating the costs of homozygosity by reproducing sexually. v Declaration This is to certify that: The thesis comprises only my original work towards the PhD except where indicated in the Preface. Due acknowledgement has been made in the text to all other material used. The thesis is fewer than 100 000 words in length, exclusive of tables, maps bibliographies and appendices. ………………………………………… Yasaman Alavi vi Preface Yasaman Alavi (YA) was the primary author for all manuscripts (presented as chapters) within this thesis. These manuscripts were written with assistance and editorial changes by supervisors Mark Elgar (MAE), Therésa Jones (TMJ), and Andrew Weeks (ARW). The ideas concerning the design of the experimental aspects of this thesis were originally conceived by YA but refined and developed in collaboration with MAE and TMJ. YA carried out the laboratory experiments and field samplings and collected all the data used within this thesis. YA performed analyses of all data with advice and suggestions provided by MAE, TMJ and ARW. Chapter 2-5 of this thesis are written and presented as separate papers. Chapter 2 has been published as Alavi, Y., Elgar, A. M., and Jones, M. T. (2016). Male Mating Success and the Effect of Mating History on Ejaculate Traits in a Facultatively Parthenogenic Insect (Extatosoma tiaratum). Ethology, 122: 1-8. Chapter 3 has been accepted for publication as Alavi, Y., van Rooyen, A., Elgar, M. A., Jones, M. T., and Weeks, A. R. (2016). Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum is automixis with terminal fusion. Insect Science, in press. The manuscripts as presented within this thesis are largely unchanged from the published material. vii Acknowledgments I would like to thank my amazing supervisors for their help and support during the production of this thesis. First of all, I am grateful to Mark Elgar, for giving me this wonderful opportunity to come to Melbourne and work on this project, and for his ongoing support and patient guidance. I have learned a lot from him, both in science and also on the philosophy of life. I am truly thankful to Therésa Jones for her constant and brilliant ideas, and for always giving me great advice and motivation. I have learned a lot from her, especially on experimental design and statistical analysis. I am also grateful to Andrew weeks for his helpful advice on chapters 1 and 3 of this thesis. I would like to thank all members of the Elgar and Jones lab for their help and support over the past years. Especially, I am thankful to Gareth Hopkins for his comments on Chapter 2 and 4 of my thesis, and to Dany Zemeitat, Qike Wang, Bernie Wittwer and Eunice Tan for their help in maintaining the insect population in my absence. Also thanks to Joanna Durrant for showing me how to run immune assays. I am thankful to Patrick Honan, Chloe Miller and Maik Fiedel from the Museum Victoria, for their help in the field collections. Many thanks to Anthony Van Rooyen for helping with all the molecular lab work during the development and use of the microsatellite markers. Thanks to Mandy Parfitt, Uoda Khammy, Stephen Frankenberg and Rohan Long for borrowing me equipment for lab work. viii Many thanks to the Holsworth Wildlife Research Endowment for funding this project, and the Melbourne School of Graduate Research and the School of BioSciences for providing funding for conferences. Last but not least, I would like to thank my parents Salimeh and Kambiz, and my brother Behrang, for their everlasting love and support, without which this would not have been possible. ix Table of contents Abstract iii Declaration vi Preface vii Acknowledgments viii Table of contents x List of Figures xiv List of Tables xviii Chapter 1 Introduction: the evolutionary significance of sex and parthenogenesis 1 Modes of reproduction 2 Parthenogenesis: modes, mechanisms and emergence 3 The paradox of sex: theories and empirical evidence 7 What is missing? 12 Facultatively parthenogenetic species as models 13 The study system, an Australian Phasmatid 14 The evolutionary significance of facultative parthenogenesis in E. tiaratum 17 References 27 Chapter 2 Variation in male mating success in a facultatively parthenogenic insect (Extatosoma tiaratum) 35 Summary 36 Introduction 37 Methods 40 Experimental animals 40 Experimental design 40 Preparation of the sperm solution 41 x Sperm density assay 42 Sperm viability assay 42 Statistical analyses 43 Results 45 Male mating history and survival 45 Effects of multiple mating on ejaculate size and quality 46 Discussion 50 References 54 Chapter 3 Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum, is automixis with terminal fusion 58 Summary 59 Introduction 60 Methods 63 Laboratory stock population 63 Field collection 63 Next-generation sequencing and de novo genome assembly 64 Microsatellite isolation and characterization 64 Patterns of inheritance 66 The mechanism of parthenogenesis 67 Results 68 Next-generation sequencing and de novo genome assembly 68 Microsatellite isolation and characterization 68 Patterns of allele inheritance 69 Mechanism of parthenogenesis 70 Discussion 77 References 81 Chapter 4 Sex versus parthenogenesis; immune function in a facultatively parthenogenetic Phasmatid (Extatosoma tiaratum) 85 xi Summary 86 Introduction 87 Methods 91 Animal maintenance and culturing 91 Determining reproductive history 92 Haemolymph collection and processing 92 Haemocyte concentration 93 Lytic activity assay 94 Phenoloxidase (PO) assay 95 Statistical analysis 95 Results 97 Determining reproductive history 97 Immune function assays 97 Discussion 106 References 110 Chapter 5 Sex versus parthenogenesis; female reproductive success in a facultatively parthenogenetic Phasmatid (Extatosoma tiaratum) 114 Summary 115 Introduction 116 Methods 121 Animal maintenance 121 Deriving F1 from F0 females 121 F1 fecundity 125 F2 Juvenile survival 125 Offspring survival of field- collected females 126 Microsatellite heterozygosity and modes of conception 127