GENETIC STRUCTURE AND MATING PATTERNS OF DIPLOID AND POLYPLOID EASTER DAISIES (TOWNSENDIA HOOKERI, ASTERACEAE) by STACEY LEE THOMPSON B.Sc, The University of Guelph, 1999 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA August 2006 © Stacey Lee Thompson, 2006 Abstract Reproduction in the Rocky Mountain genus Townsendia involves a complex interplay of polyploidy and apomixis. Molecular markers were used to assess genetic structure in relation to patterns of mating mode and ploidy at four hierarchical levels: within the genus, within T. hookeri, within populations of this species, and among progeny from these populations. Phylogenetic analyses of rDNA repeats indicated that polyploid apomixis has evolved multiple times within the genus. Pollen studies and analyses of cpDNA demonstrated that sexual diploids of T. hookeri are found in both northern and southern unglaciated regions, polyploid apomicts have evolved at least once in the north and thrice in the south, and these polyploid apomicts have colonized the post-glacial landscape from two refugia, suggesting that glaciation and not latitude influenced the distribution of apomicts. Pollen studies, flow cytometry, and multilocus tests on AFLP marker genotypes from four Yukon standing populations of T. hookeri indicated sexuality in one male-fertile diploid population, clonality in two male-sterile tetraploid populations, and a combination of sexual and clonal reproduction in one male-sterile polyploid population. This latter population of mixed mating mode included triploids and tetraploids and showed that evidence of cryptic sex may linger in the genomes within a morphologically asexual population. Finally, a new method for mating system analysis, which jointly estimates the rates of outcrossing, selfing, automixis and apomixis, was developed and applied to dominant AFLP marker genotypes from progeny whose mothers arose from three Yukon populations of T. hookeri, one consisting of male-fertile diploids, the other two of male-sterile tetraploids. Despite indications of sexuality in some standing populations, progeny analyses revealed that apomixis is the predominant mating mode in all populations. Levels of outcrossing were moderate in the diploid population and very low in the tetraploids. Selfing/automixis was absent in the diploids and moderate in tetraploids. These findings suggest that the correlation between ploidy and apomixis is not strict when observed on a fine scale, that polyploidy alone does not induce apomixis, and perhaps it is asexuality that selects for polyploidy within this system. Table of contents Abstract ii Table of contents iii List of Tables iv List of Figures v Acknowledgements vi Dedication vii Co-authorship statement viii 1 Intoduction 1 1.1 References 8 2 Recurrent evolution of polyploid apomixis, endemism, and acaulescence in the Rocky mountain agamic complex Townsendia (Astereae, Asteraceae) 11 2.1 Introduction 11 2.2 Material and methods 14 2.3 Results 16 2.4 Discussion 19 2.5 References 24 3 Patterns of recurrent evolution and geographic parthenogenesis within apomictic polyploid Easter daises (Townsendia hookeri) 35 3.1 Introduction 35 3.2 Material and methods 36 3.3 Results 39 3.4 Discussion 42 3.5 References 48 4 Detection of clonality and sexuality in diploid and polyploid populations of the Easter daisy, Townsendia hookeri 60 4.1 Introduction 60 4.2 Material and methods 61 4.3 Results 66 4.4 Discussion 68 4.5 References 73 5 A novel mating system analysis for modes of self-oriented mating applied to diploid and polyploid arctic Easter daisies (Townsendia hookeri) 80 5.1 Introduction 80 5.2 Material and methods 81 5.3 Results 85 5.4 Discussion 87 5.5 References 91 6 Conclusions 97 IV List of Tables Table 2-1 List of accessions and DNA regions sequenced 27 Table 2-2 Summary of pairwise likelihood distances 28 Table 3-1 Designation, locality information, and pollen data for populations of Townsendia hookeri included in this chapter 53 Table 3-2 Haplotype designations based on chloroplast DNA sequences from Townsendia hookeri, showing the constituent polymorphisms at respective alignment positions 55 Table 4-1 Overrepresentation statistics for four sub-arctic populations of the Easter daisy (Townsendia hookeri) 76 Table 4-2 Association statistics, measures of identity, and estimates of effective rate of long-term sexuality and mutation within four populations of the Easter daisy, Townsendia hookeri 76 Table 5-1 Probabilities of gametes from autotetraploid parents assuming double reduction, under co-dominance and dominance, respectively 93 Table 5-2 Probabilities of tetraploid offspring genotypes with dominance and assuming no double reduction, under outcrossing, selfing, automixis and apomixis, respectively) 93 Table 5-3 Theoretical variances and correlations estimates per individual sampled when selfing rate, automixis and outcrossing rate are simultaneously estimated, for two frequencies of the recessive marker q, and three levels of sample size 93 Table 5-4 Estimates of outcrossing t, selfing s, automixis u, and apomixis a, for each of the three populations, under various hypotheses 94 V List of Figures Figure 2-1 Beamans' hypothesis of phylogenetic relationships within Townsendia with indicated characteristics for species 29 Figure 2-2 Strict consensus of 440 equally-parsimonious trees, based on the external transcribed spacer (ETS) of Townsendia 30 Figure 2-3 Strict consensus of 133 equally-parsimonious trees of 171 steps, based on the internal transcribed spacer (ITS) of Townsendia 31 Figure 2-4 Maximum likelihood tree (-InL = 1886.22) based on the external transcribed spacer (ETS) of Townsendia with character states indicated 32 Figure 2-5 Maximum likelihood tree (-InL = 1835.73) based on the internal transcribed spacer (ITS) of Townsendia 33 Figure 2-6 Maximum likelihood tree (-InL = 3780.71) based on the combined analysis of the external and internal transcribed spacer (ETS and ITS) of Townsendia 34 Figure 3-1 Localities of sexual diploid and apomictic polyploid Easter daisy (Townsendia hookeri) populations collected from western North America 56 Figure 3-2 Plot of mean pollen diameter by mean pollen stainability for populations of the Easter daisy (Townsendia hookeri) 57 Figure 3-3 Chloroplast haplotype variation within 3 geographically proximal populations of Easter daisies (Townsendia hookeri) 58 Figure 3-4 Intraspecific chloroplast phylogeny of sexual diploid and apomictic polyploid Easter daisies (Townsendia hookeri) 59 Figure 4-1 DNA content per 2C nucleus as determined through flow cytometry for Yukon populations of the Easter daisy, Townsendia hookeri 77 Figure 4-2 Most parsimonious trees from Yukon populations of the Easter daisy, Townsendia hookeri, based on AFLPs 78 Figure 4-3 Incompatibility distributions for four Yukon populations of the Easter daisy, Townsendia hookeri 79 Figure 5-1 Gene frequency distribution for AFLP markers from one diploid (Tantalus Butte) and two tetraploid (Mile Thirteen and Tachal Dhal) sub-arctic populations of the Easter daisy, Townsendia hookeri 95 Figure 5-2 Log-likelihoods across a range of outcrossing rate t, for other parameters (s, u, a) jointly estimated 96 VI Acknowledgements Thanks to my committee members: • Mary Berbee, Sally Otto, Kermit Ritland and Jeannette Whitton Helpful colleagues and research facilities: • Institut de recherche en biologie vegetale: Anne Bruneau, Simon Joly, Madoka Misumone • Universite de Montreal: Francois-Joseph Lapointe, and Labo LEMEE • University of Colorado at Boulder: Richard D. Noyes, Nan Lederer • University of Guelph: Paul Kron • University of California at Santa Cruz: Katrina Dlugosh • University of Lethbridge: Joanne Golden • University of British Columbia: Linda Jennings, Gina Choe, NAPS Unit, FACS Facility Supportive friends: • Doreen Haven Thompson and the Deline Clan, Jesse Dylan Thompson, Ryan Marshall Driver, Dylan Leblanc, Teika Newton and Enzo Michel Lhermitte Pirelli Collecting permits: • Jasper National Park • Banff National Park • Waterton Lakes National Park • Kluane National Park • Writing-on-Stone Provincial Park • City of Boulder Open Space • Boulder County Open Space • United States Forestry Service (Rocky Mountain Region) • Territorial Government of the Yukon Financial support: • Post-Graduate Scholarship A/B, The Natural Science and Engineering Research Council of Canada • University Graduate Fellowship, The University of British Columbia • Challenge Grants in Biodiversity • The Alberta Conservation Association • Northern Student Training Program • Botanical Society of America • Department of Botany, The University of British Columbia • Several anonymous backers vii Dedication This thesis is dedicated to the enduring memory of Laika, the Soviet spacedog. She was the first living organism to enter orbit, launched into space inside Sputnik II on 3 November, 1957. A stray from the mean streets of Moscow, and dubbed "Muttnik" by the Americans, Laika's genetic ancestry likely comprised a Nordic breed and part terrier. Dear Laika died a few hours after launch from stress and overheating. Her coffin circled the earth 2,570 times, then incinerated upon reentering the Earth's atmosphere on 4 April, 1958. Her true cause of death was not made public until more than 40 years after the flight, with officials stating at the time that she was painlessly euthanized with poisoned food. Russian bureaucrats have since