Genetic variation in New Zealand abalone, Haliotis iris A thesis submitted in partial fulfillment of the of the requirements for the Degree of Doctor of Philosophy in Biological Sciences at the University of Canterbury by Margaret Will University of Canterbury Christchurch, New Zealand 2009 Table of Contents ABSTRACT ...............................................................................................................................1 1. INTRODUCTION..................................................................................................................3 GENE FLOW.............................................................................................................................3 ABALONE ................................................................................................................................7 Systematics .........................................................................................................................9 Assessing genetic structure...............................................................................................11 Genetic structure of abalone.............................................................................................12 NEW ZEALAND ABALONE......................................................................................................20 Aims .................................................................................................................................22 2. GENETIC STRUCTURE ACROSS COOK STRAIT.........................................................25 ABSTRACT .............................................................................................................................25 INTRODUCTION ......................................................................................................................25 Genetic splits around Cook Strait.....................................................................................26 Cook Strait as a barrier .....................................................................................................28 H. iris genetic structure ....................................................................................................31 MATERIALS AND METHODS...................................................................................................32 Marker choice...................................................................................................................32 Samples.............................................................................................................................33 DNA extraction, PCR amplification, and sequencing......................................................33 Population genetic analyses..............................................................................................34 RESULTS................................................................................................................................40 Haplotypes: Relationships and distributions ....................................................................41 Cluster analyses ................................................................................................................41 A priori hypothesis testing................................................................................................42 Nested clade phylogeographic analysis............................................................................43 Mismatch distributions and neutrality tests......................................................................44 DISCUSSION ...........................................................................................................................44 Chatham Islands vs. North and South Island....................................................................45 North Island vs. South Island ...........................................................................................47 Sample sizes .....................................................................................................................50 Future work ......................................................................................................................51 3. ISOLATION AND CHARACTERIZATION OF HALIOTIS IRIS MICROSATELLITES 53 ABSTRACT .............................................................................................................................53 INTRODUCTION ......................................................................................................................53 Haliotis and conserved microsatellites.............................................................................54 H. iris and microsatellites.................................................................................................55 MATERIALS AND METHODS....................................................................................................56 Cross-species amplification..............................................................................................56 Microsatellite development and screening .......................................................................57 Allele scoring....................................................................................................................59 Genotyping error rate........................................................................................................59 Locus characterizations ....................................................................................................60 RESULTS................................................................................................................................60 Cross-species amplification..............................................................................................60 Screening H. iris loci........................................................................................................60 Genotyping error rate....................................................................................................... 61 Characterization of AB14, AB21, and AB31 .................................................................. 61 DISCUSSION .......................................................................................................................... 62 Cross-species amplification ............................................................................................. 62 H. iris microsatellites ....................................................................................................... 63 Ongoing projects.............................................................................................................. 67 4. POPULATION GENETIC STRUCTURE BASED ON THREE MICROSATELLITE LOCI ........................................................................................................................................ 69 ABSTRACT ............................................................................................................................ 69 INTRODUCTION ..................................................................................................................... 69 Molecular markers and the Cook Strait ........................................................................... 70 MATERIALS AND METHODS ................................................................................................... 71 Population genetic analyses ............................................................................................. 71 RESULTS ............................................................................................................................... 73 Genetic structure .............................................................................................................. 74 DISCUSSION .......................................................................................................................... 74 Inbreeding and abalone .................................................................................................... 77 5. VARIATION IN GAMETE RECOGNITION PROTEINS ................................................ 79 ABSTRACT......................................................................................................................... 79 INTRODUCTION ..................................................................................................................... 79 Abalone lysin ................................................................................................................... 81 MATERIALS AND METHODS.................................................................................................... 83 Haplotype inference ......................................................................................................... 84 Population genetic analyses ............................................................................................. 84 RESULTS ............................................................................................................................... 85 DISCUSSION .......................................................................................................................... 86 Lysin exon variation......................................................................................................... 87 Lysin intron variation....................................................................................................... 90 Population genetic structure............................................................................................