CRYPTIC HOST-ASSOCIATED AND FREQUENCY-DEPENDENT PATTERNS OF HOST SPECIES SELECTION OF A CANDIDATE WEED BIOLOGICAL CONTROL AGENT IN ITS NATIVE RANGE by CHANDRA ELAINE MOFFAT B.SC., THE UNIVERSITY OF VICTORIA, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (BIOLOGY) THE UNIVERSITY OF BRITISH COLUMBIA (OKANAGAN) AUGUST 2012 © CHANDRA ELAINE MOFFAT, 2012 ABSTRACT At least nine species of European hawkweeds in the genus Pilosella Vaill. (Asteraceae) are invasive in western North America, where they are a detriment to natural and managed lands and have vast economic impacts. A promising candidate biological control (biocontrol) agent for multiple species of Pilosella is the gall inducing wasp Aulacidea pilosellae Kieffer (Hymenoptera: Cynipidae). Initial studies of this candidate agent revealed the potential for two biotypes of the insect that differed in physiological host range, voltinism, and reproductive mode. It was hypothesized that these differences were related to geographic separation of the populations and it was further observed, within one subset of the distribution, that different host species were being used at nearby sites, despite similar host species being present. The overarching goal of this thesis was to increase the understanding of the patterns of host species use by A. pilosellae in order to inform the biocontrol programme for invasive Pilosella hawkweeds. The specific objectives were to (i) test the hypothesis that A. pilosellae has definitive preferences for species of Pilosella within its ecological host range, (ii) further quantify the ecological host range of A. pilosellae by conducting systematic surveys across a broad geographic distribution, and (iii) test the hypothesis that differences between the purported biotypes were due to cryptic genetic differentiation, predicted to be based primarily on geographic location of populations and secondly on host-association. By utilizing an integrative approach of thorough and widespread field surveys and molecular methodologies, this thesis presents findings that substantially increase the understanding of patterns of host species use by A. pilosellae in its native range. Specifically, (i) the hypothesis of host preferences of A. pilosellae is rejected in favor of frequency-dependent host species selection, (ii) five species of Pilosella invasive in North America are confirmed as hosts of A. pilosellae in its native range and (iii) the hypothesis that cryptic genetic differentiation exists within the species A. pilosellae was supported. However, the prediction that differentiation was based primarily on geographic separation was rejected in favor of differentiation based on a combination of disjunct host- associations, infection with the bacterial endosymbiont Wolbachia, and to a lesser extent, on geography. ii PREFACE With guidance from my co-supervisors Jason Pither and Bob Lalonde, my committee members Rose De Clerck-Floate and John Klironomos, and collaborator Gitta Grosskopf-Lachat, I was responsible for the design, implementation and analysis of the research presented in Chapter 2: Quantifying patterns of host plant species use by Aulacidea pilosellae, a candidate weed biological control agent for Pilosella hawkweeds, in its native range. I was assisted in this aspect of the work by field assistants Dave Ensing and Jeff Padgham. With guidance from my co-supervisors Jason Pither and Bob Lalonde, my committee members Rose De Clerck-Floate and John Klironomos, and collaborator Kevin Floate, I was responsible for the design and analysis of the research presented in Chapter 3: Cryptic diversity of a candidate weed biocontrol agent – Due to geography, host-association or Wolbachia infection? The implementation of the molecular work (DNA extraction and PCR amplification) was primarily conducted in the lab of Kevin Floate by technician Paul Coghlin, although I performed initial DNA extractions. Paul Coghlin also sent the PCR product for sequencing, performed the initial BLASTn which revealed the CO1 sequence JN288739, and provided the raw sequence data to me, which I analyzed with advice from and modified analysis code obtained from Michael Russello. I was responsible for the writing of this thesis in its entirety, with helpful suggestions and editing that improved the thesis from my co-supervisors. iii TABLE OF CONTENTS ABSTRACT ................................................................................................................................. ii PREFACE .................................................................................................................................. iii TABLE OF CONTENTS ........................................................................................................... iv LIST OF TABLES ...................................................................................................................... vi LIST OF FIGURES .................................................................................................................. viii ACKNOWLEDGEMENTS ....................................................................................................... x DEDICATION ............................................................................................................................ xi CHAPTER 1: GENERAL INTRODUCTION ............................................................................ 1 INTRODUCTION ................................................................................................................ 1 Biological control of invasive plant species................................................................... 1 Application of molecular methods to weed biological control ...................................... 6 Wolbachia infection and potential impacts on its hosts ................................................. 8 STUDY SYSTEM ............................................................................................................... 12 Invasive hawkweeds..................................................................................................... 12 History of the hawkweed biological control program.................................................. 13 Aulacidea pilosellae, a candidate biological control agent for invasive Pilosella ....... 14 RESEARCH AIMS............................................................................................................. 16 CHAPTER 2: QUANTIFYING PATTERNS OF HOST PLANT SPECIES USE BY AULACIDEA PILOSELLAE, A CANDIDATE WEED BIOCONTROL AGENT FOR PILOSELLA HAWKWEEDS, IN ITS NATIVE RANGE .......... 19 SYNOPSIS ........................................................................................................................... 19 METHODS .......................................................................................................................... 21 Study area ...................................................................................................................... 21 Sampling period ........................................................................................................... 21 Site selection and characteristics .................................................................................. 22 Estimation of Pilosella host plant availability ............................................................. 22 Estimation of Pilosella host plant use by Aulacidea pilosellae ................................... 24 Analysis of host preference .......................................................................................... 25 Geographic distribution of Pilosella host species ........................................................ 25 RESULTS ............................................................................................................................ 25 Distribution of Pilosella species availability ............................................................... 25 Patterns of Pilosella species use by Aulacidea pilosellae ............................................ 26 Analysis of host preference .......................................................................................... 27 DISCUSSION ...................................................................................................................... 28 Host preference of Aulacidea pilosellae ...................................................................... 28 The ecological host range of Aulacidea pilosellae ...................................................... 30 Summary and future directions .................................................................................... 32 iv CHAPTER 3: CRYPTIC DIVERSITY OF A CANDIDATE WEED BIOCONTROL AGENT – DUE TO GEOGRAPHY, HOST-ASSOCIATION OR WOLBACHIA INFECTION? .............................................................................. 42 SYNOPSIS ........................................................................................................................... 42 METHODS .......................................................................................................................... 44 Specimen collection ..................................................................................................... 44 DNA extraction, amplification and sequencing ........................................................... 45 Sequence alignment and phylogenetic analyses .......................................................... 46 RESULTS ...........................................................................................................................