UNIVERSITY OF CALIFORNIA RIVERSIDE

Associational Susceptibility of a Native Shrub, Atriplex canescens, Mediated by an Invasive Annual Forb, Brassica tournefortii, and Invasive Stinkbug, Bagrada hilaris

A Dissertation submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

in

Entomology

by

Sarah Lillian

September 2017

Dissertation Committee: Dr. Matt Daugherty, Chairperson Dr. Rick Redak Dr. Erin Rankin

Copyright by Sarah Lillian 2017

The Dissertation of Sarah Lillian is approved:

Committee Chairperson

University of California, Riverside

ACKNOWLEDGEMENTS

Over the past 5+ years, I have received so, so much help…with every part of graduate school and dissertate-ing, with decisions about my career, research, and everything else, with my personal life transitions and the occasional crisis, and with a million things I needed help with or just didn’t want to do alone. I have found myself surrounded by a community of people that are generous, funny, helpful, kind and lovely.

Thank you to everyone who has helped me, in big and small ways, once or many times, thank you.

And…

Dr. Matt Daugherty: Thank you first and foremost, for serving as my advisor, for the privilege (and perhaps, risk) of being your first graduate student. You have provided guidance, support, sympathy and honesty; you’ve pushed me when I needed it and when I didn’t want it. You’ve allowed me the freedom to fail, graciously let me know when my ideas were overly ambitious, and still helped me pursue them so I learned it for myself. You’ve mostly refrained from saying “I told you so” (but you totally wore it on your face a few times). Your door has always been open, your responses to my questions and last-minute requests for recommendation letters always generous and prompt.

Selfishly, I’d have preferred if you wouldn’t have moved to Berkeley during my time in grad school. But even with that distance, you were still attentive and available when I needed help or encouragement, checked in on me regularly, and provided the extra time and involvement I needed during my last year, and it made all the difference. You

iv granted me autonomy over my work, allowed me to manage my own team of undergraduate student research assistants, complimented and defended my efforts as a mentor. In your lab, I’ve always felt free to pursue any career path I wanted, and your advising, mentorship, and (especially) statistical prowess, prepared me for the non- academic, non-research journey I now find myself starting. I have repeated to others the advice you’ve given me. You allowed me to present at every conference I wanted to, and made it financially possible to do so. You’ve supported and even participated in the varied extra-curricular activities I’ve committed myself to, obliged my requests for lab social events, and you have fed me a lot of food over the past 5 years. You were kind and forgiving when I spilled champagne on your desk, and when I made a toner cartridge explode inside the lab printer, and when Duchess ripped up the underside of your office futon. Thank you.

Dr. Rick Redak: Thank you for serving as my co-advisor. Before I applied to graduate school, you responded to my inquiry with a long and informative reply full of generous advice and insight into the graduate school politics and the admissions process.

From you I learned that I was more likely to be admitted as a PhD student than a MS student, this changed how I approached potential advisors, and effectively put me on the path to a PhD. (Is it fair to say that it’s your fault I did this?) I’ve come to you countless times since then with questions and requests for help, and you haven’t let me down; you have more than one answer to most questions I ask, opinions I trust and have relied on often, and you always seem to know who has what I need. You have provided guidance, support, snarky commentary, criticism, and a lab full of abundant resources – in terms of

v space, storage, people to ask for help, and random lab supplies that have served me well in both my fruitful and fruitless endeavors. Each time we had a meeting to discuss designing an experiment, how to interpret results or write a paper, and even at my oral qualifying exam, you made a point of saying how much you had enjoyed that time, that it was the most productive thing you’d do all day, I appreciated hearing that each time. It’s been encouraging and uplifting at times when I’ve felt low, and unsure about whether I could do this thing. Meetings with you were entertaining sources of campus and departmental news, and having you as a co-advisor has pushed me to learn how to analyze all my data in so many more ways than I would have if I only had one stats whiz on my committee, rather than three (honestly, sometimes I hated this, but it was really good for me). Thank you for feeding Shithead all of the treats in your office; I’m sorry he’s still afraid of you. But I also think it’s funny. Thank you.

Dr. Erin Wilson-Rankin: Thank you serving as my committee member, and for kindly treating me as an honorary member of your lab. Your enthusiasm for my ideas and projects gave me confidence when I felt lost, and gently pushed me to continue, to not give up on my sometimes-too-ambitious goals. Attending and presenting in lab meetings with you and your lab gave me structure in my schedule, confidence in what I had accomplished, and a way to recognize in myself how much I knew and had learned through this process. It was empowering to realize I had something to offer the newer graduate students in your lab, and rewarding to share my experience and perspective with them. You have been generous with your time and resources (training me in PCR and barcoding, reviewing my work, and advising me on professional next steps, networking,

vi and strategizing how to advance my career). Your qualifying exams were my favorite to prepare for, because you gave me a very well organized study guide and an interesting, enjoyable reading list. Being a part of your lab gave me the opportunity to visit San

Clemente Island, an experience I may never get to repeat, and that I will always remember fondly. I’ve felt welcome in your lab, and this has been a source of support, friendship, scientific and personal community, good food, and often just a whole lot of fun. Thank you.

Diane Soto: Thank you for so, so much help. You have spent many, many hours in the field with me (and en route to/from field work), you’ve managed and coordinated assistants and supplies and vehicle use, you’ve anticipated things I’d need before I knew

I’d need them, helped with so much monotonous data collection and processing, and colluded with me to make lab social events happen. You’ve helped me troubleshoot and navigate minor catastrophes, all with a calm, patient attitude toward problem-solving. In general, you’ve played a huge role in making things go smoothly, truly above and beyond. Your sweet, quiet demeanor and efficient work ethic have been a huge asset to the lab, and I’m so glad that Matt stole you. Thank you for everything.

Dr. Andy Sanders: Thank you for permitting me to audit the lab sections of

“Taxonomy of Flowering Plants,” for indulging my plant identification requests, and for providing your input on my experimental designs. You provided extensive assistance in identifying seeds and seedlings for me and for undergraduate assistants, without your help I would have been lost. Your approach to plant identification, your perspectives on plant community ecology have informed and improved my own skills in these areas, as

vii well as the quality of my research. I appreciate your generosity, your wisdom and insight, and the indispensable UCR Herbarium. Thank you.

Dr. Christiane Weirauch: Thank you for sharing your expertise in Hemiptera, and for assisting me with access to scientific papers, for complimenting me to my advisor during my first year of grad school, and for being a kind face in the department.

Dr. Dan Hare: Thank you for allowing me use of your laboratory space and equipment.

Dr. Richard Stouthamer: Thank you for allowing me use of your laboratory space and equipment during ENTM 203. You provided criticism of early research ideas, and insight into relevant experimental limitations. Since then, you’ve been a source of entertaining criticism of poor seminars, insufficient amounts of job talk food, and teasing those who deserve it.

Dr. Darcy Reed: Thank you for extensive assistance with early experimental design and development of experimental methods. You provided so much assistance with learning to rear and care for Bagrada hilaris, and granted access to microscopes and lab equipment which have been invaluable. You helped me learn how to be a Teaching

Assistant, and granted me the freedom to develop my own lecture as a brand new instructor.

Dr. Tim Paine: Thank you for patient, thoughtful, helpful advice and guidance with questions on science, research, teaching, graduate school, academia, entomology, and all the other topics I came to you with questions about. You helped me track down

Rick numerous times, and you were kind and gracious when I replaced you on my

viii guidance committee and I was really nervous about it. Since then, I’ve come to trust you with questions that don’t have easy answers. Thank you.

Dr. Doug Yanega: Thank you for advice, guidance, and resources on methods and best practices in insect sampling, pollinator surveys, and investigations of plant- insect communities. Thank you for generous access to the Entomology Research Museum and use of outreach and teaching resources for my coaching Science Olympiad students.

And thank you for accepting my gazillions of arthropod specimens into the Museum!

Dr. Alec Gerry: Thank you for your role as my quarterly advisor during my first few years of graduate school. Checking in with you quarterly gave me reassurance that I was on the right track, and trust that I was being mentored and advised well.

Grace Hartt: Thank you for all of your time and work with me: countless hours at (and travelling to) Snow Creek, sitting at the lab microscope, plant and bug propagation, counting so many bugs, and setting up field cages at Ag Ops. Your effectiveness, attention to detail, and conscientious work ethic led me to trust you to work independently, to handle things without my presence. This was rewarding on multiple levels – I felt good about myself as a mentor, and pride in you as my assistant, and so pleased with how much work we were all able to achieve. You are an excellent independent worker, collaborative team player, and immensely capable. I look forward to learning how you choose to navigate your own career path.

Jacob Smith: Thank you for hours upon hours of work outside in the Riverside summer heat and out in the desert at Snow Creek. I gave you so many diverse tasks and projects to work on, and you rolled with it. Your engineering background and skills were

ix integral to executing so many experiments, and your disciplined military brain kept other experiments on regimented, predictable schedules – I always knew where to find you!

Your easygoing attitude, cheerful demeanor and general curiosity were an asset to our team, and will serve you well in all of your pursuits. I’m sorry for that time I led you to nearly driving your truck into the Ag Ops water canal, so grateful for your quick reflexes.

Thank you for your work in data collection in Chapters 1 & 2, plant propagation and insect propagation, for training others and being an excellent team player.

Seth Freitas: Thank you for so, so, so much work contributing to data collection in Chapter 1 & Chapter 2, many hours in the field at Snow Creek, setting up the experiment in Chapter 3, and insect and plant propagation. You’ve been a dedicated, curious and enthusiastic team player, a natural born entomologist, and a delight to work with. You eagerly tackled every task I set before you, devoured every journal article I sent you, and meticulously revised each paper, poster, and presentation you created on your research with me until it was impeccable. Your intelligence, curiosity, and strong work ethic will serve you well, and I look forward to seeing you succeed.

Gabriela Bobadilla: Thank you for your help in executing and troubleshooting new experiments and methods, for your work in soil seedbank assessments, assistance in setting up Snow Creek plots and traps. You were an excellent first mentee, you helped me learn how to be a mentor and how to guide someone else through a research project

(as I was figuring it out myself!). Thank you for being my guinea pig. Your enthusiasm is infectious, your work ethic and dedication have already taken you far, and your diverse interests and talents will serve you well.

x James Phillips: Thank you for all of your work on so many varied projects, in the lab, greenhouses, all over Ag Ops and in Snow Creek and the Box Springs Reserve. You showed up ready to work and ready to learn, your maturity, curiosity, and personal interest led you to completing several independent projects, and to teaching other team members rather quickly. You were reliable, trustworthy and honest, your strategic, critical and clear thinking about experiment methods, scientific papers, and even your own career have been an asset to my research, my communication and mentorship styles.

Thank you.

Amy Michael: Thank you for your assistance in soil seedbank assessment projects, for your organization and leadership that were critical to the success of that work. Thank you for your cheery disposition which brightened the lab, your assistance on early morning field days and occasional sweet treats. You became a friend, and I’m so glad for it. Thank you for kind words and shared meals and for videos of your ferrets being adorable. Thank you.

Stephanie Grande: Thank you for your work on many Bagrada hilaris projects, a literature survey, and your assistance with field days, insect rearing, plant growing, and field cage set-up. I relied on your organization, critical analysis skills, and attention to detail, and I so enjoyed watching you develop some fondness for Bagrada bugs, I looked forward to your bright cheerful face when visiting the greenhouses. Thank you for your hard work, including in the Riverside summer heat.

Julian Rasco: Thank you for your assistance with insect rearing, plant propagation and insect identification and sorting.

xi Pedro Montes: Thank you for your assistance and company on Temecula field days, insect sorting and identification.

Tim Lewis: Thank you for your advice and guidance in insect rearing, experimental methods, and equipment use.

Crystal May: Thank you for extensive advice and guidance on Bagrada hilaris propagation, so many Bagrada hilaris donations, and your willingness and availability to help me get Bagrada hilaris colonies going.

Dr. Anuar Morales-Rodriguez: Thank you for critique of my proposal writing which strengthened its quality, for guidance on experimental and statistical methods, and for letting me borrow equipment. For cheeriness and checking in on me regularly, for encouragement and support, I’ve appreciated it immensely.

Jessica Garcia, Clarisa Leon & Jorge Vasquez: Thank you for assisting me with a variety of projects and diverse (maybe even random) tasks, from identifying insects to labeling pots to field days at Snow Creek to erecting and disassembling those enormous field cages. You all have been available to me at many critical times when I needed extra help, and have been reliable, trustworthy and dependable. Thank you.

Dr. Rebeccah Waterworth: Thank you for your guidance and assistance right from the beginning of graduate school. You helped me design experiments, learn how to propagate Bagrada hilaris, write a proposal, analyze data, create a presentation and troubleshoot everything along the way. You’ve been kind and encouraging, and one of the sweeteset people I’ve ever met. Thank you.

xii Dr. Adam Zeilinger: Thank you for so much guidance and orientation to the lab, to UCR and the Entomology department, to Ag Ops and the greenhouses, and not least of all, to R. Much of my proficiency in using R and statistical analysis is build on a foundation that you provided, and it has served me well.

Dr. Sheena Sidhu: Thank you for lab meetings, for writing groups, for paper critiques, for the trip to San Clemente Island. For sharing the process of what to do with a dissertation AFTER you’ve written it. For helping me become a more thoughtful scientist, a better writer. For encouraging and complimenting me along the way, and for being honest about your experiences in graduate school and science. You’ve been a role model in a way that has helped me feel comfortable being honest about my own career path and goals. Thank you.

Korie Merrill: Thank you for the trip to San Clemente Island, for lab meetings and paper critiques and writing groups.

Robert Bonnett, Richard Zapata, Ramon Noriega, Sue Yamada, Mark

Tankersley and everyone at Ag Ops: Thank you for all you’ve done to make my work possible and successful at Ag Ops and in the greenhouses. You’ve been amazing and accommodating, and none of this would have been possible without you. Thank you.

Dr. Amelia Lindsey, Dr. Eric Smith, Dr. Emily McDermott, Nick Dvorscak,

Judith Herreid, Levi Zahn, Eric Gordon, Ryan Perry, Evin Lambert: Thank you for being my friends. For weird friend vacations, happy hours, and backyard beers and barbecues, cats and butter, football (sports-balls), parties, dog park trips, girls days/nights. For telling me you loved me and that I looked pretty. For letting me live with

xiii you. For sharing your holidays with me and treating me like part of your family. For laughing at my jokes, and for not. For cleaning up my messes, and not getting mad when

I accidently threw a waffle on your couch or left your car door open in the Bevmo parking lot in the rain. For understanding, and not asking before I was ready. For not drowning in the ocean that one time. For lots of manual labor on my research, feedback on my writing and presentations, and teaching me how to do science-things. For our new year’s kiss. For being a good sport about all the pineapple. For videotaping me at my best on my last birthday, and for taking care of me the next day. For goofy stupid jokes, for that company we should totally start one day. For all your pets, I’ve grown to love them all and now miss them terribly. Thank you for being good people, and for being there. I love you.

Sarah Morrison and Dr. Keenan Morrison and Elizabeth Rosen: Thank you for being my friends, and for being my Chicago people. For drunk Sundays in the pool and puppy classes and doggy dates. For letting me sleep on your couch and sharing your holidays with me, for treating me like family. For helping me move across the country.

For listening, for crying with me, and for making me laugh when I didn’t feel like it. For understanding, and for fort night. For surprising me on my birthday that one year, and for going out and getting stupid the last year. For throwing ice cubes at a wall, frozen yogurt, and so much champagne. For being a home base. Thank you for being there for me, I needed it. I love you.

Matt O’Neill: Thank you for being my grad school husband. For lots of manual labor, accompanying me to campus on weekends and evenings, critiquing papers and

xiv seminars with me, proofreading my writing, and troubleshooting my experimental designs, helping me learn my plants and how to think like a botanist. Today, it is hard to thank you for more than that…but I learned a lot from you, and from the marriage we had. I wish you all the best.

Robin Davenport, my mom, Peter Davenport, my dad, Cydnie Davenport, my sister: Thank you for being my family, for being there, for telling me I could. Thank you for a childhood full of Joshua Tree and Lake Tahoe and California beaches and San

Gabriel mountains. Thank you for books and cats and gardening and girl scout camp.

Thank you for listening, for pointing out what I was good at, for believing that this is all worth something in the end. For feeding me and visiting me and helping me, for being there when I really needed it. I love you.

Jackie Milot, my grandma: Thank you Grandma, for being my Grandma. For asking questions, for listening, for telling me it would be okay to quit when I sometimes wanted to, and for pushing me to do what needed to be done. For always welcoming me for a visit, and talking with me about all the things we aren’t supposed to talk about.

Thank you for your no-nonsense attitude, and for wine with ice cream. I love you.

Ray Milot, my grandpa: Thank you Grampies, for being my Grandpa. For telling me I’m smart, for telling me you’re proud of me…I sometimes felt like you were too easily impressed with me simply continuing to be in school; I appreciate that you were, it felt good to hear. I miss you, and I love you.

xv Jacob Smith, Seth Freitas, and James Phillips assisted with insect and plant propagation, experimental set-up and maintenance, and data collection in Chapter 1.

Jenny Hazlehurst, Kevin Loope, Jacob Cecala, and Michelle Miner provided feedback on an early draft of Chapter 1, which improved its quality.

Jacob Smith, Seth Freitas, Stephanie Grande, and Bryan Vanderveer assisted in insect and plant propagation, experimental set-up and maintenance, and data collection in

Chapter 2.

Seth Freitas, Stephanie Grande, Grace Hartt and Matt O’Neill assisted in insect and plant propagation, experimental set-up, planting, and maintenance, and data collection in Chapter 3. Anuar Morales-Rodriguez, Clarisa Leon, Jessica Garcia, Seth

Freitas, Matt O’Neill, Nick Dvorzcak, and Eric Smith disassembled field cages in

Chapter 3.

Funding was provided by the National Science Foundation Graduate Research

Fellowship Program, UCR Shipley-Skinner Reserve Endowment, the Southern California

Academy of Sciences, the Anza-Borrego Foundation, the California Native Plant Society, and the Community Foundation Desert Legacy Fund.

xvi DEDICATION

To Grampies, who told me I was smart;

To Grandma, who listened when I thought it was too hard;

And to Umma, for my surname, it is perfect for this new beginning.

xvii

ABSTRACT OF THE DISSERTATION

Associational Susceptibility of a Native Shrub, Atriplex canescens, Mediated by an Invasive Annual Forb, Brassica tournefortii, and Invasive Stinkbug, Bagrada hilaris

by

Sarah Lillian

Doctor of Philosophy, Graduate Program in Entomology University of California, Riverside, September 2017 Dr. Matt Daugherty, Chairperson

Indirect interactions have increasingly been recognized as important forces influencing population dynamics and structuring communities. Associational susceptibility is a form of indirect effect in which a focal plant experiences greater herbivore damage due to neighboring plant identity or diversity. These interactions remain poorly understood in the context of invasion ecology, though they may be responsible for huge impacts of invasive species on native communities. This dissertation investigates the potential mechanisms and consequences of associational susceptibility of a native perennial shrub, Atriplex canescens, driven by an invasive annual forb, Brassica tournefortii, and an invasive herbivorous stinkbug, Bagrada hilaris. In Chapter 1, a potential associational effect is experimentally demonstrated and a phenologically-driven trait is identified as a potential mechanism for this interaction. In Chapter 2, relative host plant quality is explored for its role in mediating the numerical response of the shared herbivore, and the herbivore’s damage impact on A. canescens. In Chapter 3, neighbor density, herbivore presence and herbivore density were manipulated to identify their

xviii impacts on spillover timing, extent, and fitness consequences for A. canescens. Overall, potential mechanisms of A. canescens associational susceptibility to Br. tournefortii and

Ba. hilaris identified include: Ba. hilaris accumulation on Br. tournefortii followed by

Br. tournefortii senescence and depletion, triggering Ba. hilaris alternative host-seeking.

Associational susceptibility of A. canescens could not be re-created under experimental conditions, but further study is required to ascertain whether this interaction is due to experimental limitations or ecological implausibility.

xix TABLE OF CONTENTS

Acknowledgements ...... iv Dedication ...... xvii ABSTRACT OF THE DISSERTATION ...... xviii Table of Contents ...... xx List of Tables ...... Error! Bookmark not defined. List of Figures ...... xxii Introduction ...... 1 References ...... 9 Chapter 1 ...... 15 Trait-mediated associational susceptibility of native shrub induced by context- dependent attraction of invasive species ...... 15 Abstract ...... 16 Introduction ...... 17 Methods ...... 22 Results ...... 27 Discussion ...... 29 References ...... 36 Tables and Figures ...... 44 Chapter 2 ...... 47 Invasive host plant quality mediates associational susceptibility of a native plant to the invasive herbivore Bagrada hilaris ...... 47 Abstract ...... 48 Introduction ...... 49 Methods ...... 54 Results ...... 61 Discussion ...... 64 References ...... 70 Tables and Figures ...... 75 Chapter 3 ...... 81

xx Direct & Indirect competitive effects of an invasive annual plant on a native shrub ...... 81 Abstract ...... 82 Introduction ...... 83 Methods ...... 86 Results ...... 89 Discussion ...... 93 References ...... 100 Tables and Figures ...... 107 Conclusion ...... 114 References ...... 117

xxi LIST OF FIGURES

Figure 1.1: Mean (±SE) cumulative number of Bagrada hilaris nymphs collected over 40

censuses from nine replicate plants of Atriplex canescens alone (Control), A.

canescens with neighboring Brassica tournefortii (Treatment) and Br. tournefortii

(Neighbors)…………………………………………………………...……………50

Figure 1.2: Mean (±SE) cumulative number of Bagrada hilaris adults collected from

Atriplex canescens alone (Control), A. canescens with neighboring Brassica

tournefortii (Treatment) and Br. tournefortii (Neighbors)…………...……………50

Figure 1.3: Proportion (±SE) of Atriplex canescens alone (Control) vs. A. canescens with

neighboring Brassica tournefortii (Treatment) with presence of Bagrada hilaris

over time. Census dates averaged by week for clarity. Neighboring Br. tournefortii

(not shown) had Ba. hilaris present on all individual plants at nearly every census

date…………………………………………………………………………………51

Figure 1.4: Proportion (±SE) of Bagrada hilaris individuals found at each of three

possible locations in choice trials: on Atriplex canescens, on Brassica tournefortii,

or on an ‘other’ surface. Trials consisted of an individual Ba. hilaris introduced into

arenas containing one A. canescens and one Br. tournefortii plant at one of three

possible ontogenetic stages: ‘rosette’ in which only basal leaves are present,

‘bolting’ in which flowers and fruits are present, and ‘senescent’ in which the plant

has discontinued growth and is browning…………………………………….……52

Figure 1.5: Proportion (±SE) of individual Bagrada hilaris found at each of three

possible locations (on Atriplex canescens, on Brassica tournefortii, on an ‘other’

xxii surface) over six censuses in choice trials between A. canescens and Br. tournefortii

in one of three ontogenetic stages: (A) Br. tournefortii in rosette stage, (B) Br.

tournefortii in bolting stage, (C) Br. tournefortii in senescent stage……………....52

Figure 2.1: Mean (±SE) maximum number of Ba. hilaris nymphs observed during any

single census and mean (±SE) total adult Ba. hilaris production on A. canescens and

Br. tournefortii……………………………………………………………………..83

Figure 2.2: Proportion of trials in which Ba. hilaris mating pairs successfully produced

nymphs and in which offspring successfully matured to adulthood when reared on

A. canescens and on Br. tournefortii……………………………………………….84

Figure 2.3: Proportion of trials in which Ba. hilaris reached adulthood on A. canescens

and on Br. tournefortii……………………………………………………………..85

Figure 2.4: Proportion of Ba. hilaris nymphs over time reaching the (a) second nymphal

instar or (b) third nymphal instar when reared in isolation on A. canescens and on

Br. tournefortii. Hash marks denoted censored observations due to nymphal

mortality……………………………………………………………………………86

Figure 2.5: Proportion of adult Ba. hilaris adults dying over time when restricted to either

A. canescens or Br. tournefortii……………………………………………………87

Figure 2.6: Mean (±SE) percent of A. canescens and Br. tournefortii plant tissue affected

by (a) yellowing of leaves and stems, and (b) starburst feeding lesions, in presence

versus absence of Ba. hilaris………………………………………………………88

xxiii Figure 2.7: Mean (±SE) percent of A. canescens plants affected by (a) yellowing of

leaves and stems, and (b) starburst feeding lesions with increasing Ba. hilaris

abundance………………………………………………………………………….88

Figure 3.1: Mean ±SE Bagrada hilaris abundance in Ba. hilaris present cages (a) in total,

and (b) on A. canescens over time………………………………………………..112

Figure 3.2: Proportion of Ba. hilaris present cages reaching: (a) peak Ba. hilaris

abundance, (b) peak Ba. hilaris abundance on A. canescens, and (c) Ba. hilaris cage

extinction over time………………………………………………………………114

Figure 3.3: Mean (±SE) Atriplex canescens (a) biomass, and (b) mean leaf production

score in cages with varying levels of Brassica tournefortii density and Bagrada

hilaris presence…………………………………………………………………...116

Figure 3.4: Structural equation model path diagram displaying regression coefficients

±SE among model variables. Arrows denote directionality of causal relationships,

black indicates positive and red indicates negative, solid lines indicate significant

relationships, dotted lines indicate non-significant relationships………………...117

xxiv INTRODUCTION

Indirect interactions have increasingly been recognized as important forces influencing population dynamics and structuring communities (Strauss 1991; Wootton

1994). In some cases, the magnitude of indirect interactions exceeds that of direct ones

(Menge 1995; Schmitz 1998). For example, shared predation is one of the most commonly identified trophic modules in empirical food webs (Milo et al. 2002;

Bascompte & Melian 2005). Among plants and their herbivores, shared predation can have negative effects on plant species’ fecundity and population growth rates

(Dangremond et al. 2010; Recart et al. 2013), recruitment (Orrock et al. 2008; Chaneton et al. 2010) and spatial distributions (Rand 2003). Negative outcomes occur when shared herbivores are food-limited; presence of preferred host plants (or increased density of preferred host plants) increase consumption of a focal host plant by eliciting a numerical or behavioral response of the shared herbivore (Holt 1977; Thomas 1986; Letourneau

1995). Of particular interest are negative outcomes which exhibit asymmetry, termed associational susceptibility, in which neighboring plants cause increased consumption of the focal host plant, but not vice versa (Thomas 1986; Letourneau 1995). Associational susceptibility is an ubiquitous and significant interaction (Chaneton & Bonsall 2000;

Barbosa et al. 2009; Underwood et al. 2014) but an understudied impact of exotic species invasions (White et al. 2006; Barbosa et al. 2009). Further, synergism among co- occurring invasive plants and invasive herbivores (Simberloff & Von Holle 1999;

Simberloff 2006) via associational susceptibility has the potential to exact important impacts on native communities (White et al. 2006; Barbosa et al. 2009).

1 Associational susceptibility among plants is predicted to occur when one or more of the following conditions are met (White et al. 2006; Bezemer et al. 2014): (1) plants experience differential attack rates, mediated by differential preference or performance of the shared herbivore (Rand 2003), (2) plants possess different levels of defense, allowing increased herbivore populations on one plant, and increased impact on the less defended plant (Rand & Louda 2004; Rand et al. 2004; Russell et al. 2007), (3) one plant provides a necessary resource at a particular time or life stage, allowing for increased impact on the other species at a later time or life stage of the herbivore (sensu Schmidt & Ostfeld

2008). In addition, spatial proximity of the two plant species, and total resource availability to the herbivore will mediate the strength and likelihood of associational susceptibility to occur. Beyond species-specific thresholds, herbivore damage influences plant fecundity, fitness and population dynamics (Halpern & Underwood 2006). Thus, associational susceptibility influences the compositional dynamics of ecological communities.

The role of herbivores in generating associational effects among plants has been convincingly demonstrated (Tahvanainen & Root 1972; Root 1973; Atsatt & O’Dowd

1976; Barbosa et al. 2009; Underwood et al. 2014). However, associational effects remain poorly understood in the context of invasion ecology. A recent meta-analysis concluded that exotic invertebrate herbivores have negligible effects on native plants, and even negative effects on exotic plants (Oduor et al. 2009), contradicting results of an earlier analysis (Parker et al. 2006). While invasive plants do experience significant rates of herbivory (Chun et al. 2010), and sometimes even higher herbivore abundances than

2 neighboring native plants (Ando et al. 2010), they usually host lower arthropod diversity than native plants (van Hengstum et al. 2014), and their populations are generally not controlled by herbivory (Keane & Crawley 2002; Chun et al. 2010). The low number of studies available to Oduor et al. 2009 (only 11), exclusive consideration of pair-wise interactions precluding indirect effects, and the short time period over which effects are considered, limit possible inferences about population level effects. Moreover, invasive herbivore impacts have rarely (especially with invertebrates) been studied in multi- trophic or multi-invader systems. Indeed, in the cornerstone review evidencing positive interactions among invasive species, only three of 254 studies considered indirect interactions (Simberloff & Von Holle 1999).

Kuebbing et al. 2013 emphasized the need for synthetic research on co-occurring invasive plants to support conservation and management of multiply-invaded systems.

However, exploration into multi-trophic invader interactions is equally important and valuable (Simberloff & Von Holle 1999; Simberloff 2006). Invasive plants, through competition with and displacement of native plants, are likely to exert negative indirect effects on many native plant and insect species and their interactions in invaded communities (Mack et al. 2000; Levine et al. 2003; Wagner & Van Driesche 2010; Vilà et al. 2011). Interactions among co-occurring invasive species generate synergistic negative impacts on native species termed “invasional meltdown,” often through indirect effects (O’Dowd et al. 2003). Indeed, invasion by herbivores is particularly threatening to native ecosystems due to their selection on plant community composition, facilitation of

3 exotic plants, and the resulting decrease in resilience to disturbance (Huntly 1991; Parker et al. 2006; Nuñez et al. 2010).

Despite the fact that co-occurring invasive species may interact in their native ranges, their interaction in the invaded range is likely to exhibit different qualities due to both abiotic factors and the biotic context in which this interaction is embedded.

Interactions among invasive species in their new ranges can result in synergistic impacts and the facilitation of future invaders (Simberloff & Von Holle 1999; Simberloff 2006).

New invasive species can accelerate the invasion process of other exotic species

(Grosholz 2005), pushing exotics present at low densities through the “lag phase” into high densities and range sizes through both direct and indirect facilitative interactions.

Further, new invasive may species change the form and strength of interactions among other invaders with native species (Montgomery et al. 2012), and among other co- occurring invaders (Ness et al. 2013). Interactions among plants and herbivores naturally vary temporally with both ontogeny and seasonality. Invasive species may exert novel impacts on the timing and strength of interactions among plants and herbivores.

Conclusions on the impacts of herbivores on native and exotic plants lack realism and predictive capacity if indirect interactions have not been considered. Thus, the objective of this dissertation research was to investigate the role of an invasive herbivore in propagating novel indirect effects of an invasive plant on a native plant, via associational susceptibility.

4 Study System: Bagrada hilaris is an invasive, herbivorous stinkbug (Hemiptera:

Pentatomidae) that invaded southern California in 2008 (Arakelian 2008), and has rapidly spread throughout the southwest U.S. Ba. hilaris is multivoltine, with a generation time of approximately 20-30 days, lifespan up to 3 months, and females can lay up to 200 eggs over their life time (Reed et al. 2013). Ba. hilaris is regularly observed feeding on

Brassica tournefortii, an invasive annual forb that has been present in southern California since at least 1927 (Minnich & Sanders 2000). Br. tournefortii seeds are readily collected in large numbers in Riverside and surrounding localities. Br. tournefortii seeds have a germination rate over 90% (O’Neill unpublished data), completes its life cycle within 3-5 months and is self-pollinating. Ba. hilaris has also been observed feeding upon a dominant native perennial shrub, Atriplex canescens. A. canescens is readily available at commercial nurseries and tolerant of harsh abiotic conditions. All three species co-occur throughout southern California and the southwestern U.S. – results obtained in the proposed research are relevant to management and conservation across several states. All three species are readily available and easily propagated under laboratory and greenhouse conditions.

Br. tournefortii is listed by the California Exotic Pest Plant Council in category A-

2 as a regionally aggressive invasive wild land pest (CalEPPC 1999), and poses a significant threat to the conservation of desert biodiversity in the southwestern U.S.

(Schiermeier 2005). Its most apparent impact has been the displacement of native annual plants (Barrows et al. 2009), though negative effects on some invertebrates have been identified (Hulton VanTassel et al. 2014). The success and dominance of Br. tournefortii

5 stems from its accelerated phenology (Marushia 2009). Following significant rain events, it monopolizes soil moisture, typically germinating 1-2 months before native annuals and grows rapidly to reach flowering and seed set in winter and early spring. Dense stands of mustard reduce native plant cover and may also obscure detection by native arthropods of the few natives which do germinate (Harvey & Fortuna 2012). Landscape-level changes in plant community diversity, species distributions, physical vegetation structure and microclimates threaten the persistence of native arthropods.

High densities of Br. tournefortii are implicated in supporting introduced populations of Ba. hilaris. Since its 2008 introduction, it has rapidly spread throughout the southwestern U.S.; it’s range currently encompasses southern California, Arizona, southern Nevada, New Mexico and western Texas, and is still expanding (Bundy et al.

2012; Reed et al. 2013). Detrimental impacts of this pest have been documented on

Brassicaceous crops in southern California and Arizona (Palumbo & Natwick 2010), with losses of up to 60% in newly planted fields (Reed et al. 2013). Despite extensive documentation of Bagrada in agricultural fields, our understanding of the factors supporting the rapid explosion in stinkbug populations and range size remains incomplete. Ba. hilaris has been commonly observed in wild lands and along roadsides feeding on Br. tournefortii throughout southern California, particularly in arid regions of

Br. tournefortii dominance. Br. tournefortii’s rapid phenology (Marushia 2009) provides abundant food and shelter for Ba. hilaris early in the year, especially when little else is present in harsh and dry desert environments.

6 In spring 2010, observations of Ba. hilaris feeding en masse on the native shrub

Atriplex canescens (Capparaceae; Four-winged saltbush) were made throughout southern

California’s Colorado Desert (C. Barrows, pers. comm.; L. Hendrickson, pers. comm.).

Following these observations, increased mortality of adult A. canescens, and decreased recruitment of A. canescens seedlings were recorded in 2011 and subsequent years in the

Coachella Valley (Barrows 2014). Atriplex canescens is a long-lived perennial shrub, and a dominant species across much of the southwestern U.S. Additionally, as a salt-tolerant shrub, it is often the only or one of just a few species growing in haline soils (e.g. dry lakes in southwest U.S. deserts). A. canescens is an essential foundation species in desert habitats, providing critical microclimates and habitat for native plants and animals, both above and belowground. It provides shade, ameliorating high temperatures and provides cover from predators to native species, including hatchlings of the endangered Coachella

Valley Fringe-toed Lizard, Uma inornata (Barrows 1997). These large shrubs promote greater biodiversity, both aboveground and belowground via “resource island” effects

(Ritchie & Olff 2005). A. canescens is dioecious; its population viability is potentially quite sensitive to increases in adult mortality rates. Threats to this single species have the potential to negatively impact many other plant and animal species through a ‘bottom-up cascade’ (sensu Kagata & Ohgushi 2005).

In the years since, such dramatic aggregations on A. canescens have not been recorded, though Ba. hilaris is found on A. canescens and other Chenopodiaceae plants, especially during summer. This inter-annual variability in species interactions may be explained by rainfall, which has been significantly below average, particularly in fall and

7 early winter, since 2010. Abundance of Br. tournefortii is significantly correlated with precipitation; low precipitation yields low Br. tournefortii abundance, and likely also smaller populations of Ba. hilaris (Barrows et al. 2009; Hulton VanTassel et al. 2014).

While high precipitation generally elicits a strong positive response from desert shrubs like A. canescens, negative indirect interactions like those described here may outweigh and nullify positive direct effects.

The prominent role of environmental variability in the strength of association among Br. tournefortii and A. canescens is expressly relevant to current concerns about the effects of global climate change on species interactions. This interaction is expected to be strongest following winters with above normal precipitation, due to sequential positive effects on the populations of Br. tournefortii and Ba. hilaris. Currently, regional predictions for the southwest U.S. under projected climate change scenarios are uncertain, though multiple processes are expected to result in changes to wintertime precipitation across North America including changes in the frequency and amplitude of

El Nino Southern Oscillation cycles (IPCC 2013). As community responses to changing climate may differ fundamentally from individual species responses (Suttle et al. 2007), explicit consideration of the mechanisms responsible for community responses is of critical importance. Specifically, herbivory is predicted to be exceptionally strong during rapid declines in water availability following periods of low water stress conditions

(McCluney et al. 2012). The interacting effects of changing environmental conditions and invasional meltdown pose a massive threat to the persistence of A. canescens, and as a result, other native species dependent upon this dominant plant.

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14 CHAPTER 1

Trait-mediated associational susceptibility of native shrub induced by context-dependent attraction of invasive species

Sarah Lillian1, Richard A. Redak1, Matt P. Daugherty1

1Department of Entomology, University of California, Riverside, CA 92521

15 ABSTRACT

Indirect interactions among native and invasive species are notoriously difficult to predict. Here, we apply theory from trait-mediated indirect effects and plant-insect interactions to explain the outcomes of multiple invader interactions. The present study investigates the roles of herbivore preference and host plant ontogeny in mediating associational effects among a southern California native shrub, Atriplex canescens, an invasive annual forb, Brassica tournefortii, and an invasive stinkbug, Bagrada hilaris.

Ba. hilaris have been observed to form dense aggregations on A. canescens in late spring following senescence of local Br. tournefortii, with subsequent increased mortality in A. canescens. A. canescens is a foundation species in arid habitats; any increase in its mortality risk is relevant to long-term population dynamics and management decisions throughout the western U.S. A manipulative experiment found Ba. hilaris recruited to A. canescens in greater numbers when neighbored by Br. tournefortii than when alone. Ba. hilaris nymph production, while minimized experimentally, occurred only on Br. tournefortii, suggesting A. canescens is not of sufficient quality for Ba. hilaris reproduction. In greenhouse preference trials, Ba. hilaris exhibited an overall preference for Br. tournefortii over A. canescens, however this preference was driven by ontogenetic stage of Br. tournefortii. Overall, results suggest significant potential for associational susceptibility mediated by a combination of: (1) Ba. hilaris preference for reproductive

Br. tournefortii and, (2) sufficient aggregation of Ba. hilaris onto Br. tournefortii and, (3) a marked decline in Ba. hilaris preference for Br. tournefortii with advancing ontogeny, triggering spillover onto neighboring plants. Annual plants can indirectly impact

16 perennial plants in complex ways relevant to invasive species management and native species conservation. The results presented here demonstrate an important role of multiple invaders and phenologically-driven trait changes in mediating associational susceptibility.

INTRODUCTION

Invasive species are among the worst threats to global biodiversity (Elton 1958;

Vitousek et al. 1996; Mack et al. 2000) and have wrought devastating impacts on native ecosystems around the world (Vitousek et al. 1997; Crooks 2002; Vilà et al. 2011). The impacts of invasive plants can be characterized generally as modifications (1) to the local abiotic environment experienced by native species (Crooks 2002), and (2) to the existing network of biotic interactions in which native species are embedded (Levine et al. 2003;

Vilà et al. 2011). In the latter case, direct interactions between an invader and a native species may also have many indirect effects on other organisms in the community. The enormity of potential indirect effects by invasive plants makes them critical to the study of invasion ecology (White et al. 2006).

Associational effects are a class of indirect effects in which consumer-resource interactions are modified by the identity or diversity of neighboring individuals (Barbosa et al. 2009; Underwood et al. 2014). For plants, associational susceptibility occurs when a focal plant experiences greater herbivore damage due to neighboring plant identity or diversity (Letourneau 1995); conversely, associational resistance occurs when herbivore damage is reduced (Tahvanainen & Root 1972; Agrawal et al. 2006). Both associational

17 susceptibility and associational resistance are thought to be ubiquitous interactions in plant communities (Chaneton & Bonsall 2000; Barbosa et al. 2009), though their importance among taxa and variability across plant communities remains poorly understood (Underwood et al. 2014).

Indirect effects of invasive species on native species are notoriously difficult to predict. To begin with, indirect interactions among co-existing species generate dense, reticulate interaction webs which produce emergent properties (Ohgushi et al. 2012).

Further, despite concerted efforts among ecologists to ascertain characteristics that predispose some species to become ‘invasive,’ reliable predictions of invasive species utilize estimates of propagule pressure over species traits (Simberloff 2009). So far, generalizable predictions based upon the traits of interacting species are not reliable. For example, the bio-control weevils, Rhinocyllus conicus and Larinus planus, introduced in

North America to control Carduus nutans (musk thistle) attack less-preferred native thistles in proximity to target weeds. These insects increase both oviposition and feeding rates on native thistles with increasing density and proximity of the invasive musk thistle

(Rand & Louda 2004; Rand et al. 2004; Russell et al. 2007). Such studies challenged an assumption of the preference-performance hypothesis (Mayhew 1997; Gripenberg et al.

2010): that reduced preference for and performance on non-target host plants minimizes the risk of impacts by introduced insect herbivores on non-target hosts (Suckling &

Sforza 2014; Catton et al. 2015). In reality, insect herbivores utilize sub-optimal plants even when preferred plants are present. This generates real fitness costs in the field, higher-order impacts on native plant density and diversity, and long-term effects on plant

18 population dynamics and community structure (Crawley 1989; Huntly 1991; Marquis

1992). Invasive herbivore attacks on native species can indirectly facilitate invasive plants, and underscores the necessity of research on co-occurring invasive species

(Simberloff & Von Holle 1999; Simberloff 2006; Kuebbing et al. 2013; Nuñez et al.

2010).

The study of associational effects in invasion ecology is nascent; much of the work to date has been conducted on plant species pairs with very similar life histories, in which direct antagonistic interactions are expected to be important (Rand 2003; Rand &

Louda 2004; Rand et al. 2004; Russell et al. 2007; Kim & Underwood 2014; Catton et al.

2015). Associational effects among dissimilar species pairs, in which both the strength and direction of interactions may vary greatly through time, and remain largely unexplored. Perennial plants persist through shifts in annual plant community composition within seasons and across years; at any time they may neighbor plants with both highly similar and highly dissimilar phenology, plant architecture, defensive chemistry, and resultant herbivore communities. Due to this temporal variability, associational effects may vary in their impact on plant populations with the phenology and ontogenetic stage of one or more plant species. Estimating and predicting plant population dynamics is critical to assessing the effect of a species on the local ecosystem, the strength of interactions with other species, and even extinction risk (Ehrlén & Morris

2015). Effective prediction of perennial plant population dynamics demands greater resolution in the role of associational effects (Underwood et al. 2014).

19 As in other indirect interactions, associational effects manifest via density- mediated and trait-mediated (e.g. behavior) interactions among herbivores and plants.

Density-mediated associational susceptibility occurs as a result of dense host plants supporting inflated herbivore populations and later ‘spill over’ onto a neighbor plant

(Rand 2003; Rand et al. 2004). Trait-mediated associational susceptibility occurs when some trait in a neighboring plant (independent of density or abundance) influences some change in herbivore behavior, increasing the herbivore impact on a focal plant (Karban

2010; Pearse et al. 2012). Density-mediated and trait-mediated interactions are not necessarily mutually exclusive, and relative herbivore preference for each plant modulates the strength of associational susceptibility (Rand 2003).

The present study investigates the role of herbivore preference in associational effects among invasive and native plants of differing life histories. This system comprises a native perennial shrub, Atriplex canescens (Chenopodiaceae; four-winged saltbush), an invasive annual forb, Brassica tournefortii Gouan (Brassicaceae; Sahara mustard), and an invasive insect herbivore, Bagrada hilaris Burmeister (Heteroptera:Pentatomidae; painted bug). While Ba. hilaris is an oligophage feeding primarily on plants in the family

Brassicaceae (Palumbo et al. 2016), including weedy mustards, they have been observed to form dense aggregations on A. canescens in late spring following seasonal depletion and senescence of local Br. tournefortii populations in southern California deserts

(Barrows 2014). Many of the attacked A. canescens in the Coachella Valley National

Wildlife Refuge and in Anza-Borrego Desert State Park suffered mortality in succeeding months (Barrows 2014). These field observations suggest that Br. tournefortii senescence

20 triggers intense Ba. hilaris herbivory on A. canescens. As a dominant shrub associated with increased native biodiversity and the endangered Coachella Valley fringe-toed lizard

(Uma inornata Cope), any increase in A. canescens mortality risk warrants close examination.

Phenology-driven host-plant switching is common among stinkbugs (Kiritani et al.

1965; Jones & Sullivan 1982; Panizzi 1997; Panizzi 2000). While these field observations are consistent with patterns of associational susceptibility, the phenomenon of annual plants triggering herbivore-induced mortality of perennial plants has not previously been documented in natural ecosystems. Further, it is unclear whether potential A. canescens susceptibility is density-mediated (i.e. herbivore spillover due to high densities and proximity of Br. tournefortii neighbors) or trait-mediated (i.e. advancing Br. tournefortii ontogeny triggers changes in Ba. hilaris relative host plant preference) (Kim & Underwood 2014; Underwood et al. 2014). Accordingly, this study used a set of greenhouse and manipulative field experiments to address the following questions:

(1) Does A. canescens experience greater Ba. hilaris recruitment when neighbored by

Br. tournefortii?

(2) Does Ba. hilaris demonstrate differential preference between Br. tournefortii and

A. canescens?

(3) Is Ba. hilaris host plant preference modified by Br. tournefortii ontogeny?

21 METHODS

Study System: Br. tournefortii is a noxious invasive forb from the Mediterranean region of northern Africa and southern Europe that has been present in California since at least 1927 (Minnich & Sanders 2000). This regionally aggressive wildland pest is associated with declines in native plant diversity, and in density of the endangered

Coachella Valley Fringe-toed Lizard (Minnich & Sanders 2000; Schiermeier 2005; Cal-

IPC 2006; Barrows et al. 2009). Br. tournefortii’s success stems from its early, rapid phenology: dense stands appear following fall rains and begin flowering as early as 40 days post-emergence (Marushia et al. 2012). By late winter, mature individuals produce hundreds of siliques containing thousands of seeds. Upon senescence in the spring, brittle siliques dehisce to expel ripe seeds and replenish the soil seedbank.

A. canescens is a trioecious, wind-pollinated, perennial shrub native to western

North America from Mexico to Canada (McArthur et al. 1992; Ogle & St. John 2005;

Baldwin et al. 2012). A. canescens exhibits a delayed phenology relative to Br. tournefortii: new leaf buds emerge in early spring, flowers appear in mid-late spring and fruits mature in late summer (Ogle & St. John 2005). As a drought-tolerant shrub in arid landscapes, A. canescens is a foundation species associated with increased density and diversity of native annual plants, and provides shelter, food and water for insects, small mammals, reptiles, granivorous birds, deer and sheep (Bestelmeyer & Wiens 2001). It is commonly planted in ecological restoration efforts following grazing, mining, and other anthopogenic disturbances (Meyer 2008).

22 Ba. hilaris is an aposematically colored, herbivorous stinkbug native to eastern and southern Africa, Middle East and Asia, where it is a major agricultural pest of brassicaceous crops and some grains (Palumbo et al. 2016). Ba. hilaris first appeared in the western hemisphere in June 2008 in Los Angeles, California (Garrison 2009;

Palumbo & Natwick 2010). Within two years, it had spread throughout southern

California into Nevada and Arizona, and caused severe economic damage to desert crops and organically grown crops (Bundy et al. 2012; Palumbo 2015). It is now also present in

Utah, New Mexico, Texas, Hawaii (Maui) and Mexico (Matsunaga 2014; Torres-Acosta

& Sánchez-Peña 2016; Palumbo et al. 2016). Ba. hilaris is oligophagous, with apparent preferences for plants in the family Brassicaceae (Singh & Malik 1993; Reed et al. 2013;

Lambert & Dudley 2014). Dense aggregations on preferred host plants comprise nymphs of all stages and adults; adults are often observed in copula (Huang et al. 2012). While active year-round, reproduction and population growth is concentrated in warm, dry months (Palumbo et al. 2016). Further, the actual distribution is likely underestimated in areas where infestations supported by non-agricultural host plants (e.g. weedy mustards) may go unnoticed (Palumbo et al. 2016).

Experiments were conducted at the Citrus Research Center and Agricultural

Experiment Station (CRC-AES), adjacent to the University of California, Riverside campus in Riverside, CA. The experiment station encompasses 510 acres of citrus orchards, greenhouses, experimental farm land and open space.

Bagrada hilaris recruitment: To determine whether A. canescens experiences greater recruitment of Ba. hilaris in the presence of neighboring Br. tournefortii (Obj. 1),

23 the response of local Ba. hilaris to A. canescens with and without Br. tournefortii neighbors was observed. Ten sites were identified throughout CRC-AES to deploy experimental plants, each site at least 50 m away from the nearest other site. Though the flight range of Ba. hilaris has not yet been clearly determined, 50 m is greater than the 3-

6 m flights commonly observed at peak daytime temperatures for this species (Huang et al. 2012). At each site, two potted A. canescens plants were placed 10 m from one another. One A. canescens was randomly designated the treatment plant, and the other the control plant. Two potted Br. tournefortii individuals were placed alongside treatment A. canescens, such that all neighboring pots were in physical contact. Control A. canescens plants had no neighbors. All plants were watered to field capacity 2-3 times weekly as needed. Mortality of any experimental plants within a site prompted replacement of all A. canescens and Br. tournefortii plants within that site. Twice weekly, all plants were examined for presence of Ba. hilaris. All Ba. hilaris detected were identified as nymphs or adults, counted, manually removed and added to greenhouse colonies for use in other experiments. To minimize the chance that plant examination may influence insect movement, at each census, plants were consistently examined in the following order within sites: control A. canescens, treatment A. canescens, Br. tournefortii.

The numerical response (i.e. reproduction) of Ba. hilaris to Br. tournefortii was explicitly limited by removing the recruited insects from plants at each census.

Reproduction and population growth would likely increase the strength of any observed associational effects by increasing the density of insect herbivores. The experimental design employed here is thus a conservative test of the potential for associational effects.

24 This design was employed to allow for detection of patterns in initial Ba. hilaris recruitment (i.e. attraction) onto experimental plants. Subsequent behaviors, as well as responses to varying plant quality and phenology, were largely excluded by design. If trait-mediated associational susceptibility of A. canescens is important, we expect greater frequencies of Ba. hilaris on A. canescens with Br. tournefortii neighbors than on A. canescens without neighbors.

Relative Ba. hilaris attraction and recruitment onto the 10 pairs of A. canescens plants over 40 censuses was evaluated in two complementary analyses. First, variation among the three plant types in the summed total of Ba. hilaris collected over the duration of the field experiment was assessed by two separate negative binomial generalized linear models for adults and for nymphs with R package ‘MASS’ (Crawley 2007; Venables &

Ripley 2002). Nymphs were excluded from further analysis due to limited data and the minimal contribution of nymphs to recruitment via dispersal (Panizzi et al. 1980). Next, variation in seasonal presence/absence of Ba. hilaris adults on A. canescens alone versus

A. canescens with Br. tournefortii neighbors was analyzed by a generalized linear mixed- effects model with a fixed effect of plant type, a random effect of replicate identity, and a binomial error distribution with R package ‘lme4’ (Crawley 2007; Bates et al. 2015).

This design was used to capture autocorrelation due to repeated measurements made on individual plants over time. Data were analyzed using R version 3.3.2 (R Core Team

2017).

Bagrada hilaris preference: To assess the relative preference of Ba. hilaris for A. canescens and Br. tournefortii (Obj. 2), a series of choice experiments was conducted (N

25 = 66 total replicates). Choice arenas were composed of a 40cm3 insect cage (BugDorm 2,

Megaview Science Co., Taiwan) containing one mature A. canescens potted in 7.5L round pot of UC mix #3 soil and one Br. tournefortii potted in 2.5L “tree pot” (Stuewe &

Sons Inc., Tangent, OR) of UC mix #3 soil (http://agops.ucr.edu/soil/). Plants were watered to field capacity three times weekly. To assess the role of Br. tournefortii ontogeny in mediating preference (Obj. 3), the ontogenetic stage of presented Br. tournefortii was manipulated as a factor with 3 levels: rosette (basal leaves only), bolting

(flowers and fruits present) and senescent (no new buds present, and plant browning). A total of 66 replicate choice trials were conducted, each with a unique Ba. hilaris individual and unique Br. tournefortii and A. canescens plants. Br. tournefortii life stages were replicated as follows: 23 ‘rosette’ with only leaves present, 30 ‘bolting’ with flowers and fruits present, and 13 ‘senescent’ or naturally deceased. A single un-mated adult Ba. hilaris, was introduced into the experimental arena within 2 days of eclosion.

The subject insect was placed on the center floor of the experimental arena, and then the insect’s position within the arena was recorded at six censuses: 30 minutes, 4 hours, 24 hours, 28 hours, 72 hours and 76 hours post-introduction. For analysis purposes, insects were recorded as being present at a given census as either on A. canescens, Br. tournefortii or in some location “other” than the provided vegetation (e.g. cage wall, cage floor, pot surface).

Ba. hilaris relative preference was assessed via a series of related analyses. First, the influence of Br. tournefortii life stage on variation in Ba. hilaris first choices (among

Br. tournefortii, A. canescens or “other” at the 30 minute post-introduction census) was

26 analyzed by multinomial logistic regression, with Br. tournefortii life stage included as a fixed effect in R package ‘MASS’ (Anon 2015; Venables & Ripley 2002). This was followed by three pairwise Fisher’s exact tests assessing variation in Ba. hilaris choices only between Br. tournefortii and A. canescens with varying Br. tournefortii life stage

(Crawley 2007; R Core Team 2017). Next, variation in Ba. hilaris’ choices upon each of six censuses was assessed by generalized estimator equations with a binomial error structure, logit link function and exchangeable correlation structure with R package

‘geepack’ (Højsgaard et al. 2006). Br. tournefortii life stage was included as a fixed effect and replicate as a random effect. Wald tests were used to assess significance of the overall model and model coefficients. Data were analyzed using R version 3.3.2 (R Core

Team 2017).

RESULTS

Bagrada hilaris recruitment: In the field study of Ba. hilaris recruitment over 40 sampling dates between July – November 2014, a total of 5,962 Ba. hilaris adults and

335 nymphs were collected from experimental plants. Data from a single site was removed prior to analysis due to a lack of Ba. hilaris detection on any experimental plants deployed at that site over the duration of the experiment, analyses were conducted utilizing data from the remaining nine sites. Over the duration of the experiment, the total number of Ba. hilaris nymphs collected was greatest on Br. tournefortii, significantly more than on A. canescens with neighbors and without (Fig. 1; χ2 = 48.471, df=2, p<0.001). Further statistical analyses assess Ba. hilaris adult abundance exclusively, as

27 nymph recruitment was explicitly limited by experimental design. The total number of

Ba. hilaris adults collected was also greatest on Br. tournefortii relative to A. canescens with neighbors and without (Fig. 2; χ2 = 31.992, df = 2, p < 0.0001). On average, Ba. hilaris abundance was 70 times greater on A. canescens with neighbors than on control A. canescens. Further, Ba. hilaris was more than threefold more abundant on Br. tournefortii than on neighbored A. canescens. Ba. hilaris was most prevalent in the field between early August and mid-October (Fig. 3). Ba. hilaris presence was recorded on A. canescens with neighbors on significantly more census dates than on A. canescens without neighbors (χ2 = 42.106, df = 1, p < 0.0001).

Bagrada hilaris preference: In 32 of 66 trials, Ba. hilaris had not chosen either plant species upon the initial 30 minute census; of these, 21 eventually chose one of the two plants. The life history stage of Br. tournefortii presented in choice experiments had a significant effect on Ba. hilaris first choices (χ2 = 12.941, df = 4, p = 0.0116). Although two of the three stages showed a trend consistent with initial preference for Br. tournefortii over A. canescens (Fig. 4), post-hoc pair-wise comparison Fisher’s exact tests comparing frequencies within each of the Br. tournefortii life stages found no significant differences in the frequency of Br. tournefortii or A. canescens choices. The longitudinal patterns of Ba. hilaris choices within replicate trials varied significantly among Br. tournefortii life history stages (generalized estimation equation; χ2 = 8.26, df = 2, p =

0.016). Ba. hilaris was present on Br. tournefortii more often than on A. canescens, this pattern was observed most strongly when Br. tournefortii were in the bolting stage, followed by rosette, then with the weakest preference for senescent Br. tournefortii plants

28 (Fig. 5). Indeed, when restricted to A. canescens and senescent Br. tournefortii, Ba. hilaris was most often found on neither plant.

DISCUSSION

This study investigated the role of variable herbivore preference in contributing to associational effects among invasive and native plants of divergent life histories, and the mediating effect of annual plant ontogeny. The patterns in Ba. hilaris relative abundances among plants and over time observed in the recruitment experiment suggest an important role of host plant ontogeny and herbivore preference in mediating associational susceptibility in this system. Choice experiments assessing Ba. hilaris relative preference suggest that a combination of preference for reproductive Br. tournefortii coupled with a marked decline in preference for Br. tournefortii with advancing ontogeny is driving this associational effect. From these results, we predict that associational susceptibility of A. canescens in the field is strongest when large populations of Br. tournefortii growing in close proximity support Ba. hilaris population growth and then senesce following Ba. hilaris herbivory. It has been suggested in agricultural systems that Ba. hilaris infest some crop plants simply due to their proximity to brassicaceous hosts (Brassica crops or weeds in field margins) (Lambert & Dudley 2014; Palumbo et al. 2016). This observation may be consistent with patterns of associational susceptibility tested in the experiments presented here.

The recruitment field experiment showed Ba. hilaris recruitment was greatest on

Br. tournefortii, and was much greater on A. canescens neighbored by Br. tournefortii

29 than on A. canescens alone. In seeking host plants, insect herbivores utilize volatile chemicals emitted by plants, as well as visual and gustatory cues to recognize and accept suitable hosts (Bruce et al. 2005; Chapman 2009; Randlkofer et al. 2010). Brassicaceae are well known for their signature defensive compounds, glucosinolates, and the associated volatile breakdown products, including isothiocyanates (Hopkins et al. 2009).

Glucosinolates have been shown to be important in host-plant recognition in many

Brassicaceae plant-herbivore systems (Renwick 2002; Hopkins et al. 2009), and are present in Br. tournefortii (Marzouk et al. 2010; Underwood 2014). The persistent aggregation of Ba. hilaris onto Br. tournefortii observed in this experiment is consistent with Ba. hilaris utilizing chemical cues emitted by Br. tournefortii plants to locate suitable habitat and host plants; further experimentation is needed to confirm this mechanism of host-plant seeking.

The differential Ba. hilaris abundance among treatments in the recruitment experiment (e.g. fewer on focal A. canescens relative to Br. tournefortii neighbors, and much less abundant on control A. canescens) is consistent with Ba. hilaris spillover from more attractive Br. tournefortii (Rand et al. 2004). Associational susceptibility may only occur following sufficient time for Ba. hilaris aggregation on Br. tournefortii, and following some trigger to switch onto A. canescens. Pestiferous stinkbugs in various crop systems are known to switch from wild or weedy plants on to agricultural crops when they become available (Kiritani et al. 1965; Jones & Sullivan 1982; Panizzi 1997; Panizzi

2000; Awuni et al. 2015). Further, phenological shifts in insect herbivore feeding and aggregation behavior have been documented in some other natural systems: Bay

30 checkerspot butterfly (Euphydryas editha bayensis) caterpillars more readily switch from the common Plantago erecta to patchy Castilleja exserta when Plantago erecta plants senesce rapidly (Hellmann 2002), and seed-feeding true bugs (Corimelaena extensa) switch from feeding on abundant Nicotiana obtusifolia in early spring to feeding in high densities on the more rare Nicotiana attenuata in late spring, resulting in increased insect fitness and decreased N. attenuata seed fitness (Stanton et al. 2016).

We found spillover to be most dramatic in late-August and September, when rapid declines in Br. tournefortii quality were also most difficult to minimize experimentally. Br. tournefortii exhibits a ruderal life history strategy (Grime 1977), completing its life cycle rapidly, especially under stressful abiotic conditions (Marushia et al. 2012). While experimental plants were promptly replaced when they began to senesce, late in the summer Br. tournefortii showed signs of senescence (e.g. wilting, yellowing) within days of deployment, likely succumbing to water and heat stress in addition to Ba. hilaris herbivory. Ba. hilaris may perceive and respond to these and other senescence cues earlier than human experimenters. We suspect this contributed to the divergence in timing of peak herbivore abundance (2-3 weeks earlier on Br. tournefortii than on A. canescens). This, along with consistently greater recruitment onto Br. tournefortii over A. canescens, is consistent with a trait-mediated indirect effect, in which the switch in Ba. hilaris foraging behavior exerts a negative impact on A. canescens.

The temporal patterns of peak Ba. hilaris abundance observed here corroborate what has been observed in the field (Barrows 2014): Ba. hilaris feed on Br. tournefortii preferentially, then subsequently spill over onto A. canescens. Br. tournefortii exhibits an

31 advanced phenology (Marushia et al. 2012) relative to A. canescens, and is available as an early food resource to Ba. hilaris. However, Br. tournefortii also exhibits rapid ontogeny (Marushia et al. 2012), reaching maturity and senescence earlier than related weeds and co-occurring native plants, including A. canescens. In this way, we predict that

Br. tournefortii inflicts both direct and indirect impacts on invaded communities. Its advanced phenology relative to native annuals confers a beneficial priority effect, largely excluding natives (Sale 1977; Seabloom et al. 2003; Wolkovich & Cleland 2011;

Barrows et al. 2009; Hulton VanTassel et al. 2014), and its rapid ontogeny may buffer Br. tournefortii populations against harsh abiotic conditions in short growing seasons and provides abundant food resources to Ba. hilaris populations (Marushia et al. 2012). Both features serve to provide Ba. hilaris with abundant food resources, but only for a short period until Br. tournefortii senesce and alternative host plants must be sought out.

In the recruitment experiment, peak Ba. hilaris abundance on A. canescens occurred with peak overall Ba. hilaris density, consistent with a density-mediated indirect effect in which associational susceptibility is driven by inflated herbivore populations spilling over onto neighboring plants. It is worth noting that in the recruitment experiment total plant density and Br. tournefortii neighbor presence are confounded.

However, if observed patterns were driven by plant density rather than identity, we would expect roughly equal insect occurrence on Br. tournefortii and on their neighboring A. canescens. Instead, the strong result of greater Ba. hilaris abundance on Br. tournefortii than on their neighboring A. canescens, along with the clear preference for bolting Br. tournefortii in choice trials, suggest that this experimental design has not impeded our

32 interpretations. In addition, Ba. hilaris nymph production, while explicitly minimized by removing insects twice weekly, occurred only on Br. tournefortii. This may be indicative that only Br. tournefortii, and not A. canescens, is of sufficient quality for reproduction.

Further experiments are necessary to assess whether Ba. hilaris reproduction and population growth influence the strength or direction of this associational effect.

While these results are suggestive of a density-mediated associational effect, the experimental design employed is insufficient to draw this conclusion. Only one level of neighbor density was utilized; it remains to be seen whether varying neighbor density will result in varying strength in the associational effect observed (Underwood et al.

2014). The Br. tournefortii density utilized here is considerably less than that observed in the field (Barrows et al. 2009; Hulton VanTassel et al. 2014), likely underestimating any density-mediated indirect effects. In addition, Br. tournefortii deployed in this study were replaced upon the detection of senescence to minimize any reduction in Br. tournefortii quality through time. Our results indicate senescence may serve as the stimulus prompting host-plant switching in Ba. hilaris, thus our design (preventing senescence) further underestimates the impacts of spillover. Given the strong results observed with these constraints in place, we expect A. canescens to experience heavier spillover herbivory under similar naturally occurring conditions. A. canescens associational susceptibility is relevant to long-term population dynamics and management decisions in the southwest U.S. where both Br. tournefortii and Ba. hilaris co-occur.

These conclusions are supported by results from the preference experiment. Ba. hilaris exhibited an overall preference for Br. tournefortii over A. canescens, however

33 this preference was driven by ‘bolting’ Br. tournefortii, and largely disappeared with senescent Br. tournefortii. Ontogenetic variation in insect herbivory has been well- documented, and is generally associated with patterns in plant defense and nutritional quality (Awmack & Leather 2002; Barton & Koricheva 2010). Senescing plant tissues rapidly decline in quality by exporting nutrients (sugars, amino acids, etc.) to storage or reproductive tissues (Thomas 2013), and most herbivorous insects are incapable of utilizing senescing and senescent plant tissue (White 2015). In some Brassicaceae species, the profiles of glucosinolates and other secondary metabolites vary ontogenetically within and among plant tissues (Bennett et al. 2006), which may contribute to declining apparency (Feeny 1976; Castagneyrol et al. 2013) to foraging Ba. hilaris.

Overall, we have shown evidence that in this system, associational susceptibility is driven by trait-mediated and possibly also density-mediated effects. Ba. hilaris exhibit strong preference for Br. tournefortii over A. canescens. However, this preference is modulated by the life history state of Br. tournefortii: ‘bolting’ Br. tournefortii is more preferred over A. canescens than other states, and senescent Br. tournefortii is the least preferred state. Ba. hilaris appear more likely to leave senescent Br. tournefortii than they are to leave bolting Br. tournefortii. Indeed, A. canescens experiences the greatest associational effect when Br. tournefortii is senescent, and being unpalatable does not protect A. canescens from Ba. hilaris herbivory. Moreover, in years of above-average precipitation promoting high Br. tournefortii density, Ba. hilaris population explosions may be more likely and A. canescens may be at drastically increased risk. These data

34 suggest that environmental conditions which would otherwise be beneficial to A. canescens (i.e. increased yearly precipitation) may instead promote extreme negative impacts on this native foundation species. Further investigations are necessary to determine population-level responses of Ba. hilaris to Br. tournefortii and to A. canescens, and the strength of direct and indirect effects on each host plant. Associational susceptibility is a complex indirect interaction, influenced by the traits of species involved (e.g. plant defense, herbivore feeding mode), as well as their numeric status

(e.g. relative density and frequency). The results presented here demonstrate that the role of annual plants in indirectly impacting perennial plants is complex, significant and important for invasive species management and native species conservation.

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43 TABLES AND FIGURES

Figure 1.1: Mean (±SE) cumulative number of Bagrada hilaris nymphs collected over 40 censuses from nine replicate plants of Atriplex canescens alone (Control), A. canescens with neighboring Brassica tournefortii (Treatment) and Br. tournefortii (Neighbors).

Figure 1.2: Mean (±SE) cumulative number of Bagrada hilaris adults collected from Atriplex canescens alone (Control), A. canescens with neighboring Brassica tournefortii (Treatment) and Br. tournefortii (Neighbors).

44

Figure 1.3: Proportion (±SE) of Atriplex canescens alone (Control) vs. A. canescens with neighboring Brassica tournefortii (Treatment) with presence of Bagrada hilaris over time. Census dates averaged by week for clarity. Neighboring Br. tournefortii (not shown) had Ba. hilaris present on all individual plants at nearly every census date.

45

Figure 1.4: Proportion (±SE) of Bagrada hilaris individuals found at each of three possible locations in choice trials investigating the effect of Br. tournefortii ontogeny: on Atriplex canescens, on Brassica tournefortii, or on an ‘other’ surface. Trials consisted of an individual Ba. hilaris introduced into arenas containing one A. canescens and one Br. tournefortii plant at one of three possible ontogenetic stages: ‘rosette’ in which only basal leaves are present, ‘bolting’ in which flowers and fruits are present, and ‘senescent’ in which the plant has discontinued growth and is browning.

Figure 1.5: Proportion (±SE) of individual Bagrada hilaris found at each of three possible locations (on Atriplex canescens, on Brassica tournefortii, on an ‘other’ surface) over six censuses in choice trials between A. canescens and Br. tournefortii in one of three ontogenetic stages: (A) Br. tournefortii in rosette stage, (B) Br. tournefortii in bolting stage, (C) Br. tournefortii in senescent stage.

46 CHAPTER 2

Invasive host plant quality mediates associational susceptibility of a native plant to the invasive herbivore Bagrada hilaris

Sarah Lillian1, Richard A. Redak1, Matt P. Daugherty1

1Department of Entomology, University of California, Riverside, CA 92521

47 ABSTRACT

Differential quality of co-occurring plant species to foraging herbivores may influence the rate at which herbivores attack each plant, resulting in associational effects among neighboring plants. High quality host plants increasing local herbivore population densities can indirectly contribute to an increase in herbivory on neighboring plants via herbivore spillover, i.e. associational susceptibility. Here we explore how a relative difference in host plant quality among an invasive forb, Brassica tournefortii, and a native shrub, Atriplex canescens, contributes to associational susceptibility by an invasive stinkbug herbivore, Bagrada hilaris. Each investigated measure of Ba. hilaris performance on Br. tournefortii and A. canescens consistently revealed greater performance on Br. tournefortii. Indeed, A. canescens is an insufficient host plant.

Preliminary assessments of herbivore damage severity found no significant difference between the two plant species, but did show increased damage on both plants with Ba. hilaris presence, and increasing damage with increasing Ba. hilaris abundance on A. canescens. A. canescens is susceptible to Ba. hilaris herbivory, though more work is required to assess fitness impacts of this novel herbivore. The results of the present study reveal the importance of initial host plant quality for driving the numerical response of a shared herbivore.

48 INTRODUCTION

Associational effects (AE) are a class of indirect interactions in which a consumer-resource interaction is affected by a neighboring third organism, usually another resource (Underwood et al. 2014). AE can generate both positive effects

(associational resistance) and negative effects (associational susceptibility) on focal organisms (Tahvanainen & Root 1972; Root 1973; Atsatt & O’Dowd 1976; Letourneau

1995). As in other forms of indirect interactions, AE are mediated by traits of the interacting organisms (Karban 1997; Ohgushi 2005), as well as their relative densities

(Kfivan & Schmitz 2004; Hambäck et al. 2014). While AE occur among a wide range of taxa engaged in consumer-resource interactions, they have been especially well-studied in plant-herbivore systems (Agrawal et al. 2006; Barbosa et al. 2009; Underwood et al.

2014). AE arise via indirect interactions among neighboring plants mediated by shared consumers (Agrawal et al. 2006; Barbosa et al. 2009; Underwood et al. 2014).

Associational susceptibility occurs when neighboring plants increase herbivory damage experienced by a focal plant (Letourneau 1995). Differential quality of neighboring plants to foraging herbivores may influence the rate at which herbivores attack each plant (Huntly 1991). For example, in the presence of a strong competitor,

Solidago altissima (Tall goldenrod), the leaves of Solanum carolinense (Carolina horsenettle) are apparently more palatable to insect herbivores, resulting in increased damage with increasing S. altissima frequency (Kim & Underwood 2014). Further, when neighboring plants are of high quality, they may increase herbivore populations locally and contribute to spillover (Rand & Louda 2004; Rand et al. 2004), sensu ‘apparent

49 competition’ (Holt 1977). Fall cankerworm (Alsophila pometaria) exhibit both greater preference for and performance on Box elder (Acer negundo), but will spillover onto cottonwood (Populus angustifolia x P. fremontii) when in close proximity to the preferred host, where they feed and develop (White & Whitham 2000).

Host plant quality is a complex suite of plant characteristics (e.g. %C, %N, C:N, trace elements, defensive metabolites, physical defenses) that affect herbivore performance (Awmack & Leather 2002). Generally, insect herbivores respond positively to increased nitrogen and negatively to increased C:N ratios; response to carbohydrates, sterols, lipids, or trace minerals is context-dependent. Defensive compounds vary greatly among plant taxa and even within plant tissues, and negatively affect naïve herbivores but may have neutral or positive effects on adapted specialist herbivores (Huntly 1991). Leaf pubescence and trichomes have been shown to reduce insect herbivore mobility, feeding rates, growth rates, larval nutrition and survivorship and specialized hooked trichomes can even physically impale herbivorous insects (Levin 1973; Johnson 1975; Haddad &

Hicks 2000). Further, exudates produced by trichomes and glandular hairs on leaf surfaces can impede insect mobility, trap herbivores, reduce growth rates and body mass of feeding insects (Wilkens et al. 1996; Kennedy 2003; Valkama 2005; Kobayashi et al.

2008). Host plant quality is generally assessed by measures of insect herbivore performance on potential host plants, and is likely an important mediator of the strength of associational susceptibility via the numerical response of shared herbivore(s).

Insect herbivore performance is a latent variable, comparable to fitness, estimated by traits related to insect survival and reproduction. As host plant quality varies, insect

50 herbivores may vary in key life history traits, including growth rates, timing of maturation, reproduction and oviposition, longevity, total fecundity, egg size, clutch size, maternal nutritional investment into eggs (vitellogenin), body size, and population growth

(Awmack & Leather 2002). Increased survival, longevity and reproduction of insect herbivores on high quality host plants may contribute to population outbreaks (Behmer &

Joern 2012). High quality host plants increasing local herbivore population densities can indirectly contribute to an increase in herbivory on neighboring plants via herbivore spillover (Catton et al. 2015), i.e. associational susceptibility. Indeed, the relative quality of host plants influences the magnitude of herbivore numerical response (Underwood

2004) and thus also the magnitude of the associational effect on neighboring plants. High quality host plants neighboring low quality plants may contribute to a rapid, dramatic increase in neighbor herbivory damage (Agrawal et al. 2006). Here we explore how relative differences in host plant quality among neighboring species contributes to associational susceptibility by an invasive stinkbug herbivore.

The painted bug Bagrada hilaris Burmeister (Hemiptera: Pentatomidae) is an oligophagous insect herbivore with host plants across 23 families, though it preferentially feeds on cruciferous plants in the family Brassicaceae (Taylor et al. 2015; Palumbo et al.

2016). Ba. hilaris is invasive in the southwestern U.S.A. where it was first recorded in

2008 in Los Angeles Co., CA (Palumbo & Natwick 2010; Bundy et al. 2012; Torres-

Acosta & Sánchez-Peña 2016). Ba. hilaris is commonly observed aggregating, feeding, and mating on Brassicaceous weeds in disturbed, agricultural, and natural landscapes, in addition to its pestiferous presence on cruciferous crops including broccoli, cauliflower,

51 kale, and cabbage (Reed et al. 2013; Lambert & Dudley 2014). Ba. hilaris appears to prefer young leaves, and also feeds on stems, flower buds and seed pods (Palumbo &

Natwick 2010; Reed et al. 2013; T.-I. I. Huang et al. 2014; Torres Acosta et al. 2016;

Palumbo et al. 2016). Feeding damage is evident by starburst-shaped chlorotic lesions at sites of repeated mouthpart insertion, characteristic of Ba. hilaris’ lacerate-and-flush method of feeding (T.-I. Huang et al. 2014; Palumbo et al. 2016).

Within two years of its apparent introduction, Ba. hilaris had established throughout Southern California and reached the deserts of Nevada and Arizona (Palumbo et al. 2016). During periods when Brassicaceae crops are unavailable (i.e. post-harvest, between seasonal plantings), Ba. hilaris appears on nearby alternative hosts which do not support population growth, including wheat, corn and tomato (Anwar Cheema et al.

1973; Reed et al. 2013; Palumbo et al. 2016). Ba. hilaris feeding can inflict significant damage on these alternative host plants in agricultural systems; it may be causing similar impacts to plants in native ecosystems. During the winter and spring of 2010, high precipitation (relative to the preceding years) supported high recruitment of annual plants, including weedy mustards that are favored host plants of Ba. hilaris (Hulton VanTassel et al. 2014). Following senescence of these and other annual plants, dense aggregations of

Ba. hilaris were observed on the native shrub Atriplex canescens in the Coachella Valley

National Wildlife Refuge and in Anza-Borrego Desert State Park (Barrows 2014).

Alternative host plant use is common among stinkbugs (Panizzi 1997), and as a dominant plant in harsh desert landscapes, A. canescens may be vulnerable to spillover herbivory due to limited alternative options (Ogle & St. John 2005). Subsequently, A. canescens

52 suffered increased mortality rates in the summer and fall months (Barrows 2014). These observations motivated investigations into whether the invasive mustard Brassica tournefortii promoted Ba. hilaris population growth and spillover herbivory on A. canescens in the region. This aligns with observations from agricultural systems, in which abundant Ba. hilaris in Brassica weeds along field margins are suspected source populations of crop pests (Lambert & Dudley 2014). Further, increased precipitation in

2010 may have dislodged and thus decreased the density of epidermal salt bladder cells on the surface of A. canescens leaves (LoPresti 2014). Salt bladders do confer some defense against chewing herbivores (Kenagy 1972; Kenagy 1973; Mares et al. 1997;

LoPresti 2014); decreased salt bladder density may also result in greater A. canescens vulnerability, though it is unclear whether piercing-sucking herbivores are deterred by the presence of salt bladders.

Previous studies have confirmed the phenomenon of Ba. hilaris spillover from Br. tournefortii onto A. canescens under experimental conditions. Further, Br. tournefortii was found to be highly attractive to Ba. hilaris, particularly during the bolting phase of producing flowers and fruits. Upon senescence, Br. tournefortii is no longer attractive, potentially instigating spillover onto neighboring plants (i.e. A. canescens). Ba. hilaris also appeared to exhibit much greater numerical response on Br. tournefortii than on A. canescens, implicating host plant quality as a contributing factor in Br. tournefortii- mediated associational susceptibility of A. canescens to Ba. hilaris. Here, we explore the role of relative host plant quality and sensitivity to herbivory in promoting associational susceptibility of A. canescens. The present study aims to answer the following questions:

53 (1) Does Ba. hilaris exhibit differential performance on Br. tournefortii and A.

canescens (i.e. are the two plant species of different quality with respect to

this insect’s feeding behavior)?

(2) Does Ba. hilaris-inflicted damage severity on Br. tournefortii and A.

canescens correspond with the respective host quality of each plant?

METHODS

Plant propagation: Br. tournefortii used in all following experiments were grown from seed that was hand-collected in Coachella Valley, CA. A. canescens were acquired from two sources. Mature plants were purchased from Mockingbird Native

Plant Nursery in Riverside, CA (http://mockingbirdnursery.com/). Seedling A. canescens plants used in some experiments were grown from seed donated by S&S Seeds

(www.ssseeds.com/plant-database/atriplex-canescens/). Pot sizes varied among experiments and are specified below; all plants were grown in UC mix #3 soil

(http://agops.ucr.edu/soil). Plants were fertilized with Osmocote Flower & Vegetable

Smart Release Plant Food following label directions. No herbicide or insecticides were applied to any experimental plants.

Ba. hilaris population growth: In a first study, we compared the relative quality of Br. tournefortii and A. canescens by measuring Ba. hilaris numerical response on mature plants of either species over approximately one generation. Within 48 hours of eclosion, Ba. hilaris adult virgin mating pairs were placed into individual 40cm3 insect cages (BugDorm 2, Megaview Science Co., Taiwan) containing an abundant supply of

54 either Br. tournefortii or mature A. canescens plants potted in 3L round pots. Cages were inspected daily for the appearance of eggs and nymphs, which were transferred to a separate 40cm3 insect cage, such that the emergence of new adult offspring was isolated from the original adult mating pair. Offspring cages were inspected daily to census the total number of nymphs and new adults. Trials were considered to be concluded when the female Ba. hilaris in the mating pair had died, all nymphs had matured to adults, and no new nymphs appeared for at least three consecutive days of inspection. A total of 18 replicates were included in the study (n=13 A. canescens, n=5 Br. tournefortii). An additional 5 replicates on Br. tournefortii were conducted that concluded upon the first offspring reaching adult, which underestimates total offspring production. These 5 replicates were excluded from cumulative offspring analyses, but were included in binomial tests of whether offspring were produced at all.

To assess relative host plant quality, the numerical response of Ba. hilaris on Br. tournefortii versus A. canescens were compared via two sets of analyses. First, we compared effects on nymph and adult abundance in a pair of analyses. Since nymphs were counted repeatedly without removal, the exact total number of nymphs produced in each replicate cage cannot be determined. Instead, the peak number of nymphs observed in each replicate cage, and the mean number of nymphs over the duration of each trial were used as response variables in a multivariate analysis of variance, with plant species as a fixed effect (Crawley 2007; R Core Team 2017). For adults, the cumulative number of adults produced is known precisely, because offspring reaching adulthood were sexed, counted once and removed. Therefore, cumulative total number of adults was compared

55 with a generalized linear model with plant species as a fixed effect, and a negative binomial error distribution to handle over-dispersed count data with zero inflation

(Crawley 2007; Venables & Ripley 2002). Next, we compared the frequency of production of any nymphs or adults in a given replicate cage. This second pair of analyses included 5 additional replicates on Br. tournefortii which were concluded upon the first offspring reaching adult. Two separate generalized linear models for nymph and adult production were analyzed with plant species as fixed effect and a binomial error distribution (Crawley 2007).

Ba. hilaris ontogeny: In a second experiment, we measured Ba. hilaris development time on either Br. tournefortii or A. canescens. Ba. hilaris eggs were collected from isolated mating pairs in 40cm3 insect cages containing both Br. tournefortii and A. canescens plants. Female Ba. hilaris were provided cotton pads as an oviposition substrate, eggs were collected daily and isolated into individual plastic petri dishes by separating cotton segments containing a single egg. Eggs were not directly exposed to the substrate of either experimental plant species (Hilker & Meiners 2006).

Upon eclosion, first instar nymphs were individually placed onto either a single ‘bolting’

Br. tournefortii plant or a single seedling A. canescens plant potted in a 0.5L round pot and then were covered by customized organza sleeve cages supported on a single bamboo stake. A total of 73 Ba. hilaris nymphs were reared on Br. tournefortii (n=33) and A. canescens (n=40). Nymphs were observed daily for evidence of molting. Dates of egg each molt, and death were recorded for each individual Ba. hilaris.

56 To assess relative host plant quality, we compared the frequency of Ba. hilaris nymphs reaching adulthood and development rate when reared on Br. tournefortii or A. canescens, as well as the longevity of newly developed adult Ba. hilaris on Br. tournefortii or A. canescens. The frequency with which Ba. hilaris nymphs successfully reached adulthood was compared by a generalized linear model with plant species as a fixed effect and a binomial error distribution (Crawley 2007; R Core Team 2017). The number of days until Ba. hilaris nymphs reached the second nymphal instar and the number of days until the third nymphal instar were compared by survival analysis log- rank test using the R package ‘survival’ (Therneau & Grambsch 2000). No comparison were conducted for later instars, because no nymphs survived on A. canesens beyond the third instar. Similarly, because no adults were produced in the A. canescens treatment, a formal comparison of Ba. hilaris lifetime longevity between plant species was not possible in this experiment.

Ba. hilaris longevity: In a third experiment, we compared the longevity of adult

Ba. hilaris confined onto Br. tournefortii or A. canescens. As in the previous experiment,

Ba. hilaris eggs were collected from isolated mating pairs in 40cm3 insect cages containing both Br. tournefortii and A. canescens plants, female Ba. hilaris were provided cotton pads for oviposition, and eggs were collected daily and placed into petri dishes. Eggs were not directly exposed to the substrate of either experimental plant species (Hilker & Meiners 2006). Upon eclosion, first instar nymphs were each individually placed onto a single Br. tournefortii plant potted in a 0.5L round pot and caged with a customized organza sleeve cage. Nymphs were observed daily for evidence

57 of molting, and upon reaching adulthood were moved on to a new caged plant, either a new Br. tournefortii or onto A. canescens. A total of 21 replicate trials were conducted in which Ba. hilaris nymphs reared on Br. tournefortii were transferred and isolated on a new plant at adulthood, either A. canescens (n=18) or Br. tournefortii (n=5). Data from the ontogeny experiment above in which Ba. hilaris were raised on Br. tournefortii following the same experimental methodology (n=17) was used to supplement this dataset. Adult Ba. hilaris were observed daily on new host plants until death, and were never mated. Dates of each molt and death were recorded for each individual Ba. hilaris that reached the adult stage. Differential longevity of Ba. hilaris adults between plant species was evaluated by survival analysis log-rank test (Therneau & Grambsch 2000;

Crawley 2007).

Plant species response to Ba. hilaris herbivory damage: A final pair of experiments compared the susceptibility of Br. tournefortii and A. canescens plants to feeding damage by Ba. hilaris. In the first experiment, individual Br. tournefortii and A. canescens plants were subjected to one of three Ba. hilaris density treatments to assess plant response to Ba. hilaris herbivory. This pilot study was intended to verify that our proposed metrics of plant condition were reliable indicators of herbivore pressure. A. canescens were grown in 7.5L round pots, and Br. tournefortii in 2.5L ‘treepots’ (Stuewe

& Sons Inc., Tangent OR). Individual plants of a single species were placed into 40cm3 insect cages and assigned to one of three Ba. hilaris density treatments. Experimental plants were chosen to be approximately the same size within species, based on initial measurement of total cumulative stem length and biomass (i.e. Br. tournefortii: 0.009

58 g/cm, A. canescens: 0.018 g /cm). Insect density treatments were: control (0 insects), low

(1 insect/g), high (40 insects/g). A total of six mature plants (n=3 Br. tournefortii, n=3 A. canescens) were subjected to Ba. hilaris herbivory for 16 days, with n=1 replicate per plant species by density treatment. Due to limitations in Ba. hilaris propagation and abundance, a single replicate of each plant species/insect density treatment was monitored over time. Ba. hilaris were introduced into cages at corresponding densities and Ba. hilaris density was maintained constant by counting and replacing dead Ba. hilaris insects three times/week. Plants were inspected approximately three times/week over a two-week period for evidence of herbivory damage, for a total of eight censuses.

The percent of the entire plant affected by yellowing (i.e. plant tissue which was previously green and turgid) or ‘starburst’ feeding lesions evident of Ba. hilaris mouthpart penetrations and feeding activity (Reed et al. 2013) was recorded at each census. Percentage of plant affected by either damage type was visually estimated in increments of 5% of total plant surface area.

To assess the effect of Ba. hilaris herbivory on each plant species, the percent of plant with yellowing tissue or starburst feeding lesions was analyzed in separate linear mixed-effects models with plant species, Ba. hilaris density and census day as fixed effects, and plant replicate identity as a random effect to capture autocorrelation stemming from repeated measurements made over time (Crawley 2007). The response variables, percent yellowing and percent starburst lesions, were square-root transformed to meet test assumptions. We also analyzed a related pair of linear mixed-effects models

59 with the same model structure, except that the low and high Ba. hilaris densities were pooled to create a two-level (insects absent or present) treatment.

In the second experiment, we quantified variation in the response of only A. canescens over a broader range of Ba. hilaris abundance and over a longer time scale. A total of 13 A. canescens plants were subjected to varying levels of Ba. hilaris abundance in two experimental blocks to assess response to Ba. hilaris herbivory pressure. In the first block, Ba. hilaris were caged on seedling A. canescens plants in 3L round plastic pots within custom organza sleeve cages at two levels of insect abundance: 5 insects

(n=1) and 10 insects (n=2). In the second block, mature A. canescens plants within individual 40cm3 insect cages were subjected to either zero insects (n=5), or 40 Ba. hilaris adults (n=5) per g estimated A. canescens biomass (300-555 total Ba. hilaris adults). In both experimental blocks, Ba. hilaris were introduced into cages and the number of insects was maintained constant by counting and replacing dead Ba. hilaris insects weekly. Plants were inspected weekly over an eight week period for evidence of herbivory damage. The percent of the entire plant affected by yellowing, i.e. any leaf or stem tissue fading from green to yellow, and ‘starburst’ feeding lesions, resulting from

Ba. hilaris stylet penetrations (Reed et al. 2013), was visually estimated in increments of

5% of total plant surface area at each census.

To assess the effect of Ba. hilaris abundance on A. canescens herbivore damage, we analyzed the percent of plant surface area with yellowing tissue or starburst feeding lesions over time. The analysis consisted of two separate linear mixed-effects models with experiment block and week as fixed effects, Ba. hilaris abundance as a covariate,

60 and plant replicate identity as a random effect. The percent of plant with yellowing was square-root transformed to meet test assumptions. For the percent of plant with starburst feeding lesions, multiple transformations were attempted, but none adequately corrected apparent violations of test assumptions. Therefore, results from the test on percent of plant with starburst lesions should be interpreted cautiously.

RESULTS

Ba. hilaris population growth: Over the duration of the population growth experiment, among all replicates, Ba. hilaris offspring production ranged between 0 and

148 nymphs observed in one census period (mean ±SE: 20.11±9.8 maximum observed nymphs), and between 0 and 182 total adults (21.22±10.8 adults). Mean nymph abundance over time was more than two orders of magnitude higher on Br. tournefortii

(31.52±1.59) than on A. canescens (0.05±0.03), and the maximum number of nymphs was over 150 times higher on Br. tournefortii than on A. canescens. A multivariate analysis of variance on the mean number of Ba. hilaris nymphs over time and maximum number of nymphs observed in any census found a significant effect of plant species

(Pillai’s trace=0.815, df=15, p<0.001). A negative binomial generalized linear model also found a significant effect of plant species on the number of adult Ba. hilaris adult offspring produced (χ2=190.22, df=1, p<0.001). No Ba. hilaris adult offspring were produced on A. canescens, versus more than 75 on average on Br. tournefortii (Fig. 2.1).

Plant species significantly affected whether Ba. hilaris nymphs were produced (χ2=24.79, df=1, p<0.001). At least one nymph was produced in all 10 Br. tournefortii replicates, but

61 only in 1 of 13 A. canescens replicates (Fig. 2.2). Similarly, plant species significantly affected the frequency with which Ba. hilaris offspring survived to adulthood (χ2=19.712, df=1, p<0.001). At least one Ba. hilaris offspring reached adulthood in 8 of 10 Br. tournefortii replicates compared to 0 of 13 A. canescens replicates (Fig. 2.2).

Ba. hilaris ontogeny: The ontogeny experiment also revealed significant differences between the two plant species with respect to the frequency of Ba. hilaris successfully reaching adulthood (χ2=31.791, df=1, p<0.001). When restricted to A. canescens only, Ba. hilaris nymphs failed to mature beyond the third nymphal instar in all of the 40 replicates, whereas Ba. hilaris nymphs reached adulthood in 17 of 33 replicates (Fig. 2.3). A log-rank test found a significant difference in the rates of Ba. hilaris nymphs reaching the second nymphal instar between plant species (χ2=4.1, df=1, p=0.044). Nymphs on A. canescens successfully molted and reached the second nymphal instar in 24 of 40 replicates, versus 27 of 33 replicates on Br. tournefortii, with more rapid development on Br. tournefortii (Fig. 2.4a). Similarly, a log-rank test also found a significant difference in the rates of Ba. hilaris nymphs reaching the third nymphal instar between plant species (χ2=17.8, df=1, p<0.001). On A. canescens, 4 of 40 nymphs reached the third nymphal instar, compared to 21 of 33 on Br. tournefortii, with more rapid development on Br. tournefortii (Fig. 2.4b).

Ba. hilaris longevity: In the Ba. hilaris adult longevity experiment, Ba. hilaris adults confined to a single plant survived as few as 0 d, up to 136 d. A log-rank test on the rate at which Ba. hilaris adults die found a significant difference among plant species

(χ2=33.9, df=1, p<0.001), with insects surviving longer on Br. tournefortii (Fig. 2.5).

62 Plant species response to Ba. hilaris herbivory damage: For the first herbivory damage experiment, we compared the percentages of plant tissue affected by yellowing and starburst feeding lesions in two ways. The first set of analyses included Ba. hilaris density as a covariate. The analysis of yellowing found a significant effect of day of census (χ2=21.1469, df=1, p<0.001), but not plant species (χ2=0.3086, df=1, p=0.579), or

Ba. hilaris density (χ2=2.6383, df=1, p=0.104). The analysis of starburst severity found a significant effect of Ba. hilaris density (χ2=5.2877, df=1, p=0.021), but not day of census

(χ2=2.357, df=1, p=0.125), or plant species (χ2=1.251, df=1, p=0.263). In a second set of analyses, we considered the effect of Ba. hilaris presence/absence as a binary treatment.

Information theoretic criteria suggest that the latter analyses with Ba. hilaris presence/absence are preferred, given lower Akaike Information Criterion values (AICy1:

150.4493, AICy2: 142.5892; AICs1: 129.9926, AICs2: 123.1508). The second analysis of yellowing found no significant effect of plant species (χ2=0.690, df=1, p=0.406), a significant effect of Ba. hilaris presence (χ2=4.799, df=1, p=0.028), and a significant effect of day of census (χ2=17.469, df=1, p<0.001). The second analysis of starbursts found no significant effect of plant species (χ2=1.808, df=1, p=0.179), a significant effect of Ba. hilaris presence (χ2=5.074, df=1, p=0.024), and no significant effect of day of census (χ2=2.347, df=1, p=0.126). These analyses reveal that yellowing increases with time (slope±SE: 0.106±0.023), and both yellowing and starbursts are more severe when

Ba. hilaris is present (Fig. 2.6).

For the second herbivory damage experiment, we compared the severity of yellowing and starburst lesions on A. canescens over a wider range of Ba. hilaris

63 abundance and over a longer time period. Percent of A. canescens plant yellowing differed significantly between experimental blocks (χ2=4.352, df=1, p=0.037), but was also significantly affected by Ba. hilaris abundance (χ2=5.560, df=1, p=0.018), and week

(χ2=104.28, df=1, p< 0.001). The severity of yellowing increased with time (slope±SE:

8.412±0.631) and with increased Ba. hilaris abundance (Fig. 7a). The percent of A. canescens plants with starburst feeding lesions was significantly affected by block

(χ2=8.011, df=1, p=0.005), Ba. hilaris abundance (χ2=14.563, df=1, p<0.001), and week

(χ2=63.635, df=1, p<0.001). The severity of starburst lesions increased with time

(slope±SE: 0.676±0.072) and with increased Ba. hilaris abundance (Fig. 7b).

DISCUSSION

This study investigated the relative quality of both the invasive forb Br. tournefortii and the native shrub A. canescens as host plants for the invasive herbivore

Ba. hilaris, as well as resulting damage severity on each potential host plant. Field observations implicate Ba. hilaris herbivory in increased mortality of A. canescens in southern California deserts in 2010 via associational susceptibility, following an increase in seasonal precipitation, and high Br. tournefortii population density (Barrows 2014).

Associational susceptibility occurs with an increase in local herbivore density and resulting increased damage (Letourneau 1995). In this system, this would require a strong numerical response of Ba. hilaris to Br. tournefortii, and a severe impact of Ba. hilaris herbivory to A. canescens. Previous work has demonstrated increased Ba. hilaris recruitment on A. canescens with neighboring Br. tournefortii, at least in part due to Br.

64 tournefortii’s attractiveness to herbivores in the local area. Here we expand investigations into the numerical response of Ba. hilaris on each plant species and associate Ba. hilaris density with plant damage response. In a series of experiments assessing Ba. hilaris performance, Br. tournefortii was consistently found to be a far superior host than A. canescens. Indeed, A. canescens is an insufficient host plant and cannot support Ba. hilaris population persistence or growth. Decreased insect performance on the less preferred host plant is consistent with the preference-performance hypothesis (Mayhew

1997; Gripenberg et al. 2010). Preliminary assessments of herbivore damage severity found no significant difference among the two plant species, but did show increased damage on both plants with Ba. hilaris presence, and increasing damage with increasing

Ba. hilaris abundance on A. canescens.

Each investigated measure of Ba. hilaris performance on Br. tournefortii and A. canescens consistently revealed greater performance on Br. tournefortii. Ba. hilaris did not successfully produce a new generation when reared on A. canescens. In population growth experiments, Ba. hilaris on A. canescens produced a small number of nymphs in only 1 of 13 replicate trials, and none survived to adulthood. Similarly, in ontogeny experiments, no nymphs reared on A. canescens reached adulthood. Ontogeny experiments revealed that Ba. hilaris develop to second and third nymphal instars significantly more quickly on Br. tournefortii than on A. canescens. Longevity experiments demonstrated a significantly longer lifespan of adult Ba. hilaris placed on

Br. tournefortii than on A. canescens. Br. tournefortii is a sufficient host for both growth and reproduction of Ba. hilaris, supporting herbivore population growth. High population

65 density of Br. tournefortii in 2010 (Hulton VanTassel et al. 2014) were likely responsible for observed high Ba. hilaris population density in southern California deserts (Barrows

2014).

Importantly, our results suggest that observed declines in A. canescens populations in the field (Barrows 2014) are likely not attributable to greater insect herbivore preference for or numerical response on the susceptible species (A. canescens).

Rather, associational susceptibility requires a host switch from Br. tournefortii, a significantly better host for Ba. hilaris. Host switching and alternative host plant use is common in stinkbugs and is often correlated with phenological availability and relative quality of various hosts (Panizzi 1997; Panizzi 2000). Ba. hilaris host shifting may be due to declining quality or availability of Br. tournefortii. In previous studies in this system,

Ba. hilaris preference was demonstrated to decline with advanced ontogeny and senescence of Br. tournefortii. Observed host switching in California deserts occurred in late spring, following senescence of Br. tournefortii and most other annual plants. This is also consistent with observations in other systems. Fall cankerworm (Alsophila pometaria) exhibits increased preference for and performance on box elder (Acer negundo) relative to cottonwood (Populus angustifolia x P. fremontii), but high herbivore densities deplete box elder and spillover onto cottonwood growing under box elder

(White & Whitham 2000).

Like other Chenopodiaceae, A. canescens plants concentrate Na+ in epidermal salt bladders on the surface of their leaves (Flowers & Colmer 2008), and these bladders can be manually removed (Pan et al. 2016). While these bladders have been primarily

66 investigated for their role in salt tolerance, they also contribute to defense against herbivory. Three species of rodents have independently evolved strategies to utilize

Atriplex spp. leaves in their diet, notably by scraping the leaf surface, removing bladders and external accumulated salts (Kenagy 1972; Kenagy 1973; Mares et al. 1997). Leaves of various Chenopodiaceae suffer increased rates of herbivory by chewing insect herbivores when leaf salt bladders are removed by brushing and rinsing the leaf surface

(LoPresti 2014). The impact of epidermal salt bladders on piercing-sucking insect herbivores remains unknown. Rinsing a leaf surface partially removes the chemical and structural defense posed by epidermal salt bladders (LoPresti 2014) – this suggests A. canescens may be more vulnerable to herbivores in certain contexts.

Our preliminary investigations into Ba. hilaris damage on Br. tournefortii and A. canescens reveal that Ba. hilaris attacks both plants, Ba. hilaris presence increases the percent of plant tissue yellowing and with starburst feeding lesions. A. canescens, though less preferred, appeared to have a greater percentage of tissue with starbursts, and increasing Ba. hilaris abundance resulted in increased plant yellowing and starburst feeding lesions. While these results should be interpreted cautiously due to low levels of replication, the observed patterns may be ecologically relevant, and are worthy of future study. Stinkbug feeding on vegetative tissues can reduce photosynthetic rate, induce wilting, cell degradation, necrosis and plant death (Panizzi 1997; Velikova et al. 2010;

Sharma et al. 2014). An apparent increase in starburst lesions suggests an increase in the number of feeding probes by Ba. hilaris individuals on A. canescens, the reason for this increase is unknown. It is unlikely to be due to differences in plant size or architecture, as

67 A. canescens possesses greater surface area than Br. tournefortii. Increased numbers of feeding probes has been associated with greater attractiveness in other stinkbugs (Molina

& Trumper 2012), but previous work has shown A. canescens is unattractive to Ba. hilaris. In the experiments described here, A. canescens mortality was not observed. This pilot study characterized Ba. hilaris damage to A. canescens by visible external symptoms on individual plants at a limited range of herbivore intensities; it remains unclear exactly what conditions are required to elicit the mortality observed in the field

(Barrows 2014). While we are able to detect some relationship between Ba. hilaris density and observed plant damage, we lack data to assess impacts on plant fitness.

Future studies should address the fitness consequences of Ba. hilaris damage to enable population-level predictions.

Overall, Br. tournefortii’s attractiveness to and high quality for Ba. hilaris, as well as it’s early and rapid phenology, are consistent with conditions promoting strong associational susceptibility of A. canescens. The results of the present study reveal the importance of initial host plant quality for driving the numerical response of a shared herbivore. Associational susceptibility of A. canescens requires high densities of Ba. hilaris, and high densities of Ba. hilaris cannot be propagated on A. canescens. Instead, high quality preferred host plants (e.g. Br. tournefortii) are required to build sufficiently high densities of Ba. hilaris to significantly impact A. canescens. A. canescens is susceptible to Ba. hilaris herbivory, though more work is required to assess fitness impacts of this novel herbivore. Future experiments should investigate the strength of spillover in this system at higher levels of herbivore pressure, and A. canescens fitness

68 consequences under more natural conditions.

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74 TABLES AND FIGURES

Figure 2.1: Mean (±SE) maximum number of Ba. hilaris nymphs observed during any single census and mean (±SE) total adult Ba. hilaris production on A. canescens and Br. tournefortii.

75

Figure 2.2: Proportion of trials in which Ba. hilaris mating pairs successfully produced nymphs and in which offspring successfully matured to adulthood when reared on A. canescens and on Br. tournefortii.

76

Figure 2.3: Proportion of trials in which Ba. hilaris reached adulthood on A. canescens and on Br. tournefortii.

77

Figure 2.4: Proportion of Ba. hilaris nymphs over time reaching the (a) second nymphal instar or (b) third nymphal instar when reared in isolation on A. canescens and on Br. tournefortii. Hash marks denoted censored observations due to nymphal mortality.

78

Figure 2.5: Proportion of adult Ba. hilaris adults surviving over time when restricted to either A. canescens or Br. tournefortii.

79

Figure 2.6: Mean (±SE) percent of A. canescens and Br. tournefortii plant tissue affected by (a) yellowing of leaves and stems, and (b) starburst feeding lesions, in presence versus absence of Ba. hilaris.

Figure 2.7: Mean (±SE) percent of A. canescens plants affected by (a) yellowing of leaves and stems, and (b) starburst feeding lesions with increasing Ba. hilaris abundance.

80 CHAPTER 3

Direct & Indirect competitive effects of an invasive annual plant on a native shrub

Sarah Lillian1, Richard A. Redak1, Matt P. Daugherty1

1Department of Entomology, University of California, Riverside, CA 92521

81 ABSTRACT

Associational effects among neighboring plants are ubiquitous, though understudied in invasion ecology. In southern California deserts, the invasive annual forb

Brassica tournefortii promotes recruitment and population growth of the herbivorous stinkbug, Bagrada hilaris. Ba. hilaris exhibits decreased preference for Br. tournefortii following senescence, which may promote spillover herbivory on to the native shrub

Atriplex canescens, as has been observed in the field. A manipulative field cage experiment was conducted to investigate the impact of Br. tournefortii density on Ba. hilaris population growth and spillover onto A. canescens, and the consequences for A. canescens fitness. Ba. hilaris total abundance over time and herbivore pressure on A. canescens were both significantly affected by Br. tournefortii density, census date and an interaction of Br. tournefortii density and census date. We observed 93-fold and 92-fold lower abundance on average, as well as more rapid Ba. hilaris extinction, in cages without Br. tournefortii relative to cages with densities of 10 or 100 Br. tournefortii, respectively. Very little A. canescens mortality was observed, in contrast to field observations. Structural equation models found Br. tournefortii presence negatively affects A. canescens final mass but not leaf production, and promotes Ba. hilaris total abundance, which promotes Ba. hilaris abundance on A. canescens, but there was no significant impact of Ba. hilaris abundance on A. canescens final mass or leaf production.

Br. tournefortii exerts an interspecific competitive effect on A. canescens, but a synergistic impact of Ba. hilaris spillover on A. canescens fitness was not observed.

82 INTRODUCTION

The presence of neighboring heterospecific plant species can influence the risk of herbivore attack and the damage experienced by a focal plant species (Barbosa et al.

2009). The indirect effect of plant neighborhood on herbivory damage (Karban 1997;

Hambäck et al. 2014; Underwood et al. 2014) (i.e. ‘associational effects’) can be positive, termed ‘associational resistance’ (Tahvanainen & Root 1972; Root 1973) or negative, termed ‘associational susceptibility’ (Letourneau 1995).

Associational effects may be driven by both density-mediated and trait-mediated interactions among herbivores and plants (Agrawal et al. 2006; Bezemer et al. 2014;

Underwood et al. 2014). Density-mediated associational susceptibility occurs as a result of neighbor host plants supporting inflated herbivore populations which later ‘spill over’ onto focal plants (Rand 2003; Rand et al. 2004). Density-mediated associational susceptibility is predicted to occur when herbivore populations become large enough to deplete their preferred host plant, and are thus forced to forage on alternative hosts

(White & Whitham 2000; Blossey et al. 2001). On the other hand, trait-mediated associational susceptibility occurs when a neighboring plant’s traits influence a shared herbivore’s behavior, resulting in increased herbivore impact on the focal plant (Karban

2010; Pearse et al. 2012).

Density- and trait-mediated associational effects are not mutually exclusive, and may occur in tandem within the same system (Kfivan & Schmitz 2004). Invasive species provide an ideal system in which to explore simultaneous density- and trait-mediated interactions because they tend to exhibit unusually large population sizes, unique

83 evolutionary history, and often a novel suite of traits relative to co-occurring native species (White et al. 2006; Bezemer et al. 2014). Indeed, for these reasons, the mechanism driving associational effects among native and invasive plants may be unclear without careful experimentation.

Invasive plants can facilitate invasion by exotic herbivores, and invasion by herbivores is particularly threatening to native ecosystems due to their selection on plant community composition, facilitation of exotic plants, and the resulting decrease in resilience to disturbance (Huntly 1991; Grosholz 2005; Parker et al. 2006; Nuñez et al.

2010). To date, much investigation into the impacts of invasive herbivores on native plants has considered only pairwise interactions, excluding possible indirect effects from detection (Parker et al. 2006; Oduor et al. 2009; Ando et al. 2010). Most communities globally are multiply invaded at multiple trophic levels; and co-occurring invasive species may exact synergistic impacts on native species, termed ‘invasional meltdown’

(Simberloff & Von Holle 1999; Simberloff 2006). New invaders may change the form and strength of interactions among other invaders with native species (Montgomery et al.

2012), and among other co-occurring invaders (Ness et al. 2013). Holistic investigation into the interactions among co-occurring invaders at multiple trophic levels is critical to effective prediction of community-wide responses and long-term dynamics (Simberloff

& Von Holle 1999; O’Dowd et al. 2003; Simberloff 2006; Wagner & Van Driesche

2010; Vilà et al. 2011). The present study investigates the dynamics of direct and indirect effects of an invasive plant and invasive herbivore on a native plant.

84 In previous studies, we have demonstrated that in a novel system, the invasive herbivorous stinkbug Bagrada hilaris Burmeister (Hemiptera: Pentatomidae) exhibits preference for and greater performance on the invasive weedy forb Brassica tournefortii

Gouan (Brassicaceae) over the native shrub Atriplex canescens Pursh (Chenopodiaceae).

Local Ba. hilaris populations recruited heavily to Br. tournefortii and to A. canescens neighbored by Br. tournefortii, but not to A. canescens alone, corroborating Ba. hilaris preference for Br. tournefortii under field conditions. Differential relative preference between the two plants disappears when Br. tournefortii senesces, and Ba. hilaris become less likely to be observed on either plant. Ba. hilaris were observed aggregating on A. canescens in areas of high Br. tournefortii density in late spring 2010 following above- average precipitation, and unusually high mortality of A. canescens was noted later that year (Barrows 2014). These observations motivated investigations into potential associational effects of the invasive Br. tournefortii on native A. canescens, mediated by the invasive Ba. hilaris. Preliminary data suggested that Ba. hilaris herbivory may affect

A. canescens, though fitness impacts remain unknown. The experiment described herein aims to address fitness impacts on A. canescens, as well as to better characterize the mechanism of associational susceptibility in this invaded plant-herbivore system by addressing the following three questions:

(1) How do Bagrada hilaris populations respond to varying Brassica tournefortii

density over time?

(2) How is Bagrada hilaris spillover onto Atriplex canescens influenced by

Brassica tournefortii density?

85 (3) How is Atriplex canescens seasonal growth and fitness affected by the

presence and/or density of both Brassica tournefortii & Bagrada hilaris?

METHODS

Experimental Design: This experiment took place on a tilled open field at the

Citrus Research Center and Agricultural Experiment Station (CRC-AES), adjacent to the

University of California, Riverside campus in Riverside, CA. The CRC-AES encompasses 510 acres of citrus orchards, greenhouses, experimental farm land and open space. A suite of 48 experimental field cages (1.8 m3) were erected in three rows of 16 each, with 2 m between rows and 0.3 m between cages within rows. Within each field cage, a single A. canescens individual was planted on December 31, 2014. A. canescens plants were purchased from Mockingbird Native Plant Nursery in Riverside, CA

(http://mockingbirdnursery.com/). No herbicides, insecticides or fertilizers were applied to any experimental plants. Within 1 m2 surrounding the focal A. canescens in each cage, seeds of Br. tournefortii hand-collected from Coachella Valley, CA were sown into the soil on January 22, 2015, and then thinned to one of three densities: 0, 10, or 100 individuals per 1 m2 on April 6, 2015. These densities are representative of what has been observed in the field, and of Br. tournefortii’s dominance under desert shrubs (Marushia et al. 2010). Plants were irrigated three times weekly until Ba. hilaris introduction for 30 minutes by 2 sprinklers mounted 1 m high on opposing corners within each field cage, saturating soil within cages to field capacity. Following plant establishment, a total of 20

Ba. hilaris mating pairs were introduced into half of all cages in a series of four

86 introduction events of five mating pairs each, two weeks apart between April 9 and May

21, 2015. Only adults were introduced, such that any nymphs observed throughout the course of the experiment can be attributed to reproduction since introduction. Each level of Br. tournefortii density and Ba. hilaris presence/absence was crossed in a balanced factorial design with eight replicates per treatment combination. Replicates with zero Ba. hilaris were included as controls against spillover herbivory, and replicates with zero Br. tournefortii were included as controls against interspecific plant competition.

Censuses of each field cage were conducted weekly May 12, 2015 through

January 22, 2016 to determine the total Ba. hilaris population size within each cage, presence and density of Ba. hilaris on focal A. canescens and on Br. tournefortii within each cage. Because Ba. hilaris lays eggs singly in soil, on plants, and on cage structures, egg production could not be estimated (Taylor et al. 2014). At each census, the phenological status of A. canescens and Br. tournefortii within each cage was assessed.

Plant phenology was assessed following protocols set forth by the U.S.A. National

Phenology Network on provided species-specific data sheets (USA-NPN 2010; USA-

NPN n.d.; USA-NPN n.d.). Phenological measurements included the number of: breaking leaf buds or initial growth, young leaves, flowers or flower buds, fruits, and recently dropped fruits or seeds, as well as the percent of: flowers that are open and fruits that are ripe. Counts of leaves, flowers and fruits were estimated and binned into the following categories: “0” = none, “1” = less than 3, “2” = 3 to 10, “3” = 11 to 100, “4” = 101 to

1000, “5” = 1001 to 10,000, “6” = more than 10,000. Percent of open flowers and ripe fruits were estimated and binned into the following categories: 0-4%, 5-24%, 25-49%,

87 50-74%, 75-94%, 95% and more. All binning was conducted separately for each variable and plant species; estimates for A. canescens were for the single individual within the cage, and estimates for Br. tournefortii were cumulative for all individuals within the cage. Upon conclusion of the experiment, all aboveground biomass of A. canescens individuals was harvested, dried and weighed.

Data Analysis: The total abundance of Ba. hilaris observed in cages was assessed by generalized linear mixed-effects models with a fixed effect of Br. tournefortii density, census date as a covariate, and their interaction, and a random effect of replicate cage to account for autocorrelation stemming from repeated measures (Crawley 2007; Fournier et al. 2012; Skaug et al. 2013). The amount of time until Ba. hilaris in a given cage reached their peak population density, and reached extinction, were assessed by separate log-rank tests on Kaplan-Meier survival curves and Cox proportional hazards models (Therneau &

Grambsch 2000; Therneau 2015; Moore 2016). We defined extinction as the second of two consecutive census dates in which 0 Ba. hilaris were observed, contingent that no more than 2 Ba. hilaris were ever subsequently observed in that cage.

Herbivore pressure, the abundance of Ba. hilaris observed to have migrated onto focal A. canescens within cages, was assessed by a generalized linear mixed-effects models with a fixed effect of Br. tournefortii density, census date as a covariate, and their interaction, and a random effect of replicate cage (Crawley 2007; Fournier et al. 2012;

Skaug et al. 2013). The amount of time until peak Ba. hilaris abundance on A. canescens, was assessed by a log-rank test on Kaplan-Meier survival curves (Therneau & Grambsch

2000; Therneau 2015; Moore 2016).

88 The overall response of A. canescens fitness to mean cumulative abundance of

Ba. hilaris observed on A. canescens was assessed by permutational multivariate analysis of variance with a response matrix including final A. canescens biomass, mean total leaf production and mean total fruit production (Oksanen et al. 2017). Follow-up analyses of variance assessed each univariate response. A. canescens mass was square-root transformed to meet assumptions of the univariate test; mean total leaf production could not be successfully transformed to meet test assumptions, thus untransformed results are presented here and should be interpreted with caution (Crawley 2007). To decouple the direct effects of Br. tournefortii presence and the indirect effects mediated via Ba. hilaris, structural equation modeling was employed. A piecewise structural equation model was evaluated to assess the system of causal relationships among (1) Br. tournefortii presence effect on final A. canescens mass, (2) Br. tournefortii presence effect on Ba. hilaris total cage abundance, (3) Ba. hilaris total cage abundance effect on the abundance of Ba. hilaris on A. canescens, and (4) Ba. hilaris abundance effect on A. canescens on final A. canescens mass (Grace 2006; Lefcheck 2015). A second piecewise structural equation model was evaluated with mean total A. canescens leaf production in the place of A. canescens mass. All analyses were performed in R version 3.4.1 (R Core Team 2017).

RESULTS

Among cages in which Ba. hilaris were introduced (“Ba. hilaris present cages”), the peak total abundance ranged from 0 insects to 1,555 insects per cage on a single census (Fig. 3.1a), and total abundance over time was significantly affected by Br.

89 tournefortii density (χ2=149.172, df=2, p< 0.001), census date (χ2=51.776, df=1, p<0.001) and an interaction of Br. tournefortii density and census date (χ2=451.368, df=2, p< 2.2e-16). Follow-up pairwise comparisons revealed significant differences in total Ba. hilaris abundance between cages with densities of 0 Br. tournefortii and 10 Br. tournefortii (χ2=124.272 df=1, p<0.001), and cages with densities of 0 Br. tournefortii and 100 Br. tournefortii (χ2=118.640, df=1, p< 0.001), but not between cages with densities of 10 Br. tournefortii and 100 Br. tournefortii (χ2=1.1703, df=1, p=0.279).

The mean cumulative abundance of Ba. hilaris observed on focal A. canescens within Ba. hilaris present cages ranged from 0 insects to 72.48 insects per A. canescens

(Fig. 3.1b), and was significantly affected by Br. tournefortii density (χ2=57.639, df=2, p< 0.001), census date (χ2=22.517, df=1, p< 0.001), and an interaction of Br. tournefortii density and census date(χ2=122.130, df=2, p< 0.001). Follow-up pairwise comparisons reveal significant differences in the abundance of Ba. hilaris on focal A. canescens between cages with densities of 0 Br. tournefortii and 10 Br. tournefortii (χ2=51.571, df=1, p< 0.001), and between cages with densities of 0 Br. tournefortii and 100 Br. tournefortii (χ2=45.6028, df=1, p< 0.001), but not between cages with densities of 10 Br. tournefortii and 100 Br. tournefortii (χ2=0.1696, df=1, p=0.680).

The number of days until peak Ba. hilaris population size within Ba. hilaris present cages ranged from 0 to 163 days. All cages with densities of 0 Br. tournefortii reached peak Ba. hilaris abundance by 28 days, while cages with densities of 10 and 100

Br. tournefortii took nearly 6 times as long to reach peak Ba. hilaris abundance (Fig.

3.2a). Time to peak Ba. hilaris population size was significantly affected by Br.

90 tournefortii density (χ2=28.1, df=2, p< 0.001). Follow-up pairwise comparisons revealed significant differences in the number of days until peak Ba. hilaris population size between cages with densities of 0 Br. tournefortii and 10 Br. tournefortii (χ2=15.2, df=1, p< 0.001), and between cages with densities of 0 Br. tournefortii and 100 Br. tournefortii

(χ2=16.6, df=1, p< 0.001), but not between cages with densities of 10 Br. tournefortii and

100 Br. tournefortii (χ2=0.7, df=1, p= 0.407).

The number of days until Ba. hilaris population extinction within Ba. hilaris present cages ranged from 28 to 255 days. In all cages with a density of 0 Br. tournefortii,

Ba. hilaris extinction had occurred by 63 days, while only ~50% of cages with densities of 10 and 100 Br. tournefortii had reached Ba. hilaris extinction by 204 days (Fig. 3.3c).

Time to Ba. hilaris extinction was significantly affected by Br. tournefortii density

(χ2=23.2, df=2, p< 0.001). Follow-up pairwise comparisons revealed significant differences in the number of days until Ba. hilaris extinction between cages with densities of 0 Br. tournefortii and 10 Br. tournefortii (χ2=11.8, df=1, p< 0.001), and between cages with densities of 0 Br. tournefortii and 100 Br. tournefortii (χ2=16.2, df=1, p< 0.001), but not between cages with densities of 10 Br. tournefortii and 100 Br. tournefortii (χ2=0, df=1, p= 0.866).

The number of days until peak Ba. hilaris abundance on A. canescens ranged from 0 to 170 days. All cages with a density of 0 Br. tournefortii had reached peak Ba. hilaris abundance on A. canescens by 42 days, while it took more than 3 and 4 times as long for all cages with densities of 10 and 100 Br. tournefortii, respectively, to reach peak

Ba. hilaris abundance on A. canescens (Fig 3.2b). Time to peak Ba. hilaris abundance on

91 A. canescens was significantly affected by varying levels of Br. tournefortii density

(χ2=21.8, df=2, p<0.001). In the absence of Br. tournefortii, Ba. hilaris peak abundance on A. canescens occurred on average at 15.75±2.88 days, while in cages with a density of

10 Br. tournefortii peak occurred at 94.63±16.48 days, and in cages with a density of 100

Br. tournefortii peak occurred at 119.75±13.03 days. Follow-up pairwise comparisons reveal significant differences in number of days until peak Ba. hilaris abundance on A. canescens between cages with densities of 0 and 10 Br. tournefortii (χ2=8.5, df=1, p=0.004) and between cages with densities of 0 and 100 Br. tournefortii (χ2=16.7, df=1, p<0.001), but only a marginal difference between cages with densities of 10 and 100 Br. tournefortii (χ2=2.9, df=1, p=0.087).

Minimal A. canescens mortality was observed: only one of 48 A. canescens plants succumbed during the experiment, though notably it was subjected to the presence of both Ba. hilaris and Br. tournefortii (at a density of 10 individuals). Sub-lethal impacts of

Ba. hilaris and Br. tournefortii were variable. Final dry A. canescens biomass ranged from 0.04 kg to 3.35 kg, and mean cumulative A. canescens leaf production ranged from a score of 2.75 to 5.27 (Fig. 3). The overall response of A. canescens final biomass and total leaf production was significantly affected by mean cumulative Ba. hilaris abundance observed on A. canescens (F=4.678, df=1, p=0.042). Similarly, the cube root of A. canescens final biomass was significantly impacted by mean cumulative Ba. hilaris abundance on A. canescens (F=4.997, df=1, p=0.030) and the mean total leaf production was marginally affected by mean cumulative Ba. hilaris abundance on A. canescens

(F=3.863, df=1, p=0.055).

92 A well-supported structural equation model was identified assessing Ba. hilaris,

Br. tournefortii and A. canescens mass relationships (Fisher’s C=5.43, df=4, p=0.246).

Br. tournefortii presence negatively affects A. canescens final mass, and promotes Ba. hilaris total abundance, which promotes Ba. hilaris abundance on A. canescens, but there was no significant impact of Ba. hilaris abundance on A. canescens final mass (Fig. 4). A second structural equation model investigating the relationships among Br. tournefortii presence and Ba. hilaris abundance found no significant relationships with A. canescens total leaf production.

DISCUSSION

This study examined variation in the strength of associational susceptibility of a native shrub, A. canescens, via an invasive weed, Br. tournefortii, and a shared herbivore,

Ba. hilaris. In this system, we expect that strong associational susceptibility of A. canescens would require: (1) high Br. tournefortii density, (2) strong numerical response of Ba. hilaris to Br. tournefortii, (3) considerable Ba. hilaris spillover from Br. tournefortii onto A. canescens, (4) Ba. hilaris herbivore pressure on A. canescens of sufficient intensity and duration.

Field observations and previous studies demonstrate that under favorable conditions, the first 3 of these 4 requirements for strong associational susceptibility are met. Br. tournefortii reaches high densities in natural A. canescens habitat (Barrows et al.

2009; Marushia et al. 2012) and is a preferred host plant of Ba. hilaris. Br. tournefortii is

93 highly attractive to Ba. hilaris and supports Ba. hilaris development, reproduction and population growth. On the other hand, Ba. hilaris cannot persist or reproduce on A. canescens alone. Ba. hilaris preference for Br. tournefortii is dramatically reduced when

Br. tournefortii senesces, which may stimulate alternative host plant seeking (Panizzi

1997) and spillover of Ba. hilaris onto neighboring A. canescens. Further, Ba. hilaris has been observed aggregating on A. canescens in heavily Br. tournefortii invaded areas following Br. tournefortii senescence, with subsequent high A. canescens mortality

(Barrows 2014). The experiment presented here investigates the fourth and final requirement for strong associational susceptibility: the intensity of Ba. hilaris herbivore pressure necessary to generate A. canescens fitness impacts or mortality.

In a manipulative field cage experiment, Ba. hilaris total abundance and density on A. canescens significantly increased with Br. tournefortii presence, but was not affected by varying Br. tournefortii density. Ba. hilaris in cages without Br. tournefortii reached their peak (and drastically lower) population size and extinction much sooner than Ba. hilaris in cages with Br. tournefortii presence, but there was no significant difference in the amount of time until Ba. hilaris presence was noted on A. canescens. A. canescens fitness, as approximated by biomass and mean leaf production, was significantly greater in treatments without Br. tournefortii presence, but unaffected by cumulative Ba. hilaris abundance.

Br. tournefortii, as well as other weedy mustards, do promote Ba. hilaris population growth and local recruitment (Reed et al. 2013; Lambert & Dudley 2014;

Palumbo et al. 2016). In this study, cages with Br. tournefortii present supported much

94 larger Ba. hilaris population sizes, and much longer Ba. hilaris population persistence than cages without Br. tournefortii. Yet unexpectedly, increasing the density of the preferred host plant, Br. tournefortii, from 10 to 100 individuals did not stimulate an increase Ba. hilaris abundance. Similarly, the timing of peak population size and population extinction did not vary between low and high density Br. tournefortii cages. It is possible that Br. tournefortii grown in higher densities may have been of lower quality than Br. tournefortii grown in lower densities. We did observe no difference in the mean number of Br. tournefortii fruits produced in cages among any Br. tournefortii treatments

– similar total cumulative numbers of fruits were produced by 10 Br. tournefortii plants as were produced by 100 Br. tournefortii plants (data not shown). While final Br. tournefortii biomass was not possible to ascertain without destructively interfering in the experiment, the authors did observe that Br. tournefortii grown in lower density treatments appeared larger and more vigorous than those grown in high density treatments. Given that Br. tournefortii is a strong competitor with rapid growth (Marushia et al. 2010; Marushia et al. 2012), this is consistent with intra-specific plant competition.

The resource-ratio hypothesis posits that the dominant competitor is better able to persist on the most limiting resource (Tilman 1985). In the case of conspecific (and thus evenly matched) competitors, each suffer and intra-specific competition intensity is expected to increase with increasing stress (e.g. water stress) (Maestre et al. 2005). Annual plants that are dominant in inter-specific competition tend to be more susceptible to intra-specific competition (Stoll 2001). For example, highly invasive and competitively aggressive garlic mustard, Alliaria petiolata, was more likely to survive to flowering, grew larger,

95 and produced more fruits when grown at low densities rather than medium or high densities (Meekins 2002).

Similarly to total Ba. hilaris abundance, the abundance of Ba. hilaris observed on

A. canescens increased only with Br. tournefortii presence, not with increasing density. A similar result was found in (Catton et al. 2015), in which a biocontrol weevil, Mogulones crucifer, attacks a non-target plant Hackelia micrantha in the presence of the target weed

Cynoglossum officinale, but non-target herbivory is not influenced local weed density, only presence. In contrast, in other systems examining associational susceptibility, increased herbivore density on focal plants is often observed with increasing neighbor density (Rand 2003; Lau & Strauss 2005; Russell et al. 2007). In this experiment, Ba. hilaris abundance on A. canescens appears to be limited only by the overall abundance of

Ba. hilaris within the cage. Peak abundance on A. canescens occurred later than peak total cage abundance, and after ~100% of Br. tournefortii fruits had ripened, depleting

Ba. hilaris’ preferred food resource.

While the magnitude of Ba. hilaris spillover onto A. canescens was not significantly affected by Br. tournefortii density, it did vary considerably with time.

Spillover peaks in which of ½ to ¾ of all Ba. hilaris residing on plants were present on A. canescens within cages with Br. tournefortii present occurred July through October. This timing is consistent with previous studies in this system which found reduced Ba. hilaris preference for Br. tournefortii when this plant senesces, perhaps triggering alternative host-plant seeking, and resulting spillover on A. canescens. In some systems, non- overlapping or sequential phenology promotes spillover of shared herbivores when one

96 plant supports herbivore population growth or provides a spatial/temporal refuge (Lau &

Strauss 2005). In other systems, overlapping phenologies are associated with greater spillover impacts, where neighbor density and spatial proximity determine the strength of spillover (Karban 1997; Rand & Louda 2004). Notably, spillover peaks never reached

100% of all Ba. hilaris residing on A. canescens even when all Br. tournefortii were senescent; rather, Ba. hilaris were observed on soil and cage surfaces in greater abundances preceding population declines. This is consistent with results of previous studies demonstrating A. canescens is a poor quality host for Ba. hilaris.

Some indicators of A. canescens fitness (leaf production and total biomass) were negatively impacted by Br. tournefortii presence, but not increasing density, and also not

Ba. hilaris cumulative abundance. This is suggestive of an inter-specific competitive effect of Br. tournefortii on A. canescens. While plant competition is often predicted to be most intense when species pairs are most similar (Tilman 1982; Johansson 1991), this is not always the case; the net impact of neighboring plants may change from positive to negative (or neutral) over varying plant life stages (Leger & Espeland 2010). Indeed, while perennial plants often exhibit a facilitative ‘nurse plant’ effect on young heterospecific neighbors (Holmgren 1997; Brooker et al. 2008), annual plants can also exert competitive impacts on perennial plants. For example, extensive research on invasion of native perennial grasslands by invasive annual grasses has found that while established mature perennial grasses are often resilient (McGlone et al. 2012; Blank et al.

2015), seedling and young perennial grasses are vulnerable to competition with annual grasses (Humphrey & Schupp 2004; Orloff et al. 2012). Another weedy invasive

97 mustard, Alliaria petiolata, is a superior competitor to Quercus montana (prinus), chestnut oak (Meekins 1999). The impact of annual Br. tournefortii competition on perennial A. canescens is an important area for future research. Br. tournefortii is already known to decrease native annual plant diversity and abundance (Barrows et al. 2009), but it may also reduce native annual plant recruitment by negatively impacting a nurse plant and foundation species, A. canescens.

Further, we failed to identify an impact of Ba. hilaris herbivory on A. canescens fitness. This may be due to limitations in experimental design. First, field cages may not have replicated the biotic stresses observed in field conditions, because Ba. hilaris population growth was limited to only reproduction, while natural populations experience immigration and emigration. Perhaps such restricted local population growth is insufficient to generate the densities of Ba. hilaris necessary to substantially impact A. canescens. Field observations of A. canescens mortality following Ba. hilaris herbivory were made in the Coachella Valley, CA, a region heavily invaded by Br. tournefortii, and also a region in which Ba. hilaris is a significant agricultural pest of cole crops including broccoli and cauliflower (Palumbo & Natwick 2010; Reed et al. 2013). Brassicaceous plants, including Br. tournefortii, are highly attractive to Ba. hilaris, and do promote local recruitment and high Ba. hilaris densities. Since A. canescens is such a poor host for Ba. hilaris, perhaps Ba. hilaris recruitment is an integral component of associational susceptibility of A. canescens via Br. tournefortii. Field cages prohibited immigration into the system, limiting Ba. hilaris abundance to only that which could be achieved by population growth, and possibly disallowing sufficient Ba. hilaris density to impact A.

98 canescens growth and fitness. Second, while herbivory and plant competition can inflict additive and even synergistic negative impacts on focal plants (Ferrero Serrano 2008;

Shabbir 2013), abiotic stress may be necessary in this system to generate fitness impacts on A. canescens (McCluney et al. 2012). Field cage conditions may not have adequately replicated the abiotic stress of field observations, e.g. heat and water stress (Klikoff

1967). Future experiments could address these limitations by manipulating water stress

(Suttle et al. 2007) and temperature stress (Post & Pedersen 2008; Schreven et al. 2017).

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106 TABLES AND FIGURES

107

108 Figure 3.1: Mean ±SE Bagrada hilaris abundance in Ba. hilaris present cages (a) in total, and (b) on A. canescens over time.

109

110 Figure 3.2: Proportion of Ba. hilaris present cages reaching: (a) peak Ba. hilaris abundance, (b) peak Ba. hilaris abundance on A. canescens, and (c) Ba. hilaris cage extinction over time.

111

Figure 3.3: Mean (±SE) Atriplex canescens (a) biomass, and (b) mean leaf production score in cages with varying levels of Brassica tournefortii density and Bagrada hilaris presence.

112

Figure 3.4: Structural equation model path diagram displaying regression coefficients ±SE among model variables. Arrows denote directionality of causal relationships, black indicates positive and red indicates negative, solid lines indicate significant relationships, dotted lines indicate non-significant relationships.

113 CONCLUSION

Associational susceptibility of native plants mediated by invasive plants and herbivores is a complex and dynamic indirect interaction. Indirect effects in general are notoriously difficult to predict – as the number of species considered increases, potential indirect interactions among them increases exponentially, generating densely inter- connected interaction webs (Wootton 1994; Menge 1995; Ohgushi et al. 2012). Among invasive species, predicting indirect interactions with novel interaction partners is even more challenging, and generalizable predictions based upon the traits of interacting species remain unreliable (Simberloff 2009). The studies presented in this dissertation clearly illustrate this challenge.

Ba. hilaris are attracted to Br. tournefortii in greenhouse and field conditions, and will recruit to Br. tournefortii plants in the field, forming dense aggregations. When neighbored by Br. tournefortii, A. canescens experience Ba. hilaris spillover, and much greater herbivore density than when neighbors are absent. While Ba. hilaris clearly prefer

Br. tournefortii over A. canescens, the relative preference difference decreases in magnitude with advancing Br. tournefortii ontogeny, and nearly disappears with Br. tournefortii senescence.

In addition to preferring Br. tournefortii, Ba. hilaris require Br. tournefortii for development, reproduction, population growth and persistence. If restricted to A. canescens only, Ba. hilaris nymphs do not develop, adults do not reproduce and die earlier, and populations do not increase. It appears that spillover and feeding on A. canescens is facultative and occurs only under limited, stressful conditions. The timing of

114 stressful, limiting conditions for Ba. hilaris may coincide with or exacerbate stressful conditions for A. canescens; estimating fitness impacts is critical for advising conservation management priorities.

In a field cage experiment designed to estimate the magnitude of Ba. hilaris population growth, spillover herbivory, and fitness consequences under varying biotic conditions, significant impacts of Ba. hilaris herbivory on A. canescens were not detected. However, competitive impacts of the annual forb, Br. tournefortii, on the perennial desert shrub, A. canescens, were revealed, suggesting additional complexities in this system. It is possible that Ba. hilaris herbivory at naturally achievable densities cannot yield fitness impacts on A. canescens, and field observations suggesting otherwise

(Barrows 2014) missed key variables. It is also possible that biotic conditions in this experiment did not replicate the magnitude of Br. tournefortii landscape abundance

(Hulton VanTassel et al. 2014), thereby limiting potential Ba. hilaris accumulation and inhibiting the potential magnitude of attack on A. canescens. Further, abiotic conditions in the field cage experiment may have ameliorated any potential A. canescens stresses, relative to what may have occurred during summer 2010 in the Coachella Valley desert region of southern California. Field observations of potentially Ba. hilaris herbivory- induced A. canescens mortality occurred following above-average winter precipitation, during the hottest months of the year. Field cage irrigation may not have adequately replicated the effects of rainfall diminishing A. canescens leaf salt bladders, an anti- herbivory defense in Chenopodiaceae (LoPresti 2014), and may not have reached sufficiently high temperatures to induce water stress in the desert-adapted A. canescens.

115 Overall, potential mechanisms of A. canescens associational susceptibility to Br. tournefortii and Ba. hilaris identified include: Ba. hilaris accumulation on Br. tournefortii followed by Br. tournefortii senescence and depletion, triggering Ba. hilaris alternative host-seeking. Associational susceptibility of A. canescens could not be re- created under experimental conditions, but further study is required to ascertain whether this interaction is due to experimental limitations or ecological implausibility.

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