UNIVERSITY OF CALIFORNIA, SAN DIEGO Pollinator Effectiveness of Peponapis pruinosa and Apis mellifera on Cucurbita pepo A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Jessica Audrey Davids Committee in charge: Professor David Holway, Chair Professor Joshua Kohn Professor James Nieh 2018 © Jessica Audrey Davids, 2018 All rights reserved. The Thesis of Jessica Audrey Davids is approved and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ Chair University of California, San Diego 2018 iii TABLE OF CONTENTS Signature Page…………………………………………………………………………… iii Table of Contents………………………………………………………………………... iv List of Tables……………………………………………………………………………... v List of Figures……………………………………………………………………………. vi List of Appendices………………………………………………………………………. vii Acknowledgments……………………………………………………………………... viii Abstract of the Thesis…………………………………………………………………… ix Introduction………………………………………………………………………………. 1 Methods…………………………………………………………………………………... 3 Results…………………………………………………………………………………......7 Estimated pollen deposition….…………………………………………..……......7 Fruit set……………………………………………………...…………………......7 Discussion………………………………………………………………………………....9 References………………………………………………………………………………. 12 Tables…………………………………………………………………………………… 15 Figures…………………………………………………………………………………... 16 Appendices……………………………………………………………………………… 20 iv LIST OF TABLES Table 1. Behaviors of bees videotaped during visits to squash flowers………….…….... 15 v LIST OF FIGURES Figure 1. Comparison of time spent on stigmas during single visits of Cucurbita pepo by Apis mellifera and Peponapis pruinosa. The bottom and top of each box represent the first and third quartiles, respectively, and the middle line represents the median. The bottom and top whiskers represent the minimum and maximum values, respectively….16 Figure 2. Pollen deposition on stigmas of Cucurbita pepo flowers as a function of time spent on stigmas during single visits by both Apis mellifera and Peponapis pruinosa. The regression line includes both species…...………………………….………………...…. 17 Figure 3. Pollen deposition on female stigmas of Cucurbita pepo as a function of Julian day by both Apis mellifera and Peponapis pruinosa. Each regression line represents data from each bee species. …………….…………….………..……….....………………….. 18 Figure 4. Comparison of single visit pollen deposition on female stigmas of Cucurbita pepo by Apis mellifera and Peponapis pruinosa bee species. Box and whisker plots as in Fig.1………………………………………………………...……………………………19 vi LIST OF APPENDICES Appendix 1. Soil Moisture Data. This table summarizes soil moisture readings collected twice a week for the duration of the project. Each plant received four soil moisture readings per date, within an hour of watering, resulting in a mean per plot per date. The mean of all soil moisture readings spanning the duration of the project ...…………………...…..….. 20 Appendix 2. Protocols for behavioral analysis of single visits. …………………..……....22 Appendix 3. Fate of squash flowers allowed to set fruit. (HB = Apis mellifera, PP = Peponapis pruinosa (♀)).……………………………………...……………………........23 Appendix 4. Single Visit Pollen Deposition Data. The data associated with every single visit sacrificed for the pollen deposition component of the study. Pollinator taxa designations: HB = Apis mellifera, PP = Peponapis pruinosa (♀). For additional information on data collection see Appendix 5…………………………......………..…...24 Appendix 5. Single visit pollen transfer procedures. A detailed summary of stigma storage & preservation and pollen counting protocols……….………………....…….....27 Appendix 6. Fruit Data. Fruit resulted from single visits by pollinators on flowers allowed to set fruit. Pollinator taxa designations: HB = Apis mellifera, PP = Peponapis pruinosa (♀). For additional information on data collection see Appendix 7………………....…....29 Appendix 7. Fruit Processing Protocols. A detailed description of fruit handling protocols including fruit harvesting, fruit volume determination, seed weighing, counting and storage, and fruit sugar content estimation. ………………………………………………31 Appendix 8. Data from Behavioral Analysis of Videos. This table includes the data associated with behavior from each experimental single visit. Where Visit Code = (Plant Number. Flower Number. Pollinator [HB = Apis mellifera, PP = Peponapis pruinosa], Pollination Date. Fate of Flower [Fruit = F, No fruit = N, Stigma = S])..…...………..…...34 Appendix 9. R Code used for statistical analyses…………….. …………...……..………38 vii ACKNOWLEDGMENTS I would like to thank my undergraduate research assistants (Aaron Ta, Andrian Krastev, Blaine Novak-Pilch, Breana Garcia, Chandler Pourvahidi, Chantal Sengsourinho, Carline Hua, Crystal Chan, Danny Rummani, Jessica Miranda, Johana Leon, Kara Powell, Kiara Suzuki, Kylie Etter, Rosalba Herrera, Tori Renfro, Valerie Inthavong, Vanessa Heredia, William Bauer) for dedicating many hours of their time to help me both in the field and in the lab. I would like to thank the members of the Holway Lab, especially Jess Gambel, who helped me have a productive field season and guided my thesis to its completion by helping me with my analyses and R coding. Finally, I’d like to thank the members of my committee for their guidance through every step of this process, especially my committee chair, David Holway. viii ABSTRACT OF THE THESIS Pollinator Effectiveness of Peponapis pruinosa and Apis mellifera on Cucurbita pepo by Jessica Audrey Davids Master of Science in Biology University of California, San Diego, 2018 Professor David Holway, Chair Differences between specialist and generalist pollinators provide insight into the evolution of specialization in plant-pollinator interactions. Plants of the genus Cucurbita (Cucurbitaceae) are visited by both generalist pollinators (e.g., Apis mellifera) and by specialist pollinators (e.g., Peponapis pruinosa). Previous studies have estimated pollinator effectiveness of Apis mellifera (honey bees), and Peponapis pruinosa (squash bees) in agricultural Cucurbita species, but none have investigated behavioral differences that underlie variation in effectiveness. In the summer of 2017, I conducted single visit pollinator effectiveness trials on 21 acorn squash (Cucurbita pepo) plants at the UC San Diego Biology Field Station in San Diego County, California to link interspecific ix behavioral differences of pollinators to their effectiveness. Female squash bees spent more than seven times longer per single visit in contact with squash stigmas and in turn deposited more than ten times more pollen compared to generalist honey bees. For all trials combined pollen deposition and fruit set increased with time bees spent on receptive stigmatic surfaces. Single visits by squash bees were more likely to result in fruit set compared to honey bees (85% vs. 12%). In terms of single visits, these results indicate that Apis mellifera are less effective at pollinating acorn squash compared to female Peponapis pruinosa. The results of this study differ from previous studies because Apis mellifera on our study site forages for pollen from a variety of different plant species in the surrounding landscape. In agricultural systems, in contrast, honey bees may have fewer options for pollen foraging. x Introduction Investigating the key differences that make some pollinators more efficient than others is important because of the potential implications for potential changes in agricultural and land management practices needed to bolster specific species of pollinators (Garibaldi 2013). The use of single visit trials is a common means by which to estimate pollinator effectiveness (Artz and Nault 2011, Cane et al. 2011, King et al. 2013, Tepedino 1981, Thomson and Goodell 2001). Previous studies have used seed set, pollen removal, fruit set, seed set, seed weight, fruit development rate to assess pollinator effectiveness (Artz and Nault 2011, King et al. 2013, Tepedino 1981, Thomson and Goodell 2001). Single visit measures of pollinator effectiveness can clarify contributions made by individual species (Canto-Aguilar & Parra-Tabla 2000, Garibaldi et al. 2013, Hung et al. 2017, Winfree 2007). Single visit measures of pollinator effectiveness are also of interest as many crops have differing dependence on insect pollinators and managed honey bees in the United States have been in decline in recent decades (Ollerton 2011, Smith et al. 2013). Studies that focus on pollinator effectiveness are particularly insightful when they include measures of pollinator behavior. Acorn squash (Cucurbita pepo) is an agricultural crop that attracts both generalist and specialist pollinators. Cucurbita pepo is monoecious and produces unisexual flowers that are visited by a diversity of pollinators (McGregor 1976, Michelbacher 1964), including super generalist honey bees and specialist squash bees. Previous studies have compared the effectiveness of single visits by bees to female flowers of C. pepo (Artz and Nault 2011, Cane 2011, Tepedino 1981), but the behavior of bees during single visits has 1 received relatively little attention in these studies, which focus on visit duration, fruit set, fruit growth, and stigma contact. A gap in understanding thus exists with respect to the visitation behavior of individual pollinators and how it relates