ABSTRACT AN EXAMINATION OF POSSIBLE CARNIVORY IN SILENE REGIA, A MEMBER OF THE CARYOPHYLLACEAE by Garrett John Dienno Silene regia, commonly known as Royal Catchfly, a member of the Caryophyllaceae, is known to ensnare small insects with its glandular trichomes. This morphological adaptation is primarily thought to deter herbivory, but it has been speculated that S. regia may also be carnivorous. To demonstrate that S. regia is carnivorous the following objectives must be shown: S. regia can attract, capture, and retain prey; secrete proteinases to facilitate nutrient absorption; and absorb/translocate the resultant nutrients. This study addressed the first two of these objectives through field observations, UV photography, SEM imaging, and a series of experiments designed to examine a capture- induced proteinase response. While S. regia was able to ensnare insects and possessed highly specialized morphological structures for doing so a form of active attractant could not be demonstrated, and as such failed to support the first objective in its entirety. Negative test results for a capture-induced proteinase response failed to support the second objective. As both objectives were unsupported it was concluded that S. regia is not carnivorous. AN EXAMINATION OF POSSIBLE CARNIVORY IN SILENE REGIA, A MEMBER OF THE CARYOHPYLLACEAE A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science by Garrett John Dienno Miami University Oxford, Ohio 2017 Advisor: R. James Hickey Reader: Alfredo J. Huerta Reader: Richard C. Moore Reader: Richard H. Munson ©2017 Garrett John Dienno This thesis titled AN EXAMINATION OF POSSIBLE CARNIVORY IN SILENE REGIA, A MEMBER OF THE CARYOHPYLLACEAE by Garrett John Dienno has been approved for publication by The College of Arts and Science and Department of Biology ____________________________________________________ R. James Hickey ______________________________________________________ Alfredo J. Huerta _______________________________________________________ Richard C. Moore ______________________________________________________ Richard H. Munson Table of Contents INTRODUCTION .......................................................................................................................... 8 MATERIALS & METHODS ....................................................................................................... 12 RESULTS ..................................................................................................................................... 21 DISCUSSION ............................................................................................................................... 30 REFERENCES ............................................................................................................................. 33 iii List of Tables TABLE 1 ..................................................................................................................................................... 21 TABLE 2 ...................................................................................................................................................... 22 TABLE 3 ..................................................................................................................................................... 23 iv List of Figures FIGURE 1 ................................................................................................................................................... 13 FIGURE 2 ................................................................................................................................................... 15 FIGURE 3 ................................................................................................................................................... 16 FIGURE 4 ................................................................................................................................................... 17 FIGURE 5 ................................................................................................................................................... 18 FIGURE 6 ................................................................................................................................................... 19 FIGURE 7 ................................................................................................................................................... 22 FIGURE 8 ................................................................................................................................................... 24 FIGURE 9: .................................................................................................................................................. 25 FIGURE 10 ................................................................................................................................................. 26 FIGURE 11 ................................................................................................................................................. 27 FIGURE 12 ................................................................................................................................................. 28 FIGURE 13 ................................................................................................................................................. 29 v Dedication This work is dedicated to my wife Katrina. Her love, tireless support, and willingness to put up with my bumbling ways are worthy of sainthood. vi Acknowledgements This study would not have been possible without funding provided by the Miami University Biology Department’s Academic Challenge Grant. I also thank Tim Osborne and the stewardship department of the Great Parks of Hamilton County for their assistance in locating site populations, answering questions relating to the care and propagation of S. regia, and providing permission to conduct this study on park property; as well as the Ohio Department of Natural Resources for granting me permission to conduct this study at the Milford Center Prairie and Bigelow Cemetery State Nature Preserves. My fellow graduate student Patrick Garrett’s knowledge of R-coding was instrumental to the statistical analysis of the data generated by this project. Additionally, Kaitlin Campbell and Mike Minnick provided initial guidance with R-coding, modeling, and brainstorming field methods. Photographer Ron Steven’s help and technical advice were vital to getting the UV photography to work properly. I also thank Matt Duley of Miami University’s Center for Advanced Microscopy & Imaging for his assistance with the SEM micrographs. The advice and guidance of my graduate committee were instrumental in helping to complete this project. In particular I have to thank Dr. Richard Moore for his extensive help editing the various drafts of this thesis; Dr. Richard Munson for being a mentor throughout my undergraduate studies; Dr. Alfredo Huerta for his positive encouragement during setbacks; and my advisor Dr. James Hickey for his help and support with this study as well as his initial suggestion to consider graduate studies. vii INTRODUCTION Though often considered a mere curiosity, carnivorous plants have served as model organisms in a wide range of studies including rapid plant movements, nutrient absorption, and food web interactions (Król et al. 2012). Yet despite their widespread use as model organisms, defining what should and should not be considered carnivorous has been problematic (Simons 1981; Rice 2011). Givnish et al. (1984) proposed that for a plant to be considered carnivorous it must, in order: (1) have at least one adaptation for attraction, capture, retention, and/or digestion of prey, and (2) that the plant must be able to absorb nutrients from captured prey. Similar defining guidelines, while not explicitly stated, were also utilized in earlier studies conducted by Charles Darwin relating to the demonstration of carnivory in plants (Darwin 1888). Other broader definitions of carnivory have also been proposed which include plants that do not secrete their own digestive enzymes but instead rely on symbiotic relationships with insects or bacteria to digest their captured prey (Bruce, Anderson, & Midgley, 2003; Darnowski et al, 2006; Rice, 2011). This broader definition of carnivory, in which a plant may fulfill some or all of the criteria of carnivory through mutualistic associations, has also given rise to the concept of protocarnivory (Rice 2011). A protocarnivorous plant is one that meets some, but not all, of the criteria commonly considered necessary to demonstrate carnivory. Members of the Eriocaulaceae (Paepalanthus bromelioides) and Roridulaceae (Roridula spp.) have been shown to fall under this category, relying upon mutualistic host associations with resident insects to digest prey captured by the plant (Anderson and Midgley 2003). As the plants are incapable of producing their own digestive enzymes, the nutritional benefit of captured prey is passed along to the plant through the feces of the resident insects (Ellis and Midgley 1996; Anderson and Midgley 2002; Anderson 2005; Nishi et al. 2013). Other studies have demonstrated the existence of “part-time” carnivores which only exhibit carnivorous adaptations during particular stages of their life history (Bringmann et al. 2002). The genus Stylidium, a member of the Stylidaceae, produces glandular proteinase-secreting trichomes