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ABSTRACT

AN EXAMINATION OF POSSIBLE CARNIVORY IN REGIA, A MEMBER OF THE

by Garrett John Dienno

Silene regia, commonly known as Royal Catchfly, a member of the Caryophyllaceae, is known to ensnare small 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 , 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,

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.

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INTRODUCTION

Though often considered a mere curiosity, carnivorous have served as model organisms in a wide range of studies including rapid 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 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 (Paepalanthus bromelioides) and Roridulaceae ( 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 , a member of the Stylidaceae, produces glandular proteinase-secreting trichomes only on its (Darnowski et al. 2006). A more extreme example of this “part-time” carnivory is seen in the peltatum of the . Before it transitions to being a T. peltatum exists as a basal rosette of highly specialized covered in glandular trichomes (Green et al. 1979). For the purposes of this project, a strict definition of plant carnivory based off the Givnish et al. (1984) study was used where in order for the plant to be considered carnivorous the following must be demonstrated: (1) it can attract, capture, and retain prey; (2) it can secrete digestive enzymes such as proteinases to facilitate nutrient absorption; (3) it can and absorb/translocate the resultant nutrients. Trying to place the development of plant carnivory within an evolutionary and systematic context has been notoriously difficult with at least five independent origins within the angiosperms and the loss of the carnivorous habit in related lineages. It was only with the advent of modern that progress could be made on understanding these relationships 8

(Chase et al, 2009). The latest work on the points towards a monophyletic origin with a clear division between two sister groups that are referred to as the core and non-core clades. The core clade comprises what has traditionally been the circumscription of the Caryophyllales while the non-core clade comprises a clade that formerly was circumscribed as the Polygonales and includes the carnivorous families , Drosophyllaceae, Dioncophyllaceae, Nepenthaceae; and the non-carnivorous Ancistrocladaceae (which is sister to the carnivorous Dioncophyllaceae and likely lost the carnivorous habit), Polygonaceae, Plumbaginaceae, Frankeniaceae, and Tamaricaceae (Heubl et al. 2006). Within the Caryophyllales the relatively frequent occurrence of the carnivorous adaptation raises questions as to what may have led to this development. Indeed, fossil and molecular evidence for the existence of the Droseraceae extends back at least 65 mya, indicating that these lineages are quite old (Cameron et al. 2002). Sessile glands are likely a synapomorphy of the Caryophyllales with many of the non-carnivorous sister taxa possessing glands that produce either mucilage or secrete salt (Judd et al, 2002). A synapomorphy is defined as a character trait that was first present in the most recent common ancestor of a monophyletic group, has subsequently been inherited evolutionarily by its descendants, and is used to distinguish a group or clade from other organisms. While sessile glands are believed to be a synapomorphy for the order, the pitted and stalked glands found throughout the Caryophyllales in the non-carnivorous sister groups and the various carnivorous lineages are believed to have arisen independently (Renner and Specht 2011, 2012, 2013). These glands are thought to function as an adaptation that limits herbivory (those which secrete or contain mucilage) as well as a mechanism that increases survival in environments with high salinity levels (those with salt secretory glands) such as saltmarshes, coastal dunes, or brackish water (Heubl et al. 2006). A survey of known terrestrial carnivorous plants reveals that the majority are found to occur in waterlogged hypoxic or even anoxic soils, typically bogs or fens. The soil conditions of these sites limit both the availability of many essential micronutrients, but also are generally unfavorable to root development (Adamec 2011). Interestingly these habitats match a cost-benefit analysis on what environmental conditions would favor the development of carnivory; namely those which are sunny, moist, and nutrient poor (Givnish et al. 1984). Given these unfavorable growing conditions the importance of essential elements derived from captured insects is not to be understated. One study with erythrorhiza showed that nitrogen acquired from captured insects accounted for up to 17% of total nitrogen uptake for the growing season (Dixon et al. 1980). When plants of D. rotundifola were grown in shade and/or with additional fertilizer the plants were found to produce not only less mucilage but that the mucilage which was produced had a lower concentration of polysaccharides resulting in a weaker adhesive, which indicates there is some plasticity in the expression of carnivorous traits in relation to their environment and the availability of essential elements (Thorén et al. 2003). A common component of the mucilage contained in these glands are a class of enzymes called chitinases. These enzymes break down the chitin polymers that are a primary component of both exoskeletons and fungal cell-walls. Multiple classes of chitinases exist in both 9

carnivorous and non-carnivorous plants. Some are produced in response to the activity of pathogens while others may always be present. In both instances they serve as important classes of enzymes related to pathogen response and plant defenses (Renner and Specht 2013). Some classes of these enzymes may potentially have yet undetermined roles in plant physiology and the development of rapidly growing tissues, particularly in inflorescences and embryo development (Libantová et al. 2009). Within the carnivorous members of the Caryophyllales some of these chitinases are known to play a role in prey digestion (Matušíková et al. 2005; Renner and Specht 2012). In particular, the 1b subclass of chitinases has lost the vacuole targeting signal which would ordinarily be found attached to the carboxyl terminal of the enzymatic protein complex. The lack of this targeting signal means that instead of being stored intracellularly within the vacuole the 1b subclass of chitinases are moved extracellularly. In non- carnivorous plants the 1b subclass of enzymes are stored intercellularly within the apoplast (Renner and Specht 2013), but in the carnivorous members of the Caryophyllales they are secreted by glandular excretory cells (Eilenberg et al. 2006). In , gene expression studies have indicated that the 1b subclass of chitinases are only produced by the secretory cells within the pitcher traps in response to the presence of chitin (Eilenberg et al. 2006). Similar studies in Drosera have also found differential expression of chitinases across tissue types (Libantová et al, 2009) and in response to the topical application of prey signaling proteins (Matušíková et al. 2005). Furthermore, while chitinases are one group of enzymes commonly found in the secretions of carnivorous plants, other enzymes that are produced include esterases, phosphatases, and proteases (Heslop-Harrison and Knox 1971; An et al. 2002; Heubl et al. 2006). Commonly known as Royal Catchfly, S. regia belongs to the Caryophyllaceae. It is associated with the North American tallgrass prairies (Swink and Wilhelm 1994) and the center of its range is the Ozark region of and with relatively isolated populations documented throughout much of the Midwest and Southeastern United States (USDA Natural Resource Conservation Service Data Team 2015). In Ohio, S. regia is currently listed as potentially threatened and remnant populations are known to occur in Adams, Champaign, Clark, Greene, Madison, Marion, and Union counties (King 1981a; Emmit, D. Cusick, A. Schneider 2000). As its common name Royal Catchfly implies, S. regia has long been known to pick up small insects with sticky, glandular trichomes (Beal 1876). These trichomes are concentrated on the , particularly the calyces and pedicels of the individual . Research focusing on other species with similar glandular trichomes have linked their evolutionary significance to anti-herbivory (Wagner 1991; Glover 2000; Adlassnig et al. 2010; Renner and Specht 2011, 2012, 2013). A study conducted by Spomer (1999) found evidence for the presence of proteinase secretion in Stellaria americana (a member of the Alsinoidea subfamiliy within the Caryophyllacae) from , stem, and calyx tissue. In the early 20th century the Italian researchers Mameli and Ascheieri (1920) examined the biochemical properties of viscaria (now reclassified as Silene viscaria) and found evidence to suggest the production and secretion of proteinases from the trichomes. Additional research has broadened our understanding of the 10

development and prevalence of the carnivorous habit in the plant kingdom, suggesting that the trend towards carnivory may not be as uncommon as previously thought (Chase et al. 2009). Based on these studies, I hypothesize that S. regia is carnivorous. The findings of this study will directly address longstanding questions regarding the habits and life history of S. regia and could also have implications in understanding the evolutionary development of carnivorous adaptations.

STUDY OBJECTIVES In order to claim that S. regia is carnivorous the following criteria must be met: 1) that it can attract, capture and retain prey; 2) secrete protein digesting enzymes to facilitate nutrient absorption; and 3) absorb and translocate the resultant nutrients.

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MATERIALS & METHODS

ADDRESSING THE STUDY OBJECTIVES To demonstrate the first criteria 59 herbarium specimens of S. regia were examined for the presence of captured insects which were classified to Order when possible. Additionally a comparative trapping experiment was conducted in the field using commercially available yellow and blue glue traps to determine if S. regia was found to be more or less attractive to insects than the glue traps. Lastly a series of near-UV photographs were made to determine if there were any visual markings which could serve as an attractant as is found in members of Nepenthes (Moran et al. 1999), Dionea, , and (Joel et al. 1985). While scent is also known to serve as an attractant among carnivorous plants, sometimes even in conjunction with optical attractants (Adlassnig et al. 2010), this study did not address this possibility due to the perceived difficulties of testing for possible olfactory cues. The second criteria, demonstrating that S. regia is capable of secreting proteinases that facilitate nutrient absorption, was addressed utilizing the methodology laid out by Hartmeyer 1997. By affixing photographic film to portions of the plant that had been primed with a yeast extract solution, which would normally elicit a prey-mediated proteinase production response in carnivorous plants, the presence or absence of enzymatic activity can easily be determined by visual examination of the film strip for degradation of the gelatin emulsion. The third criteria, determining if S. regia is able to absorb and translocate the nutrients produced by digesting prey, was dependent upon the findings of the second objective. As these findings failed to support S. regia being able to secrete proteinases to facilitate nutrient absorption this objective was not addressed.

STUDY SPECIES DESCRPTION Silene regia is a member of the Caryophyllaceae and was first formally described by John Simms in 1815. It is a long lived, taprooted perennial possessing an upright habit and a typical height between 0.7 and 1.5 m. The terminal inflorescence is cymose with anywhere from 15-25 individual flowers (King 1981a). The pedicels and calyces of the flowers are densely covered with glandular trichomes which have been documented to trap and ensnare small insects. It is currently believed the primary function of these glandular trichomes is anti-herbivory (Wagner 1991; Glover 2000; Adlassnig et al. 2010; Renner and Specht 2011, 2012, 2013). The corolla is a striking scarlet red in coloration (Figure 1) and the flowers are almost entirely pollinated by Archilochus colubris, commonly known as the Ruby-throated hummingbird. The flowers are perfect and self-fertile but exhibit temporal dichogamy which promotes outcrossing (Menges 1995). Silene regia is associated with tallgrass prairie and open woodland environments. Like many members of the tallgrass prairie community, S. regia is a fire-adapted species with that require open patches of soil and sunlight for germination (Menges and Dolan 1998). Its range extends from the Ozark region of Missouri and Arkansas, where it is fairly common, to isolated eastern populations in Ohio, , , , and (USDA Natural 12

Resource Conservation Service Data Team 2015). The populations found in Ohio are believed to be holdouts from when the prairies reached their maximum eastern extent during the post-glacial warming period approximately 4,000 ya (Transeau 1935).

FIGURE 1: An individual open of S. regia and two unopened floral buds. The elongated corolla tube is commonly seen in other members of Silene; the red coloration of the are typical of hummingbird pollinated flowers. The central portion of the petals in S. regia are elongate and arch slightly backwards forming a corona like structure. The encircling persistent calyx is fairly tough and covered with glandular trichomes that are denser along the midvein of the individual calyx lobes. Photograph was taken at Bigelow Pioneer Cemetery.

STUDY SITE DESCRIPTIONS

MIAMI WHITEWATER FOREST (MWF) Encompassing approximately 1,600 hectares, Miami White Water Forest is managed by the Great Parks of Hamilton County system. It includes examples of restored wetlands, mature beech maple complex forest, and 300 hectares of managed tallgrass prairie. The population of S. regia used in this study is located off Oxford Rd at 39°16'49.7"N, 84°44'26.3"W near New Haven, OH (Figure 2). 13

SHAKER TRACE NATIVE NURSERY (STNSN) Situated within Miami White Water Forest, Shaker Trace Native Seed Nursery is located at 8667 New Haven Road (39°16'31.7"N 84°43'38.0"W) and consists of 20 hectares dedicated to the propagation and production of seeds from over 200 different species of wetland and prairie plants native to the area. A nursery production bed of S. regia was used in this study.

MILFORD CENTER PRAIRIE STATE NATURE PRESERVE (MCP) Located approximately 4 km south of Milford Center, OH (40°09'27.7"N, 83°27'26.3"W) this site was historically part of the once extensive Darby Plains. This 2.5 km long remnant follows a former railroad right-of-way and supports approximately 60 species of plants that would have been common to the area prior to settlement. It has been jointly managed by the Dayton Power and Light Company and the Ohio Division of Natural Areas and Preserves since 1987. (ODNR 1996)

BIGELOW CEMETERY STATE NATURE PRESERVE (BPC) Located 13 km west of Plain City, OH and approximately 10 km from Milford Center Prairie State Nature Preserve (40°06'34.6"N 83°25'08.8"W), Bigelow Cemetery State Nature Preserve, previously known as Bigelow Pioneer Cemetery, was the best preserved remnant of the Darby Plains in Ohio at the time of this study. The half-acre cemetery was in active use from 1814 until 1892 and presently preserves the largest original population of S. regia in the state in addition to many other plant species associated with the historic Darby Plains. (ODNR 1996)

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FIGURE 2: Study sites in Southwestern and Central Ohio. Upper left insert shows the study area in relation to the whole state of Ohio.

SPECIMEN EXAMINATION Herbarium specimens (N=59) of S. regia from the William Sherman Turrell Herbarium at Miami University, The Herbarium of the Missouri Botanical Gardens, and The Herbarium of the Chicago Field Museum were examined for the presence of trapped insects. The total number of insects present on each specimen were counted and identified to order using a dissecting scope. Trichome density was estimated using methods based on those described by Valverde et al. 2001. The number of trichomes were counted along a 1.5 mm long transect along the pedicel beginning from the base of the calyx. This was done for the terminal, lowest left, lowest right, and uppermost left (excluding the terminal) flowers of the inflorescence for each specimen. These values were then averaged to provide an estimate of trichome density. The total number of flowers and developing floral buds for each specimen were also counted. Insects found on the herbarium specimens of S. regia were qualitatively compared to insects found on herbarium specimens of Drosera rotundifolia for signs of enzymatic degradation.

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COMPARATIVE TRAPPING Comparative trapping tests were conducted at Miami Whitewater Forest, Bigelow Pioneer Cemetery, and Milford Center Prairie State Nature Preserve. 6.45 cm2 sections, approximating the outer surface area of one S. regia calyx, of yellow and blue unscented glue traps (Seabright Laboratories, Emeryville, CA, USA) were affixed to wooden dowel rods measuring 1 m in length utilizing paper clips. The traps were set 0.3 m on either side of the flowers being used for comparison (Figure 3). Commercially, these glue traps are used to monitor for greenhouse pests such as thrips and lacewings (Davidson et al. 2015). Yellow traps are effective in attracting and capturing a diversity of insects whereas blue traps are generally more attractive to members of the Chironomidae (Dirrigl 2012). The traps were left in the field for one week and then collected along with the flower and taken back to the lab for examination using a dissecting scope. Insects found on the surface of the traps and calyx were counted and grouped into size classes based on length: small (<1.0 mm), medium (1.0 -3.0 mm), and large (>3.0 mm). The choice to group the insects by size class as opposed to order was made because the damaged condition of the insects on the traps made accurately determining what order they belonged to impractical. Additionally, grouping by size class could determine if there was a size range that S. regia was most likely to capture, as smaller insects typically make up the majority of the prey captured by carnivorous plants (Adlassnig et al. 2010)

FIGURE 3: Example of the layout and spacing used in the comparative trapping experiment

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UV PHOTOGRAPHY Near-ultraviolet (Near-UV) (300-400 nm) photographs of the flower and calyx or S. regia were taken using a Nikon D70 fitted with the UV5035B 50MM F/3.5 UV LENS (UKAOptics), BAADER Planetarium U-filter -2” (Item No. 2458291), and 55-48 mm step down adapter ring (Adorama). All photographs were taken in.RAW format as this format least processes the data received from the image sensor of the camera. A tripod was also required due to long exposure times which typically ranged anywhere between 1/16 sec to 4 sec depending on ambient light intensity. Camera focus was first manually adjusted without the UV-pass filter and then with the UV-pass filter in place. The lens was then readjusted to correct for UV phase-shift by utilizing a predetermined lens position with final fine focus adjustments made using trial and error. To ensure there was no IR contamination of the image, test photographs of the common Dandelion, Taraxicum officinale, were taken as the plant has documented guides visible only in the UV spectrum (Guldberg and Atsatt 1975). Photographs in the visible spectrum were made using the Nikon D70 fitted with the UV5035B 50MM F/3.5 UV LENS (Figure 4). Additional test photographs were made of False Sunflower, Heliopsis helianthoides, to further confirm the equipment was functioning properly (Figure 5). These methods were based on those utilized by Rørslett (2004). Afternoon ambient light was sufficient for field photography. Digital image post-processing of the UV photographs was done in AdobePhotoshop and consisted of desaturating the image of color using all three RGB channels to produce a grayscale image which made the identification of UV patterns easier.

FIGURE 4: Utilizing the set-up described above along with bright natural lighting and longer exposure times were sufficient for generating photographs in the UV spectrum. Photo credit Katrina Dienno.

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FIGURE 5: Test images of Helianthus helianthoides. (A) In the visible spectrum. (B) In the near-UV spectrum with nectar guides, the dark inner portion of the corolla, readily visible. (C) Digital composite of the visible and UV images.

FILM TEST The methodology for testing proteinase activity on film followed previously described guidelines with high speed black & white film (Kodak) used as the film substrate (Kalina 1968; Hartmeyer 1997; Darnowski et al. 2006). The test utilizes the light sensitive gelatin emulsion layer of the film as a substrate for proteinase activity. A portion of the plant is treated with a 10% yeast extract solution and after 24 hours a section of film strip is held emulsion side down in contact with the treated area in a high humidity environment. The yeast extract solution elicits the same response from carnivorous plants as insect capture, resulting in the secretion of digestive enzymes. “Priming” the plant with yeast extract and maintaining a humid environment are not necessary to trigger an enzymatic response but it does speed up the test and provides clearer results. The two positive controls used were McCormic© meat tenderizer, which contains the protein digesting enzyme bromelain, and leaves from the known D. rotundifolium treated with a foliar spray of 10% yeast extract 24 hours prior to the application of the film strip. Negative controls consisted of unprimed (not treated with yeast extract) S. regia calyx, unprimed D. rotundifolia leaves, and distilled water. A procedural control of 10% yeast extract solution applied directly to the film was also used. The experimental group consisted of S. regia treated with the 10% yeast extract solution. After 24 hours a small pre-moistened section of film was applied to the calyx and held in place with a paper clip. The film strips were marked using notches cut into their side which 18

corresponded to their test group to aid with identification after they were collected. A plastic locking bag was then placed over the treated flowers and sealed, providing a humid environment for the test and a degree of protection from the elements (Figure 6). After 24 hours the film was removed, allowed to air dry, and placed into a protective film sheet. The procedure for the unprimed S. regia tissue was the same as the primed S. regia group except the 10% yeast extract solution was not applied. In the lab the film strips were scanned and converted into high resolution digital grayscale images using a scanner. This was done to more easily see slight changes in surface contrast indicative of enzymatic degradation of the film substrate. Film strips were qualitatively assigned into either a degraded (+) or non-degraded (-) category. These decisions were based on the appearance of the film compared to the positive and negative controls, as well as visual differences in film degradation caused by enzymatic processes versus physical damage.

FIGURE 6: Example of the set up used for the primed and unprimed S. regia film tests. In this test two flowers within the locking bag have had a strip of film applied to their calyces.

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SEM Calyx tissue from S. regia was collected from the Shaker Trace Native Seed Nursery in the morning to assure maximum turgor pressure due to nighttime hydration from the soil. After removal, the calyx tissue was immediately placed for at least 30 minutes into a primary fixation solution consisting of 2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffered to a pH of 7.2. In the lab the tissue was then rinsed 4 times, for 10-15 minutes each, with a solution of 0.05 M sodium cacodylate buffered to a pH of 7.2. The tissue was then dehydrated in 25%, 50%, and 75% absolute ethanol for 20 minutes per treatment, 95% absolute ethanol for 30 minutes, and two treatments of 100% ethanol for 60 minutes. At this stage samples were critical-point dried, mounted on specimen holders, sputter coated with 10 nm of gold and imaged using a ZEISS 35VP Scanning Electron Microscope.

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RESULTS

EXAMINATION OF CAPTURED INSECTS ON HERBARIUM SPECIMENS Representative members from 10 Orders of insects were found on the herbarium specimens of S. regia. The most frequently encountered insects were thrips, followed by small wasps (Table 1).

TABLE 1: Insect diversity and abundance on herbarium specimens of Silene regia. Specimens with insect refers to the number of herbarium specimens, out of 59 sheets, with a specific insect type. Total is the number of all insects of that type from the 59 specimens. “Mean” is the average number of a specific insect found on the herbarium specimens. The category “Unidentified” consisted of insects present on the herbarium specimens that were either too damaged or positioned in such a way as to make accurate identification impossible. Thrips Wasp Diptera Unidentified Specimens with insect (N=59) 32 29 10 9 39 Total 168 73 14 14 120 Mean 2.85 1.24 0.24 0.24 2.03

Qualitative comparisons made between insects found on herbarium specimens of S. regia and D. rotundifolia revealed that insects captured by D. rotundifolia were reduced to transparent chitinous exoskeletons while those found on S. regia did not exhibit any signs enzymatic degradation.

COMPRARTIVE TRAPPING Across all of the sites examined the number of insects caught per calyx was less than the number caught by either the yellow or blue glue traps (Table 2). Insects in the medium size class (1.0-3.0 mm) were the most commonly caught across all test groups (Figure 7). Two tailed T-tests assuming unequal variance were used to statistically compare trap data between test groups and size classes. There was a statistically significant difference in the total number of insects captured between the calyx and blue, calyx and yellow, and blue and yellow glue traps with the traps capturing more insects than the calyx groups. When examined by size class there was a statistically significant difference between the calyx and yellow as well as calyx and blue traps for all size classes with the traps again capturing more insects than the calyx groups. The one exception was the calyx and yellow trap in the small size class which despite capturing more insects than the calyx 21

groups was found to not be significant. The blue and yellow traps were also found to have a statistically significant difference in both the total number of insects caught as well as the small and medium size classes. The large size class was found to not be statistically significant between the blue and yellow traps (TABLE 3).

TABLE 2: Number of insects caught by test group and size class. Small Medium Large Total Calyx MWF (n=10) 0 0 0 0 Blue MWF (n=10) 51 236 13 300 Yellow MWF (n=10) 25 88 23 136 Calyx BPC (n=6) 4 3 0 7 Blue BPC (n=6) 18 51 11 80 Yellow BPC (n=5) 4 59 15 78 Calyx MCP (n=3) 10 0 0 10 Blue MCP (n=3) 21 69 14 104 Yellow MCP (n=3) 7 66 7 80

45 40 35 30 25 20 15 10 Large

Insects Caught Insects 5 Medium 0 Small

Test Groups

FIGURE 7: The number of insects caught over the comparative trapping test groups. Size classes were broken into small (dark gray, <1.0 mm), medium (gray, 1.0 -3.0 mm), and large (light gray, >3.0 mm). No insects were found on the calyces of the Miami Whitewater Forest test group. Standard error for each size class is indicated by the whiskers.

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TABLE 3: Results of two tailed T-tests assuming unequal variance between test groups and size classes. All but two comparisons were found to be statistically significant (< 0.05). Small Medium Large total Calyx/Blue 3.80497E-05 3.60329E-05 5.65055E-05 3.25426E-07 Calyx/Yellow 0.054352172 3.60329E-05 1.59582E-07 3.65331E-06 Blue/Yellow 0.004690532 0.044319177 0.307542167 0.025681341

EXAMINATION OF POSSIBLE ATTRACTANT PATTERNS IN NEAR-UV SPECTRUM Photographs in the near-UV spectrum (300-400 nm) revealed that the petals and calyces of Silene regia were highly UV absorptive. When the photograph was converted to grayscale by desaturating all three RGB channels in digital post-processing darker areas were indicative of high UV absorption whereas lighter areas indicated higher UV reflectivity. The corona did not appear to exhibit any increased UV reflectivity compared to the rest of the corolla (Figure 8) except when photographed from a top down angle (Figure 9). This apparent increased reflectivity was likely due to the angle of the corona and incoming light in relation to the angle of the camera as opposed to differences in the concentration of any UV absorbing/reflecting pigments throughout the corolla. It was found in test photographs made of Taraxicum officinale and Helianthus helianthoides that the angle from which the photographs were taken did not impact the visibility of the near-UV nectar guides. Overall, the corolla and entire inflorescence of S. regia can be said to be highly UV absorbent.

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FIGURE 8: Comparative side view of S. regia inflorescence in visible and near-UV spectrum. Photograph in the visible spectrum (right side) was taken in ambient afternoon lighting. The left side of the photograph is in the UV spectrum. Dark areas correspond with high UV absorption whereas light areas indicate UV reflectivity.

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FIGURE 9: Comparative top-down view of S. regia inflorescence in visible and near-UV spectrum. Photograph in the visible spectrum (right side) was taken in ambient afternoon lighting. The left side of the photograph is in the UV spectrum. Dark areas correspond with high UV absorption whereas light areas indicate UV reflectivity.

SILENE REGIA FILM DEGRADATION ABILITY Both primed and unprimed calyces failed to exhibit proteolytic activity in the film degradation experiment (Figure 10). Conversely, all D. rotundifolia test groups exhibited proteolytic activity. Negative results were observed in the distilled water and yeast extract solution trials, which was expected as they were procedural controls. Enzymatic degradation was also observed in the McCormic© meat tenderizer test groups (Figure 11).

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FIGURE 10: (A) Film strip from the D. rotundifolia primed test group showing positive results. (B) Film strip from the H2O test group exhibiting a negative result. (C) Film strip from the primed S. regia test group. The outline of the calyx is visible because the glandular trichomes stuck to the surface of the film but the lack of degradation to the film emulsion indicates a negative result.

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Film Test Results 100 90 80 70 60 50 40 30

20 % of Test Group Group Test of%

Showing Damage Damage Showing 10 0

Test Groups

FIGURE 11: Percentage of film strips in a test group which exhibited enzymatic degradation (% Damaged) over each test group with sample size given.

GLANDULAR TRICHOME MORPHOLOGY Scanning electron micrographs indicate that the glandular trichomes are concentrated on the ridges of the calyx and that their structure consists of a stalk 2-7 cells in length ending in an enlarged, spherical, capitate cell. Cell rupture appears to be the mechanism by which the capitate cells release their contents. Glandular trichomes with longer stalks are most common along the midline of calyx ridges while those with shorter stalks are typically found on either side of the ridges (Figure 12). Smaller non-glandular trichomes are also apparent; their structure consists of a stalk 2-3 cells in length that lack an enlarged capitate cell (Figure 13).

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FIGURE 12: Scanning electron micrograph of a calyx of Silene regia. A thrip is trapped amongst the glandular trichomes along a calyx ridge. Several non-glandular trichomes, mostly found between calyx ridges, are visible as well. (Photocredit Matt Duley, Center for Advanced Microscopy & Imaging, Miami University of Ohio)

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FIGURE 13: Scanning electron micrograph of a calyx of Silene regia. The increased concentration of glandular trichomes along the calyx ridges is evident as well as their general decrease in length with increasing distance from the ridges. Non-glandular trichomes are more prevalent between the calyx ridges. (Photocredit Matt Duley, Center for Advanced Microscopy & Imaging, Miami University of Ohio).

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DISCUSSION

THE CASE AGAINST SILENE REGIA BEING CARNIVOROUS The findings of this study support the contention that Silene regia is capable of capturing and retaining insects and that glandular trichomes are involved in this capture. This assertion is evidenced by the presence of captured insects on both herbarium and field specimens as well as other studies (King 1981a, 1981b; Menges 1995; Menges and Dolan 1998; Emmit, D. Cusick, A. Schneider 2000). Comparative trapping experiments demonstrated that S. regia was less of an attractant to insects than commercial glue traps. Near-UV photography revealed that S. regia is highly UV absorbent and lacks any patterns in the UV spectrum which could serve as an insect attractant as found in other known carnivorous genera such as Nepenthes and Sarracenia (Joel et al. 1985; Moran et al. 1999). Furthermore the negative results from the film test points towards S. regia as being incapable of secreting proteolytic enzymes in response to insect capture. This lack of a capture-induced proteolytic response was also supported by the examination of insects found on herbarium specimens of S. regia as they lacked any sign of enzymatic degradation when compared to insects found on herbarium specimens of D. rotundifolia. As such Silene regia failed to pass the first criteria of this study; despite having highly modified structures for capturing objects, the comparative trapping and near-UV photography experiments failed to demonstrate any form of active insect attractant. Additionally, as S. regia was found to be incapable of secreting proteinases to facilitate nutrient absorption it failed to meet the second criteria required to be considered carnivorous. Based on these findings S. regia does not meet the criteria necessary to be considered carnivorous.

ALTERNATIVE HYPTOHESISES FOR THE PRESENCE OF S. REGIA’S GLANDULAR TRICHOMES Given that Silene regia is not carnivorous, what other functions might its glandular trichomes be fulfilling? It is entirely possible that the glandular trichomes are merely a retained synapomorphy that is of neutral benefit in terms of evolutionary fitness in S. regia. The closely related species S. virginica and S. rotundifolia are known to produce sterile crosses with S. regia and share overlapping ranges, but have differing habitat requirements (Margaret 1951; King 1981a). All three of these species possess conspicuous glandular trichomes. As was mentioned in the introduction, sessile glands are likely a synapomorphy of the Caryophyllales, though the pitted and stalked glands found in various carnivorous lineages are believed to have arisen independently (Judd et al. 2002; Renner and Specht 2011, 2012, 2013). It is known that trichomes function in a number of beneficial roles that improve plant survivability including altering water relations, as a plant pathogen defense and herbivory deterrent, and an aid in surviving high salinity environments through salt excretory glands (Levin 1973; Wagner 1991; Valverde et al. 2001; Heubl et al. 2006). In the case of S. regia these 30

trichomes likely represent a significant resource investment and it would follow that as a trait it would have been, or be in the process of being, selected against unless it improved evolutionary fitness. One potential role of these glandular trichomes is in deterring nectar robbing behavior. Nectar robbing occurs when an animal, usually a member of the Anthophila clade such as a , bypasses a plant’s normal route to obtain nectar. This usually involves chewing, sawing, or piercing through the petals or calyx of the flower to reach the nectary. Nectar robbing is very common in plants that are pollinated by hummingbirds or butterflies as they tend to offer large nectar rewards (Roubik 1982; Irwin et al. 2004, 2010). It has been documented in Ipomopsis aggregata, a plant which relies on hummingbirds for pollination, that nectar robbing by insects substantially reduces the reproductive potential of the plant (Irwin and Brody 1999). In another study nectar robbing was found to negatively impact the fitness of Pavonia dayspetala by reducing pollinator visitation and subsequent seed set (Roubik 1982). Nectar robbing has also been shown to be a driving evolutionary force in controlling flower morphology (Irwin et al. 2010). The morphological adaptations which deter nectar robbing include thickening and enlargement of the and calyx (Irwin et al. 2004). In oxyanthera the presence of nectar robbing behavior has been shown to cause directional selection for larger spur circles, which are highly modified sepals or petals that extend behind the flower in the form of an elongated hollow spike that contains nectar (Wang et al. 2013). The flower morphology of Silene regia with its elongate corolla and red coloration are consistent with what has been observed in other hummingbird pollinated syndromes (Irwin 2000; Irwin et al. 2010). Previous studies have firmly established that the primary pollinator of S. regia is Archilochus colubris (King 1981a) and that seed set is directly influenced by hummingbird visitation (Menges 1995). Informal observations made during this study also supported that S. regia was frequently visited by A. colubris. Additionally, of the 59 S. regia herbarium specimens examined, no damage to the calyces caused by the chewing, sawing, or piercing actions of insect mouthparts was evident. The glandular trichomes may not be enough to immobilize larger insects such as those of the order Bombidae (indeed larger insects such as these were not found trapped on any of the herbarium specimens) but they may limit the nectar robbers’ actions. The highly UV-absorptive nature of the inflorescence could also serve as a form of camouflage from nectar robbers. Unlike hummingbirds which are known to be able to see into the near-UV spectrum (Curé and Palacios 2009) but are not known to rely on UV patterns for finding flowers (Rodríguez-Gironés and Santamaría 2004), bees readily rely on the near-UV spectrum and UV-patterns known as nectar guides. As such a highly UV-absorptive red flower would be hard to discern from a background of green vegetation due to the wavelength sensitivities of bee vision and might increase foraging time to the point of not being cost effective (Guldberg and Atsatt 1975; Rodríguez-Gironés and Santamaría 2004). In Silene regia, glandular trichomes are only present within the inflorescence and do not fully develop until bud set. This suggests that they are in some way linked evolutionarily to the deterrence of floral herbivory and are worth the trade-off between their cost of production and reproductive fitness. The positioning and density of glandular trichomes along the calyces and 31

base of the pedicels in S. regia would certainly pose a challenge to any small insects climbing along the inflorescence or attempting to enter the flower. Considering that thrips were the most commonly found insect on the herbarium specimens of S. regia this is a conjecture that has some support. Silene regia is also known to shed its lower cauline leaves at anthesis (Flora of North America Editorial Committee 2005) and this leaf shedding would certainly increase the importance of photosynthetically active tissues in the inflorescence such as the enlarged calyces and subtending sepals. Studies on other plants with persistent photosynthetically active perianths have demonstrated that these structures can contribute significantly to the energetic requirements of development and seed set and increase reproductive fitness (Hetherington et al. 1998; Smillie et al. 1999; Aschan and Pfanz 2003; Herrera 2005). The inflorescence of S. regia is typically a meter in height and has anywhere from 15-25 individual flowers (King 1981a). Keeping in mind that each individual flower within the inflorescence of S. regia contains a persistent photosynthetic calyx and large subtending bracts it is very likely given what has been shown in the above mentioned studies that these structures are supplying a large proportion of the energetic requirements needed for fruit and seed development. This in part would help to explain the benefits of the glandular trichomes in terms of their associated costs as well as their developmental timing during the life cycle of S. regia, providing not only a defensive role to the flowers and seeds but also protecting the very structures which are supporting their development as well. Future studies could focus on further resolving the possible functions of the glandular trichomes in S. regia and examining the chemical composition of their secretions, determining if nectar robbing behavior occurs in S. regia and the closely related S. virginica and S. rotundifolia and if so what impact it may or may not have in regards to fitness, or examining the UV reflectivity and absorption patterns of other genera such as Impatiens or Viola known to be targeted by nectar robbing bees.

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