ABSTRACT

PREY SELECTIVITY IN UTRICULARIA GIBBA

Suction feeding is one of the primary methods aquatic organisms use to capture prey. Suction feeding in aquatic organisms is well understood in adult fish, but poorly understood in fry. Hydrodynamic theory predicts that suction feeding is not effective in smaller organisms, where a minimum gape diameter is required for a successful suction event. This minimum gape diameter is the lower limit where suction feeding is still viable. Studies have shown that fish larvae have low capture success, but there are few data on similar sized plant suction feeders. Aquatic bladderwort species (Utricularia gibba, U. vulgaris) capture microscopic prey using suction feeding in underwater bladder-shaped traps at dimensions typically less than 1 mm. This project examines how bladderworts suction feed by quantifying the capture success, trap morphology and prey morphology to address the following questions: (1) do smaller traps catch smaller and fewer prey; (2) do smaller traps have a relatively larger gape (characterized as gape diameter relative to total trap size) than larger traps to limit the negative effects of being small on capture success. Bladderwort traps capture prey relative to gape diameter, with smaller bladders catching smaller-sized and fewer prey overall compared to larger bladders. Smaller bladderwort traps display isometric allometry, with smaller traps having relatively same gape length as larger traps.

Nolan Lynn Avery May 2017

PREY SELECTIVITY IN UTRICULARIA GIBBA

by Nolan Lynn Avery

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science and Mathematics California State University, Fresno May 2017 APPROVED For the Department of Biology:

We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.

Nolan Lynn Avery Thesis Author

Ulrike Muller (Chair) Biology

Otto Berg Chemistry

Katherine Waselkov Biology

For the University Graduate Committee:

Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS

X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.

Permission to reproduce this thesis in part or in its entirety must be obtained from me.

Signature of thesis author: ACKNOWLEDGMENTS I would like to give thanks to my mentor and advisor, Dr. Ulrike Muller. Without her guidance and support throughout this project, I would not be where I am today. I would also like to thank my committee members Dr. Otto Berg and Dr. Katherine Waselkov with their help in this project. In addition, I would like to thank all the people who have helped me on this research project for the last few years: Alejandra Tapia, Rayhan Kabir, Jennifer Espinosa, Juan Villalobos, Edgar Munoz, Fatima Hildago, Ricardo Rameriz, Ronnie Oldea, Cory Mayfield, and Andrew Jones. Lastly, I would like to thank the Biology Department for FSSRA funding, graduate travel grants, and IRA funding to help fund this project and/or support me during this project. TABLE OF CONTENTS Page

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

The Mechanics of Suction Feeding: State of Current Knowledge ...... 1

The Effects of Gape Size on Suction Feeding ...... 3

Predator-Prey Interactions in Bladderwort ...... 4

Utricularia vulgaris and Utricularia gibba ...... 5

HYPOTHESES AND SPECIFIC AIMS ...... 8

Hypotheses ...... 8

Aims ...... 9

EXPERIMENTAL DESIGN ...... 10

Methods to Address Aims 1, 2, and 3 ...... 10

Rationale for Using Ostracods as Prey ...... 11

Predator-Prey Interaction Experiments ...... 11

Scanning Setups for Prepared Predator and Prey...... 12

Predator-Prey Trial Experiment ...... 12

Post-Trial Data Collection ...... 13

Data Analysis – Processing of the Images Collected ...... 13

Methods to Address Aim 2 ...... 14

Methods to Address Aim 3 ...... 15

RESULTS ...... 17

Utricularia gibba and U. vulgaris Morphology ...... 17

Utricularia gibba and U. vulgaris Allometry Graphs ...... 17 vi vi Page

Utricularia gibba and U. vulgaris Prey Size Selectivity ...... 18

DISCUSSION ...... 20

FIGURES ...... 23

TABLES ...... 43

REFERENCES ...... 51

LIST OF TABLES

Page

Table 1-1: Utricularia gibba Shapiro-Wilk Test for body ...... 43

Table 1-2: Normal Q-Q plot of Utriculaira gibba body ...... 44

Table 2-1: Utricularia vulgaris Shapiro-Wilk Test for body ...... 45

Table 2-2: Normal Q-Q plot of Utriculaira vulgaris body ...... 46

Table 3-1: Utricularia vulgaris Shapiro-Wilk Test for gape ...... 47

Table 3-2: Normal Q-Q plot of Utriculaira vulgaris gape ...... 48

Table 4-1: Utricularia gibba Shapiro-Wilk Test for gape ...... 49

Table 4-2: Normal Q-Q plot of Utriculaira gibba gape ...... 50

LIST OF FIGURES

Page

Figure 1. Confocal image of a Utricularia vulgaris bladderwort...... 23 Figure 2. U. vulgaris before (left) & after (right) triggering. Image taken in lab using a Phantom v12.1 camera...... 24 Figure 3. A diagram showing how the velocity profile of a fluid changes as you move away from the surface of the pipe...... 24 Figure 4. A comparison between bladderwort species of Utricularia gibba, and Utricularia vulgaris. U. vulgaris is on top, U. gibba is on below. .. 25 Figure 5. A strand of Utricularia gibba post prey trial scanned using a CanoScan 8600F flatbed scanner...... 26 Figure 6. A graphic displaying the different morphological features of Ostracods (Green, 1959)...... 27 Figure 7. An image showing a bladderwort strand (Utricularia gibba) in a post-prey experimental setup...... 28 Figure 8. A scanned image of an Ostracod prey pool before being added to a predator-prey experimental setup...... 29 Figure 9. A scanned image of a bladderwort strand (Utricularia gibba) before being added to a predator-prey experimental setup...... 30 Figure 10. An image of Utricularia gibba post prey trial scanned using a CanoScan 8600F flatbed scanner...... 31 Figure 11: An image of the ostracods used for the predator-prey interaction trials, taken using a color microscope...... 31

Figure 12. Histogram of 3553 U. vulgaris gape measurements...... 32

Figure 13. Histogram of 2179 U. gibba gape measurements...... 33

Figure 14. Histogram of 2179 U. gibba bladderwort length measurements...... 34

Figure 15. Histogram of 2179 U. vulgaris bladderwort length measurements. .... 35 Figure 16. Graph of the allometry of U. gibba. Data plotted is the length of the bladderwort (mm) against the gape (mm) as a log10 function...... 36 Figure 17. Graph of the allometry of U. vulgaris. Data plotted is the length of the bladderwort (mm) against the gape (mm) as a log10 function...... 36 ix ix Page

Figure 18. A scatterplot of the gape of the bladder trap with the corresponding length of ostracod captured for U. gibba and U. vulgaris...... 37 Figure 19. A histogram showing the total number of ostracods present in the predator-prey interaction trials (blue) vs. ostracods captured by U. gibba bladder traps (orange)...... 38 Figure 20. A histogram showing the total number of ostracods present in the predator-prey interaction trials (blue) vs. ostracods captured by U. vulgaris bladder traps (orange)...... 39 Figure 21. Graph showing the percentage of total captures by gape length of U. vulgaris and U. gibba (Captures/Total Prey Pool)...... 40 Figure 22. Graph showing the percentage of total captures by gape length of U. vulgaris snd U. gibba accounting for number of bladder traps present ...... 41 Figure 23. U. vulgaris-Ostracod Predator-Prey Capture Matrix. X-axis is intervals of given bladder trap gape length...... 42 Figure 24. U. gibba-Ostracod Predator-Prey Capture Matrix. X-axis is intervals of given bladder trap gape length ...... 42

INTRODUCTION

Suction feeding is one of the primary methods aquatic organisms use to capture prey (Nelson 1976). Suction feeding has been studied extensively in adult fish and hence is well understood in large aquatic organisms (for a review see Wainwright et al., 2007). However, there are few studies on small suction feeders (e.g. Deban and Olsen, 2002; Pekkan et al., 2016). Theoretical studies in suction feeding suggest that small suction feeders are inefficient (Drost et al., 1988); experimental studies show that they are ineffective (China et al., 2014). Yet bladderworts are successful suction feeders. The main goals of this study are to quantify the capture success and to characterize the trap morphology of two bladderwort species in order to better understand what makes small suction feeders successful. In the following, I will explain what we know about the fluid mechanics of suction feeding and how size affects suction feeding; then I will introduce what we know about bladderwort predator-prey interactions and their trap mechanics.

The Mechanics of Suction Feeding: State of Current Knowledge Suction feeding is common and unique to aquatic organisms and employed by more than half the fish species (Nelson, 1976). Large suction feeding fish use a combination of suction, jaw protrusion and swimming towards the prey (ram feeding) to catch prey (Wainwright et al., 2007). Fish jaws use complex four-bar linkage systems to generate a close-to-spherical mouth opening, closing the sides of their mouths by protruding their premaxilla and maxilla (Westneat, 1990). Their mouths open quickly, with time to peak gape ranging from 5 to 25 ms (Holzman et al., 2007). While they open their mouth, they also expand their mouth cavity to 2 2 generate a strong negative suction flow to entrain the prey (Muller and Osse, 1984). The water ingested with the prey is typically expelled through the gill slits after the mouth closes. Capture success of evasive prey correlates with gape size and the amount of jaw protrusion: fish with a larger gape are more likely to capture their prey (Holzman et al., 2012). Fish typically catch prey that is considerably smaller than their gape size (Scharf et al., 2000). Yet increasing gape not only increases capture success, but also the average size of the prey and the size range of caught prey (Scharf et al., 2000). While suction feeding is well-studied in adult fish (Aerts, 1990; Ferry- Graham, 2001, 2002; Higham, 2006; Lauder, 1980; Muller, 2002; Motta, 1984; Van Wassenbergh, 2010; Wainwright, 2007), much less is known about small suction feeders (Deban, 2002; Drost, 1988; Van Wassenbergh, 2009). Among the smallest known suction feeders are fish larvae (Budick, 2000; Coughlin, 1996; Hunter, 1981; Legget, 1994) tadpoles (Deban and Olsen, 2002) and bladderwort (a carnivorous plant) (Adamec, 2011; Joeyux, 2011; Sydenham and Findlay, 1973) all of which capture evasive zooplankton. Bladderworts (genus Utricularia) capture prey in modified leaf structures (Adamec, 2011) (Fig. 1). Although the trapping mechanism is sophisticated in its implementation, it is simple in concept: bladders are loaded by osmotically pumping water out of a sealed bladder cavity (Lloyd, 1929; Sydenham and Findlay, 1973); negative hydrostatic pressure is maintained by elastic deformation of the bladder’s walls (Fig. 2, left) (Sydenham and Findlay, 1973) until prey unwittingly trigger a trap door (Joyeux et al., 2011) and are entrained by the sudden inward flow while the trap inflates (Fig. 2, right) (Adamec, 2011) (Fig 2). The feeding strikes of fish, in contrast, are a complex 3 3 interplay of muscle-driven suction force and deformation of the mouthparts (Wainwright et al., 2007), plus whole-body propulsion (Weihs, 1980).

The Effects of Gape Size on Suction Feeding How objects interact with water depends on among other things their size. This effect can be described by the dimensionless Reynolds number (Re) (Fig. 3), which is defined as a ratio of inertial and viscous forces exerted by the water on an object in a flow. In the context of suction feeding, Re expresses how the relative importance of inertial and viscous forces changes with the organism’s size and the speed of the generated suction flow. Fluid dynamics describes an inertial flow regime, in which viscous forces can be neglected, when Re exceeds 1000; when Re drops below 100, it is a viscous flow regime, in which inertial forces can be neglected; the regime between Re 100 and 1000 is called the intermediate flow regime, in which neither force can be neglected. In the context of suction feeding, at high Re, flow is dominated by inertial forces and a suction feeder will spend most of its energy on accelerating the flow. When Re is low, viscous forces dominate and the suction feeder will spend most of its energy on overcoming the viscous friction of the water. In the viscous flow regime, the boundary layer forming along the inner walls of the mouth becomes so thick that it effectively blocks the mouth; this leads to very low flow speeds, which makes it difficult for small suction feeders to entrain and capture evasive prey. In contrast, in the inertial flow regime, the boundary layer forming along the walls inside the mouth is negligibly thin compared with the width of the mouth cavity and hence does not significantly slow down the flow. 4 4 Predator-Prey Interactions in Bladderwort With more than 200 known species, bladderwort (Utricularia spec.) make up roughly half of all known carnivorous plant species (Albert, 2010). Carnivorous plants typically grow in wet, boggy, acidic areas with low nutrient availability. Bladderworts capture phytoplankton and zooplankton, such as rotifers, copepods, and gastrotriches, to absorb nitrogen and phosphorus lacking in the local environment (Albert, 2010). Bladderwort are highly modified angiosperms, in particular the aquatic species, which lack roots altogether and live as free-floating strands near the water surface. Bladderwort traps have been described as modified leaf structures (Lloyd, 1929), but this interpretation is controversial (Chormanski and Richards, 2012). The bladders are miniscule, ranging in size from 0.5 to 5 mm (Friday, 1991). The traps comprise a bladder region, which is evacuated to generate the negative pressure required for suction feeding, and a mouth region with a trap door. The mouth region is highly variable among species (Lloyd, 1929): for example, U. vulgaris has its door close to the outer edge of the mouth, whereas the trap door of U. gibba is deep within the mouth, protected by a vestibule. Some species have extensive clusters of bristles protruding from the outer edge of mouth (Lloyd, 1929; Reifenrath et al., 2006). Like the traps of the Venus flytrap, the traps of bladderwort are active: they are loaded by the bladder cells osmotically pumping out the water from the sealed bladder (Sydenham and Findlay, 1973), which creates a negative hydrostatic pressure inside the bladder of roughly 5 to 15 kPa and causes the bladder walls to become elastically loaded (Singh et al., 2014). In this evacuated state, the trap is set, with the negative pressure sealed in by a trap door near the mouth of the trap. The trap is triggered when prey touch the trigger hairs on the trap door, causing 5 5 the trap door to swing inward, causing the bladder walls to expand outward and water plus prey to be sucked into the trap. The trap door closes again within a few milliseconds, and the trap then reloads over the next 15 to 30 min (Lloyd, 1929). During this reloading period of the bladder trap ‘setting’, the trigger hairs are inactive (Sydenham and Findlay, 1973). The trigger hairs will only become active once a pressure difference between the inside of the trap and the outside environment is established (Sydenham and Findlay, 1973). The ingested prey is digested over a period of days. However, because their bladders can reset in less than 30 minutes after a trigger, bladderworts feed repeatedly during their functional lifetime (approximately two weeks) (Friday, 1991). Unlike pitcher plants, bladderwort are generalist predators, catching a wide range of planktonic species (Gordon and Pacheco, 2007). The most common prey groups are rotifers, copepods, and cladocerans (Gordon and Pacheco, 2007). Prey capture experiments show that bladderworts capture multiple prey items in each bladder (Harms, 1999).

Utricularia vulgaris and Utricularia gibba This study focuses on two species of the genus Utricularia (bladderwort), part of the Lentibulariaceae family: Utricularia vulgaris (common bladderwort) and Utricularia gibba (humped bladderwort) (Fig. 4). Both these Utricularia species are rootless, free-floating aquatic plants that flower according to an annual or perennial life cycle (Gordon and Pacheco, 2007). Ecologically, Utricularia have importance in a trophic food chain by capturing small prey, such as phytoplankton and zooplankton (Gordon and Pacheco, 2007). Both bladderwort species live in nutrient poor, acidic environments, and use modified leaves to 6 6 capture prey for nitrogen supplementation (Sorenson and Jackson, 1968). The modified leaves are in the shape of a bladder with a trapdoor used to capture prey. U. vulgaris has a larger bladdertrap gape diameter, with trigger trichomes protruding from the trap opening. Developed traps are dimorphic (Friday, 1991): small traps are near the outer edges of the strand and larger traps are near the main stem. Size is an indicator of the amount of biomass and nutrients invested in production of individual bladder traps (Friday, 1991), but it is not currently known if prey caught by large traps accommodate for the cost of production. U. gibba has a smaller gape diameter, with the trap door and trigger trichomes found inside of a hood (or vestibule), thus downstream of the external mouth (Fig. 5). Bladderwort traps are among the smallest suction feeding structures. Previous theoretical and experimental studies have shown that suction feeding is generally not effective in small suction feeders, but bladderworts are an exception (China and Holzman, 2014). Data collected in a previous study show that the bladderwort species Utricularia vulgaris is an effective suction feeder able to catch prey with success rates well above those observed in similar-sized fish larvae (China and Holzman, 2014). There exist more than 200 species of bladderwort, several of which have smaller traps than Utricularia vulgaris (Gordon, 2007). The long-term goal of this research line is to identify the lower size limit of suction feeding. Size of the prey may influence capture events for differently sized traps, as larger ostracods swim faster and hence exerts greater force on the trigger trichomes. If larger ostracods exert more force on the trigger trichomes than a smaller ostracod and hence are more likely to trigger traps, this could lead to more captures of comparatively larger ostracods. The goal of this 7 7 current project is to compare the performance of a bladderwort species that has large traps with the performance of a species with smaller traps.

HYPOTHESES AND SPECIFIC AIMS

Hypotheses Hypothesis 1 and Prediction: Smaller traps should be at a hydrodynamic disadvantage because of their small gape, which increases viscous drag and hence reduces flow speed while increasing the energetic cost of suction feeding. The smaller species Utricularia gibba has lower capture success than the larger U. vulgaris because it has smaller traps, when compared to a fixed prey species. Null Hypothesis 1 and Prediction: Smaller traps are not at a hydrodynamic disadvantage because of their small gape. The smaller species Utricularia gibba does not have lower capture success than the larger U. vulgaris because it has smaller traps, when compared to a fixed prey species. Hypothesis 2 and Prediction: Prey size distribution and size range correlates with the predator’s gape size. The smaller species Utricularia gibba captures smaller prey and has a narrower size range of prey than the larger U. vulgaris because it has smaller traps. Null Hypothesis 2 and Prediction: Prey size distribution and size range does not correlate with the predator’s gape size. The smaller species Utricularia gibba does not capture smaller prey and does not has a narrower size range of prey than the larger U. vulgaris because it has smaller traps. Hypothesis 3 and Prediction: Hydrodynamic performance is determined by gape size more than trap size; hence smaller traps can partially compensate for their smaller size by having relatively larger gapes than larger traps. The smaller species Utricularia gibba has a relatively larger gape than the larger U. vulgaris to compensate for its smaller size. 9 9

Null Hypothesis 3 and Prediction: Hydrodynamic performance is not determined by gape size more than trap size. The smaller species Utricularia gibba does not have a relatively larger gape than the larger U. vulgaris.

Aims Aim 1: Collect data on capture performance and trap morphology on a bladderwort species with smaller traps than Utricularia vulgaris. Our first choice is U. gibba. Aim 2: Compare the prey capture performance of a small and large bladderwort species (as defined by their trap size), Utricularia gibba (small traps) and U. vulgaris (large traps). Aim 3: Compare the trap morphology of a small and large bladderwort species (as defined by their trap size), Utricularia gibba (small traps) and U. vulgaris (large traps).

EXPERIMENTAL DESIGN

Methods to Address Aims 1, 2, and 3

Bladderwort Culture Setup Stock of U. gibba and U. vulgaris were cultured in 20 gallon tanks (stock and growth) in a separate lab room (Room 124 Animal Wing) submerged in sphagnum (peat moss) to create a semi-dystrophic environment. Wire mesh was used to keep the sphagnum at the bottom of the tank, and was weighed down with rocks. Sphagnum plays a vital role in culturing bladderworts, because sphagnum releases sufficient amounts of nutrients which allows the bladderwort to survive. Full spectrum 32-watt fluorescent lights on 12-hour timers were hung above the tanks. The tanks were filled with deionized water, kept at room temp (21 °C) and pH slightly above 7.0 to replicate boggy conditions. Bladderworts were evenly distributed throughout the tank, and water levels were kept constant by topping the tanks up with deionized water as needed to compensate for evaporation losses.

Prey Culture Setup The prey we used were Ostracoda from the genus Cypridopsis (Ferguson, 1964). Ostracods were identified using a key in Thorp and Covich (1991). Ostracoda are distinguished from other Crustacea by having a laterally compressed body, seven or less thoracic appendages, undifferentiated head, and a bivalved, perforate carapace lacking growth lines. The genus Cypridopsis is identified as having shells tumid, valves nearly equal, with the ultimate podpomere of maxiallary palp cylindrical, being longer than wide. This genus is commonly found in the United States (Fig. 6) (Ferguson Jr, 1968), and is found in freshwater ponds. The prey were maintained in Room 124 of the Animal Wing, in 1-gallon 11 11 glass jars containing pond water from the CSU Fresno Biology greenhouse. Prey jars were kept at room temp (21 °C) and at pH slightly above 7.0. A fluorescent light on a 12-hour timer was hung above the jars, and three pellets of TetraMin Tropical Granules Fish Food were added every other day.

Rationale for Using Ostracods as Prey Ostracods ranged in size from 0.2 mm to 3 mm (Danielpol et al., 2002). Utricularia gibba bladder traps ranged in size from 1 mm to 2.5 mm (Chormanski et al., 2012), while U. vulgaris traps ranged in size from 1 to 5 mm long (Friday, 1991). The prey size distribution covered both predators’ bladder trap size ranges, allowing species comparison of prey size selectivity. Ostracods were used to compare the prey size ranges bladderworts capture if different sizes of prey are available. The largest Utricularia gibba traps (Chormanski et al., 2012) are comparable in size to the smallest Utricularia vulgaris traps (Friday, 1991), allowing a comparison of trap efficiency for a given bladder trap size range.

Predator-Prey Interaction Experiments

Prey Preparation Before every predator-prey interaction experiment, the prey (ostracods) were prepared. Using a pipette, prey were removed from the culturing jars and placed into an empty Petri dish. Ostracods tend to gather around the top of the jar, so a sufficient number (approx. 100-150) were removed by pipetting near the edges (Fig. 8). Once a sufficient number of ostracods were placed into the Petri dish, the dish was placed into a -20°C freezer for two minutes to immobilize the prey. Immobilized prey was then scanned using a flatbed scanner (see below). 12 12 Predator Preparation Predators (bladderworts) were prepared before every predator-prey interaction experiment. Using tweezers, a single strand of bladderwort was removed from the culturing tank and placed into a Petri dish. The bladderwort strand was then submerged in pond water inside the Petri dish (Fig. 9). Selected strands were scanned using a flatbed scanner (see below).

Scanning Setups for Prepared Predator and Prey Prey- Immobilized Prey were placed in a CanoScan 8600F flatbed scanner. A millimeter ruler was then set next to the Petri dish as a means of recording scale. An image was then scanned using Adobe Photoshop at 1200 dpi. The image was saved as a TIF file (.tif) with date scanned, plant species, dpi, and current operator in the file name. Predator- The Petri dish with the single bladderwort stand was placed into a CanoScan 8600F flatbed scanner with a millimeter ruler in the frame. As in the prey setup, the ruler provided a scale. A circular glass cover was gently placed over the bladderwort strand in the Petri dish to flatten the bladders. An image was scanned using Adobe Photoshop at 3600 dpi. The image was then saved as a TIF file (.tif) with date scanned, plant species, dpi, and current operator in the file name.

Predator-Prey Trial Experiment With a prepared prey pool and predator strand, an experimental trial was conducted. An empty glass jar (500 mL) was filled with 100 mL of pond water. The prepared bladderwort strand was gently added to the jar using tweezers. The prepared prey pool was then pipetted into the jar. Once both the prey and predator 13 13 had been added, the jar was placed under the same lighting as the predator stock tank for 50 hours (Fig. 7).

Post-Trial Data Collection After 50 hours, the predator-prey trial was completed. The predator strand was removed from the 500 mL glass jar into a Petri dish. The Petri dish was then covered with a circular glass cover to flatten the bladders, and placed into a flatbed scanner with a millimeter ruler. An image was scanned using Adobe Photoshop at 3600 dpi, (higher dpi improves image resolution and detection of captured prey) and saved as a TIF file (.tif) with date scanned, plant species, dpi, and current operator in the file name.

Data Analysis – Processing of the Images Collected All images scanned were processed using ImageJ to measure total amount of prey and longest dimension (pre-trial prey), trap gape and length (pre-trial predator), and prey captured (post-trial) (Fig. 10). An image was opened using ImageJ, and the scale was set using the millimeter ruler in frame (Fig. 11). Images taken at 3600 dpi were set to 142 pixels/mm, and images at 1200 dpi were set to 47 pixels/mm. For pre-trial prey images, the total number of prey present, along with the length of their longest dimension was recorded and saved as an Excel sheet. In pre-trial predator images, the length and gape of the bladder was recorded. Measurements taken using post-trial images were the lengths of prey captured in bladder traps, along with the bladder gape. Images scanned with U. gibba as the predator provided data for aim 1 of our objectives. Images scanned with U. vulgaris as the predator provided data for aims 2 and 3. To ensure measurements were collected precisely between individual students (that is, all students were measuring the same dimensions for the same 14 14 image), a training period was conducted. New students assigned to the project were given images that had been previously measured by experienced students, and the measurements were compared. Once new students were able to precisely measure images comparatively to experienced students, measurements by the new student were added to the master data. Approximately two weeks of training was conducted for those inexperienced with the software involved.

Methods to Address Aim 2

Data Analysis Measurements of the bladder trap length, gape, and prey length captured was stored as Excel files with the date of each experimental trial, total number of bladders, and prey pool total recorded. A predator-prey capture matrix was generated, showing for a given predator gape size range, the amount of prey captured by total prey available. This heat map of prey capture frequency indicated which size ranges of predator gapes capture most frequently, and showed the ideal conditions of predator-prey interaction. The matrix was also used as a model for other predator-prey interaction trials, and is not predator or prey species dependent. Prey capture frequency graphs were also created to determine if prey size selectivity occurs for a given predator gape range, or if given predator gape size ranges capture prey at relatively higher frequencies.

Analyzing size selectivity of the traps. Histograms of trap gape against captured prey length, the total amount of prey captures for a given gape size range, and the total amount of prey available were created to determine if species of bladderwort exhibit size selectivity. A standardized size range of the total bladder 15 15 gapes available, prey captures, and prey lengths available, permitted comparisons of capture performance for a given size range. This was measured by taking the total bladder gape captures for a given size range, dividing by the total potential prey captures for the gape size, and then dividing by the total number of bladders available for the given size range. This gave ostracod prey capture frequency, with ostracod captures divided by the total prey pool available. This graph revealed which size range of bladder gape proportionally captures more prey. The ostracod prey capture frequency graph did not account for total number of predators present with the given gape size range, potentially inflating a response for those gape size ranges with more bladders present, and underrepresenting the response for bladder gapes with fewer predators present. We corrected for this variable, by adjusting the formula to be ((Captures/Total Prey Pool)/Total Bladder Traps with Given Gape Range). Dividing by the total number of predators present of a given gape size range, we measured relative capture success rate on average for each bladder gape present. Only ostracods close to the relative size of the bladder trap gape were counted in the pool of potential captures using this formula. A histogram of the output generated by the preceding calculation was the measure of size-based bladder trap performance and prey size selectivity.

Methods to Address Aim 3

Analyzing Trap Morphology Allometry of the bladder traps for a given species was determined by plotting the log scale of the longest dimensional length vs. the gape of each bladder trap per trial. Transforming the measurements as a logarithmic function expressed the proportional change between values. A slope of 1 indicated isometry, a slope greater than or less than 1 indicated allometry. 16 16

Histograms were created; one comparing the gape of bladder traps against captured prey length, the total amount of prey captures for a given gape size range, and the total amount of prey available as a fixed size range. SPSS statistic software was used to determine if bladderwort gape and length is normally distributed.

RESULTS

Utricularia gibba and U. vulgaris Morphology Histograms showing measurements of bladder gape and length for U. gibba, (Fig 13, 14) and U. vulgaris (Fig 12, 15) are shown below. SPSS software was used to conduct the Shapiro-wilk statistical test for normality (Tables 1,2). Test results indicate neither the gape or length of either bladderwort species U. vulgaris and U. gibba was normally distributed. Mode of bladder gape and length for U. gibba was 0.174-0.193 mm, with a standard deviation of 0.070 mm, and for bladder length, 0.822-0.878 mm mode, with a standard deviation of 0.208 mm (Tables 1,2). Total number of bladders measured was 2180 (n=2180). Mode of bladder gape and length for U. vulgaris was from 0.500 to 0.590 mm, with a standard deviation of 0.208 mm, and for bladder length, 1.900-1.990 mm mode, with a standard deviation of 0.368 mm (Tables 3,4). Total number of bladders measured was 2030 (n=2030). Figures 12-15 describe the morphology of two species of bladderwort, U. gibba and U. vulgaris. Average gape and length of bladder was provided, with U. vulgaris being larger in both gape and length on average compared to U. gibba.

Utricularia gibba and U. vulgaris Allometry Graphs Allometry graphs of U. gibba and U. vulgaris bladder trap length and gape are displayed below (Fig. 16, 17). A slope of 1 indicates isometry, greater than 1 or less than 1 indicates allometry in bladder trap gape and length development. Measurements from 2875 Utricularia vulgaris and 1890 U. gibba bladder traps show slopes of 0.89 and 0.93 respectively. 18 18 Utricularia gibba and U. vulgaris Prey Size Selectivity Figure 18 shows the size of the prey captured vs. the gape of the bladder that captured it, and this graph reveals two clusters of prey capture events, one with U. gibba at a gape of 0.2 mm, and one with U. vulgaris at a gape of 0.6 mm. Figures 19 and 20 show the total prey populations compared to prey caught by bladder traps. Figure 21 is a graph of ostracod prey capture frequency, with ostracod captures divided by the total prey pool available. This graph reveals which size range of bladder gape proportionally captures more prey: for U. gibba prey capture success was highest at a gape of 0.2 mm, and for U. vulgaris, prey capture success was highest at 0.6 mm. For U. gibba, bladders with a gape at 0.2 mm capture ostracods 60% more often than gapes of other size ranges, and for U. vulgaris bladders with a gape at 0.6 mm capture ostracods 25% more often compared to other bladder gape size ranges. This graph does not correct for total number of predators available with the given gape size range, potentially inflating a response for those gape size ranges with more bladders present, and underrepresenting the response for bladder gapes with fewer predators present. Figure 22 corrects for number of predators available, by adjusting the formula to be ((Captures/Total Prey Pool)/Total Bladder Traps with Given Gape Range). Results indicated for U. gibba, gapes of 0.1 mm were not more capture- effective than bladder gapes of other size ranges, while for U. vulgaris, gapes of 0.6 mm were still roughly 25% more capture-effective. Only ostracods close to the relative size of the bladder trap gape were counted in the pool of potential captures. The predator-prey capture matrix was created (Figures 23, 24) comparing bladder gape size range to the corresponding size of ostracod captured. The 19 19 colored heat map represents areas where capture frequency was highest, with dark green areas indicating areas of higher prey capture, and dark red where areas of prey capture were lowest. For figures 23 and 24, U. vulgaris prey capture effectiveness is highest at 0.50-0.65 mm bladder gape, and for U. gibba prey capture effectiveness is highest at 0.16-0.25 mm bladder gape.

DISCUSSION

For our Hypothesis 1, results support our hypothesis that U. gibba has lower capture success than the larger U. vulgaris because it has smaller traps, when compared with a fixed prey species. Figure 20 shows initially that the smaller bladderwort species, Utricularia gibba appears to capture prey at a higher frequency, but correction for number of predators present in Figure 21 shows that Utricularia vulgaris captures prey more frequently. Prey used in the predator-prey interaction trials were from the same stock grown under lab conditions, and duration for each experiment was kept constant. Presented with similar prey pools under similar circumstances, a comparison of capture frequency between bladderwort species for a given prey species is possible. Hydrodynamics may provide a potential explanation of the smaller bladderwort species lower capture success. The smaller bladders should be at a hydrodynamic disadvantage because of their small gape, which increases viscous drag and hence reduces flow speed while increasing the energetic cost of suction feeding. Further experimentation measuring flow speed and drag during capture events would need to be done to confirm this hypothesis. For Hypothesis 2, results support our hypothesis that the smaller species U. gibba captures smaller prey and has a narrower size range of prey than the larger U. vulgaris because it has smaller traps. Figures 22 and 23 show the predator-prey capture matrix for U. vulgaris and U. gibba, and for the smaller bladderwort species U. gibba both the total number of prey caught and size range of prey caught is less than U. vulgaris. Prey size distribution and size range correlates with the predator’s gape size as shown in Figure 17. Limitations on bladderwort size for U. gibba prohibits capture of larger size prey when compared to U. vulgaris. Low 21 21 capture success for U. gibba may be because Ostracoda are not the ideal prey type for this species of bladderwort. Additional predator-prey interaction trials with additional prey species would help to elucidate this. For Hypothesis 3, results contradict our hypothesis that the smaller species U. gibba has a relatively larger gape than the larger U. vulgaris to compensate for its smaller size. We initially hypothesized that hydrodynamic performance is determined by gape size more than trap size; hence smaller traps can partially compensate for their smaller size by having relatively larger gapes than larger traps. A proportionally larger gape would compensate for hydrodynamic forces reducing capture success. Results indicate there is an isometric relationship between bladder gape and length for both bladderwort species, contradicting our hypothesis. Measurements from 2875 Utricularia vulgaris and 1890 U. gibba bladder traps show slopes of 0.89 and 0.93 respectively, and these values are too close to unity to suggests an allometric relationship. Isometry in bladderwort shape may hold because neither bladderwort species produces traps with a gape close enough to the lower limit of effective suction feeding size as predicted by hydrodynamic theory. If this is true, then neither bladderwort species would be inhibited by viscous drag and reduced flow speeds within the gape during suction events. Further prey trials with another species of bladderwort producing smaller traps than U. gibba would explore the relationship of gape size in these plants to the hypothesized lower limit in hydrodynamic theory, which is currently unknown. Definite determination of the bladder trap ‘function’ is currently being debated. Conventional view holds that the plants benefit from nutrients derived from digestion of trapped prey (Friday, 1989), but calculations of return from investment in bladder development in terms of phosphorous and nitrogen uptake 22 22 indicates a 25-50% return (Richards, 2001). A large bacterial community is cultivated within the traps (Sirova et al., 2009), and it is suspected this bacteria plays a mutualistic role in plant development, although not definitively shown. Further quantification of the number and types of prey captured in natural environments would provide an evaluation of bladder trap function (Richards, 2001), as well as examination of trap bacterial function and chemicals released from the bladder trap. Chemical signaling of bladder trichome secretions are known to attract epibiotic rotifers (Wallace, 1978), and there may be other chemical signals being released that have not yet been detected. Further areas of study for Utricularia include but are not limited to, plant physiology associated with carnivory, metagenomic surveys of periphyton communities, novel plant nutrient utilization pathways, the ecology of prey attraction, and whole-plant and bladder comparative ontogeny (Victor et al. 2010). This study explored the relationship between bladder gape size to prey size captured in predator-prey interactions with a single species; multiple prey species will need to be utilized in future studies to accurately define the lower limit as proposed by current hydrodynamic theory.

FIGURES

Figure 1. Confocal image of a Utricularia vulgaris bladderwort. Prey coming into contact with the trigger hairs located on the mouth of the trap ‘activates’ the trap, causing the trapdoor to open and a suction event to occur, drawing in nearby water and prey.

24 24

Figure 2. U. vulgaris before (left) & after (right) triggering. Image taken in lab using a Phantom v12.1 camera.

Figure 3. A diagram showing how the velocity profile of a fluid changes as you move away from the surface of the pipe. Flow closest to the body is subject to viscous forces, and the point where inviscid flow forces dominates marks the boundary layer thickness. 25 25

Figure 4. A comparison between bladderwort species of Utricularia gibba, and Utricularia vulgaris. U. vulgaris is on top, U. gibba is on below. 26 26

Figure 5. A strand of Utricularia gibba post prey trial scanned using a CanoScan 8600F flatbed scanner. Numbering of the bladderworts was done using ImageJ. 27 27

Figure 6. A graphic displaying the different morphological features of Ostracods (Green, 1959). 28 28

Figure 7. An image showing a bladderwort strand (Utricularia gibba) in a post- prey experimental setup. 29 29

Figure 8. A scanned image of an Ostracod prey pool before being added to a predator-prey experimental setup. Ruler for scale is visible in the image. 30 30

Figure 9. A scanned image of a bladderwort strand (Utricularia gibba) before being added to a predator-prey experimental setup. Ruler for scale is visible in the image.

31 31

Figure 10. An image of Utricularia gibba post prey trial scanned using a CanoScan 8600F flatbed scanner. Shown is a Utricularia gibba bladderwort with an ostracod near the gape of the bladderwort. Colored lines indicate where measurements were taken; red line indicates length of bladder, green line indicates gape of bladder, and blue line indicates length of ostracod.

Figure 11: An image of the ostracods used for the predator-prey interaction trials, taken using a color microscope. 32 32

Figure 12. Histogram of 3553 U. vulgaris gape measurements. Average bladderwort gape length is 0.509 mm, with a mode ranging from 0.500-0.590 mm. See Table 2 for descriptive statistics. 33 33

Figure 13. Histogram of 2179 U. gibba gape measurements. Average gape length is 0.226 mm, with a mode ranging from 0.174-0.193 mm.

34 34

Figure 14. Histogram of 2179 U. gibba bladderwort length measurements. Average bladderwort length is 0.921 mm, with a mode ranging from 0.822-0.878 mm. Descriptive statistics in Table 1. 35 35

Figure 15. Histogram of 2179 U. vulgaris bladderwort length measurements. Average bladderwort length is 1.68 mm, with a mode ranging from 1.90-1.99 mm. 36 36

Figure 16. Graph of the allometry of U. gibba. Data plotted is the length of the bladderwort (mm) against the gape (mm) as a log10 function.

Figure 17. Graph of the allometry of U. vulgaris. Data plotted is the length of the bladderwort (mm) against the gape (mm) as a log10 function. 37 37

Figure 18. A scatterplot of the gape of the bladder trap with the corresponding length of ostracod captured for U. gibba and U. vulgaris. 38 38

Figure 19. A histogram showing the total number of ostracods present in the predator-prey interaction trials (blue) vs. ostracods captured by U. gibba bladder traps (orange). 39 39

Figure 20. A histogram showing the total number of ostracods present in the predator-prey interaction trials (blue) vs. ostracods captured by U. vulgaris bladder traps (orange). 40 40

Figure 21. Graph showing the percentage of total captures by gape length of U. vulgaris and U. gibba (Captures/Total Prey Pool). 41 41

Figure 22. Graph showing the percentage of total captures by gape length of U. vulgaris snd U. gibba accounting for number of bladder traps present ((Captures/Total Prey Pool)/Total Bladder Traps with Given Gape Range). 42 42

Bladder/Ostracod U. vulgaris Ostracod Prey Capture Matrix Bins Intervals 0.79 0.76-0.79 0.75 0.70-0.75 0.69 0.66-0.69 1 0.65 0.60-0.65 8 8 4 1 0.59 0.56-0.59 16 16 16 5 2 2 1 1 2 0.55 0.50-0.55 10 40 32 16 9 1 1 1 0.49 0.46-0.49 3 21 12 9 7 5 2 1 2 1 0.45 0.40-0.45 22 26 46 27 39 9 10 1 2 0.39 0.36-0.39 4 18 10 24 8 16 10 3 3 0.35 0.30-0.35 4 14 12 22 10 13 5 2 0.29 0.26-0.29 2 3 4 1 10 5 4 3 1 1 0.25 0.20-0.25 1 6 6 2 6 4 3 2 0.19 0.16-0.19 1 1 1 2 1 3 0.15 0.10-0.15 1 1 1 0.09 0.06-0.09 2 0.05 0-0.05 Bins 0.05 0.09 0.15 0.19 0.25 0.29 0.35 0.39 0.45 0.49 0.55 0.59 0.65 0.69 0.75 0.79 0.85 0.89 0.95 0.96+ Intervals 0-0.05 0.06-0.09 0.10-0.15 0.16-0.19 0.20-0.25 0.26-0.29 0.30-0.35 0.36-0.39 0.40-0.45 0.46-0.49 0.50-0.55 0.56-0.59 0.60-0.65 0.66-0.69 0.70-0.75 0.76-0.79 0.80-0.85 0.86-0.89 0.90-0.95

Figure 23. U. vulgaris-Ostracod Predator-Prey Capture Matrix. X-axis is intervals of given bladder trap gape length, Y-axis is length of ostracod captured. Frequency of prey caught is listed within the matrix. Dark green highlights areas of comparatively higher frequency of capture for a given bladder trap gape length, dark red highlights areas of comparatively lower frequency.

Ostracod Bins Intervals 0.49 0.46-0.49 1 1 0.45 0.40-0.45 2 1 1 0.39 0.36-0.39 2 2 0.35 0.30-0.35 2 2 1 1 0.29 0.26-0.29 1 2 4 1 1 1 0.25 0.20-0.25 2 10 18 3 3 1 0.19 0.16-0.19 4 15 16 2 1 1 1 0.15 0.10-0.15 5 13 16 4 1 1 0.09 0.06-0.09 1 3 2 1 0.05 0-0.05 1 Bins 0.05 0.09 0.15 0.19 0.25 0.29 0.35 0.39 0.45 0.49 0.55 0.59 0.65 0.69 0.75 Intervals 0-0.05 0.06-0.09 0.10-0.15 0.16-0.19 0.20-0.25 0.26-0.29 0.30-0.35 0.36-0.39 0.40-0.45 0.46-0.49 0.50-0.55 0.56-0.59 0.60-0.65 0.66-0.69 0.70-0.75 Bladder gape Figure 24. U. gibba-Ostracod Predator-Prey Capture Matrix. X-axis is intervals of given bladder trap gape length, Y-axis is length of ostracod captured. Frequency of prey caught is listed within the matrix. Dark green highlights areas of comparatively higher frequency of capture for a given bladder trap gape length, dark red highlights areas of comparatively lower frequency. 43 43

TABLES

Table 1-1: Utricularia gibba Shapiro-Wilk Test for body

Statistic Std. Error Body Mean .9206 .00446 95% Confidence Lower Bound .9118 Interval for Mean Upper Bound .9293

5% Trimmed Mean .8973

Median .8590

Variance .043

Std. Deviation .20825

Minimum .43

Maximum 2.15

Range 1.72

Interquartile Range .15 Skewness 2.269 .052 Kurtosis 6.785 .105 Shapiro-Wilk Statistic df P-Value .781 2179 .000

44 44

Table 1-2: Normal Q-Q plot of Utriculaira gibba body

45 45

Table 2-1: Utricularia vulgaris Shapiro-Wilk Test for body

Statistic Std. Error Gape Mean .2096 .00150 95% Confidence Lower Bound .2067 Interval for Mean Upper Bound .2126

5% Trimmed Mean .2028

Median .1960

Variance .005

Std. Deviation .07024

Minimum .06

Maximum .71

Range .65

Interquartile Range .07 Skewness 2.311 .052 Kurtosis 9.518 .105 Shapiro-Wilk Statistic df Sig. .832 2179 .000

46 46

Table 2-2: Normal Q-Q plot of Utriculaira vulgaris body

47 47

Table 3-1: Utricularia vulgaris Shapiro-Wilk Test for gape

Statistic Std. Error Body Mean 1.6802 .00818 95% Confidence Lower Bound 1.6641 Interval for Mean Upper Bound 1.6962

5% Trimmed Mean 1.6837

Median 1.7320

Variance .136

Std. Deviation .36848

Minimum .80

Maximum 2.88

Range 2.08

Interquartile Range .56 Skewness -.233 .054 Kurtosis -.716 .109 Shapiro-Wilk Statistic df Sig. .979 2029 .000

48 48

Table 3-2: Normal Q-Q plot of Utriculaira vulgaris gape

49 49

Table 4-1: Utricularia gibba Shapiro-Wilk Test for gape

Statistic Std. Error Gape Mean .5088 .00280 95% Confidence Lower Bound .5033 Interval for Mean Upper Bound .5143

5% Trimmed Mean .5088

Median .5180

Variance .016

Std. Deviation .12629

Minimum .20

Maximum .91

Range .71

Interquartile Range .19 Skewness -.068 .054 Kurtosis -.680 .109 Shapiro-Wilk Statistic df Sig. .987 2029 .000

50 50

Table 4-2: Normal Q-Q plot of Utriculaira gibba gape

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