Ant-Mediated Seed Dispersal in Neotropical Pioneer Tree Species

LINKING MECHANISM TO PATTERN IN COMMUNITY ASSEMBLY: ANT-MEDIATED SEED DISPERSAL IN NEOTROPICAL PIONEER TREE SPECIES

SELINA ARIEL RUZI

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Ecology, Evolution, and Conservation Biology in the Graduate College of the University of Illinois at Urbana-Champaign, 2019

Urbana, Illinois

Doctoral Committee:

Professor Andrew V. Suarez, Chair and Director of Research Professor James W. Dalling Professor Adam S. Davis Professor Lawrence M. Hanks

ABSTRACT

Dispersal is a fundamental process that affects all aspects of an organism’s biology including its distribution, genetic structure, demography, and reproduction. Ant-mediated seed dispersal has evolved multiple times in many biogeographical locations. Most research in this area has focused on myrmecochorous plants that are known to elicit seed dispersal by providing a specialized lipid rich food reward called an elaiosome attached to their seeds. However, seeds that are not known to have this food reward may still be attractive to ants. The aim of my dissertation was to describe the movement of seeds of Neotropical pioneer tree species that are not known to have elaiosomes by ants. For my research, I chose Neotropical pioneer tree species that are commonly found in the soil seed bank of Barro Colorado Island (BCI), Panama. The seeds of these tree species do not provide an elaiosome food reward to their ant removers, but varied in mass, primary dispersal mode (animal or wind), dormancy type (physical, physiological, or quiescent), and ability to persist in the soil in the absence of predators. My dissertation research from BCI had four parts: (1) I quantified seed removal rates among 12

Neotropical pioneer species in two locations of the soil seed bank (soil surface and two cm within the topsoil) to determine which seed characteristics best explained variation in seed removal rates. (2) I identified seed-removing ants and determined whether variation in ant communities / activity correlated with differences in seed removal rates among sites. (3) I determined that chemical cues played a role in mediating some seed-ant interactions for one

Neotropical pioneer seed species. (4) Lastly, I estimated where and how far ants moved seed of one pioneer species, Zanthoxylum ekmanii Urb. (Alain) (Rutaceae) and whether the seeds would survive their deposition location. My dissertation research supports that ants will interact with

ii seeds of some Neotropical pioneer tree species without any type of food reward on Barro

Colorado Island, Panama. This indicates that ants may alter the recruitment dynamics of more plant species than previously thought broadening the impact of this important ecosystem service.

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ACKNOWLEDGMENTS

This work would not have been possible without the input of many people throughout my graduate career. I would like to thank my advisor, Andrew Suarez, for his advice, guidance, editing, and support through my research, teaching, and grant writing efforts. Additionally, I would like to thank to other members of my committee Jim Dalling, Adam Davis, and Larry

Hanks for their insight and support, as well as for pushing me to grow as a researcher delving into topics such as tropical biology, statistics, and chemical ecology. Without this committee, this thesis would look very different and my PhD process would not have been as rewarding as it has been. Thank you to my labmates, both past and present: Rafael Achury, Andrea Belcher,

Kim Drager, Joshua Gibson, Dietrich Gotzek, Jo-anne Holley, Fred Larabee, Michael Rivera,

Adrian Smith, and Bill Wills, who have helped create a supportive laboratory culture, giving me advice and comments, and helping me with fieldwork, labwork, and statistical analysis. I have been fortunate to work with many amazing undergraduates including but not limited to: Paul

Kang, Marlee Ryerson, Jelena Verkler, Adam Skrzekut, Katelyn Detweiler, Brandan Brew, and

Ellen Andrews. I must specifically thank Francisco Juarez, whose help analyzing video interactions and preparing chemical extracts has been invaluable for data collected in my fourth chapter. Additionally, I must thank two wonderful Smithsonian Tropical Research Institute

(STRI) – Butler University interns, Abigail Robson and Adam Beswick, who collected data during the wet season for three of my chapters when I was unable to be in Panama. Thank you to the Panamanian researchers and assistants who helped from setting up greenhouses, letting me know where trees were fruiting, and particularly to Ernesto Bonadias for help in the field. Thank you to all my wonderful co-authors, some of which fall under previously mentioned categories,

iv but also include Daniel Roche and Camilo Zalamea, for not only their help in idea generation, fieldwork, and writing, but for their friendship. Additionally, I want to thank Bill Wcislo, Owen

McMillan, Allen Herre, and Oscar Puebla for their help, guidance, and planning of my first semester visit in Panama and research help through additional field seasons.

I thank all the logistical and support staff both at the University of Illinois and at STRI.

Within the SIB Business Office, Norma Treakle, Penny Broga, Teresa Acosta, Jessica

Katternhenry, James McGrawth, and Ted Hermann among others have provided valuable assistance in budgeting, purchasing various research supplies, and paying for flights and housing for research and conferences in the U.S. and other countries. I would also like to thank all the

PEEC and SIB secretaries and support staff, especially Carol Hall, Kim Leigh, Lisa Smith Tara

Heiser, and Liz Barnaby for all their help ranging from answering questions, scheduling logistics, and proofreading my dissertation. From STRI, I thank the visitor’s office, the permit’s office, the housing offices for both Gamboa and Barro Colorado Island (BCI), and BCI’s scientific coordinators. Specifically, Orelis Arosemena, Lil Camancho, Zurenayka Alain,

Adrianna Bilgray, Taisha Parris, Hilda Castaneda, Norisa Soto, Oris Acevedo, Belkys Jimenez, and Melissa Cano have been invaluable to conducting research in Panama. I would also like to thank the chefs, maintenance crew, boat drivers, and cleaning personnel of Barro Colorado

Island.

I am indebted to the many friends I have made throughout this PhD process, from those based at my home institution at University of Illinois to those I met during my many field seasons at Barro Colorado Island, Panama, and to the field courses and conferences I have attended. They have not only acted as sounding boards for my research and edited my numerous grants and emails, but have reminded me to maintain a work-life balance. Here I include some of

v these friends but know that there are many more that remain unnamed: Kelsey Witt Dillon,

Amanda Owings, Carolina Sarmiento, Nicholas Sly, Harriet Downey, Nick Gardner, Alan Ward,

Lourdes Hernández, Brian Harvey, Allison Gardner, Jennifer Jones, Halie Rando Robson, Beryl

Jones, and Cassidy Martin.

This research was made possible through a variety different funding sources coming from the National Science Foundation, STRI, the University of Illinois, and the STRI – Butler

Internship. Within the University of Illinois, funding came from the Graduate College, School of

Integrative Biology, a U.S. Department of Agriculture grant to Natural Resources and

Environmental Sciences, and through the Program of Ecology, Evolution, and Conservation

Biology. Additional funds came from the Association for Tropical Biology and Conservation to present work from this dissertation at conferences both within the United States and internationally.

Lastly, I must thank my family. My mom, dad, and brother who may not always understand what I was doing during my time in Illinois and Panama, but they constantly encouraged me and supported me all the same. Finally, I thank my fiancée, Timothy Dinh, who has always supported me from afar, pushing me to grow and do more than I thought I was capable of while also reminding me that it is okay to place boundaries and say no.

Para mi Abuela,

Por inculcarme el amor por las plantas y animales

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TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: TREE SPECIES IDENTITY INFLUENCES SECONDARY REMOVAL OF SEEDS OF NEOTROPICAL PIONEER TREE SPECIES ...... 9

CHAPTER 3: CAN VARIATION IN SEED REMOVAL PATTERNS OF NEOTROPICAL PIONEER TREE SPECIES BE EXPLAINED BY LOCAL ANT COMMUNITY COMPOSITION? ...... 41

CHAPTER 4: PRELIMINARY IDENTIFICATION OF CHEMICAL CUES THAT MEDIATE SEED REMOVAL BY THE ANT ECTATOMMA RUIDUM ROGER IN A NEOTROPICAL PIONEER TREE SPECIES ...... 78

CHAPTER 5: QUANTIFYING SEED FATE IN ANT-MEDIATED DISPERSAL: SEED DISPERSAL EFFECTIVENESS IN THE ECTATOMMA RUIDUM (FORMICIDAE) – ZANTHOXYLUM EKMANII (RUTACEAE) SYSTEM ...... 110

CHAPTER 6: CONCLUSIONS ...... 147

APPENDIX A: SUPPLEMENTARY TABLE FOR CHAPTER 2 ...... 152

APPENDIX B: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER 3 ...... 153

APPENDIX C: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER 4 ...... 163

APPENDIX D: SUPPLEMENTARY TABLES AND FIGURE FOR CHAPTER 5 ...... 171

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CHAPTER 1: INTRODUCTION

BACKGROUND

Plants face a variety of obstacles to the recruitment of new individuals in a population which ultimately influence the structure of plant communities. They are limited by the number of seeds they produce (source limitation), whether those seeds survive dispersal (both primary and secondary dispersal), and whether those seeds reach suitable microsites for germination and establishment (Nathan and Muller-Landau 2000). Neotropical pioneer tree species produce large quantities of seeds (Dalling et al. 2002) and require high light microsites (e.g. light gaps; Hubbell et al. 1999, Swaine and Whitmore 1988) to germinate and grow. To reach those suitable microsites, seeds either need to disperse far from the parent tree or remain viable in the soil seed bank (Dalling and Brown 2009) until a tree fall occurs, allowing for increased light penetration to the soil. For example, seeds of some pioneer species can remain viable in the soil seed bank for over 30 years (Dalling and Brown 2009). While secondary dispersal of seeds into the soil profile is frequently reported, it is unknown whether seeds experience the same potential for further movement from both the soil surface and in the topsoil. We also do not know if different species in the same location experience the same potential for secondary dispersal. Animal vectors are not homogenously distributed throughout the landscape (Mull and MacMahon 1997), therefore local assemblages of animal vectors may alter seed dispersal frequency. Ants are particularly important seed dispersers in many ecosystems (Beattie 1985, Handel and Beattie

1990), playing a role in both short and long-distance dispersal (Gómez and Espadaler 2013), and may incorporate seeds into the topsoil.

Most studies of ant-mediated seed dispersal focus on the dispersal of myrmecochorous plants. Myrmecochorous plants tend to provide their ant dispersers with elaiosome food rewards

(Beattie 1985, Handel and Beattie 1990). These elaiosomes were thought to be nutritious, making this interaction a mutualism where the seeds gain benefits from direct dispersal, protection from predators, and dispersal away from the parent plant (reviewed in Giladi 2006).

However, not all elaiosomes benefit ants (e.g. Warren II et al. 2019); they may contain chemical cues that manipulate ants into moving them taking on a more parasitic relationship. For example, elaiosomes may be perceived as dead insects, as their fatty acid compositions are similar to seven broad taxa of insect prey (Hughes et al. 1994). These chemicals, which include oleic acid and its dimer, 1,2-diolein, (Skidmore and Heithaus 1988, Brew et al. 1989) may then act as behavioral releasers inducing ants to removing seeds (Marshall et al. 1979). However, known myrmecochorous plants make up only approximately 4.5% of all angiosperm species (Lengyel et al. 2010), which leaves a knowledge gap in whether the other ~95% of angiosperms may also engage in seed dispersal by ants. Plants that lack elaiosomes could partake in ant-mediated seed dispersal by mimicking elaiosomes or insect prey by having chemicals on their seed coat that act as behavioral releasers. Therefore, this dissertation hypothesizes that more plants engage in ant- mediated seed dispersal despite than previously thought and that there is a chemical mechanism that promotes this interaction. Specifically, I focus on Neotropical pioneer tree species, describing the movement of their seeds, the ant species and chemical compound(s) that play a role in this interaction, and whether this interaction is likely to be effective dispersal rather than predation.

STUDY SYSTEM

The dissertation research took place at five sites within the Smithsonian Tropical

Research Institute’s Barro Colorado Island (BCI, 9º10’N, 79 º51’W), Republic of Panama field location. I chose 12 Neotropical pioneer tree species for Chapters 2 and 3 (below), with subsets used for Chapters 4 and 5. None of the seed species provide an elaiosome food reward to their ant removers, but they varied in size, primary dispersal mode (animal or wind dispersed), and dormancy type (physical, physiological, or quiescent).

OVERVIEW

In Chapter 2, I compared seed removal rates among species and determine whether those rates are similar in different locations in the soil seed bank (soil surface vs. top two cm of topsoil). I investigated whether season (soil surface only), primary dispersal mode (how seeds are initially moved from the tree), and dormancy type (how seeds delay germination) influenced seed removal rates for 12 pioneer tree species. I also investigated if removal rates differed between seeds located on the soil surface and seeds located within the topsoil. I placed seed caches on the forest floor during both the 2013 dry and the wet season and caches of six species within the first two cm of topsoil during the 2013 wet season. Caches on the soil surface were collected after 47 hours while caches within the topsoil remained for four weeks prior to collection. Tree Species identity significantly influenced secondary removal rate of seeds both on the soil surface and within the topsoil. However, there was no correlation between seed removal between the two environments (soil surface vs. topsoil). Therefore, species identity did not always influence removal the same way on both the soil surface as it did within the topsoil, indicating that the identity of the secondary dispersers maybe different between these two locations in the soil seed bank. Tree species identity accounted for more variation in secondary removal than both primary dispersal mode and dormancy type implying that there may be a species-specific trait, such as chemical cues, that drives this removal interaction through either

deterring or attracting removal agents. This work was co-authored by Daniel P. Roche, Paul-

Camilo Zalamea, Abigail C. Robison, and James W. Dalling and published in Plant Ecology.

In Chapter 3, I examined the role of variation in ant community structure on intra- and interspecific patterns of seed removal for 12 Neotropical pioneer tree species on Barro Colorado

Island, Panama. Chapter 2 revealed variation in seed removal patterns among these 12 tree species both between species at the soil surface and in the same species at different locations in the soil seed bank. Therefore, I asked if variation in ant community composition at five sites and two micro-habitats (soil surface and in the topsoil), and the morphological differences that characterize these ant communities, predict differences in seed removal within and among tree species. I first determined if ant communities varied by season and by location in the soil seed bank. Similar to seed removal patterns discovered in Chapter 2, ant communities did not differ by season but did by micro-habitat. Within a micro-habitat, ant communities clustered into three distinct groups both at the soil surface and within the topsoil. However, variation in ant communities could not predict seed removal with high accuracy. The ant species that were observed removing seeds at the soil surface were a subset of the ant assemblage captured aboveground, though they were not morphologically distinct from the larger ant community. The species richness of ants associated with buried seed caches were not different from the species richness of ants captured in control or unbaited subterranean traps when differences in sample size was accounted for. Overall, ant communities may be functionally redundant in terms of seed removal for pioneer species on Barro Colorado Island, Panama. This work was co-authored by

Paul-Camilo Zalamea, Daniel P. Roche, Rafael Achury, James W. Dalling, and Andrew V.

Suarez.

In Chapter 4, I examined the role of chemical compounds in mediating this ant-seed dispersal interaction. I investigated which chemical compounds from the seed coat elicited seed- carrying by ants. I extracted seed coat chemicals with two different solvents (hexane or methanol) for 30 minutes and then transferred the extract to silica bead dummy seeds determining that Ectatomma ruidum attempted to remove seeds or dummy seeds of one,

Zanthoxylum ekmanii, of the six tree species tested in greater amounts than the others. For Z. ekmanii, there was a trend of E. ruidum foragers attempting to remove seeds and dummy seeds treated with the Z. ekmanii hexane extract in similar amounts. I also used laboratory assays to test fractions of the Z. ekmanii hexane extract and determine candidate chemical compound(s) that may mediate seed removal by ants. The hexane extract was fractioned using column chromatography to separate the apolar compounds from the polar compounds. These fractions were transferred to filter paper discs. While E. ruidum colonies varied in their responses to chemical treatments, workers moved dummy seeds treated with polar compounds from the original location placed (the foraging arena) at similar levels to the Z. ekmanii hexane extract.

All other chemical treatments received little to no movement from the original location. This suggests that chemical cue(s) that may be responsible for promoting some E. ruidum-plant interactions depends on both the tree species and on which extract is presented to the ants.

In Chapter 5, I characterized the effectiveness of E. ruidum as a seed disperser of Z. ekmanii in the Neotropics by (a) determining where and how far ants move seeds, (b) whether seeds can survive their deposition location, and (c) the quality of ant handling (i.e. if ants damaged seeds). I placed 125 Z. ekmanii seeds on the forest floor and subsequently measured dispersal distances and the short-term seed fate of these seeds by determining where seeds were deposited. Foragers of E. ruidum dispersed 41 of the 73 seeds that were dispersed by ants, moved

them approximately one meter in a first distance movement, and brought over half of those seeds into a colony and deposited them there. I used wax casts to determine the possible fate of cached seeds and determined seeds were deposited within the shallowest three chambers of E. ruidum colonies. Zanthoxylum ekmanii seeds should be able to emerge successfully from depths up to seven cm (based on a formula by Bond et al. 1999). However, results from a seedling emergence study indicated that Z. ekmanii seeds are unlikely to emerge from depths greater than a few cms.

In Chapter 6, I summarize my research and explore its implications as well as future directions. Dispersal is a key ecological process that influences the structure of communities, yet it remains a “black box” for many organisms. This is particularly true for plants that rely on animal vectors for secondary dispersal. Ants have received a lot of attention as seed dispersers, but most research concerns seeds that provide an elaiosome food reward. However, these plants make up only 4.5% of all angiosperm species (Lengyel et al. 2010) leaving a knowledge gap in ant-mediated seed dispersal for the vast majority of plants. In my dissertation, I have established that seeds of non-myrmecochorous plants (ones without an elaiosome) may still be dispersed by ants. This indicates that ants may alter the recruitment dynamics of more species than previously thought and should be included in restoration and conservation efforts.

REFERENCES

Beattie, A.J. 1985. The evolutionary ecology of ant-plant mutualism. United States of America:

Cambridge University Press. pp. 196.

Bond, W.J., M. Honig, and K.E. Maze. 1999. Seed size and seedling emergence: an allometric

relationship and some ecological implications. Oecologia 120: 132-136.

Brew, C.R., D.J. O’Dowd, and I.D. Rae. 1989. Seed dispersal by ants: Behaviour-releasing

compounds in elaiosomes. Oecologia. 80: 490-497.

Dalling, J.W., and T.A. Brown. 2009. Long-term persistence of pioneer seeds in tropical rain

forest soil seed banks. Am. Nat. 173: 531-535. doi:10.1086/597221

Dalling, J.W., H.C. Muller-Landau, S.J. Wright, and S.P. Hubbell. 2002. Role of dispersal in the

recruitment limitation of Neotropical pioneer species. J. Ecol. 90: 714- 727.

doi:10.1046/j.1365-2745.2002.00706.x

Giladi, I. 2006. Choosing benefits or partners: A review of the evidence for the evolution of

myrmecochory. Oikos 112: 481-492. doi:10.1111/j.0030-1299.2006.14258.x

Gómez, C., and X. Espadaler. 2013. An update on the world survey of myrmecochorous

dispersal distances. Ecography. 36: 001-009. doi:10.111/j.1600-0587.2013.00289.x

Handel, S.N., and A.J. Beattie. 1990. Seed dispersal by ants. Sci. Am. 263: 58-64

Hubbell, S.P., F.B. Foster, S.T. O’Brien, K.E. Harms, R. Condit, B. Wechsler, S.J. Wright, S.

Loo de Lao. 1999. Light-gap disturbances, recruitment limitation, and tree diversity in a

Neotropical forest. Science. 283: 554-557. doi:10.1126/science.283.5401.554

Hughes, L., M. Westoby, and E. Jurando. 1994. Convergence of elaiosomes and insect prey:

evidence from ant foraging behaviour and fatty acid composition. Fun. Ecol. 8: 358-365.

Lengyel, S., A.D. Gove, A.M. Latimer, J.D. Majer, and R.R. Dunn. 2010. Convergence

evolution of seed dispersal by ants, and phylogeny and biogeography in flowering

plants: A global survey. Perspect. Plant Evol. Syst. 12: 43-55.

doi:101016/j.ppees.2009.08.001

Marshall, D.L., A.J. Beattie, and W.E. Bollenbacher. 1979. Evidence for diglycerides as

attractants in an ant-seed interaction. J. Chem. Ecol. 5: 335-344.

Mull, J. F., and J. A. MacMahon. 1997. Spatial variation in rates of seed removal by harvester

ants (Pogonomyrmex occidentalis) in a shrub-steppe ecosystem. Am. Nat. 138: 1-13.

Nathan, R., and H.C. Muller-Landau. 2000. Spatial patterns of seed dispersal, their determinants

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CHAPTER 2: TREE SPECIES IDENTITY INFLUENCES SECONDARY REMOVAL OF

SEEDS OF NEOTROPICAL PIONEER TREE SPECIES1

ABSTRACT

Primary dispersal agents move seeds from the maternal plant to the soil surface where they are often moved again by secondary dispersal agents. However, the extent to which different species in the same location experience secondary dispersal is often unknown despite the importance of this mechanism for determining recruitment opportunities and consequently community structure. Here we examine the secondary removal rates of 12 Neotropical pioneer species placed either on, or two centimeters below the soil surface at five locations in lowland tropical forest on Barro Colorado Island, Panama. We investigated whether species identity, primary dispersal mode (animal or wind), dormancy type, seed mass, and capacity to persist in the seed bank were correlated with removal rate. We also investigated whether season (dry or wet) influences removal from the soil surface. In general, both superficial and buried seeds were highly mobile. We found an effect of primary dispersal mode and dormancy type on removal rates both on (12 species) and beneath the soil surface (six species). However, this pattern was largely driven by species identity which accounted for most of the variation in the models

(24.3% and 10.4% at the soil surface and within the topsoil respectively). Season had no influence on seed removal rates from the soil surface. The dispersal of small-seeded pioneer species is highly species dependent, indicating that generalizations made using broader categories, such as primary dispersal mode or dormancy type, do not accurately describe

1 The final publication is available at Springer via http://dx.doi.org/10.1007/s11258-017-0745-7

observed patterns hindering our understanding of community assembly within even a single functional group of plants.

INTRODUCTION

Plants face a wide variety of obstacles to the recruitment of new individuals into the population. Plants are limited not only by the number of seeds that they can produce (‘source limitation’), but also whether those seeds escape pre-dispersal predation, survive dispersal, and reach a suitable microsite for germination, while surviving to emergence and establishment

(Nathan and Muller-Landau 2000). In tropical forests, some pioneer tree species produce sufficient numbers of small seeds to overcome source limitation (Dalling et al. 2002), yet only a small fraction of those seeds reach microsites suitable for onward growth. This indicates that they are either limited in their ability to effectively disperse seeds (‘dispersal limitation’) or that constraints imposed by reducing source limitation negatively affect the range of sites favorable for seedling establishment (Dalling and Hubbell 2002).

Dispersal away from the parent plant or conspecifics is important for seedling recruitment in plant communities, both to increase the probability of encountering suitable microsites, and to avoid predators and pathogens that act in a density-dependent manner (Janzen 1970; Connell

1971; Comita and Hubbell 2009; Mangan et al. 2010; Bagchi et al. 2014). In most forests, treefall gaps provide the microsites required for germination and establishment for pioneer trees

(Swaine & Whitmore 1988; Hubbell et al. 1999). However, treefall gaps are uncommon, they exist for short periods of time, and their spatial location and time of formation are fairly unpredictable (Young & Hubbell 1991; Schnitzer et al. 2000). As a consequence, pioneer tree species are under selection to disperse seeds widely, and/or for their seeds to persist for decades in the seed bank (Dalling and Brown 2009).

Primary dispersal, the initial movement of seeds away from the parent tree (Nathan and

Muller-Landau 2000), can be accomplished through a variety of means, including animal, wind, gravity, and ballistic dispersal (Seidler and Plotkin 2006). After primary dispersal has occurred, seeds may experience additional movement events (see Vander Wall et al. 2005) resulting in secondary dispersal or predation. The activity of these secondary removal agents ultimately influences how many seeds are available for germination and can be important at structuring plant communities (Chambers and MacMahon 1994). Potential benefits of secondary dispersal include protection from predation, reduction of competition with conspecifics, and movement to microsites beneficial for germination (Vander Wall and Longland 2004). Overall, whether secondary dispersal activity is beneficial or not to seeds is context-dependent and relies on many different factors (Chambers and MacMahon 1994). For example, secondary dispersal often comes with a price: many secondary dispersers also consume some of the seeds they remove

(Vander Wall and Longland 2004). However, the benefits accrued by the seeds that survive may outweigh the loss of seeds due to predation (Chambers and MacMahon 1994).

Incorporation of seeds into the soil seed bank (defined as the viable seeds present both on the soil surface and in the soil profile; Long et al. 2015) is a critical part of dispersal activities.

The seed bank is often referred to as a safe site for seeds; however, the seed bank is dynamic with many factors including seed, species, and environmental characteristics influencing whether seeds persist or exit the seed bank, either through decay or germination (reviewed in Long et al.

2015). Seed characteristics, such as dormancy type, can influence when seeds are able to respond to the environmental cues they require for germination, while seed size, seed coat, and the presence of appendages or exudates can influence the vulnerability of seeds to predators and pathogens (Long et al. 2015). For example, some physically dormant seeds may avoid predation

while in the seed bank because their impermeable seed coat reduces the diffusion of olfactory cues used by rodents to detect them (Paulsen et al. 2013). Spatial and temporal variation in environmental conditions associated with climate seasonality and microsite heterogeneity can also influence whether a seed is likely to persist (Long et al. 2015), and these conditions may be altered through additional dispersal events into new microsites. While secondary dispersal of seeds into the soil profile is frequently reported, it is unknown whether seeds experience the same potential for further movement at both the soil surface and in the topsoil.

In this study, we examine whether secondary removal rates of seeds vary among pioneer species found in lowland forest of central Panama. For 12 tree species, we tested whether the individual species identity, season, primary dispersal mode (animal or wind), and dormancy type

(physical, physiological, or quiescent) influenced the rate of seed removal from the forest floor.

We also examined if two seed traits, seed mass and seed persistence in the soil seed bank

(defined here as the proportion of viable seeds that survive 18 months of burial enclosed in mesh bags), were correlated with removal rates. For six species, we further tested whether seed removal occurred once seeds were incorporated into the top two centimeters of soil. We tracked the fate of 3000 seeds to test the following alternative, but not mutually exclusive, hypotheses that: (1) primary dispersal mode would serve as a strong predictor of secondary dispersal rates, predicting that animal-dispersed seeds would experience higher above-ground removal rates than wind-dispersed seeds (Fornara and Dalling 2005a); (2) seeds with physical dormancy, and that are therefore impermeable to water, would also have lower removal rates, reflecting reduced availability of olfactory cues for seed predators (Paulsen et al. 2013); and (3) the capacity for long-term survival in the soil seed bank is contingent on avoiding seed predators, resulting in a

negative correlation between seed removal rate and seed persistence in the soil in the absence of predators.

METHODS

Study Site and Species

The study was carried out in 2013 on Barro Colorado Island (BCI) (9°10’N, 79°51’W) in the Republic of Panama. BCI supports seasonal semi-deciduous forest at an average elevation of

70m above sea level with an annual rainfall of 2600mm (Windsor 1990). The forest experiences a pronounced dry season starting late-December or early-January and continuing until late-April or early-May (Windsor 1990). Seed removal experiments were located at five sites at least 350m apart, and at least 20m from conspecific trees, within either old-growth or secondary forest and representing a range of soil types (Zalamea et al. 2015; Table 2.1).

We selected 12 pioneer species found on BCI that vary in seed size, primary dispersal mode, dormancy type, and seed persistence capacity for experiments during the dry and wet season (Table 2.2). The wet season experiments also included artificial seeds (30.5 ± 0.038mg, mean ± SE, mass silica beads). Ripe fruits were collected directly from the parent tree or from beneath the crown and then cleaned to remove seeds from fruit pulp (animal-dispersed species except Zanthoxylum ekamanii (Urb.) Alain) or kapok-like fibers (Cochlospermum vitifolium

Willd. and Ochroma pyramidale Urb.). None of the species involved in this experiment have elaiosomes to attract ants.

Seed Removal Experiments

The experiments were divided into two parts: an above-ground removal experiment that investigated seed removal from the soil surface and a below-ground removal experiment that investigated the removal of seeds buried two cm beneath the soil surface. The above-ground

experiment was conducted once in the dry season and once in the wet season of 2013, and the below-ground experiment was conducted once in the wet season of 2013.

The above-ground removal experiment used similar methods to Fornara and Dalling

(2005a). Experiments were located along a 12m transect established at the edge of five rectangular (9m x 15m) plots where seeds of the same species were buried inside mesh exclosures to determine seed persistence capacity (Zalamea et al. 2015). Leaf litter was partially removed, and nine centimeter diameter Petri dish lids were inverted and placed on the ground one meter apart. Ten seeds of one species were placed in each dish, with the position of each species assigned at random. No vertebrate exclosures were used, as most seed removal observed had been previously attributed to invertebrates (Fornara and Dalling 2005a). A 1.0 m wide x 1.0 m long x 0.5 m tall transparent plastic shelter was centered over each dish to protect seeds from being washed away by rain or failing debris.

Observations of seed removal were initiated at 10h00. The number of seeds present was recorded at hourly intervals for each species until 16h00. The seeds were left out overnight.

Observations continued 24 hours after the first time point at 10h00 and again until 16h00 of the second day. One final observation was made at 09h00 on the third day. Overall, seeds were left out for 47 hours.

Two trials were performed at each of the five plots. Each trial consisted of one 47 hour period of seed removal for all twelve species along one side of each experimental plot chosen at random. A second trial was performed at least one week after the first trial along a different randomly selected plot edge. Dry season sampling was conducted from mid-March to mid-April

2013, and wet season sampling from mid-July and mid-August 2013.

The below-ground removal experiment used six of the 12 species in the above-ground study (Table 2.2). Two trials were randomly assigned to sides of the same five plots as in the above-ground experiment. Holes approximately two centimeters deep and two and a half centimeters wide were dug into the soil along the plot one meter apart. Ten seeds of one species were mixed with sieved, forest soil (autoclaved at 121°C for 2 h) and then buried in each hole.

Plastic shelters were not used for below-ground trials. As a control, we buried ten silica beads in a seventh hole to estimate ‘background’ rates of seed loss including loss by rainfall.

Each of the two trials at a plot was buried simultaneously, though plots were buried on separate days (Table A.1). Seeds were left buried for four weeks from mid-July through late-

August 2013. During collection, an area larger than the original burial holes (approximately five centimeters deep and eight centimeters wide) was dug up to ensure collection of all seeds remaining. The soil was sieved, and any remaining seeds were recovered and counted.

Statistical Analysis

We used general linear mixed-effects models (LMMs) to analyze the proportion of seeds removed to seeds placed among species for both the above- and below-ground experiments with the assumption that missing seeds had been actively removed. We also used LMMs to test for the fixed effects of season (above-ground experiment only), primary dispersal mode, and dormancy type. These models were calculated with and without species as a random effect to determine the added value of including species (model selection values reported in Table 2.3). Above-ground and below-ground data were analyzed separately. We also tested whether controls (silica beads) had a lower removal rate than seeds for both the wet season above-ground data and below- ground data. All LMMs for both experiments consisted of the proportion of the total number of seeds removed as the response variable and location within the forest (plot) and trial as nested

random effects. Tukey post-hoc means separation tests were conducted for significant fixed effect factors in the above-ground experiment and below-ground experiment. Separate analysis of variance (ANOVAs) were used to determine if dispersal mode or dormancy type influenced seed mass. Spearman rank correlation tests were conducted to determine if species highly removed in the above-ground experiment were also highly removed in the below-ground experiment, as well as whether seed mass was correlated with seed removal. Pearson correlation tests were also used to determine the relationship between the proportion of buried seeds that survive in the soil for 18 months (P-C. Zalamea, unpubl. data) and removal. We conducted variance partitioning (Borcard et al. 1992) to analyze the relative importance of season (above- ground experiment only), species identity, primary dispersal mode, dormancy type, and location within the forest (plot) on seed removal. For these analyses, above- and below-ground data were treated separately and linear models were used to obtain R2 values. All analyses were completed using R version 3.3.0 (R Developmental Core Team, 2016) using the nlme (version 3.1-127,

Pinheiro et al. 2015), and modifying the varPart function from ModEvA (version 1.3.2, Barbosa et al. 2016) to work with four or five factors.

RESULTS

For the above-ground experiment, we found that seeds were more frequently removed than silica beads (F1,119 = 12.92, P < 0.001). Combining all the species and plots together during the wet season, on average 46 ± 3.8 percent (n = 1200 seeds) (mean ± SE) of the seeds were removed from the dishes while 2 ± 1.3 percent (n = 100 beads) of the silica beads were removed.

For the below-ground experiment, we found a similar result; 59 ± 4.4 percent (n = 600 seeds) of the seeds removed versus 16 ± 7.8 (n = 100 beads) percent of silica beads (F1,59 = 14.42, P <

0.001).

Species Effect

Species identity had a significant effect on total removal both above-ground (F11,219 =

13.08, P < 0.001; Fig. 2.1a) and below-ground (F5,45 = 5.11, P < 0.001; Fig. 2.1b). For the above- ground experiment, Z. ekmanii had the highest average seed removal (85.5 ± 5.4%, and Trema micrantha (L.) Blume black seed morph had the lowest average seed removal (16.5 ± 6.1%). In contrast, for the below-ground experiment, T. micrantha black seed morph had the highest average seed removal (81 ± 9.2%) and Jacaranda copaia (Aubl.) D. Don. had the lowest average seed removal (27 ± 8.8%). There was no significant correlation between species removal rates above- and below-ground (Spearman’s r = 0.26, P = 0.66). Species alone, as well as models that included species, tended to account for most of the variation in the data for both above- (Fig.

2.2a) and below-ground experiments (Fig. 2.2b).

Effect of Seasonality

We tested if there was a seasonality effect on seed removal for the above-ground experiment and found no differences between the number of seeds removed during the dry season (39 ± 3.8%) and the wet season (46 ± 3.8%; without species: F1,226 = 1.92, P = 0.17; with species as a random effect: F1,227 = 2.39, P = 0.12).

Effect of Primary Dispersal Mode

Animal-dispersed seeds had higher mean seed removal rates than wind-dispersed species for both above-ground (F1,226 = 13.7, P < 0.003; Fig. 2.3a) and below-ground experiments (F1,47

= 5.13, P = 0.028; Fig. 2.3b). However, including species as a random effect masks this effect for both above-ground (F1,8 = 2.32, P = 0.17) and below-ground experiments (F1,2 = 3.12, P =

0.22).

Effect of Dormancy

We found an effect of dormancy type on mean seed removal for both above- (F2,226 =

10.58, P < 0.001; Fig. 2.4a) and below-ground experiments (F2,47 = 3.91, P = 0.027; Fig. 2.4b).

In the above-ground experiment, physically dormant seeds (n= 5 species) had a slightly higher mean seed removal rate than physiologically dormant seeds (n = 3 species) and greater removal rate than quiescent seeds (n = 4 species). In the below-ground experiment, physically (n = 3 species) and physiologically dormant seeds (n = 1 species) were removed in greater amounts than quiescent seeds (n = 2 species). However, including species as a random effect masks this effect for both above-ground (F2,8 = 1.80, P = 0.23) and below-ground experiments (F2,2 = 2.38,

P = 0.30).

Seed Mass

Neither dispersal mode (ANOVA: F1 = 0.26, p = 0.62) or dormancy type (ANOVA: F2 =

1.78, P = 0.22) were associated with seed mass. Seed mass was not correlated with above-ground

(Spearman’s r = 0.36, P = 0.26) or below-ground removal (Spearman’s r = 0.14, P = 0.80).

Persistence

Seed persistence in the soil seed bank after being buried within two centimeters of topsoil for 18 months was not correlated with either above-ground removal (Pearson’s r = 0.27, P =

0.39) or below-ground removal (Pearson’s r = 0.57, P = 0.24).

DISCUSSION

In this study, we examined the secondary removal rates of seeds of 12 common

Neotropical pioneer trees in the dry and wet season from the soil surface at five locations on

Barro Colorado Island in the Republic of Panama. We found no effect of seasonality on seed removal for the above-ground experiment. Consistent with both our prediction and previous

results (Fornara and Dalling 2005a), seed dispersal mode influenced removal rates. However, seeds adapted for primary dispersal by animal vectors did not on average have higher removal rates than seeds adapted for wind-dispersal when species identity was included as a random effect in our model. A similar effect to dispersal mode was seen with dormancy type. Species identity accounted for a majority of the variance in removal rates captured by the models. With additional species sampling it is possible that the amount of variation attributed to species identity would be minimized and generalizations based on seed characteristics would emerge.

In addition to above-ground removal rates, we investigated the removal rates of seeds of six species that were buried two centimeters beneath the soil surface. Clear seasonal changes in density of viable seeds present in the soil have been documented (Fornara and Dalling 2005b), particularly for seeds in upper three centimeters of soil (Dalling et al. 1997). These seasonal dynamics occur beneath closed canopy forest between fruiting seasons and therefore can be attributed to either seed movement or seed mortality. While we did not measure below-ground seed removal in the dry season, our wet season results indicate that seeds remain highly mobile below-ground even over short periods (four weeks), perhaps providing a mechanism for seasonal changes in seed density. It is unclear whether this mobility extends to deeper soil layers. For seeds larger than those used in this study (5 – 17.5 mm), Estrada and Coates-Estrada (1991) found that rodents were able to detect and eat an average of 92 percent of seeds located in the one to two and a half centimeter depth range, though their ability to detect seeds rapidly decreases with increasing depth. Previous work, however, has shown that surface removal of seeds of some species used in this study can be attributed to invertebrates rather than vertebrates

(Fornara and Dalling 2005a). We also found no support for our hypothesis that long-term

persistence in the seed bank is contingent on avoiding seed predation as seed removal rates were uncorrelated with seed persistence capacity of 18 mo.

We found that when variation associated with species identity was not accounted for, our results matched our predictions as species adapted for primary dispersal by animals had a higher average removal rate than species adapted for primary dispersal by wind both when seeds were placed on the soil surface and buried in the soil (see also Fornara and Dalling 2005a). This dispersal mode effect persists even though animal-dispersed seeds were not presented in dung, excluding possible secondary dispersal by dung beetles (Andreson and Levey 2004). However, it is possible that adaptations to attract animals during the first dispersal event may also be acting at the secondary dispersal level. For example, even though seeds were cleaned of fruit pulp, small pieces of pulp may have remained adhered to the seed. Seed movement due to wind gusts, rain, or falling debris was kept to a minimum through the use of seed shelters leaving mainly animals as the vector for seed removal in both seasons. As we found no effect of seasonality on above- ground seed removal, secondary disperser activity may be relatively uninfluenced by season or alternatively, the identity of dispersers may have changed between seasons while net removal rates remained similar. However, comparison of models with and without species identity demonstrates that dispersal mode is not as important an indicator of secondary removal potential as species identity, though this result is only significant for the above-ground experiment. This result is further supported by the variance partitioning where species identity accounts for most of the variance in seed removal both in the above-and below-ground experiments.

Similar to the results of dispersal mode, there is an effect of dormancy type on seed removal when not including species. However, our hypothesis that physically dormant seeds would have the lowest removal rates based on the decreased diffusion of olfactory cues to seed

predators (Paulsen et al. 2013) was not supported. Instead, physically dormant seeds were removed in the greatest numbers both in above-and below-ground experiments, with physiologically dormant and quiescent seeds having the lowest removal rates for the above- ground and quiescent seeds for the below-ground experiments respectively. However, species identity again masked this effect indicating that the predictive power of functional and life- history traits is weak relative to species-specific characteristics, such as seed chemical and physical traits. Seed polyphenols, important for seed defense, have been shown to be ubiquitous though variable in concentration among tropical tree species, correlating negatively with seed size and seed physical defenses (Gripenberg et al. 2017, but see Tiansawat et al. 2014). While pioneer species tend to have fewer of these defensive compounds (Gripenberg et al. 2017), it is possible that there is variation among pioneer species that could relate to differences in species- specific removal rates by deterring removal agents, though future work will need to be done to say this conclusively. Alternatively, seeds may contain attractive chemicals that act as behavioral-releasers to elicit seed-removal responses in organisms (e.g. 1,2-diolein acts as a behavioral releaser in elaiosome bearing systems by eliciting removal responses in ants, Marshall et al. 1979). However, future studies will need to investigate the chemistry of these species to know how chemistry influences secondary removal.

Species identity did not always influence removal the same way above-and below- ground, as the rank order of removal rates was different between above-and below-ground experiments. For the above-ground experiment Z. ekmanii experienced the greatest amount of removal (85.25 ± 5.4%), while T. micrantha black seed morph experienced the lowest (16.5 ±

6.1%). In contrast, T. micrantha black seed morph experienced the highest average total below- ground seed removal (81 ± 9.2%). This suggests that different seed removal agents and seed

traits affect removal rates above- and below-ground. For above-ground removal rates, we found more removal for Apeiba membranacea Aubl. and Luehea seemannii Triana and Planch than previously recorded on BCI for both the same time frame and approximately time of year (75.5 ±

6.4% and 17 ± 6.5% this study compared to 35 ± 3% and 5 ± 8% for A. membranacea and L. seemannii respectively in May-June 2001, Fornara and Dalling 2005a). It is possible more pulp remained on A. membranacea seeds post cleaning making seeds more attractive for removal, while not removing the wings on the L. seemannii seeds may have led to gusts of air removing them from the petri dishes, though this is unlikely. Additionally, studies investigating other

Cecropia species have found variable rates of seed removal at the genus level (C. longipes 47.0 ±

9.7%, this study; Cecropia peltata L. 17 ± 3%, Fornara and Dalling 2005a; Cecropia obtusifolia

Bertol. and O. pyramidale together 40% of seeds removed in 48 hours, Garcia-Orth and

Martinez-Ramos 2008).

Ants (Formicidae) were the only taxa observed removing seeds during our observation periods. Out of 158 daytime (between 10h00 and 16h00) intervals that had a recorded change in hourly seed count, ants were present at 47.5 percent (75/158) of these daytime seed count changes and were observed removing seeds 38 percent (60/158) of the time (S.A. Ruzi, unpubl. data). Both Z. ekmanii and O. pyramidale stand out for their high overall removal rates (85.25 ±

5.4% and 66.9 ± 8.0%, respectively). Zanthoxylum ekmanii seeds are dispersed from capsules and do not contain any pulp or other conspicuous agent that could attract ants, yet they had the highest removal rate in the above-ground experiment. Preliminary data from ongoing projects suggests that there are chemicals on the seed coat of Z. ekmanii seeds that elicit the seed removal response for one of the common ants Ectatomma ruidum (Roger) observed removing these seeds

(S.A. Ruzi, unpubl. data). Ectatomma ruidum have also been observed moving Z. ekmanii seeds

into their colony, though whether the seeds are ingested, cached, or later removed remains unclear (S.A. Ruzi, unpubl. data). Ochroma pyramidale has a swollen spongy area in the funicle region whose function is unclear (Vazquez-Yanes and Perez-Garcia 1976). Ants in the genus

Trachymyrmex were observed to approach O. pyramidale seeds, remove and carry away the funicle leaving the rest of the seed in the petri dish, though they did occasionally remove the entire seed (S.A. Ruzi, pers. obs.). Other ant genera (Solenopsis and Pheidole) have also been recorded as removing Ochroma seeds (Garcia-Orth and Martinez-Ramos 2008), though removal of the funicle was not mentioned. It is possible that other tree species traits are driving this pattern, such as seed specific chemical profiles that ants cue in on to remove seeds.

Although ants were the only taxa observed removing seeds from the soil surface, it is difficult to tell whether ants were the taxa responsible for the removal of seeds within the topsoil.

The common ants observed removing seeds are known to forage at the soil surface (E. ruidum,

Franz and Wcislo 2003; Trachyrmrmex, Leal and Oliveira 2000). It is likely that the ant community below-ground consists of different species than those that forage at the soil surface.

For example, soil probes captured a significantly different assembly of ants than sampling methods traditionally used to sample ground, leaf litter, and arboreal ants in Amazonian Ecuador

(Wilkie et al. 2007). This difference in ant community could account for the difference in the observed rank removal rates between above- and below-ground experiments if different ant species have preferences for seeds of different tree species; however, further investigation will be needed to determine if this is the case.

While species identity accounts for most the variance in seed removal both above- and below-ground (0.243 and 0.104 respectively) in the current models, there is still a large amount of variance unaccounted for in both the above- (0.566) and below-ground (0.679) experiments.

Other factors that potentially influence seed removal rates, but that were not explicitly studied, include incorporation of seeds into the soil by rain, and habitat variables that alter foraging patterns of the removal agents. While direct removal of seeds by rainfall from the above-ground experiment was controlled for using seed shelters, rainfall could have affected the below-ground experiment, though the amount of vertical movement will also depend on the soil structure, material, and pore size (Chamber and MacMahon 1994). Habitats with higher moisture levels tend to have higher foraging activity as it reduces the risk of desiccation (Kaspari and Weiser

2000). Litter cover also alters the size of the ants most likely to forage (smaller ants forage below the leaf litter, Kaspari and Weiser 2000), potentially affecting ant species interactions that could influence dispersal (Horvitz and Schemske 1986). The presence or abundance of other seed or plant species could also have indirect effects on focal species by attracting seed removal vectors

(apparent competition, Holt and Kotler 1987; Garb et al. 2000; Veech 2000) or alternatively reducing seed removal rates if vectors preferentially remove seeds of other tree species. In an attempt to reduce these confounding effects, the experiment locations were selected to be >20 m away from adult conspecifics of the tree species. For the below-ground experiment, however, seeds of conspecifics were buried in mesh bags within the plots sampled (see Zalamea et al.

2015) and could have attracted density-dependent removal agents increasing our observed below-ground removal rates.

Conclusions

Overall, our results suggest that seeds show a striking amount of variation in removal rates both above- and below-ground. Our results indicate that seed removal rates are primarily associated with traits that vary at the species-level. These may include characteristics such as chemical cues or physical seed structures that attract ants. We found that incorporation of seeds

into the topsoil did not lead to the loss of seed mobility. Future studies should determine whether the high mobility in the topsoil can be generalized to all Neotropical pioneer species, whether mobility remains high over longer periods of time, and how this mobility is correlated with seed fate to ultimately understand how this influences the recruitment patterns of Neotropical pioneer species.

ACKNOWLEDGMENTS

We thank the Smithsonian Tropical Research Institute (STRI) for providing facilities, logistical support, and permission to conduct the project. We thank the University of Illinois

Integrative Graduate Education and Research Traineeship-Vertically Integrated Training with

Genomics training fellowship National Science Foundation (NSF) Department of Graduate

Education grant-1069157, NSF Department of Environmental Biology grant-1120205 to JWD, and the Smithsonian Tropical Research Institute-Butler University internship program for funding. We also thank two anonymous reviewers for their comments.

TABLES

Table 2.1 Soil and flora characteristics of sampling areas (plots) within Barro Colorado Island. Forest age is approximated from classifications mapped in Mascaro et al. (2011) that were generated based on land-use data obtained from Enders (1935). Soil type, soil form, and parent material information are from Baillie et al. (2006).

Plot / Location Name Forest Age (years) Soil Type/ Soil Form/ Parent Material

25 Ha 80-120 Marron/ brown fine loam/ Andesite Armour 80-140 AVA/ red light clay/ Andesite Drayton 400+ Fairchild/ red light clay/ Bohio Pearson 400+ Standley/ brown fine loam/ Bohio Zetek 400+ Marron/ brown fine loam/ Andesite

Table 2.2 Pioneer tree species used throughout the study. All seeds were used for the above-ground experiment, while a subset was chosen for the below-ground experiment.

Tree Species Family Species Fruiting Primary Dormancy Seed Mass Seed Used in Used in (Species code) Distributi Period Dispersal Type (mg) Persistenc Above- Below- on Mode e (18 ground ground months)

Apeiba Tiliaceae Mexico- February- Animal Physicalb 13.58±1.23 0.75c x x membranacea Aubl. Brazila Marcha (Ape) Cecropia longipes Urticaceae Panama- July- Animal Quiescentc 0.9±0.07 0.85c x x Pitt. (Cec) Colombiad Auguste Cochlospermum Bixaceae Mexico- February- Wind Physicalc 24.4±2.27 0.24c x vitifolium Willd. Brazila Aprila (Coc) Ficus insipida Willd. Moraceae Mexico- Variableg Animal Quiescentc 1.56±0.12 0.46c x (Fic) Brazilf Guazuma ulmifolia Sterculiacea Mexico- March- Animal Physicalb 3.38±0.28 0.86c x Lam. (Guz) e Colombiah Aprilh

a Zalamea et al. 2015 b Sautu et al. 2007 c P-C. Zalamea unpubl. data d Zalamea et al. 2012 e Zalamea et al. 2011 f Phillips 1990 g Milton et al. 1982 h Janzen 1975

Table 2.2 (cont.) Tree Species Family Species Fruiting Primary Dormancy Seed Mass Seed Used Used in (Species code) Distributi Period Dispersal Type (mg) Persistenc in Below- on Mode e (18 Above- ground months) ground Hieronyma Euphorb- ___ January, Animal Physiologica 6.55±0.33 0.98c x alchorneoides iaceae June, July, lb Allemao (Hie) Decemberi Jacaranda copaia Bignoniacea Belize- July- Wind Quiescentc 1.16±0.18 0.33c x x (Aubl.) D. Don. (Jac) e Brazilj September k

Luehea seemannii Tiliaceae Mexico- February- Wind Physicalb 1.85±0.3 0.88c x Triana and Planch. Venezuela Aprila (Lue) a Ochroma pyramidale Malvaceae Mexico- March- Wind Physicalb 5.73±0.29 0.88c x x Urb. (Och) Brazila Maya Trema micrantha Cannabacea ___ August- Animal Physiologica 3.2±0.21 0.97c x x (L.) Blume“black” e Octoberi lb (TrBl) Trema micrantha Cannabacea ___ August- Animal Quiescentc 1.71±0.1 1.00c x (L.) Blume“brown” e Octoberk (TrBr) Zanthoxylum Rutaceae ___ February- Animal Physiologica 16.2±0.81 1.00c x x ekmanii (Urb.) Alain Marchi lc (Zan)

i Zimmerman et al. 2007 j Scotti-Saintagne et al. 2013 k Dalling et al. 1997

Table 2.3 Models testing for the effects of season (above-ground only), dispersal mode, and dormancy type both with and without species identity included as a random effect for above-and below-ground experiments. P-values are from comparing models without and with species identity as a random effect for the above-or below-ground experiments separately.

Experiment Model DF AIC BIC LogLik P- value

Above- Proportion~Season+Dispersal+Dormancy+(1|Plot/Trial) 8 236 264 -110 ___ ground

Above- Proportion~Season+Dispersal+Dormancy+(1|Plot/Trial)+(1|Species) 9 202 233 -92 < 0.001 ground

Below- Proportion~Dispersal+Dormancy+(1|Plot/Trial) 7 42.8 57.4 -14.4 ___ ground

Below- Proportion~Dispersal+Dormancy+(1|Plot/Trial)+(1|Species) 8 43.7 60.5 -13.9 0.312 ground

FIGURES

Figure 2.1 Average seed removal by species for above- (n = 200 seeds) (a) and below-ground (n = 100 seeds) (b) experiments. Error bars correspond to SE. Letters denote significance differences as determined by Tukey HSD means separation tests. Light gray and dark gray bars represent species categorized as being primarily animal- or wind-dispersed respectively. See Table 2.2 for species codes.

a Dispersal Animal−dispersed Wind−dispersed b Mode 100 100 A A AB

AB AB ABC AB 75 ABC 75

BC BC BCD 50 50 CD C

CD CD

CD Seed Removal (%) Total

Total Seed Removal (%) Total CD 25 25 D

0 0 Zan Ape Guz Cec Fic Hie TrBr TrBl Och Coc Jac Lue TrBl Ape Cec Zan Och Jac Species Species

Figure 2.2 Variance partitioning results for above-(a) and below-ground (b) experiments. Values were obtained by modifying formulas in the varPart function in the ModEvA package and obtaining the R2 values from linear models. All factors were treated as fixed effects for these analyses.

Figure 2.3 Average seed removal by dispersal mode for above-(a) and below-ground (b) experiments. Error bars correspond to SE.

Figure 2.4 Average seed removal by dormancy type for above-(a) and below-ground (b) experiments. Error bars correspond to SE. Letters denote significance differences as determined by Tukey HSD means separation tests from models without species identity as a random effect.

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CHAPTER 3: CAN VARIATION IN SEED REMOVAL PATTERNS OF

NEOTROPICAL PIONEER TREE SPECIES BE EXPLAINED BY LOCAL ANT

COMMUNITY COMPOSITION?

ABSTRACT

Ants are important seed dispersers, moving seeds into suitable microsites for germination and growth. Myrmecochorous plants (i.e., those that provide an elaiosome food reward) are the primary focus of research on ant-mediated seed dispersal. However, some plants, including many species of Neotropical pioneer trees, have seeds that are attractive to ants yet are not known to provide a food reward. We examined if heterogeneity in ant community composition among sites, between above- and below-ground foraging guilds, or between seasons predicts observed variation in seed dispersal rates for 12 Neotropical pioneer tree species on Barro Colorado

Island, Panama. We also investigated if ants associated with removing seeds differed in specific morphological characters from the larger ant community. We observed ant-seed interactions at caches to determine which ants dispersed seeds of 12 tree species. We also sampled ant community composition by placing 315 pitfall traps and 160 subterranean traps across five sites where seed removal rates were quantified. Above-ground ant community composition varied by site but not season. However, this among-site variation in ant composition was unable to predict accurately seed removal patterns at these same sites. Below-ground ant communities differed from above-ground ant communities, but were similar at all sites studied. Finally, ants that removed seeds did not differ morphologically from the broader ant community. Overall, our results suggest ant communities vary over relatively small spatial scales, but may be functionally redundant in terms of seed removal services provided for Neotropical pioneer tree species.

INTRODUCTION

Plant recruitment is limited by many factors including the number of seeds produced (e.g. source limitation), whether seeds survive primary and secondary dispersal, and whether seeds reach suitable microsites for germination and establishment (Nathan and Muller-Landau 2000).

Neotropical pioneer tree species produce large quantities of small seeds (Dalling et al. 2002), which require high light microsites such as canopy gaps to germinate and grow (Hubbell et al.

1999, Swaine and Whitmore 1988). To maximize success, seeds must be dispersed away from the parent tree to a suitable microsite, or remain viable in the soil seed bank until a tree fall occurs creating a suitable microsite (Dalling and Brown 2009). While treefall gaps can occur relatively frequently in forests, their formation can be unpredictable and spatially heterogeneous

(Martinez-Ramos et al. 1988). Seeds of some pioneer species are buffered from this uncertainty by remaining viable in the soil for 30 years or more (Dalling and Brown 2009). Moreover, entry into the soil seed bank does not mean that seeds lose their mobility (Ruzi et al. 2017). Seeds can experience secondary dispersal via animal vectors, though these dispersal agents may vary among sites and depth within the seed bank (e.g. Sanchez-Cordero and Martinez-Gallardo 1998,

Mull and MacMahan 1997). In addition to differences in seed characteristics that may affect their attractiveness to animal vectors (Fornara and Dalling 2005), the heterogeneous distribution of seed dispersers makes it difficult to predict secondary dispersal distributions for Neotropical pioneer trees (Dalling et al. 2002, Ruzi et al. 2017).

Ants are important seed dispersers in many ecosystems (Beattie 1985, Handel and Beattie

1990), playing a role in both short (< 2 m) and long-distance (> 175 m) dispersal (Gómez and

Espadaler 2013). By carrying seeds into their colonies or depositing them in nutrient-rich refuse piles, ants remove seeds from the soil surface where they are more likely to be eaten by

granivores (O’Dowd and Hay 1980, Bond and Slingsby 1984, Christian and Stanton 2004), and provide suitable microsites for germination and growth (Culver and Beattie 1980, Davidson and

Morton 1981, Hanzawa et al. 1988). Most work on ant mediated seed dispersal focuses on myrmecochorous plants whose seeds provide an attached food reward called an elaiosome.

However, it is not clear if ants benefit from elaiosomes despite a long history of research suggesting so (Warren II et al. 2019). Many other plant species may participate in ant-mediated seed dispersal but are not currently listed as myrmecochorous because ants have yet to be observed dispersing seeds. For example, seeds of some plants that that have been previously thought to lack an elaiosome have now been identified as being dispersed by ants as they have an aril (e.g. Pizo and Oliveira 1998, Passos and Oliveira 2002, Christianini and Oliveira 2010,

Magalháes et al. 2018) or fruit (e.g. Barroso et al. 2013). Even among ants that use seeds as resources, not all ants are attracted to equally seeds of different plant species. In some communities, foragers of individual ant species may have disproportionally large effects on plant communities through their dispersal services (Youngsteadt et al. 2009, Barroso et al. 2013).

Vertical stratification of ant communities is well documented in tropical forests

(Yanoviak and Kaspari 2000, Weiser et al. 2010). In addition to differences between arboreal and surface ant assemblages, ant communities can vary greatly among samples collected at the soil surface, from the leaf litter, and within the top layers of soil (Ryder Wilkie et al. 2007, Ryder

Wilkie et al. 2010, Jacquemin et al. 2016). These assemblages differ in key traits including food preferences (Yanoviak and Kaspari 2000, Hahn and Wheeler 2002), mode of resource defense

(Yanoviak and Kaspari 2000), and morphology (e.g. body size distribution, Kaspari and Weiser

1999). Variation in ant microhabitat preferences, morphological, and ecological traits could result in widely different patterns of seed dispersal over very small scales. For example, in

addition to varying with microhabitat preference (Kaspari and Weiser 1999), ant body size is often correlated with seed size (Kaspari 1996, Pfeiffer et al. 2006) and seed dispersal distance

(Ness et al. 2004, Gómez and Espadaler 2013). Therefore, ant mediated seed dispersal should vary both by tree species and the microhabitat seeds end up in after primary dispersal due to the differences in ant morphological traits in different microhabitats.

In a recent study of 12 Neotropical pioneer species, Ruzi and colleagues (2017) found secondary seed removal rates varied among tree species both for seeds deposited on the soil surface and when seeds were placed two cm below the surface in the topsoil. However, removal rates did not vary between the wet and dry seasons. Here, we combined observations of ants removing seeds with a characterization of the above- and below-ground ant communities at five sites to determine if variation in ant community structure predicts observed seed removal rates.

Specifically, we tested the following five hypotheses regarding seed removal rates of Neotropical pioneer tree species: (1) A lack of an effect of season on seed removal rates reflects similar ant community composition between the wet and dry season at these sites; (2) Differences in removal rates between seeds on the soil surface and the topsoil reflect differences in ant community composition above- and below-ground; (3) The composition of the ant community predicts seed removal rates; (4) Differences in removal rates among tree species correspond to differences in their attractiveness to individual ant species; and (5) Ants that are responsible for removing seeds will differ in specific morphological traits relative to ants from the community at large.

METHODS

Study site and species

The study was performed at five sites on Barro Colorado Island (BCI) (9°10’N,

79°51’W) in Lake Gatun in the Republic of Panama. BCI is characterized as a seasonal semi- deciduous forest with annual rainfall of 2600 mm/y with a pronounced dry season starting late

December or early January until late April or early May (Windsor 1990). Each location was located along a range of soil types in either old-growth or secondary deciduous forest (Zalamea et al. 2015, Ruzi et al. 2017, Sarmiento et al. 2017).

Ripe fruits were collected from below parent trees of 12 pioneer species (Table 3.1).

Except for Guazuma ulmifolia (mucilage, Escobar-Ramírez et al. 2012), seeds of these

Neotropical pioneer tree species are not known to have elaiosomes to attract ants and vary in several seed characteristics including defense syndromes (Zalamea et al. 2018), mass, primary dispersal mode, and dormancy type (Ruzi et al. 2017).

Above-ground seed removal and ant community sampling

The above-ground seed removal experiment was conducted once in the dry season and once in the wet season of 2013 following methods in Fornara and Dalling (2005) (Ruzi et al.

2017). At each of five sites, seeds of the 12 pioneer tree species were placed in seed caches every meter along a 12m transect along the edge of a rectangular plot (9-m by 15-m) (see Zalamea et al. 2015). The order along the transect was randomized using a list randomizer. Each cache consisted of an inverted Petri dish lid (9 cm diam, and 8 mm deep) with 10 seeds of one of the 12 different species or 10 silica beads as a control (mass 30.5 ± 0.038 mg, mean ± SE; wet season only). Caches were placed under transparent plastic shelters (1.0 m wide x 1.0 m long x 0.5 m tall) to reduce passive removal of seeds by raindrops and forest debris.

Caches at each site were observed over a 47 h period. Observations were initiated at

10h00 on the first day and retrieved at 09h00 on the third day. Observations of ant removal and number of seeds remaining were recorded hourly on the first and second days from 10h00 to

16h00. Samples of ants were collected only once ants exited the inverted Petri dish lid or if they were present in large numbers. As a result of collecting ant samples, it is possible that overall removal rates were reduced (see Kaspari 1993). As observations spanned the 12 m transect, some ant removal was likely missed. However, the lip of the Petri dish prevented ants from quickly exiting with a seed, increasing handling time and the probability that seed removal would be observed.

We used pitfall traps to sample above-ground ant community composition in each of the five sites. Pitfall traps consisted of 50 ml conical tubes (28 mm diam opening) buried so that the lip was flush to the soil surface and containing 20 ml of preserving fluid (aqueous saturated NaCl solution with a drop of detergent). Each site was sampled twice during the wet season (2013) and twice during the dry season (2014). During each sample period, we placed 15-16 pitfall traps at regular intervals inside the experimental plots. The traps were collected after 48 h and all ants sorted into 95 percent ethanol. Traps were pooled within seasons as the sampling locations within the plot were the same and not independent of each other.

Below-ground seed removal and ant community sampling

The below-ground seed removal experiment was conducted once in the wet season of

2013 and used seeds of six tree species (Table 3.1; Ruzi et al. 2017). At each of the five sites, seven seed caches were buried along a randomly chosen edge of the same rectangular plot in the previous section and left for four weeks. Each cache consisted of a 2 cm deep by 2.5 cm wide hole that had 10 seeds of one of the tree species or 10 silica beads with sieved sterile soil

(autoclaved at 121ºC for 2h). We replicated this set up twice at each of the five sites; each site had a total of 14 caches, two for each of six tree species and the silica bead control cache, for a total of 70 caches across all sites.

While useful for estimating seed removal rates, below-ground seed caches do not allow for direct observations of ants interacting with seeds of each tree species. To sample ants that may be attracted to seeds of each tree species, we placed two subterranean traps within 10 cm of each cache. We placed an additional two subterranean traps away from the seed caches for a total of 16 traps each sample period, or 32 total subterranean traps at each of the five sites.

Subterranean traps consisted of 50 ml conical tubes with eight approximately two through four mm holes equidistantly spaced around the side of the tube approximately two cm below the top of the lid. The traps were buried so that the cap was flush with the soil surface and contained 20 ml of preserving fluid. Traps were placed at the same time as the seeds caches and also left out for four weeks (12 July –24 August 2013). After collection, all ants were sorted and preserved in

95 percent ethanol.

Ant identification and size measurements

Ants were identified to genus using Palacio and Fernández (2003) and identified to species whenever possible using published keys, online resources for identifying ants of BCI and

Costa Rica, AntWeb.org (AntWeb 2017). For each of the most common species or morphospecies at the soil surface (found in at least 10% of the pitfall traps at a single site), we measured head width (across the eyes: HW), head length (HL), eye position (EP; calculated as head width minus intraocular ocular width), mandible length (ML), scape length (SL), eye width

(EW), eye length (EL), hind femur length (HF), and Weber’s length (WL) (Fig. B.1). These measurements relate to specific ecological functions and how these ants interact with their

environment (summarized in Table 3.2). Each measurement was repeated for two ants of each species and three times per specimen, and the average value across all measurements was used

(Table B.1). All ant species observed removing seeds at the soil surface had the same measurements recorded in the same way. All measurements were done on point-mounted individuals using a Semprex Micro-DRO digital stage micrometer that was accurate to the 0.005 mm (Semprex Corporation, Campbell, U.S.A.) connected to a stereomicroscope (Leica MZ 12.5,

Switzerland).

Statistical analyses

All statistical tests were conducted in R (version 3.4.2, R Core Team 2017) unless otherwise noted. Ant communities were characterized by the presence and frequency of ant species in traps either at the plot level (pitfall traps) or per cache type per plot (subterranean traps). This considers both the identity of ant species present and the relative activity of foraging ants as higher frequencies of presence indicate more common or more active ant species. Species rarefaction and extrapolation curves were generated using EstimateS (version9.1.0, Colwell

2013) to determine the completeness of sampling.

Non-metric multidimensional scaling (NMDS) and the multi-response permutation procedure (MRPP, with 999 permutations) were used to assess if the composition of the ant community varied with season (above-ground experiment only), location within the soil seed bank (wet season only), and by tree species (above- and below-ground separately). NMDS depicts relationships between assemblages in n-dimensional space (McCune and Grace 2002).

Additional axes were added if stress was reduced by at least five to improve the goodness-of-fit of the model (McCune and Grace 2002). MRPP calculates within group means weighted by sample size and then randomly redistributes specimens to recalculate the weight within group

means due to random chance (McCune and Grace 2002). Specimen randomization was blocked by site location unless otherwise noted as sites served as replicates. NMDS and MRPP were conducted using the metaMDS and mrpp functions in the vegan package respectively (version

2.4-5, Oksanen et al. 2017). The distance matrix was compiled using frequency of presence of ant species in traps and the Bray-Curtis index, which accounts for both presence / absence of species and their relative abundance. As the number of hand-collected samples of ant species observed removing species was low, hand samples of ants removing seeds were not included in

NMDS or MRPP analyses unless otherwise noted.

We used partial least squares regression (PLS-R, Mevik and Wehrens 2007) to assess the effect of ant community on seed removal using the plsr function in the pls package (version 2.7-

0, Mevik et al. 2018). The ant community (frequency of presence of ants in traps) was used at the predictor variables with the percent seed removal as the response variable. In the above-ground analysis, the ant community represented the frequency of presence at the plot level, while in the below-ground analysis, the ant community represents the frequency of presence in traps associated with each cache type (tree species or silica bead controls) at each plot. Partial least squares regression is robust to many predictor variables with few observations (StatSoft 2013).

The number of components were chosen based on whether they increased the amount of explained variance in the response variable by at least five percent to maximize the predictive ability of the models, while keeping the root mean square error of prediction (RMSEP) low.

PLS-R can also pick out individual variables (e.g. ant species) that contribute to differences in removal rates across sites. In addition, we used agglomerative hierarchical cluster analyses using the beta-flexible method (b = -0.25) and Bray-Curtis index to assess whether ant communities at the sites clustered. These analyses were done using the agnes function in the cluster package

(version 2.0.7-1, Maechler et al. 2018). Agglomerative hierarchical cluster analyses join individual objects, in this case, ant communities from one site or communities associated with one type of cache, together with communities from other sites until there is one large group

(Quinn and Keough 2002). Final dendrograms were visualized using ggplot2 (version 3.0.0,

Wickham 2016), ggdendro (version 0.1-20, de Vries and Ripley 2016), and dendextend (version

1.8.0, Galili 2015) packages in R. Once visual groups were determined with the dendrogram,

MRPPs were conducted to determine if the visual groupings were significantly different from each other.

Morphological traits were compared across ant species that were observed removing seeds at the soil surface to those commonly found in pitfall traps using a Principal Components

Analysis (PCA) using the prcomp function in the base stats package. Both the center and scale parameters in prcomp were set to true so that the values would be centered around zero and so that the variables would be scaled to have unit variances. All nine morphological measurements were used at the species level, and species were categorized into two groups: 1) those removing seeds and 2) those common in pitfall traps (present in at least 10% of traps at one site) but not observed removing seeds. Differences between the groupings were compared using significant principal components and an analysis of variance (ANOVA; aov function in the base stats package in R). Measurements were log transformed prior to analyses to improve normality.

RESULTS

We identified a total of 77 ant species belonging to 29 genera and 6 subfamilies from samples taken of ants removing seeds (dry and wet season, 14 species), pitfall traps (dry and wet seasons, 66 species), and subterranean traps (wet season only, 22 species) (Table B.2). There were 158 daytime (between 10h00 and 16h00) intervals that had a recorded change in hourly

seed count. Ants were present at 75 of these 158 (47.5%) times and observed removing seeds 60 of these 158 (38%) times.

Effect of season on ant communities

There was no effect of season on the ant assemblages captured in pitfall traps during the dry (5 sites) and wet season (5 sites) (NMDS: stress = 0.072; MRPP: permutations = 999, strata

= fixed by site, A = -0.01, P = 0.125; Fig. B.2).

Effect of location in the soil seed bank on ant communities

There was an effect of trap location (i.e. pitfall traps above-ground versus subterranean traps below-ground) on ant community composition (5 pitfall sites vs. 36 cache types across 5 below-ground sites) during the wet season (NMDS: stress = 0.124; MRPP: permutations = 999, strata = fixed by site, A = 0.08, P = 0.001; Fig. 3.1). The five most common ant species found above-ground (based on averaging frequency of presence at all sites over both seasons) were

Ectatomma ruidum (57.50%), Pachycondyla harpax (29.38%), Pheidole multispina (16.88%),

Sericomyrmex amabilis (11.88%), and Labidus praedator (10.63%). The six most common ant species found below-ground (based averaging frequency of presence at all sites) were Labidus coecus (22.50%), Solenopsis cf. vinsoni (17.50%), Tranopelta gilva (16.88%), Solenopsis cf. bicolor (15.63%), P. harpax (3.13%) and Pheidole sp.002 (3.13%) (Table B.2).

Effect of site on seed removal and ant community composition

PLS-R analyses used a subset of the variation in ant community composition to explain a majority of the variation in above-ground seed removal for all tree species pooled together, and for each species was tested separately (Table 3.3). Conversely, the below-ground analysis only explained approximately half of the variation in seed removal. In all cases, RMSEP remained

high indicating the predictive power of using ant communities to predict seed removal had low accuracy both at the soil surface and within the topsoil.

Hierarchical cluster analyses depicted three main clusters for ant communities both at the soil surface (Fig. 3.2A) and within the topsoil (Fig. 3.2B). MRPP determined these clusters were significantly different from each other at the soil surface (MRPP: permutations = 999, strata=fixed by season, A = 0.17, P = 0.008) as well as within the topsoil (MRPP: permutations =

999, A = 0.18, P = 0.001). However, groupings within the topsoil contained assemblages collected from traps at different sites, and assemblages collected from different cache types were found in each of the three groupings.

Seed identity and ant species

Ant species observed removing seeds from the surface caches (pooling both the dry and wet season) were primarily a subset of the species collected in pitfall traps with only four species not also found in pitfall traps (Fig. 3.3A). The five most common ant species observed removing seeds during time intervals when there was an hourly change in seed number were E. ruidum (22 intervals), Trachymyrmex cornetzi (11), Pheidole sussanae (4), and both S. amabilis and

Trachymyrmex bugnioni (3 each). Ants removing seeds of the most commonly visited tree species during the first day (with at least three ant samples at a given site: G. ulmifolia,

O.chroma pyramidale, and Zanthoxylum ekmanii) were distinct from the ant assemblages captured in pitfall traps at those same sites (NMDS: stress = 0.074; MRPP: permutations = 999,

A = 0.09, strata = fixed by site, P = 0.017; Fig. 3.4A). These three tree species also differed from one another and from passive samples in terms of ant identity visiting the caches when not pooled (MRPP: permutations = 999, A = 0.10, strata = fixed by site, P = 0.047; Fig. 3.4B). Three ant species—Aphaenogaster araneoides, E. ruidum, and T. cornetzi—significantly separate out

the assemblages at the different tree species and passive samples, with pitfall traps and Z. ekmanii seeds having a higher abundance of E. ruidum and occasionally a higher abundance of

A. araneoides, and both G. ulmifolia and O. pyramidale having higher abundances of T. cornetzi

(Fig. 3.5B). These three tree species also had some of the highest first day removal (10h00 to

16h00 removal; Fig. 3.3A, Table 3.1).

As we could not watch ants interacting with seeds buried beneath the soil, we used subterranean traps to detect differences in ants near the different tree species seed caches, silica bead controls, or in unbaited traps. Most ant species were found in traps associated with seed caches, with a few additional species found in traps associated with bead controls or not associated with any seed cache (Fig. 3.3B). Most ant species were associated with many tree species and we found no differences in the assembly of ants collected at subterranean traps placed next to specific tree species seed caches (NMDS: stress = 0.085; MRPP: permutations =

999, A=-0.04, strata = fixed by site, P = 0.44, Fig. B.3). Rarefaction and extrapolation of all sites pooled suggest that the subterranean traps had the same species richness regardless of whether the traps were next to seeds or control silica beads / unbaited traps (Fig. B.4). The five most common ant species found in subterranean traps associated seed caches (all seed caches pooled) were L. coecus (25.83%), T. gilva (16.67%), S. cf. vinsoni (15.83%), S. cf. bicolor (14.17%), and

P. harpax (4.17%). While the four most common ant species found in silica beads / unbaited traps S. cf. vinsoni (22.50%), S. cf. bicolor (20.00%), T. gilva (17.50%), and L. coecus (12.50%) were all also common in traps associated with seed caches.

Morphological traits of ants removing seeds

PC1 weighted all morphological characters in similar amounts and accounted for 91.95 percent of the variation in the morphological data (Table 3.3). In contrast, PC2 accounted for

only 4.82 percent in the data and mainly consisted of EW, EL, and ML. Ant morphological traits did not differ with respect to whether ants were categorized as removing seeds or common in pitfall traps without being observed removing seeds (ANOVA: PC1 loadings, F2,23 = 0.41, P =

0.53; PC2 loadings, F2,23 = 3.50, P = 0.07; Fig. 3.5).

DISCUSSION

We examined if variation in seed removal rates of Neotropical pioneer trees corresponded to differences in above- and below-ground ant communities at five sites on Barro Colorado

Island, Panama. We also observed ants removing seeds at the soil surface to determine which ants were attracted to seeds of each tree species. Consistent with our first two hypotheses and predictions, we found ant assemblages above-ground were not different between dry and wet seasons, but above- and below-ground assemblages did vary in composition. We also found that ant species observed removing seeds above-ground were a subset of the ant species found in the above-ground ant community. The ant assemblages observed removing seeds of three of the most commonly removed tree species (G. ulmifolia, O. pyramidale, and Z. ekmanii) were distinct from each other and from the larger above-ground community. However, ant species observed removing seeds at the soil surface did not differ morphologically from the most commonly collected ant species found in pitfall traps. Overall, there was little support for our hypothesis that spatial variation in ant assemblages would predict differences in seed removal patterns among our five sites.

Even within distinct vertical assemblages (e.g., arboreal, ground / litter, subterranean), ant community structure can vary spatially. Studies comparing ant activity in plots across moisture gradients on Barro Colorado Island found increasing ant activity with moisture, though there was not necessarily a change in activity based on season or time of day (Kaspari and

Weiser 2000). Ant communities are also influenced by patchy disturbances, such as those between gap and canopy locations, which alter microclimate variables (e.g. moisture, light penetration), though these differences decrease with time (Feener and Schupp 1998). We found support for similar levels of variation among our plots in the above-ground ant communities which clustered into three distinct groupings based on dissimilarities in presence / frequency of capture at a site. These differences in ant community structure could impact the amount of dispersal services plants receive at local scales (e.g. Horvitz and Schemske 1986), and consequently seed fate. Below-ground ant communities also clustered, but the groups included both sites and different cache types (ie. associated with seeds, silica bead controls, or unbaited traps) among sites. If ant communities were primarily structured spatially, we would have expected the sites to cluster together regardless of cache type.

Variation in secondary seed removal rates can be seen among forests that vary in seasonality and / or altitude (Fornara and Dalling 2005). In a comparison between five forests located in Panama, Barro Colorado Island was found to have the highest secondary removal rates

(Fornara and Dalling 2005). Within this comparison of five different forests, ants in the subfamilies Myrmicinae and Ponerinae were responsible for nearly all the observed seed removal; however, the ant species observed removing seeds were not always the same at each forest studied (Fornara and Dalling 2005). Here, we found that even within a single forest there is substantial variation in ant communities, both at the soil surface and within the topsoil. This finding is consistent with other studies (e.g. Mull & MacMahon 1997), indicating that animal vectors, including ants, are not distributed homogeneously through the habitat. This could potentially influence seed removal based on these different communities. However, PLS-R analyses using these communities (represented as the frequency of presence of each ant species

in traps associated with a site or with a certain cache type) as predictor variables predicted seed removal rates with low accuracy. This could imply that there may be functional redundancy in seed removal among the different ant communities on BCI for these tree species.

While ants were observed removing seeds above-ground, we lacked direct observations of below-ground ant-seed interactions with the assumption that ants are the responsible seed removing agent. It is likely that ants below the soil surface are passively encountering seeds rather than actively searching for seeds given the general lack of known seed specialists in the community of ants captured within the topsoil. Rarefaction of ant species accumulation curves from subterranean traps with and without associated seed caches suggests that differences in the number of species captured among cache types is likely due to differences in sample size rather than ants being more attracted to seed caches than controls.

Ants most commonly found removing seeds at the soil surface varied in their diet preferences. Of the five most common, E. ruidum was also among the most common ant species found in the above-ground pitfall traps. Ectatomma ruidum is considered an insect predator, but will also consume plant-based resources including fruit, honey-dew, and floral nectar (Pratt

1989, Lauchaud 1990), and has been recorded as moving seeds in other systems (e.g. Zelikova and Breed 2008, Escobar-Ramírez et al. 2012). Trachymyrmex cornetzi, T. bugnioni, and S. amabilis are all members of the Attini tribe and tend fungus gardens, collecting fresh and decaying plant material (e.g. leaves, twigs, fruits/berries, seeds and their husks) and insect frass to feed their gardens (de Fine Licht and Boomsma 2010). Pheidole spp. in general are omnivorous and include many seed harvesters consuming most of the seeds they move, but some seeds avoid being eaten and are instead placed in refuse piles where seedlings have lower mortality and increased growth relative to seedlings in nearby soil (Levey and Bryne 1993).

While only one of the five ant species commonly seen removing seeds above-ground was also frequently detected in pitfall traps, four of the five most common ant species collected in subterranean traps associated with seed caches were also common in the control silica beads / unbaited traps. Labidus coecus, and other army ants, are known as important predators in tropical forests and are commonly found raiding underground in tropical forests (O’Donnell et al. 2007,

Ryder Wilkie et al. 2010). Though, less is known about their specific prey preferences compared to other army ant species. Similarly, there is very little known about the biology of other subterranean foraging ants including T. gilva (AntWeb 2017). Most interactions between

Solenopsis spp. and seeds have been recorded for Solenopsis geminata, which tends to reduce seed dispersal and will consume fruit pulp or arils attached to seeds without further dispersing the seed (Horvitz and Schmenske 1986, Zelikova and Breed). They also interfere with other ant species that would otherwise disperse seeds such as P. harpax (Horvitz and Schmenske 1986), which was another common ant species in subterranean traps associated with seeds in this study.

Pachycondyla harpax was the only species not also found in the silica beads / unbaited traps.

The ants observed removing seeds from the soil surface were a subset of the species captured by pitfall traps, but they did not have distinct morphological characteristics relative to ants that were not removing seeds. This lack of association could be due to the seeds attracting generalists rather than specialist seed predators. For example, the chemical cues used by the plants to attract ants to the seeds may attract scavenging / insectivorous species to collect seeds even though they are incapable of consuming them (e.g. Youngsteadt et al. 2010). Subsequently, many of the ants would likely share morphological features with other generalist or predatory species in the community. Even ants in this system that are seed predators may not have distinct morphological characteristics. For example, small worker body size could be offset by

communal foraging (e.g. Pheidole sussanae worked together to carry Z. ekmanii seeds up a tree,

SAR personal observation). We also saw fungus growing ants (Cyphomyrmex and

Trachymyrmex) carrying seeds, and while further research remains to determine if the seeds collected by these genera can be broken down by their fungi, their morphology is likely driven / constrained by factors related to specialized life style. This does not preclude fungus growing ants from being important seed-dispersers; in the Brazillian cerrado, fungus growing ants increase seed germination by removing fruit and arils (Leal and Oliveira 1998). Future work linking morphological traits to diet, for example through stable isotope analyses, will help elucidate the role of functional groups on variation in seed dispersal rates.

Seed dispersal by ants can affect plant populations by influencing fitness of dispersed seeds (Hanzawa et al. 1988) and by increasing seedling recruitment following disturbance

(Gallegos et al. 2014). Individual ant species may differ in their effectiveness of dispersal moving seeds longer or shorter distances based on ant size (Ness et al. 2004, Gómez and

Espadaler 2013). While the ant communities studied here did not exhibit a strong trait-based association with seed-removal, there could be lingering effects of disperser identity on post- dispersal seed fate. For example, a study comparing the fate of seeds dispersed by either the tapir

(Tapirus terresstris) or the muriqui (Brachyteles arachnoides), determined that there were lasting effects on seed fate based on the identity of the primary disperser (Lugon et al. 2017). Seeds dispersed by tapirs were less likely to be buried by dung beetles than seeds dispersed by muriquis due to differences in feces traits. This resulted in tapir dispersed seeds being more vulnerable to mortality due to predation. Therefore, future work aimed at examining the fates of these seeds once they are moved by ants and the chemical cues that the plants use to make these seeds attractive to ants (Youngsteadt et al. 2010) should be a priority.

ACKNOWLEDGMENTS

We thank the National Science Foundation (NSF) Department of Environmental Biology grant-120205 to JWD, the University of Illinois (UIUC) Integrative Graduate Education and

Research Traineeship-Vertically Integrated Training with Genomics training fellowship NSF-

Department of Graduate Education grant-1069157, NSF Doctoral Dissertation Improvement grant-1701501 to AVS and SAR, and the Dissertation Travel Grant from UIUC for funding. This work was partially supported by the Cooperative State Research, Education, and Extension

Service, US Department of Agriculture, under project number ILLU 875-952. We also thank the

Smithsonian Tropical Research Institute for permission to conduct the project, as well as, for logistical support. We thank Abigail C. Robison for help in the field and Adam S. Davis for advice on statistical analyses.

TABLES

Table 3.1 Tree species used in the different experiments (above-ground/below-ground) as well as seed removal percentages (mean and standard error) for each experiment. Above-ground average percent seed removal and standard error (SE) per tree species (pooled over dry and wet seasons) was also broken down to represent percentage of seeds placed removed from 10h00 to 16h00 on day 1 (Day 1), between 16h00 day 1 and 10h00 day 2 (Night 1), 10h00 to 16h00 on day 2 (Day 2), and from 16h00 day 2 to 09h00 day 3 (Night 2).

Experiment Above-ground Below- Seed Removal Mean ± SE (%) ground Seed Removal Mean ± SE (%) Tree Species Above- Below- Total Day 1 Night 1 Day 2 Night 2 Total (species code) ground ground Apeiba x x 75.50 ± 6.43 5.00 ± 3.03 49.50 ± 8.03 1.50 ± 1.09 19.50 ± 5.45 78.00 ± 6.11 membranacea Aubl. (Ape) Cecropia longipes x x 47.00 ± 9.65 8.00 ± 5.11 24.00 ± 7.41 8.00 ± 3.45 7.00 ± 3.33 63.00 ± 8.44 Pitt. (Cec) Cochlospermum x 20.00 ± 7.81 1.50 ± 1.50 13.50 ± 6.97 0.00 ± 0.00 5.00 ± 4.50 — vitifolium Willd. (Coc) Ficus insipida x 43.00 ± 9.46 3.50 ± 2.09 26.50 ± 7.89 3.50 ± 3.02 9.50 ± 5.26 — Willd. (Fic) Guazuma ulmifolia x 71.00 ± 8.24 42.50 ± 8.61 25.50 ± 5.87 0.00 ± 0.00 3.00 ± 1.79 — Lam. (Gua) Hieronyma x 32.50 ± 8.55 0.50 ± 0.50 29.50 ± 8.51 1.00 ± 0.69 1.50 ± 0.82 — alchorneoides Allemao (Hie)

Table 3.1 (cont.) Experiment Above-ground Below- Seed Removal Mean ± SE (%) ground Seed Removal Mean ± SE (%) Tree Species Above- Below- Total Day 1 Night 1 Day 2 Night 2 Total (species code) ground ground Jacaranda copaia x x 20.00 ± 7.40 1.00 ± 0.69 16.00 ± 7.34 2.00 ± 1.56 1.00 ± 0.69 27.00 ± 8.23 (Aubl.) D. Don. (Jac) Leuhea seemannii x 12.00 ± 4.84 0.50 ± 0.50 8.50 ± 4.55 0.00 ± 0.00 3.00 ± 1.47 — Triana and Planch. (Lue) Ochroma x x 66.89 ± 8.01 31.17 ± 8.46 31.72 ± 7.91 0.50 ± 0.50 3.50 ± 2.09 65.00 ± pyramidale Urb. 10.67 (Och) Trema micrantha x x 16.50 ± 6.08 5.50 ± 3.20 7.50 ± 5.02 2.50 ± 2.04 1.00 ± 0.69 81.00 ± 9.24 (L.) Blume “black” (TrBl) Trema micrantha x 21.00 ± 7.61 6.00 ± 4.99 5.50 ± 2.94 2.50 ± 1.43 7.00 ± 5.03 — (L.) Blume “brown” (TrBr) Zanthoxylum x x 85.50 ± 5.35 29.50 ± 8.78 53.00 ± 7.85 1.00 ± 0.69 2.00 ± 1.38 41.00 ± ekmanii (Urb.) 11.87 Alain (Zan)

Table 3.2 Ant morphological traits and their proposed ecological functions based on the literature (summarized in Parr et al. 2017 and references within). Eigenvectors calculated from the log transformed morphological measurements as well as the standard deviation, contribution and total observed variation for the first two principal components.

Principal Components Trait Related Function PC1 PC2 Head Width (HW) Indicative of how much musculature there is for the mandibles, -0.3382 -0.2457 worker body size, and the size gaps workers can pass through Head Length (HL) May relate to diet and also indicates worker body size -0.3364 -0.2884 Eye Position (EP; calculated as Indicative of hunting ability and habitat used -0.3306 0.2851 HW – intraocular width) Mandible Length (ML) Related to diet -0.3306 -0.4037 Scape Length (SL) Sensory ability -0.3381 0.0313 Eye Width (EW) Food searching behavior and activity times -0.3290 0.4653 Eye Length (EL) Food searching behavior and activity times -0.3166 0.5699 Hind Femur (HF) Foraging speed and complexity of habitat -0.3383 -0.1471 Weber’s Length (WL) Corresponds to worker body size which also related to metabolic -0.3414 -0.2195 traits Standard deviation — 2.8767 0.65874 Proportion of Variance — 0.9195 0.04822 Cumulative Proportion — 0.9195 0.96772

Table 3.3 Partial least squares-regression summary statistics including the response variable (seed removal), experiment (either above- or below-ground), number of ant species in the ant community predictor, number of components in the final model, root mean square error of prediction (RMSEP) for leave-one-out cross-validation, and the amount of variation explained in the ant community and seed removal. For species abbreviations in the response variable see Table 3.1

Explained Variance

(%)

Response Experiment # of Ant # of RMSEP Ant Seed

Variable Species Components (adjusted) Community Removal

Species Above 66 3 18.45 62.41 96.72 pooled (17.53) Ape Above 66 3 19.66 69.63 94.98 (18.77) Cec Above 66 3 45.56 64.41 95.23 (43.38) Coc Above 66 3 44.83 66.14 95.13 (42.56) Fic Above 66 3 49.44 67.23 97.16 (46.91) Gua Above 66 3 39.06 69.87 96.64 (37.16) Hie Above 66 3 30.55 60.69 98.27 (29.00) Jac Above 66 4 40.31 79.97 98.59 (38.27) Lue Above 66 3 24.66 63.07 97.06 (23.41) Och Above 66 3 22.22 58.12 96.70 (21.11) TrBl Above 66 3 18.71 68.93 96.24 (17.83) TrBr Above 66 4 28.17 74.53 96.57 (26.80) Zan Above 66 4 28.87 76.15 98.74 (27.41) All Below 21 3 26.84 42.91 51.14 Species (26.59) and Bead Controls

FIGURES

Figure 3.1 Nonmetric multidimensional scaling (NMDS) depicting the relationship between ant communities found in the wet season pitfall or subterranean traps for NMDS axes 1 and 2 (A), axes 1 and 3 (B), and 2 and 3 (C). Ellipses represent the multivariate t distribution. Sites: 25 = 25Ha, A = AVA, D = Drayton, P = Pearson, Z = Zetek

Figure 3.2 Hierarchical clustering analyses with beta-flexible method (b = -0.25) using the Bray- Curtis index on ant communities above-ground (A), and below-ground (B). For above-ground, leaves represent communities at the site level. For below-ground leaves represent communities captured in different cache types per site.

Figure 3.3 Average seed removal percent by species of all sites pooled together with representative ant heads of ant species observed removing each species above-ground (A) or in seed associated traps below-ground (B). Above-ground data has been pooled from both dry and wet season while below-ground data is only for the wet season. Colors represent the breakdown of percentage of seeds removed during different time periods (Day 1: 10h00 of first day to 16h00 on first day; Night 1: 16h00 on first day to 10h00 on second day; Day 2: 10h00 on second day to 16h00 on second day; Night 2: 16h00 on second day to 09h00 on third day) with standard error bars around the average total seed removal after 47 h. Letters denote significant differences as determined by Tukey’s HSD means separation tests conducted in Ruzi et al. (2017). Ant heads are either from AntWeb.org if they were identified to species or from z-stacked images if the ant was identified to morphospecies. Ape = Apeiba membranacea, Cec = Cecropia longipes, Coc = Cochlospermum vitifolium, Fic = Ficus insipida, Gua = Guazuma ulmifolia, Hie = Hieronyma alchorneoides, Jac = Jacaranda copaia, Lue = Luehea seemannii, Och = Ochroma pyramidale, TrBl = Trema micrantha “black” seed morph, TrBr = Trema micrantha “brown” seed morph, Zan = Zanthoxylum ekmanii

Fig. 3.3 (cont.)

Figure 3.4 NMDS depicting the relationship between ant assemblages collected removing seeds of tree species (Gua, Och, and Zan pooled) and those found at pitfall traps (A) and with species separated (B). Both figures have the same axes shown. Ellipses represent the multivariate t distribution (A) or are polygons of the data (B). A = AVA, D = Drayton, Z = Zetek, P = pitfalls, Gua = Guazuma ulmifolia, Och = Ochroma pyramidale, Zan = Zanthoxylum ekmanii, AphAra = Aphaenogaster araneoides, EctRui = Ectatomma ruidum, TraCor = Trachymyrmex cornetzi

Figure 3.5 Principal components analysis (PCA) of the ant species found in at least 10 percent of pitfall traps at one site and all the ant species observed removing seeds at the soil surface categorized into two groups (1) those removing seeds and (2) those common in pitfall traps but not observed removing seeds (A) and boxplots of the loadings on the first two principal components (PCs).

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CHAPTER 4: PELIMINARY IDENTIFICATION OF CHEMICAL CUES THAT

MEDIATE SEED REMOVAL BY THE ANT ECTATOMMA RUIDUM ROGER IN A

NEOTROPICAL PIONEER TREE SPECIES

ABSTRACT

Ant-mediated seed dispersal has evolved repeatedly and is geographically widespread, affecting plant distributions through both primary and secondary dispersal. Research investigating chemical cues associated with ant-mediated seed dispersal primarily focuses on dispersal of myrmecochorous plants. Myrmecochorous plants have lipid-rich food bodies called elaiosomes attached to their seeds that contain chemicals cues, and these cues may be as important as nutritional benefits for eliciting seed removal. Seeds without elaiosomes can also be attractive to ants resulting in chemically-mediated behavioral manipulation by the plant where ants disperse seeds without receiving a reward. To examine the chemical cues that play a role in seed dispersal, I field-tested hexane and methanol extracts from seed coats of six Neotropical pioneer tree species on Barro Colorado Island, Panama. Seeds of five of the six tree species are not known to have elaiosomes, but three commonly elicit a seed carrying response from ants. I measured rates at which ants of the generalist species Ectatomma ruidum Roger removed, or attempted to remove seeds, and tested the response of ants to silica beads or filter paper treated with solvent extracts from seeds. Only one of the six tree species tested (Zanthoxylum ekmanii

Urb. (Alain)) had chemical cues that elicited a removal response in E. ruidum when applied to beads or paper in the absence of the seed itself, with the chemical(s) responsible residing in a polar fraction of the hexane seed extract.

INTRODUCTION

Plant chemistry mediates many plant-animal interactions. It is often examined in the context of defensive and attractive properties for defense against herbivores (reviewed in

Mithöfer and Boland 2012) and floral attractive compounds for pollination purposes (Pichersky and Gershenzon 2002, González-Teuber and Heil 2009, Dötterl and Vereecken 2010).

Additionally, plant chemistry acts as a defense against microbial infection (Bednarek and

Osbourn 2009), mediates associations with mycorrhizal symbioses (Bednarek and Osbourn

2009), and with other plants in the form of allelopathy (Mithöfer and Boland 2012). Some of these secondary compounds in the form of plant volatiles may also be used to attract organisms such as parasitic wasps that help defend the plant from their herbivores (see Mithöfer and Boland

2012, Pichersky and Gershenzon 2002). Seed chemistry has also recently received attention, particularly from the perspective of its role in defense against pathogens (e.g. in tropical systems,

Gripenberg et al. 2017, Zalamea et al. 2018). Research on seed and fruit chemistry is often related to frugivory and the role of chemistry on seed dispersal. Several hypotheses relating the role of fruit chemistry on which fruit, and therefore seeds, are dispersed have been put forward

(reviewed in Cipollini and Levey 1997). These generally tend to focus on chemistry being important in the attraction or repulsion of different dispersal agents and how long the diaspore remain in the gut (Cipollini and Levey 1997). However, these studies tend to leave out invertebrate dispersal agents as they tend to be too small to consume seeds whole and even if they remove the fruit and leave the seed intact the importance of chemistry is often overlooked.

An exception to the lack of information on chemistry in term of seed dispersal by invertebrates is the study of myrmecochorous plants. Myrmecochorous plants invoke seed dispersal by providing an elaiosome (a fatty acid rich food reward) attached to their seed (Beattie

1985; Handel and Beattie 1990; Hughes et al. 1994). Foraging ants retrieve the diaspore (seed and attached elaiosome), feed the food body to their brood, and discard the seed in a waste pile in or outside the nest (Handel and Beattie 1990, Giladi 2006). The conditions in and around the nest are thought to provide a favorable microsite for seed germination and growth (e.g. Davidson and

Morton 1981, Sankovitz et al. 2019). Two hypotheses have been proposed as to why ants are attracted to the elaiosome including (1) elaiosomes are nutritious for the ants, and (2) elaiosomes may simply be chemically perceived as dead insects (Carroll and Janzen 1973). Some short-term laboratory-based research provides evidence of benefits when colonies are provided with elaiosome bearing seeds including increased alate production (e.g. short-term experiment in

Warren II et al. 2019), and increased larval mass (e.g. Gammans et al. 2005). However, other aspects of ant colony fitness are not affected by elaiosomes (e.g. no change in worker number or males produced in Gammans et al. 2005; no change in worker or alate abundance in Warren II et al. 2019), or experience negative effects (e.g. long-term removal of myrmecochorous plants resulted in increased colony health in Warren II et al. 2019). Chemically, the fatty acid compositions of elaiosomes are more similar to seven broad taxa of insect prey than to the seed they are attached to, specifically in levels of palmitic, palmitoleic, stearic, and oleic acids

(Hughes et al. 1994). Oleic acid also induces corpse-carrying behavior in many ant species

(Haskins and Haskins 1974), which could be responsible for the seeds being moved into waste piles. The dimer of oleic acid, 1,2-diolein, is also found in elaiosomes and is the chemical that is most commonly thought to elicit the seed carrying response in ants (Marshall et al. 1979;

Skidmore and Heithaus 1988; Brew et al. 1989).

Myrmecochorous plants only make up only 4.5% of all angiosperm species (Lengyel et al. 2010), leaving a gap in knowledge in ant-mediated seed dispersal for a majority of

angiosperms. Plants that participate in ant-mediated seed dispersal but lack an elaiosome food reward are often overlooked. Plants that lack an elaiosome, termed non-myrmecochorous plants, may also engage in ant-mediated seed dispersal, but have been overlooked as ants have not previously been observed removing seeds. For example, some plant species previously termed non-myrmecochorous have since been discovered to be dispersed by ants with the aril (e.g. Pizo and Oliveira 1998; Christianini and Oliveira 2010) or fruit (e.g. Barroso et al. 2013) mediating the interaction. Alternatively, they may mimic elaiosomes by having chemicals on their seed coat that acts as behavioral releasers. This would work similarly to how the presence of 1,2-diolein acts as a behavioral releaser in elaiosome bearing systems by eliciting removal responses in ants

(Marshal et al. 1979). Without providing a food reward, plants may be exploiting the behavior of ants, particularly those that are not capable of processing the seeds themselves for food (e.g. non- granivorous species). Neotropical pioneer tree species are an excellent system to examine the cues responsible for ant dispersal in the absence of a food reward as many are not known to have elaiosomes yet are dispersed by a wide variety of ants (CHAPTER 3).

I investigated the seed removal rates of 12 Neotropical pioneer tree species that are not known to have elaiosomes from the soil surface and found that tree species varied in removal rates by ants (CHAPTERS 2 and 3). In this chapter, I begin to fill a knowledge gap in understanding the role of seed chemistry in a nontraditional ant-mediated seed dispersal interaction. I do this by using a subset of the Neotropical pioneer tree species studied in

CHAPTER 3 that were found to vary in seed removal rate by ants. I test the hypothesis that ants respond to specific chemicals on the seed coat which mediate ants to remove seeds.

METHODS

Ectatomma ruidum was the focal ant of this study as it is the most common species at the study site (CHAPTER 3). Ectatomma ruidum was also observed removing seeds of four of the six focal Neotropical pioneer tree species, though other ants were also responsible for some removal (CHAPTER 3, Table C.1). While this ant in usually considered an insect predator / scavenger, it will also consume plant-based resources (e.g. fruit and floral nectar), tend insects for honey-dew (Pratt 1989, Lauchaud 1990), and has been recorded moving seeds in other systems (e.g. Zelikova and Breed 2008). It is a relatively large ant, being the second largest ant collected ant in pitfall traps at the same study locations (E. ruidum Weber’s length = 2.780 mm,

Odonotmachus bauri is larger at 3.658 mm, CHAPTER 3). This large size allowed for easy observation of interactions between the ant and the dummy seeds (e.g. silica beads) used in the study. While E. ruidum were unable to pick up the dummy seeds controls, they were easily observed biting and attempting to remove the dummy seeds. Therefore, counts of removal attempts were used as the response variable in statistical analyses.

Field bioassay

The study was performed in 2014 at on Barro Colorado Island (BCI) (9°10’N, 79°51’W), the Republic of Panama. BCI has an annual rainfall of 2600 mm/y, which primarily falls during the wet season (late or early May to late December or early January (Windsor 1990). Chemical trials were performed at five previously established sites (e.g. see Zalamea et al. 2015, Ruzi et al.

2017, Zalamea et al. 2018, CHAPTER 3) that range in forest age and soil type (see Table 2.1 in

CHAPTER 2).

Ripe fruits were collected from below parent trees of the six common pioneer species

(Apeiba membranacea, Cecropia longipes, Guazuma ulmifolia, Ochroma pyramidale, Trema

micrantha “brown” seed morph, and Zanthoxylum ekmanii; Table 4.1). Except for G. ulmifolia

(mucilage may act as an attractant, Escobar-Ramírez et al. 2012), seeds of these tree species are not known to have elaiosomes. Seeds of these tree species also vary in several seed characteristics (e.g., size, primary dispersal mode, and dormancy type; Ruzi et al. 2017). Apeiba membranacea, C. longipes, and T. micrantha “brown” seed morph all had pulp removed prior to the experiment using cleaned forceps or by gently massaging the pulp away with distilled water and a glass mortar and pestle. Glassware was cleaned following the glass cleaning protocol mentioned below. Guazuma ulmifolia and Z. ekmanii seeds were removed from the intact fruit with sterile forceps. Seeds of A. membrenacea, C. longipes, T. micrantha “brown” seed morph, and Z. ekmanii were rinsed with distilled water and allowed to dry in an air-conditioned, dehumidified dark room (~22ºC) to prevent molding. Only A. membranacea and Z. ekmanii seeds that sank in water were used in the study as these are likely to be viable seeds. Ochroma pyramidale and G. ulmifolia seeds were not rinsed prior to being used in the study. Guazuma ulmifolia produces a mucilaginous film when wet (Escobar-Ramírez et al. 2012) that glues it to other surfaces when the mucilage dries (S.A. Ruzi, personal observation).

Seeds were collected using sterilized forceps underneath parent trees in the Barro

Colorado Nature Monument (BCNM) straight into glassware that had previously been cleaned by washing with alconox (White Plains, NY, USA) solution, rinsing with acetone, and then left to dry in a drying oven for at least 24 h between 50-60°C in an oven. Silica bead dummy seeds (3 mm diameter, 30.5 ± 0.012 mg, mean ± SD mass; University of Illinois Chemical Store Room,

Urbana, IL), which were used in the field trials described below, were cleaned in a similar method.

Chemicals on seed coats were collected by extracting 20 seeds with either 500 µl of methanol (ACS grade; for polar compounds) or hexane (ACS grade; for apolar compounds) in glass vials for 30 min. Extracted chemicals were applied to ten silica bead dummy seeds by adding the beads to the vial and allowing the solvent to evaporate under a fume hood. Solvent control dummy seeds were created in the same fashion using neat methanol or hexane (ie., no additional chemicals were added to the solvent).

The extracts created from the six tree species were tested during the same season in which the trees of the different species fruits. Therefore, responses of ants to seed coat chemicals were tested for four tree species during the dry season (A. membranaceae, G. ulmifolia, O. pyramidale, and Z. ekmanii) and two species during the wet season (C. longipes and T. micrantha “brown” seed morph; Table 4.1). Each species was tested individually at five sites with previously established study plots. There were two trials per site for each tree species. The response of ants to experimental treatments was tested for each tree species by placing caches of

10 seeds or dummy seeds (silica beads) in an inverted petri dish lid (9 cm diameter, 8 mm deep), the treatments being the two experimental extracts, the two solvent controls, a dummy seed control, and the seeds themselves. Treatments were deployed one m apart along a randomly chosen side of previously established study plots. The behavior of ants was observed from 10h00 to 14h00 and the number of seeds or beads remaining was recorded on every half hour. I also recorded whether ants attempted to remove a seed or bead within each half hour interval (coded

0 = no attempts, 1 = any attempt). Ants of other species also were collected from in caches, however due to their small size it was difficult to observe their behavior and determine if these ants were attempting to remove the beads.

Laboratory bioassay of seed coat chemicals

Eight colonies of E. ruidum in 2015 and eight colonies in 2016 were excavated by digging a hole approximately 30 to 40 cm deep 10 cm away from the colony entrance before carefully digging closer to the colony to expose the nest chambers. All colonies were collected from Pipeline Road and Parque Soberania in the Republic of Panama. Laboratory colonies consisted of a single Petri dish nest (9 cm diameter) with dental plaster (Midwest Dental, Wichita

Falls, TX, USA) lining the bottom to keep colony chambers moist / humid. Colonies had access to ad lib water and sugar solution and were fed cut up frozen crickets (Armstrong Crickets, West

Monroe, LA, USA) two or three times a week until the experiment began. Colonies were starved an average period of 3.4 d (min = 2, max = 5, mode = 3) days prior to experiments to increase likelihood of ants interacting with dummy seeds (filter paper discs).

Seed coat extracts were collected by placing 10 seeds in a glass vial, adding one ml of hexane (HPLC grade), and removing the seeds from the solvent after 30 min. Seed coat hexane extracts were fractioned by column chromatography to separate apolar and polar components using Pasteur pipettes plugged with clean glass wool (Supleco, Signma Aldrich) and containing

~0.5 g of silica gel impregnated with silver nitrate (Sigma Aldrich). These pipettes were prepared by capping with aluminum foil and baking at 50 – 60°C for 24 hours. Columns were first wetted with two 1 ml aliquots of hexane, allowing excess to drain, the hexane extract was then added, followed by 2.5 ml of 20% cyclohexene in hexane solution (yielding the apolar fraction), and 5 ml of 100% ethyl ether (yielding the polar fraction). Neat hexane was used for control extracts following the same protocol.

The response of ants to the fractionated extracts of seed coats was tested by applying fractioned chemicals to discs of filter paper and allowing the solvent to evaporate under a fume

hood. The filter paper discs were approximately six mm diameter and were cut with a standard hole punch. Solvent control filter paper discs were created in the same fashion using neat hexane fractions and neat hexane. The paper discs were first crumpled with sterile forceps so that they could be more easily carried by ants. The four chemical treatments were tested simultaneously, one for each of four ant colonies, by presenting each colony with five filter paper discs of the same treatment assigned randomly (list randomizer: https://www.random.org/lists/). Filter paper discs were always placed in the foraging arena (Fig. C.1) and the final location of the discs was recorded after ants were allowed 64 to 134 min (70 ± 9 min, average ± SD; mode = 65 min) to interact with the dummy seeds. Ant colonies were allowed at least one day to recuperate between trials.

Chemical analysis of laboratory seed coat chemicals

Attractive fractions and their respective solvent controls were prepared to run on the gas chromatogram-mass selective detector (GC-MS) by first having their solvent (20% cyclohexene in hexane solution or 100% ethyl ether) evaporated by running a gentle stream of nitrogen gas over them. The chemicals were then re-suspended into one ml of hexane for analyses on the GC-

MS. The respective solvent control was used to determine which chemicals were background noise and, therefore, were not unique to the seed coat chemical fraction.

One ml of each sample was analyzed using a Hewlett Packard 5890 GC with a DB-17 column (30 m x 0.25 mm x 0.25 micron film) connected to a 5973 MS (scans a range of 40-400 daltons, 70eV). The injector temperature was set to 250ºC with the column initially set to 40º for one minute, then set to increase by 10ºC per minute until reaching 280ºC which was held for 20 minutes. The transfer line from the GC to the MS was set to 280ºC.

Statistical analyses

Analyses were all done in R (version 3.4.2, R Core Team 2017). I compared seed removal data from the field study with similar data of seeds removal of the same six tree species collected during a previous study (Ruzi et al. 2017) to assess whether removal was roughly consistent between years. Seed removal for this analysis was conducted by ants belonging to the entire ant community and was not restricted to only foragers of E. ruidum. The proportion of seeds removed to seeds placed (response variable) were compared with a linear mixed model

(LMM) that included year and year*tree species interaction as fixed effects, with site and replicate as a nested random effect. The LMM was conducted using the nlme package (version

3.1-131; Pinheiro et al. 2017). Tukey post-hoc tests of the proportion of seeds moved per year were conducted using the glht function in the multcomp package (version 1.4-8; Hothorn et al.

2008).

Field data were analyzed with generalized linear mixed models (GLMMs), zero-inflated models, and hurdle models using the glmmTMB function in the glmmTMB package (version 0.2.3 from github; Brooks et al. 2017) to determine if the number of time intervals ants attempted to remove seeds or dummy seeds differed by chemical treatment. glmmTMB is able to fit GLMMs with multiple error distributions which are typical for count data, including Poisson, and negative binomial distribution models (Brooks et al. 2017). The advantage of the glmmTMB package is that it is also able to incorporate overdispersion of the data and zero-inflated adjustments into the same models, and as all models are calculated with the same log likelihood method, akaiki information criteria (AIC) can be used to select the most parsimonious model (Brooks et al.

2017). For the field data, the models included the number of time intervals in which a removal attempt by E. ruidum occurred as the response variable, chemical treatment as a fixed effect, and

site and trial as nested random effects. I fitted models with Poisson and negative binomial distributions both with and without zero-adjusted parameters. If none of the GLMMs, zero- inflated models, or hurdle models converged, I then conducted a generalized linear model

(GLM). I compared models using AIC values using the AICtab function in the bbmle package

(version 1.0.20; Bolker and R Core Team 2017) to determine the model with the best fit

(summarized in Table C.2, and Table C.3). Tukey post-hoc tests and linear contrasts were conducted on significant fixed effects.

The response of ants to seed coat chemicals in the laboratory bioassay was tested using

LMMs to analyze the differences between treatments (fixed effect) in the proportion of filter paper discs moved from the foraging arena to filter paper discs presented (response variable), including colony identity (random effect). A Tukey’s post hoc means separation test was conducted on significant effects. Data were visualized using the ggplot2 package (version 2.2.1;

Wickham 2009).

RESULTS

Field bioassay

There was no interaction effect of year studied and species identity on seed removal (F5,99

= 1.61, P = 0.16). There was no difference in rate of seed removal by year (2013 vs. 2014) for the same four-hour time interval during the season that the seeds were presented in 2014 (F1,99 =

0.02, P = 0.90; Fig. 4.1A). However, tree species differed in the rates at which their seeds were moved by ants, with means significantly higher for Z. ekmanii and G. ulmifolia, and an intermediate value for O. pyramidale (F5,99 = 7.08, P < 0.001; Table 4.1, Fig. 4.1B).

Tree species differed in the number of time periods in which E. ruidum attempted to remove seeds or beads (Fig. 4.2; GLMM: distribution = negbin2, X2 = 32.08, df = 5, P < 0.001).

Zanthoxylum ekmanii had the greatest number of time periods in which E. ruidum attempted to remove seeds or dummy seeds, with O. pyramidale having intermediate removal attempts due to a high variance, and all other tree species experiencing a low number of removal attempts (Fig.

4.2). All other species were not significantly different from each other.

There was no effect of Z. ekmanii chemical treatment on whether E. ruidum would attempt to remove seeds or dummy seeds for the most parsimonious model tested, though the second most parsimonious model did find an effect of chemical treatment (Table 4.2, Table C.3).

E. ruidum workers appeared to attempt to remove dummy seeds treated with the hexane extract at rates similar to the Z. ekmanii seeds themselves (Fig. 4.3). The other tree species did not show this trend; however, E. ruidum workers did not attempt to move many seeds or dummy seeds

(Fig. C.2). Linear contrasts with Bonferroni adjustments found that E. ruidum workers attempted to remove, seeds similar amount of times to hexane extracts, seeds more often than methanol extracts, and hexane extracts more often than methanol extracts (Table 4.3).

Laboratory bioassay of seed coat chemicals

The ant colonies differed in the degree to which they moved the filter paper discs (Fig.

C.3). Nevertheless, ants moved a greater percentage of discs treated with the ethyl ether extract of seed coat chemicals than discs treated with the other fraction or controls) with the mean for the hexane extract being intermediate (F5,75 = 6.05, P < 0.001; Fig. 4.4). This finding suggests that ants were responding to polar compounds in the seed coat.

Chemical analysis of laboratory seed coat chemicals

The ethyl ether extract of seed coat chemicals shared some chemicals in common with the ethyl ether solvent control (Fig. 4.5). However, there were at least five compounds present in the ethyl ether seed coat fraction that were unique to that fraction. These chemicals have

tentatively been identified as aldehydes though comparisons to chemical standards are needed to confirm identifications (Table 4.4).

DISCUSSION

In this study, I examined whether ants would attempt to remove or remove dummy seeds treated with certain chemical cues on the seed coat of Neotropical pioneer tree species. Only one tree species, Z. ekmanii, appeared to potentially have seed chemistry play a role in removal attempts in both field and laboratory assays. This however, assumes that all chemicals were extracted from the seed coat using both hexane and methanol solvents. It is possible that some chemicals were missed, or that there may have been a synergistic effect of compounds found in these two separate extractions that was not considered as the hexane and methanol extracts were not pooled together. If either or both assumptions are not true, then other tree species may also have chemistry play a role in ant-mediated seed dispersal.

Seed removal in the field was not different by year, nor was there an interaction effect of tree species identity by year. However, seed removal did vary by tree species identity, when data from both years were pooled together. More data from additional years is needed to determine if seed-removal patterns remain consistent or if there is annual variation in these rates.

Tree species differed in the number of times that E. ruidum workers attempted to remove seeds or dummy seeds. This finding is consistent with the hypothesis raised in Ruzi et al. (2017) that there is a species-specific trait that mediates seed removal. Zanthoxylum ekmanii, which provides no visible food reward in terms of pulp or elaiosome, had the highest removal of seeds

(47-hour period, Ruzi et al. 2017; 4-hour period, 2013 and 2014 data combined) and the highest amount of time periods in which E. ruidum attempted to remove seeds or dummy seeds.

Zanthoxylum ekmanii was also the only species for which chemical cues appeared to play a role

in removal attempts in a field setting. Visually, E. ruidum attempted to remove dummy seeds treated with the Z. ekmanii hexane extract at similar amounts to the seeds themselves and to a lesser degree the Z. ekmanii methanol extracts, and did not attempt to remove either solvent controls or the dummy seed control. These patterns were based on the second most parsimonious model however, suggesting that increased replication is needed before robust conclusions can be drawn. In addition, these results may also be influenced by the silica bead dummy seeds themselves which were difficult for ants to handle. If a different, smaller medium was used for application of the extracts, I may have better been able to characterize removal rates and include additional, smaller ant species.

I further investigated the potential for the role of seed chemistry in mediating ant removal in the laboratory. In contrast to the field experiment with silica bead dummy seeds, E. ruidum workers could easily pick up the filter paper discs allowing for comparison of filter paper disc dummy seeds actually moved per treatment instead of removal attempts. Though colonies differed in their responses to these treatments, overall, treatment did influence the number of filter paper discs moved from the foraging arena into either the colony chamber or the waste pile.

Sometimes filter paper discs that went into the colony chamber were later moved to the waste pile (S.A. Ruzi personal observation). This behavior may be similar to that observed in elaiosome bearing systems, where ants consume elaiosomes attached to the seeds in the nest and then discard the seed in a waste pile (Handel and Beattie 1990, Giladi 2006). However, unlike in elaiosome bearing systems, if the attractant is a part of the seed coat then there is the potential for further dispersal movements whereas once the elaiosome is removed then further seed dispersal would be unlikely. The ethyl ether fraction is the fraction that elutes the more polar compounds from the original extract and it has been thought that polar lipids, such as oleic acid and its

derivatives are the important compounds in promoting the interaction of ants and elaiosome bearing seeds (Marshall et al. 1979; Skidmore and Heithaus 1988; Brew et al. 1989). However, oleic acid and its derivatives do not always increase seed removal by ants, suggesting that there may be a larger group of compounds that elicit ants to remove seeds (Midgley and Bond 1995).

It is also important to keep in mind that the presence of a chemical alone may not be enough to elicit the response, but may depend instead on the concentration of the chemical or the surrounding context in which the chemical is present. For example, Odontomachus brunneus workers only respond to the fertility signal in egg laying queens or workers if the surrounding nestmate hydrocarbon signal is also present (Smith et al. 2015).

Of the unique chemical compounds present in the ethyl ether fraction of Z. ekmanii seed coats, they have tentatively been identified as aldehydes. Some of these compounds (nonanal and

2-decenal) have been found to be components of essential oils. Nonanal, for example, is found in essential oils including essential oils from plants in the Citrus genus (Kim et al. 2019), which Z. ekmanii shares the Rutaceae family with. Others (e.g. 2-undecenal) are found in animal based foods (Kim et al. 2019); therefore, they could aid in perceiving seeds as prey. All five unique compounds to the ethyl ether seed coat fraction, or isomers of these compounds, have been identified as playing a role in chemical communication either of insects or mammals (El-Sayed

2019). Therefore, it is possible that these compounds are using established sensory systems within ants to elicit seed removal behavior as these compounds may already be important for insect communication in general.

Both G. ulmifolia and O. pyramidale experienced high to mid-levels of seed removal in the field assays; however, my results did not show that chemistry may play a role in seed removal attempts by E. ruidum workers. It is possible that chemistry does play a role, yet those

chemicals had not been extracted by the hexane and methanol solvents used. Alternatively, ants belonging to other species than E. ruidum may have driven these seed removal patterns as all seed removal was not limited to only E. ruidum foragers. For example, at the seeds of G. ulmifolia alone, there were an additional nine ant species, many of which included attine ants which have been recorded removing G. ulmifolia seeds (CHAPTER 2). Ochroma pyramidale had three additional ant species found at seeds. It is possible that if the behavior of these other ants were included in the analyses that a pattern in removal attempts would have been observed.

As these ants are small, interpreting their behavior at silica bead dummy seeds was difficult and therefore data from the foragers of these other ant species were not included in the analyses.

However, most ant samples were collected from the seeds of these two tree species, with much fewer samples being collected at the dummy seed treatments.

Myrmecochory in the traditional sense is considered a classic form of mutualism, in which seeds obtain a safe location to germinate and grow away from the parent plant and ants obtain a food reward called an elaiosome (Beattie 1985, Handel and Beattie 1990, but see

Warren II et al. 2019). However, in this non-traditional system of tree species that are not known to have evolved seed dispersal by ants. Zanthoxylum ekmanii, in specific, does not provide any sort of fruit pulp or aril, it is possible that this interaction is no longer a mutualism. Instead, this interaction could be more of a parasitic one with the plant taking advantage of a chemical cue that is important in some other aspect of ant ecology acting as a behavioral releaser.

Alternatively, the ants may be using or harvesting the chemicals from the seeds to perform other functions. For example, the seed chemistry may have defensive properties. For example,

Zalamea et al. (2018) found that Z. ekmanii was the only pioneer tree species studied out of 10 species tested (five species in common with this study, only C. longipes was not included) that

decreased the growth of two fungal pathogens (Fusarium sp. 1 and Fusarium sp. 2). More research is needed to determine what these specific chemical cue(s) is(are), the functional significance of these chemicals, and the nature of this interaction (ie. where it falls on the mutualism—parasitism spectrum).

TABLES

Table 4.1 Tree species used in the field experiment, their fruiting period, what was cleaned from the seeds, whether seeds were placed in water prior to study, and mean percentage of seeds removed (± standard error; SE) from 10h00 to 14h00 in 2013 and 2014.

Tree Species Family Fruiting Cleaned Seeds Season Seed Seed (species code) Period Floated Tested Removal Removal 2013 (Mean ± 2014 (Mean ± SE) (%) SE) (%) Apeiba Malvaceae February- Pulp removed Yes Dry 1.00 ± 1.00 4.00 ± 2.21 membranacea Marcha Aubl. (Ape) Cecropia Urticaceae July-Augustb Pulp removed No Wet 9.89 ± 8.83 11.00 ± 7.06 longipes Pitt. (Cec) Guazuma Malvaceae March-Aprilc No Dry 48.00 ± 12.54 33.00 ± 10.86 ulmifolia Lam. (Gua) Ochroma Malvaceae March-Maya Filaments No Dry 16.00 ± 9.21 25.00 ± 10.33 pyramidale removed Urb. (Och) Trema Cannabaceae August- Pulp removed No Wet 0.00 ± 0.00 20.00 ± 10.33 micrantha (L.) Octoberd “brown” (Tre) Zanthoxylum Rutaceae February- Yes Dry 51.00 ± 12.95 29.00 ± 10.80 ekmanii (Urb.) Marche Alain (Zan)

a Zalamea et al. (2015) b Zalamea et al. (2011) c Janzen (1975) d Dalling et al. (1997) e Zimmerman et al. (2007)

Table 4.2 Summary statistics of the most parsimonious models for the effects of chemical treatment (treatment) on counts of time periods in which E. ruidum attempted to remove a seed or silica bead for each tree species tested (codes explained in Table 4.1). Significant effects of chemical treatment are in bold. Model formulas are written as response variable ~ fixed explanatory variables + (1|random variables). * denotes the second most parsimonious model for Z. ekmanii (Zan). GLM = generalize linear model, GLMM = generalized linear mixed model

Tree Species Model Model Type Error Distribution Chisq DF P Ape Count ~ treatment GLM Poisson 0 5 1.00 Cec Count ~ treatment + (1|site/trial) GLMM Negbin2 0 5 1.00 Gua Count ~ treatment + (1|site/trial) GLMM Poisson 3.34 5 0.65 Och Count ~ treatment + (1|site/trial) GLMM Poisson 8.40 5 0.14 Tre Count ~ treatment GLM Poisson 0.32 5 1.00 Zan Count ~ treatment + (1|site/trial) GLMM Negbin2 7.08 5 0.21 Zan* Count ~ treatment + (1|site/trial) GLMM Poisson 13.36 5 0.02

Table 4.3 Summary statistics for linear contrasts with Bonferroni adjustments comparing the number of time periods E. ruidum workers attempted to remove seeds or silica bead dummy seeds with Z. ekmanii seed coat extracts. Linear contrasts were completed on the second most parsimonious model. Hypotheses in bold are supported by the significant P-value.

Null Hypothesis Alternative Hypothesis Z-value P Seeds ≤ Hexane extract Seeds > Hexane extract -0.40 0.92 Seeds ≤ Methanol extract Seeds > Methanol extract 3.33 < 0.01 Hexane extract ≤ Methanol extract Hexane extract > Methanol extract 3.60 < 0.001

Table 4.4 Tentative chemical compound identifications found in the ethyl ether seed coat fraction and the ethyl ether solvent control fraction. Compounds in bold are unique to the ethyl either seed coat fraction. Retention time refers to the time at which the compound passed through the column on the gas chromatograph-mass selective detector.

Retention Time Tentative Chemical Identification 3.62 3-hexanone or a branched isomer 3.70 2-hexanone or a branched isomer 3.78 Hexanal 4.85 Unknown 5.17 Unknown, but related to 4.85 5.44 Unknown, but related to 4.85 5.60 Unknown, but related to 4.85 6.28 Unknown 8.00 Nonanal 10.50 2-decenal 11.18 Unknown 11.25 2,4-decadienal isomer 11.63 2,4-decadienal isomer 11.88 2-undecenal 13.46 Impurity, derivative of BHT stabilizer 13.56 Derivative of BHT stabilizer 13.69 Unknown 14.32 BHT stabilizer

FIGURES

Figure 4.1 Average seed removal by year (A) and boxplots of seed removal by year and by tree species (B). Error bars represent standard error. Letters denote significance differences as determined by Tukey’s HSD means separation tests between species based on a LMM without the interaction term between species identity and year. The line in the boxplot represents the median value, the bottom and top of the boxplot represent the 25th and 75th percentiles of the data respectively, the whiskers extend to the most extreme data point that is not an outlier, and the points represent outliers (Wickham 2009). Outliers are those that are 1.5 x the inter-quartile range which is the distance between the 25th and 75th percentiles.

Figure 4.2 Boxplots of the counts of time periods in which E. ruidum attempted to remove seeds or treated silica beads related to each species trial of the six different tree species tested (all chemical treatments pooled; n = 60). Letters denote significant differences based on a Tukey post-hoc means separation test from the most parsimonious generalized linear mixed model (GLMM with a negative binomial error distribution). The bottom and top of the boxplot represents the 25th and 75th percentiles of the data respectively, the whiskers extend to the most extreme data point that is not an outlier, and the points represent outliers that are 1.5 x the inter- quartile range which is the distance between the 25th and 75th percentiles (Wickham 2009).

B B B AB B A 100 ●

●

75 ●

● ●

50 ● ●

● Time Points (%) Time

25 ● ● ●

● ● ● ●

0 Ape Cec Gua Och Tre Zan

Species

100

Figure 4.3 Average percentage of time points that ants attempted to remove seeds or silica beads of different experimental seed extracts and associated controls (n = 10). Error bars represent standard error. The line in the boxplot represents the median value, bottom and top of the boxplot represents the 25th and 75th percentiles of the data respectively, whiskers extend to the most extreme data point that is not an outlier, and the points represent outliers that are 1.5 x the inter- quartile range which is the distance between the 25th and 75th percentiles (Wickham 2009).

100 ●

●

● Time Points (%) Time

25 ●

0 Seeds Hexane Methanol Hexane Methanol Beads Extract Extract Control Control

Treatment

101

Figure 4.4 Boxplots of the percentage of filter paper discs moved by chemical treatment. Letters denote significant differences as determined by Tukey’s HSD means separation tests. The line in the boxplot represents the median value, bottom and top of the boxplot represents the 25th and 75th percentiles of the data respectively, whiskers extend to the most extreme data point that is not an outlier, and the points represent outliers that are 1.5 x the inter-quartile range which is the distance between the 25th and 75th percentiles (Wickham 2009).

AB B A B B B 100

● ●

● ● ●

25 ● ● ● Percentage of Filter Paper Discs Moved 0 Hexane 20% Cyclohex Ethyl Ehter Control Control Control Extract Hexane 20% Cyclohex Ethyl Ehter Treatment

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Figure 4.5 Chromatograms of ethyl ether seed coat fractions (A, B) and ethyl ether solvent control fractions (C, D). Arrows point to the five compounds that appear to be unique to the ethyl ether seed coat fraction.

103

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CHAPTER 5: QUANTIFYING SEED FATE IN ANT-MEDIATED DISPERSAL: SEED

DISPERSAL EFFECTIVENESS IN THE ECTATOMMA RUIDUM

(FORMICIDAE) – ZANTHOXYLUM EKMANII (RUTACEAE) SYSTEM

ABSTRACT

Ant-mediated seed dispersal has evolved repeatedly in many regions around the world.

Most research on ant-mediated seed dispersal focuses on removal rates and dispersal distances of elaiosome bearing seeds. Less is known about post dispersal seed fate, especially for plants that lack elaiosomes yet are also attractive to ants. For example, seeds of the pioneer tree species

Zanthoxylum ekmanii does not provide a food reward on its seeds yet is dispersal limited requiring light gaps that are unpredictable in time and space. Ants are attracted to Z. ekmanii seeds, but the consequence and potential effectiveness of ant-mediated seed dispersal are unknown. Ants may act primarily as seed predators, or they may bury and incorporate seeds into the seed bank, caching them until conditions suitable for germination occur. To assess the effectiveness of ants as a seed disperser of Z. ekmanii, I utilized the seed dispersal effectiveness framework, which incorporates both the quantity of seeds moved and the quality of the dispersal services. Tracking the movement of seeds from seed caches on the forest floor revealed that foragers of Ectatomma ruidum, the most common visitor to seed caches, moved 32.8% of seeds an average first movement distance of 99.8 cm (SD: ± 91.1cm) with a 68.3% being brought into a colony. Further dispersal may occur however as E. ruidum engages in intraspecific thievery.

The quality of deposition location was assessed using a seedling emergence study where freshly germinated seeds were buried at 0, 2, 5, and 7 cm depths, though seedlings were primarily able to emerge from the 0 and 2 cm depths. Wax casting of E. ruidum colonies demonstrated that

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seeds brought into the colony were deposited in chambers that also had larvae present and experienced more damage than seeds that had not been moved by ants. Foragers, however, did not have a strong enough bite force to break through the hard layer of the Z. ekmanii seed coat which is likely based on how their muscle morphology is not structured to maximize force generation. Overall, E. ruidum may help fine tune deposition location, incorporating seeds into the topsoil, though few seeds will likely emerge if deposited within the colony if soil bioturbation is low.

INTRODUCTION

Ant-mediated seed dispersal is both phylogenetically and geographically widespread with an estimated 101 to 147 convergent evolutions (Lengyel et al. 2010). Traditionally, this interaction is considered a mutualism, where plants benefit in multiple ways and the ants receive a nutritious food reward. However, the nature of this interaction as a mutualism has more recently come under question, particularly from the perspective of the ants (e.g. Gammans et al.

2005, Warren II et al. 2019). Research often fails to find an effect of foraging on elaiosome bearing seeds on various components of colony fitness (e.g. worker number or number of males produced, Gammans et al. 2005; growth, Warren II et al. 2019), and some plants fail to provide any food resource to the ants at all (CHAPTER 3). In contrast, there are many hypothesized benefits of ant dispersal to plants including directed dispersal into favorable microsites, increased dispersal distance, and protection from predators and pathogens (reviewed in Giladi 2006).

The benefits of ant-mediated dispersal to plants vary depending on how the benefit is defined, the plant species studied, and the ecosystem it is studied in. The stated benefits of directed dispersal are typically due to ant nests providing favorable microsites for germination or growth of the plants (e.g. Davidson and Morton 1981, Sankovitz et al. 2019). Giladi (2006)

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found about 46% of 46 studies supported this hypothesis based on evidence that ant nests benefited seeds by providing a favorable nutritional environment. Dispersal away from the parent plant is beneficial if seeds experience species-specific negative density dependent mortality near the maternal plant (though these negative effects can also be felt near conspecific trees; Janzen 1970, Connell 1971, Augspurger 1983, Comita and Hubbell 2009, Mangan et al.

2010, Bagchi et al. 2014). Typically, ants act as short-distance dispersers that may fine tune seed deposition location after primary dispersal by a vertebrate or bird. However, ant dispersal distances vary greatly depending on the species being investigated and range from a few centimeters up to 180 m (Gómez and Espadaler 2013). The dispersal distance hypothesis was supported by 76% of 17 studies examined (Giladi 2006). A third proposed benefit comes from predator-avoidance when ants incorporate seeds into the soil where they are less likely to consumed (Giladi 2006). Burying seeds may reduce olfactory cues that rodents use to detect them (Paulsen et al. 2013). For example, rodents detected 92% of seeds 8-20 mm max length when buried 1-2.5 cm within the soil, though the detection rate rapidly decreased with increasing burial depth (Estrada and Coates-Estrada 1991). Giladi (2006) found 81% of 27 studies supported the predator-avoidance hypothesis. Despite the evidence supporting each hypothesis comes from diverse taxa, research from tropical forests or from trees are generally underrepresented.

Research on ant-mediated seed dispersal has focused on the dispersal of myrmecochorous plants that mediate seed dispersal by having an elaiosome attached to the seed (Beattie 1985,

Handel and Beattie 1990). While myrmecochorous plants occur world-wide in several plant lineages, together they make up only 4.5% of all angiosperm species (Lengyel et al. 2010), leaving a knowledge gap in ant-mediated seed dispersal for the vast majority of angiosperms.

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Many other plant species may participate in ant-mediated seed dispersal but are currently overlooked because ants have yet to be observed interacting with them. For example, seeds of some plants that that have been previously thought to lack an elaiosome have now been identified as being dispersed by ants as they have an aril (e.g. Pizo and Oliveira 1998, Passos and

Oliveira 2002, Christianini and Oliveira 2010, Magalháes et al. 2018) or fruit (e.g. Barroso et al.

2013). Alternatively, chemical cues from the seed coats of some species may elicit seed removal

(CHAPTER 4). These chemicals may mimic food rewards by having chemical cues on their seed coat that act as a behavioral releaser (e.g. 1,2-diolein acts as a behavioral releaser in elaiosome bearing systems by eliciting removal responses in ants, Marshal et al. 1979). Without providing a food reward, plants may be exploiting the behavior of ants, particularly those that are not capable of processing the seeds themselves for food (e.g. non-granivorous species).

Neotropical pioneer tree species are an excellent system to examine the cues responsible for ant dispersal in the absence of a food reward as they lack elaiosomes yet are dispersed by a wide variety of ants (Pizo and Oliviera 1998, CHAPTER 3). Pioneer species face many barriers to recruitment of new individuals into a population. While they overcome source limitation by producing many small seeds, only a fraction of seeds reach suitable microsites for germination and establishment (Dalling et al. 2002). Pioneer species require high light microsites for seeds relying either on diel temperature fluctuations or the ratio of red to far red light to germinate depending on seed size (Pearson et al. 2002). Treefall gaps are examples of suitable microsites

(Hubbel et al. 1999) and can be relatively common in forests though spatially heterogeneous

(Martinez-Ramos et al. 1988). Seeds of pioneer species reach these gaps by either being good colonizers (e.g. dispersing long distances) or remaining viable for long periods of time waiting for conditions to become favorable. For example, Zanthoxylum species produce many seeds with

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a short (0.8 m) median dispersal distance (Dalling et al. 2002) but can remain viable in the seed bank for at least 18 years (Dalling and Brown 2009). Like other Zanthoxylum, the seeds of the

Neotropical pioneer tree Zanthoxylum ekmanii Alain. lack an aril, pulp, and are not known to provide an elaiosome food reward, yet are attractive to ants, especially the common, ground dwelling ant Ectatomma ruidum (CHAPTER 3).

Here I use the dispersal effectiveness framework (Schupp 1993; reviewed in Schupp et al.

2010) to examine the effectiveness of the ant E. ruidum as a dispersal agent for the pioneer tree species Z. ekmanii. Dispersal effectiveness was first developed for endozoochorous dispersal in which animals consume fruit or seeds (Schupp et al. 2010). This framework breaks down seed dispersal effectiveness into quantitative and qualitative components (Fig. 5.1). The quantitative component consists of the number of visits a dispersal agent makes and the number of seeds dispersed per visit, while the qualitative component consists of the quality of the deposition location and the quality of handing (Schupp 1993, Schupp et al. 2010). In endozoochorous systems more than one seed is dispersed per visit, and the quality of handling relates to gut passage time. Adapting this framework to ants, ants may need to visit seeds multiple times before seeds are successfully dispersed, and ants typically only disperse one seed at a time. As seeds do not pass through the gut of ants intact, the quality of ant handling refers to any processing / damage the seeds may accrue either during dispersal by foragers or by larvae attempting to break down and eat the seeds. The aim of this study was to characterize the short- term seed fate of Z. ekmanii by assessing the quality of dispersal by E. ruidum. Specifically, the following questions are addressed: (1) Does seed removal vary by year? (2) Does E. ruidum conduct directly dispersal, deposit seeds in their colonies at distances that are biologically relevant (i.e. potentially far enough away to escape negative density dependent drivers of

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mortality)? (3) Can seeds survive and emerge from the deposition location? And (4) to what extent do E. ruidum damage the seeds that they disperse?

METHODS

Study site and species

Seed removal rate and dispersal distance experiments were carried out at five sites > 350 m apart from each other (and at least 20 m from conspecific trees; Zalamea et al. 2015) on Barro

Colorado Island (BCI) (9°10’N, 79°51’W), Republic of Panama. BCI is a seasonal lowland tropical rainforest, experiencing most of its annual 2600 mm rainfall during its wet season (late

April or early May to late December or early January; Windsor 1990). Wax casting of ant colonies were conducted at Parque Soberania near Gamboa (9°11’N, 79°70’W), Republic of

Panama.

The Zanthoxylum genus is widespread, with most species being found pantropical and some species found in temperate regions (Appelhans et al. 2018). Zanthoxylum ekmanii

(formerly Z. belizense Lund.) is found ranging from Southern Mexica through Panama and potentially into Colombia (Croat 1978). It is a dioecious pioneer species that ranges from 13 to

30 m tall, can reach one meter in diameter at breast height, flowers in the August to October, and fruits between January and March (Croat 1978). They are recorded in the literature as having animal dispersal as their primary dispersal mode (Muller-Landau et al. 2008) with birds mentioned as likely dispersers and monkeys as potential dispersers (Croat 1978). Seeds generally fall still attached to their infructescences beneath the parent crown, are about 3.5 to 5 mm long

(Croat 1978) but are not known to have an elaiosome, aril or fruit to act as a food reward to ants

(S.A. Ruzi personal observation). The only potential food reward comes from the outer layer of

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the seed coat, the sarcotesta, which is present in all members of the Zanthoxylum genus (Hartley

2001).

Seeds of Z. ekmanii were collected during the dry season, roughly February through April in the Barro Colorado Nature Monument (BCNM) (Zimmerman et al. 2007). Seeds were rinsed with distilled water and allowed to dry in an air-conditioned (~ 22°C), dehumidified dark room to prevent molding. Only seeds that sunk in water were used in the study as these are likely to be viable seeds.

Ectatomma ruidum is the most common ant at the five sites in this study and was frequently observed removing Z. ekmanii from the soil surface in both 2013 and 2014

(CHAPTERS 3 and 4). Ectatomma ruidum is a diurnal foraging omnivorous ant known to consume insects and plant-based resources, including tending insects for honey-dew (Pratt 1989,

Lauchaud 1990). Ectatomma ruidum is a relatively large ant (Weber’s length of 2.780 mm) and tends to forage individually though can recruit other foragers if the resource is large or if it is a high-quality resource (e.g. sugar baits, Pratt 1989).

Quantitative component – seed removal rates

To determine the number of seeds dispersed by ants, seed caches were placed on the forest floor during the dry season in 2013, 2014, 2015, and 2016 and observed from 10h00 to

12h00. For each year, only seeds collected in that year were used in the study. In 2013 and 2014,

10 Z. ekmanii seeds were placed per cache (n = 10 caches per year). In 2015 and 2016, caches consisted of five Z. ekmanii seeds (2015 = 15 caches, 2016 = 10 caches). Approximately 0.75 m2 of leaf litter was cleared around the caches and baited from 09h30 to 10h00 with Quaker oatmeal cookies (granola flavor). This was done to increase visibility around the cache to track ants / seeds and to encourage ant activity back into the area after the litter was cleared. I expect that

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seed removal in 2015 and 2016 will be greater than in other years as a consequence of increased ant activity due to baiting. If a seed was dropped or brought into a colony, but then picked up and carried to another location by a different ant, I split these observations into “first” and “second” movements (Fig. 5.2). Ants removing seeds were either identified on site or by collecting vouchers after the dispersal event.

Qualitative component – deposition location and ant handling

Distance to seed deposition location - The same caches of five Z. ekmanii seeds were used to estimate seed removal distances to deposition location. Seeds were each marked with an individual colored dot of enamel modelling paint (Testors®) to facilitate tracking individual seeds. This paint has had no observable impact on seed removal by ants of other species (see

Passos & Oliveira 2002, Magalháes et al. 2018). A screw with the same color paint as the seed was placed wherever the seed was dropped or at the colony entrance if it was taken into a colony.

At 12h00, the straight-line distances of the seed movements were measured with respect to the original location for the first movement and from screws for subsequent movements. The locations of screws were categorized as either being dropped, brought to a colony, or location unknown (e.g., if not observed). Occasionally multiple seeds were being removed at once and so not all possible movements, distances, and deposition locations were observed. Distances and deposition location were recorded for all movement numbers (i.e. first, second and third; described in quantitative component; Fig. 5.2).

Seed deposition location - To assess the quality of the deposition location by determining chamber depths, location of seed deposition within nests, and distribution of ants within colonies,

13 colonies were wax casted in 2015 and 2016 following methods by Tschinkel (2010). These casts were subsequently melted down after chamber depths were measured. Six of the 13 casts

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were provisioned with Z. ekmanii seeds on the same day as casting. Chambers were numbered by following the tunnel from the nest entrance deeper into the soil; therefore, shallower attached chambers were given lower numbers than deeper chambers. Chamber depths were measured as the distance from the soil surface, marked by the entrance to the colony, to the bottom of the chamber.

Seedling emergence - Chamber depths were compared to the proposed maximum emergence depth for this seed species. This value was estimated using the following formula from Bond et al. (1999):

maximum emergence depth = 27.3 ∗ 6778 97:;ℎ=>.??@

Ten replicates of 50 fresh seeds each were massed on a microbalance to the nearest 0.1 mg

(Table D.1). To assess whether Z. ekmanii could emerge from burial depths in ant nests, a seedling emergence study was conducted in the 2018 dry season. Soil was collected from beneath two mature Z. ekmanii trees on BCI and sieved through a 0.2 mm sieve to collect seeds.

Seeds that sunk in water and did not have holes were placed in paraffin sealed petri dished lined with two layers of moistened paper towel in an ambient air greenhouse (temperature: approximate 22-34 ºC; humidity: approximately 45-94%) on BCI. These seeds were checked daily for germination. Seeds were buried in clear plastic tubing (2.5 cm diameter) either the same day as germination or the day after germination. Plastic tubing with seeds were placed in a plastic shelf that had a white Styrofoam sheet above and below it. This was then covered with two layers of shade cloth to reduce sunlight. Each tube was filled with sieved soil to the five cm mark before seeds were gently placed at regular intervals along the circumference of the tube and then more sieved soil was carefully poured over the seeds until the desired burial depth was reached. Burial depths consisted of 0 (n replicates = 4), 2 (n = 14), 5 (n = 12), and 7 cm (n = 8)

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below the soil surface. Each tube was massed using a microbalance before and after soil and seeds were added so that the density of the soil could later be calculated and compared to the reported bulk density of soil from Gigante peninsula (0-10 cm depth = 0.74 g per cm-3 and 10-20 cm depth = 0.73 g per cm3; Cavelier 1992), which is located within the Barro Colorado Nature

Monument (BCNM). The soil used for burial was the same soil the seeds were originally collected in. Tubes were watered with distilled water as needed. To assess if soil microorganisms or pathogens influenced the success of seedling emergence there was a non-sterilized and a sterilized soil treatment for the 2 (non-sterilized: n = 5; sterilized: n = 9), 5 (non-sterilized: n = 4; sterilized: n = 8), and 7 cm (non-sterilized: n = 4; sterilized: n = 4) depths. Sterilized soil was autoclaved at 121°C for 2 h.

Assessment of seed damage - To assess the handling damage caused by ants to seeds, the percentage of seed coat damaged was compared between seeds that presumably have never encountered ants and seeds that had been retrieved from wax casts. Seeds that had presumably never encountered ants were collected from infructescences that had fallen under the parent crown. Images were made using Leica Application Suite (LAS) Core (version 4.9.0, Leica

Microsystems, Switzerland), a Leica M205 C stereo microscope (467 nm resolution) that had an attached Leica DFC 425 digital camera (5 megapixel). Multiple images per seed taken at different focal depths were stacked using Zerene Stacker (version 1.04, Zerene Systems LLC) using the align and stack all (PMax) setting. Seeds were oriented in the same plane so that as much surface area as possible would be visible in the stacked images. Stacked images were then assessed in ImageJ (version 1.52a, Schneider et al. 2012) by calculating the surface area of the seed and any damaged areas on the seeds. The percentage of damaged area per seed was used for later analyses so that the amount of damage was standardized to seed size.

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To determine if E. ruidum workers have the mandible strength to damage seeds, bite force measurements on four workers were taken using a modified piezoelectric sensor and software (FlexiForce, ELF system, Tekscan) to record real time mandibular pressure (10 g force required to start the recording). The sensory was modified by removing excess plastic around the edge to better allow for ants to access to the sensor while biting. The exposed edge of the sensor was covered with parafilm to provide some protection. The maximum force reached was recorded for three to five bites, each 20 sec long. All bites were recorded in a single session and ants were not returned to the colony. The average fracture resistance of seeds was taken from

Zalamea et al. (2018) and is 68.3 N (SD = 24.3, n = 100) and has the second largest seed coat thickness of (mean = 268.8 micrometers, SD = 58.0, n = 35) out of 16 Neotropical pioneer tree species measured (Zalamea et al. 2018).

One individual of E. ruidum with bite force data was fixed in alcoholic Bouin’s solution

(Signma Aldrich Corporation, Steinheim, Germany) to assess muscle volume with regards to the bite force generated. After 48 h in Bouin’s solution, the ant was washed and dehydrated in an increasing concentration of ethanol for at least 20 min in each concentration (70%, 80%, 90%,

95%, and twice in 100%). The ant was then stained overnight in a two percent iodine in 100% ethanol solution before being washed and transferred back to 100% ethanol. The musculature of the ant’s head was then assessed using an X-ray microtomography (microCT) using an Xradia

MicroXCT-400 scanner (Carl Zeiss, Oberkochen, Germany) after the individual was critical point dried (AutoSamdri-93.1GL Supercritical Point Dryer, Tousimis Research Corporation,

Rockville, MD). The machine was set to a source voltage of 25 kV with a source power of 5 W and an exposure time of 5 sec. The source-RA distance was 28.05 mm and the detector-RA distance was 30.05 mm. A total of 1441 images were taken while rotating 360 degrees.

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Segmenting of muscles and determination of relative muscle volume was produced using Amira

5.4.5 (FEI, Hillsboro, OR).

Statistical analyses

Analyses were done in R (version 3.4.2, R Core Team 2017) unless otherwise noted. All graphics were visualized using the ggplot2 package (version 2.2.1; Wickham 2009).

To determine if seed removal differed by year studied, seed removal data over the same two-hour period (10h00 to 12h00) in the dry season in 2013 (Ruzi et al. 2017) and 2014

(CHAPTER 4) were included with seed removal data from this study (2015 and 2016). This was assessed using a linear mixed effects model (LMM) using the nlme package (version 3.1-131;

Pinheiro et al. 2017) with the proportion of seeds removed to seeds placed as the response variable, and site and trial as a nested random effect. A Tukey HSD means separation test was performed using the emmeans function in the emmeans package (version 1.3.1, Lenth 2018).

To assess dispersal distances and deposition locations of seeds, trials from 2015 and 2016 were pooled together. To describe the distribution of seed distances moved by E. ruidum, the skewness and kurtosis functions in the e1071 package was used (version 1.7-0, Meyer et al.

2018). Skewness is a measure of how the distribution differs from a symmetrical normal distribution (StatSoft 2013), therefore a negative value indicates the distribution is skewed left and a positive value indicates the distribution is skewed to the right. Kurtosis is a measure of the sharpness of the peak in a distribution and that value for a normal distribution is zero (StatSoft

2013).

A one sample t-test was used to compare chamber depth to the estimated maximum emergence depth. Each chamber number in which seeds were found in at least one wax cast was

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tested independently from one another. Shapiro-Wilk tests were conducted prior to t-tests to check for the assumption of normality on chamber depth.

A LMM was used to assess whether the fixed effects of burial depth (as a factor) and soil sterilization (either sterilized or not) impacted the ratio of seedling emergence to seeds placed

(response variable). Tukey post-hoc means separation tests were conducted on significant fixed effects. The mean soil bulk density was compared to both reported soil densities from Cavelier

(1992) using a one-sample Wilcoxon signed rank tests as soil bulk density was not normally distributed (W = 0.92, P < 0.01).

A Welch two sample t-test assuming unequal variances was used determine if there was a difference in mean surface area percentage that exhibited processing or damage between seeds that were never placed in contact with ants and ones that were recovered from colony wax casts.

A one sample t-test was used to determine if the maximum bite force E. ruidum workers produced was different than the 68.3 N force (Zalamea et al. 2018) required to rupture Z. ekmanii seeds. A Shapiro-Wilk test was first conducted to check the assumption of normality on maximum bite force data.

RESULTS

Quantitative component

There was a significant difference in 2-hour seed removal rates for Z. ekmanii by year studied (F3,27 = 4.76, P < 0.01). Both 2015 and 2016, which were baited with cookies, had 58% seed removal in two hours (2015 = 58.67% mean ± 11.46% SE, 44 seeds out of 75 placed; 2016

= 58.00% ± 13.81%, 29 seeds out of 50 placed). In contrast, 2013 and 2014 had the lower seed removal rates (2013: 31.00% ± 10.69%; 2014: 15.00% ± 6.37%) (Fig. 5.3). Combining data from

2015 and 2016, 32.8% of placed seeds (41/125) experienced a first movement by E. ruidum,

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7.2% (9 seeds) experienced a second movement by E. ruidum, and 0.8% (1 seed) experienced a third movement by E. ruidum.

Qualitative component

The Z. ekmanii seeds dispersed by E. ruidum in 2015 and 2016 experienced a first movement distance of 99.8 ± 91.1 cm (mean ± SD, n = 39 seeds, 2 seeds were lost during tracking) and a second movement distance of 61.5 ± 70.9 cm (n = 9 seeds, Fig. 5.4A). The only seed moved a third time by E. ruidum was moved an additional four cm. Both first and second movement distances were characterized as being skewed to the right (statistics summarized in

Table D.2). However, the sharpness of their peaks was higher than that of a normal distribution for the first movement and lower than that of a normal distribution for the second movement

(statistics summarized in Table D.2). Pooling all movements together, a total of 51 seed destinations were recorded: 32 seeds were taken into an E. ruidum colony, 15 seeds were dropped while being followed, and 5 seeds were lost while following other ants moving seeds

(Fig. 5.4B). Most of the times seeds were moved into a colony from a cache, they went into one

E. ruidum colony, though they occasionally were taken into two different colonies (e.g. Fig. 5.2) or three different colonies.

Wax casts revealed colonies typically had at least three (up to a max of six) chambers at average depths of 7.72 (min = 3.8), 9.92 (min = 5.7), and 13.58 (min = 6.7) cm. Notably, chambers one through four sometimes were shallower than the estimated maximum emergence depth of 70.26mm from Bond et al. (1999) depending on the colony (Fig. 5.5B, Table D.3).

Workers tended to be present in all chambers while larvae were mostly present in chambers 1-3 and occasionally in chamber 4, and Z. emanii seeds were present in chambers 1-3 (Table D.3,

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Fig. 5.5A). Of the chambers where Z. ekmanii seeds were found, the first chamber was the only one not significantly different from the estimated maximum emergence depth (Table 5.1).

The soil bulk density (0.82 g per cm3 ± 0.04, mean ± standard deviation) used in the study was significantly greater that either of the reported 0.74 g per cm3 (V= 731, P < 0.001) or

3 0.73 g per cm (V = 741, P < 0.001) from Cavelier (1992) (Table D.4). Only burial depth (F3,20 =

7.68, P < 0.01) significantly influenced the likelihood of seedling emergence, while whether the soil was sterilized (F1,20 = 0.98, P = 0.33) did not. Both the 0 cm and 2 cm depths had the highest proportion of seedlings emerging (Fig. 5.6), with only one seedling emerging from the 5 cm depth.

Thirty-seven seeds in total were recovered from the six colonies that were provisioned with seeds prior to wax casting (Table D.3). As mentioned previously, these seeds were found mainly in chambers 1-3 of casts, with only one seed not being found in a chamber (Fig. 5.5A).

Of these, only one seed was recovered with an exposed embryo and this seed was not included in the analysis of surface area damage. There was a significant difference in the percentage of seed surface area damaged by ant handling between seeds recovered from inside a colony (i.e. from 6 wax castings, n = 36) or from seeds that have presumably never interacted with ants (n = 41) (t- test: t = -5.04, df = 48.9, P < 0.001; Fig. 5.7A). Seeds that were damaged by foraging ants had a pitted look (Fig. 5.7B and C) while unhandled seeds were smooth (Fig. 5.7D). Eighty-three percent of seeds recovered from chambers in the wax casts were found in chambers that also had larvae present.

The maximum bite force from the four ants ranged from 0.060-0.092 N (Table 5.2).

Ectatomma ruidum workers produced a much smaller bite force than the force recorded in

Zalamea et al. (2018) needed to rupture Z. ekmanii seeds (t-test: t = -8990, df = 3, P < 0.001) and

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those bite forces met the assumption of being normally distributed (Shapiro-Wilk: W = 0.86, P =

0.25). One of the E. ruidum workers that had a maximum bite force of 0.092 N had its muscle volumes summarized in Table D.5 (Fig. D.1).

DISCUSSION

I used the seed dispersal effectiveness framework (Schupp 1993, Schupp et al. 2010) to assess the short-term seed fate for Z. ekmanii seeds moved by E. ruidum foragers. In general, E. ruidum foragers were responsible for moving over half of seeds moved by ants (41 of 73 seeds), highlighting the importance of this species and a seed dispersal agent in this system. Seeds were not moved far (approximate one meter on average) but did experience directed dispersal as most were brought into a colony. Once deposited in a colony, seeds were typically placed in chambers at depths too deep for seedling emergence to occur reliably indicating that seeds would likely need to remain dormant, be further dispersed, or experience bioturbation for effective dispersal to occur. Ectatomma ruidum workers did not produce enough force to easily crush Z. ekmanii seeds, but were able to remove the outer layer of the seed coat. Together, the data suggest that while E. ruidum is an effective secondary dispersal agent over relatively short distances, seeds cached within the colony will not likely increase seedling emergence rates if the colony is the final place of deposition.

Seed removal of Z. ekmanii varied by year, with higher removal rates in 2015 and 2016 years (over 50% both years) than in 2014. The only taxa observed removing Z. ekmanii seeds from all years were ants (Formicidae). In 2015 and 2016 combined, 41 of the 125 seeds placed were removed initially by E. ruidum. The higher removal in 2015 and 2016 is not unexpected as sites were first baited with cookies to increase ant activity in the area where leaf litter was cleared and seeds were placed. Alternatively, in 2013 and 2014, there were other caches placed

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one meter away from the seed cache consisting of either other seeds (2013; Ruzi et al 2017) or chemical extracts from the same seed species (2014; CHAPTER 4) which could have reduced attraction at the seed caches.

Ants are not typically thought of as long-distance dispersers, and are more likely responsible for fine-tuning the deposition location of seeds (reviewed in Giladi 2006). Therefore, they are unlikely to disperse seeds far enough away from the parent to escape negative density dependent causes of mortality. Ectatomma ruidum fits this model of short distance dispersal and moved seeds an average straight-line distance of 99.8 cm with a maximum first movement distance of 405.7 cm. These values are similar to those reported for other dispersal distances by

E. ruidum (70 cm +/- 81 cm, maximum = 385 cm in Zelikova and Breed 2008) and other

Ectatomma spp. (Ectatomma edentatum: mean = 0.71cm, max = 400 cm, Magalhães et al. 2018).

Though this dispersal distance is larger than the 42 cm reported for the Ectatomma genus in

Gómez and Espadaler (2013). Our dispersal distances may reflect the high E. ruidum colony densities on BCI which have been estimated at 1.06 entrances per m2 (Pratt 1989). These distances are also straight line distances, and E. ruidum workers did not always walk in a straight path but took more meandering ones, making the overall distance they travelled carrying seeds much longer (S.A. Ruzi personal observation). Additionally, E. ruidum occasionally moved seeds that were initially moved by other ants, and even took seeds out of one E. ruidum colony in another direction (4 observations) with one seed going from one E. ruidum colony into another

E. ruidum colony (S.A. Ruzi personal observation). Ectatomma ruidum have been demonstrated to engage in thievery, where foragers from one colony are able to successfully infiltrate another colony and intercept food items being brought into the nest (Guénard and McGlynn 2013), which could potentially explain our observations that seeds removed into one colony do occasionally

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reappear and are moved to another colony. If seeds remain attractive over time then there is the potential for further dispersal similar to what was found by Jansen et al. (2012) with rodents stealing cached seeds from other rodents. These tertiary dispersal mechanisms can increase the overall dispersal distance of the seeds, especially as these seeds can remain viable in the soil for decades (Dalling and Brown 2009).

Consistent with the directed dispersal, Ectatomma ruidum workers brought over half the seeds they moved into a colony. However, colonies themselves vary in their suitability regarding seedling emergence as chamber depths are variable. Chambers 1-3 of some colonies were shallower than the estimated maximum emergence depth of 7.26 cm; however, these are also the chambers that tend to have larvae present, which are the food digesting systems of the colony

(Cassil et al. 2005, Dussutour and Simpson 2009). Using both the depths at which seeds may be cached and the estimated maximum emergence depth with a seedling emergence study suggests that freshly germinated Z. ekmanii seeds may have a much shallower maximum emergence depth than expected. A similar finding was described by Renard et al. (2010) for seeds of Manihot esculenta subsp. flabellifolia, a myrmecochorous plant found in French Guiana savannas that is dispersed by Ectatomma brunneum. The predicted maximum emergence depth (11.7 ± 1.4 cm) was deeper than the depths seedlings could emerge from though seeds were often found buried deeper than the predicted maximum emergence depth from within colonies of E. brunneum.

Bond et al. (1999) generated the formula for maximum seedling emergence depth by planting seeds of 17 species ranging in seed mass from 0.1 to 100 mg. However, they tested burial depth in sand, which was not added to the substrate in which Z. ekmanii seeds were grown during this experiment. Additionally, seeds of Z. ekmanii have thick seed coats (Zalamea et al. 2018), therefore, the mass of the intact seeds does not accurately represent the amount of stored energy

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available to the seedling for growth before photosynthesis occurs. Using the embryo mass may have more accurately estimated the maximum emergence depth, which would have been shallower than the estimated depth used here. Fresh seeds of Z. ekmanii seeds also have low germination probability though the probability of germination increases with time buried in the soil (P-C. Zalamea unpubl. data). Also, 100% of Z. ekmanii seeds that are initially viable can remain viable even after 30 months in the topsoil (Zalamea et al. 2018) and some seeds have been recorded as viable up to 18 years (Dalling and Brown 2009). If E. ruidum workers are engaging in thievery and moving seeds, potentially discarding them in waste piles, it is possible that seeds may not remain in colonies long enough to germinate. Therefore, colonies may not be the final deposition place of seeds and may not dictate the microhabitat that seedlings must be able to emerge from. Alternatively, the tubes used in the seedling emergence study may not accurately represent the conditions freshly germinated seeds experience in nature. These tubes were filled with soil that had been sieved through a two mm mesh sieve, which could have decreased pore size and contributed to the density of the soil being greater than that measured in the field.

Regardless of whether the colony was the final deposition location, seeds that had been taken into E. ruidum colonies had more damage or processing marks on the surface area of the seed than those that had never encountered ants. However, all the damage observed was confined to the sarcotesta being removed, while the inner layer of the seed coat (the sclerotesta) remained intact. Ectatomma ruidum foragers were observed biting the seed multiple times to get a better hold on the seed prior to dispersing it from the cache location (S.A. Ruzi personal observation) and may have scraped off some of sarcotesta of the seed coat during that handling time.

Alternatively, this damage could come from handling by larvae, as larvae conduct most of the

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food digestion in ant colonies (Cassil et al. 2005) and seeds were almost always found in chambers that also had larvae. Some seeds that were in the control group (i.e. ones that presumably have never encountered ants) did have damage. This could have occurred during seed collection as seeds were plucked from their capsules using forceps that have ridges on the end. Only one seed recovered from the wax castings had an exposed embryo. This seed was cut in half and was most likely damaged when processing of the wax casts occurred, as casts were cut and partitioned into their different chambers for processing.

Though E. ruidum workers were able to remove the outside of the seed coat, workers do not produce enough bite force to rupture Z. ekmanii seeds. In the genus Pheidole, there are both minor and major worker castes, and the differences in head size between these castes is greater in seed harvesting species than in species that have other diet preferences (Holley et al. 2016).

These major workers have often been proposed as specialized seed millers (e.g. Wilson 1984) as their larger heads fit larger mandible closer muscles (Paul and Gronenberg 1999). The angle at which these closer muscles attach to a region of the mandible called the apodeme determines whether speed (0º) or force (41 - 44º) is maximized. For E. ruidum this angle is 22.7 ± 3.4º, while Pogonomyrmex badius (a dimorphic ant known for milling seeds) is 38.4 ± 8.2º (data from

Paul and Gronenberg 1999). Therefore, E. ruidum’s closer muscle is not configured in a way to maximize force output for crushing seeds.

Overall, I have demonstrated that ants of E. ruidum will disperse seeds of Z. ekmanii that were previously unknown to provide an elaiosome food reward on BCI. While E. ruidum consistently remove seeds away from the parent plant, these seeds do not move far through single dispersal events. However, these dispersal events were tracked over a two-hour window of time, which is extremely short compared to the amount of time these seeds have been recorded

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as remaining viable in the soil seed bank (Dalling and Brown 2009). If these seeds remain attractive to ants for years, there is the potential through intraspecific thievery that these single dispersal events poorly estimate the absolute distance a seed may move after experiencing multiple dispersal events. Seeds are often brought into the colony, but whether seeds remain there long enough to germinate in unknown. If they were to germinate, it is unlikely that seedlings would survive this burial depth. However, diel temperature fluctuations are needed for seeds of larger pioneer tree species (> 2 mg) to germinate (Pearson et al. 2002). Therefore, seeds of Z. ekmanii are to not likely to germinate at the deeper burial depths indicated by the wax casting as the magnitude of diel temperature fluctuations decreases with increasing depth

(Pearson et al. 2002). As a result, seeds will likely only germinate if the soil has been perturbed.

For example, tip-up mounds caused from fallen trees have a higher abundance of pioneer tree seedlings than other areas within tree fall gaps with the species composition of pioneer tree seedlings reflecting the composition of seeds in the soil in the same location (Putz 1983). Both the potential negative impacts (e.g. damage and caching at depths too deep for seedling emergence) or positive impacts (e.g. incorporation into the seed bank and further dispersal) of ant dispersal of seeds increases our awareness that ants may influence the seed bank of

Neotropical pioneer tree species. However, these interactions need to be studied in the context of other seed traits, such as dormancy cues and long-term seed viability, and ecological processes, such as bioturbation of soil. Additionally, climate change could have severe consequences on the recruitment dynamics of these long-lived seeds as changes in temperature, humidity, or litter depth could impact both ant dispersers and conditions needed for germination.

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TABLES

Table 5.1 Summary statistics from Shapiro-Wilk test for normality and one sample t-tests for chamber depths versus estimated maximum emergence depth of 7.26 cm.

Shapiro-Wilk T-tests Chamber n Mean depth ± W P t df P SD (cm) 1 13 7.72 ± 2.29 0.90 0.14 1.10 12 0.29 2 13 9.92 ± 9.80 0.93 0.30 4.38 12 < 0.001 3 10 13.58 ± 12.05 0.91 0.29 5.25 9 < 0.001

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Table 5.2 Bite force data for four E. ruidum workers in grams (g). Values in bold and italic are the max bite for that individual in grams. Both max force and standard deviation on bite force generated converted into newtons (N). * indicates the ant that was CT scanned.

Bites (g) Individual # of 1 2 3 4 5 Max bite SD bites Bites (N) (N) SAR851_01 5 4.69 6.09 2.81 5.16 6.09 0.060 0.013 SAR948_01* 3 9.37 8.44 8.44 NA NA 0.092 0.005 SAR961_01 5 5.62 5.16 6.56 9.37 2.34 0.092 0.025 SAR1314_01 3 7.97 5.62 5.16 NA NA 0.078 0.015

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FIGURES

Figure 5.1 Seed dispersal effectiveness framework broken down to its two main components both consisting of two subcomponents (Schupp et al. 2010) and how these relate to ant-mediated seed dispersal.

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Figure 5.2 Representative drawing of straight line dispersal distances and deposition locations of Z. ekmanii seeds relative to north (N). The pink seed demonstrates a first movement of 221 cm and a second movement of 60 cm. Two different E. ruidum colonies took seeds. Color refers to the paint marking on the seed. Drawing is not to scale.

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Figure 5.3 Boxplots of seed removal by year. Letters denote significance differences as determined by Tukey’s HSD means separation tests between years based on a LMM. The line in the middle of the boxplot represents the median value, the bottom and top of the boxplot represents the 25th and 75th percentiles of the data respectively, the whiskers extend to the most extreme data point that is not an outlier, and the points represent outliers (Wickham 2009). Outliers are those that are 1.5 x the inter-quartile range which is the distance between the 25th and 75th percentiles.

AB B A A 100 ●

● 75

50 Seed Removal (%) 25

0 2013 2014 2015 2016

Year

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Figure 5.4 Histogram of straight line dispersal distances by E. ruidum by movement number (bin width = 15 cm; first movement number: n = 39; second movement number: n = 9; third movement number: n = 1) (A) and percentages of seeds that went into either colonies, were dropped, or were lost based on the total number of seed destinations recorded (n = 51; based on frequency of all deposition locations recorded) (B).

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Figure 5.5 Boxplots of the relative abundance of seeds per chamber averaged across six wax castings (A) and chamber depths both by colony (circles) and average (black lines) (B). Red circles are an example of the chamber depths from one colony (SAR843). Boxplots are plotted the same way as described in Figure 5.2

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Figure 5.6 Boxplots of percentage of seedlings emerging by burial depth. Letters denote significant differences based on a Tukey post-hoc means separation test. Boxplots are plotted the same way as described in Figure 5.2

A A B B 100

Seedlings Emerged (%) 25

●

0 0 2 5 7

Depth

138

Figure 5.7 Boxplots of percentage of seed surface area damaged by E. ruidum that were either recovered from a colony wax caste (n = 36 seeds) or had presumably never interacted with ants (control: n = 41 seeds) (A). Image of E. ruidum damaged seeds, one of which still has some wax on the seed (B, C). Image of a control seed that still shows some damaged (D). Boxplots are plotted the same way as described in Figure 5.2

139

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146

CHAPTER 6: CONCLUSIONS

SUMMARY, SIGNIFICANCE, AND FUTURE DIRECTIONS

This dissertation examines ant-mediated seed dispersal in non-myrmecochorous plants, specifically on Neotropical pioneer tree species that either provided no food rewards or were cleaned of fruit pulp. Non-myrmecochorous plants, or plants that are not known to have evolved seed dispersal by ants, make up the vast majority of angiosperm species (~94.5%, Lengyel et al.

2010). However, some plant species previously thought to be non-myrmecochorous have been identified as being ant-dispersed (e.g. Pizo and Oliveira 1998, Passos and Oliveira 2002,

Christianini and Oliveira 2010, Barroso et al. 2013, Magalháes et al. 2018). In these systems, seeds are often first dispersed by other animals, such as birds, and then secondarily dispersed by ants. Eight of the 12 tree species that I tested in this dissertation were observed being removed by ants, which were the only taxon observed removing seeds during the observation periods.

I sampled 12 pioneer tree species to try to determine the relative importance of different factors on secondary removal and determine generalizations for this important functional group of tropical forests. Despite this attempt, the results contrasted with predictions, finding that tree species accounted for most of the variation in the data. As tree species explained most of the variation, including it as a variable in the models masked the effects of primary dispersal mode and dormancy type. It is possible that the amount of variation attributed to species identity would be minimized and generalizations based on seed characteristics emerge (e.g. primary dispersal mode) if additional species were sampled. Therefore, future studies should include more pioneer species to determine if primary dispersal mode is an important factor for secondary removal of seeds of this functional group. Additionally, I found that incorporation of seeds (6 species) into

147

the topsoil did not lead to a loss of seed mobility. This leads to the question of whether high mobility in the topsoil is a general feature of Neotropical pioneer species and for how long this high mobility lasts. Understanding the cause of this mobility and how that interacts with seed fate will ultimately lead to better understanding of how mobility influences the recruitment of pioneer seed species.

My third chapter focused on whether variation in ant communities correlated with variation in seed removal, finding that while a portion of the sampled ant communities explained much of the variation in seed removal, the communities themselves could not predict seed removal rates with high accuracy. Therefore, ant communities may be functionally redundant in terms of seed removal. However, this redundancy in seed removal does not necessarily translate to a redundancy in the effectiveness of dispersal that these seeds face. For example, Lugon et al.

(2017) demonstrated that two mammal primary dispersers that were thought to be functionally redundant, differ in terms of seed fate due to altering secondary dispersal patterns. Additionally, ant dispersers may differ in the quality of seed dispersal they perform (e.g. Passos and Oliveira

2002, Magalháes et al. 2018). Therefore, future studies should investigate the short- and long- term fate of seeds that are moved by different animal vectors to determine if seed fate is also functionally redundant in this system.

I investigated if seed chemistry was the species-specific trait that elicited the seed- removal response in the common ground dwelling ant, Ectatomma ruidum, which was observed removing four of the 12 seed species studied and has been recorded removing seeds in other systems (e.g. Zelikova and Breed 2008). Like seed-removal, removal of chemical caches appears to be tree species-specific. Therefore, some tree species (e.g. Zanthoxylum ekmanii) may use chemicals to promote ant-seed interactions in the absence of a food reward. By testing fractions

148

of an attractive compound, I have been able to tentatively identify candidate chemicals to field test in the future. Future studies that can confirm the identity of these chemical cues that elicit the seed removal response can be used along with factors already known to constrain seed dispersal by ants (i.e., the size of both the ant and the seed) to provide a framework to modeling dispersal potential in plant species that have not yet been studied. This could create predictive models that would serve as a worldwide resource to understand which plant species should have ants included in conservation or restoration plans. Additionally, once chemicals that mediate seed removal by ants are identified, these chemicals can be applied to diodes or other trackers, facilitating the study of dispersal kernels of small-seeded tree plant species.

I assessed the short-term seed fate of the mostly highly removed seed species, Z. ekmanii that also does not provide any type of food reward, by E. ruidum. Ectatomma ruidum moved seed short distances into colony chambers. Though these chambers are too deep for seedlings to emerge and survive burial if they germinate, seeds of Z. ekmanii often form a persistent seed bank, remaining viable in the soil for 18 years (Dalling and Brown 2009). This long persistence time coupled with how E. ruidum workers may engage in intraspecific thievery (Guénard and

McGlynn 2013), may lead to seeds not remaining in colonies for long potentially increasing the dispersal distance travelled and perhaps leading to seeds being discarded in waste piles that are generally thought to be beneficial microsites for germination and later growth (reviewed in

Giladi 2006). Additionally, E. ruidum was not the only ant species to remove Z. ekmanii seeds and therefore other ant species may also be providing a dispersal service to this species. The quality of that dispersal provided by other ant species currently has not been explored for Z. ekmanii. The inclusion of other ant species in future studies, the exploration of further dispersal through thievery, whether seeds remain attractive for long periods of time, and the incorporation

149

the long-term fate of these seeds through important life stage transitions (ie. seed to seedling, seedling to sapling, etc.), will provide a better understanding of how ants influence the recruitment dynamics of Z. ekmanii.

Overall, my dissertation begins to lay the foundation for understanding ant-mediated seed dispersal of this important functional group (pioneer tree species) on Barro Coloardo Island, panama. It indicates that ants be important in the recruitment dynamics of more pioneer species than previously thought. As the successful dispersal of seeds is important for forest regeneration, ants should be included in restoration and conservation efforts. For example, understanding the subset of ant species that conduct seed removal, and sampling for these species could help indicate whether seeds are likely to have their deposition locations fine-tuned and incorporated into the soil seed bank. More in depth studies of seed fate, both short- and long-term, are needed though to fully reveal the complexity of this ant-seed dispersal interaction of Neotropical pioneer tree species.

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Christianini, A.V., and P.S. Oliveira. 2010. Birds and ants provide complementary seed

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Dalling, J.W., and T.A. Brown. 2009. Long-term persistence of pioneer species in tropical rain

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150

Guénard, B., and T.P. McGlynn. 2013. Intraspecific thievery in the ant Ectatomma ruidum is

mediated by food availability. Biotropica 45: 497-502. doi:10.111/btp.120031

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plants: A global survey. Perspect. Plant Evol. Syst. 12: 43-55.

doi:101016/j.ppees.2009.08.001

Lugon, A. P., M. Boutefeu, E. Bovy, F. Z. Vaz-de-Mello, M-C. Huynen, M. Galetti, and L.

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Magalhães, V.B., N.B. Espíritosanto, L.F.P. Salles, H. Soares Jr., and P.S. Oliveira. 2018.

Secondary seed dispersal by ants in Neotropical cerrado savanna: Species-specific effects

on seeds and seedlings of Siparuna guianensis (Siparunaceae). Ecol. Entomol. 43: 665-

674. doi:10.1111/een.12640

Passos, L., and P.S. Oliveira. 2002. Ants affect the distribution and performance of Clusia criuva

seedlings, a primarily bird-dispersed rainforest tree. J. Ecol. 90: 517-528.

Pizo, M.A., and P.S. Oliveira. 1998. Interaction between ants and seeds of a

nonmyrmecochorous neotropical tree, Cabralea canjerana (Meliaceae), in the Atlantic

forest of southeast Brazil. Am. J. of Bot. 85: 669-674.

Zelikova, T.J., and M.D. Breed. 2008. Effects of habitat disturbance on ant community

composition and seed dispersal by ants in a tropical dry forest in Costa Rica. J. Trop.

Ecol. 24: 309-316. doi:10.1017/S0266467408004999

151

APPENDIX A: SUPPLEMENTARY TABLE FOR CHAPTER 2

Table A.1 Burial and collection calendar for the below-ground seed removal experiment. Two trials at each plot were buried at the same time. Site (or plot) locations on Barro Colorado Island were named 25Ha, Armour, Drayton, Pearson, and Zetek.

Sunday Monday Tuesday Wednesday Thursday Friday Saturday 1 2 3 4 5 6

7 8 9 10 11 12 13 Zetek burial

14 15 16 17 18 19 20 July 2013 Armour Pearson burial burial 21 22 23 24 25 26 27 Drayton 25Ha burial burial 28 29 30 31

1 2 3

4 5 6 7 8 9 10 Zetek

collected 2013 August 11 12 13 14 15 16 17 Armour Pearson collected collected 18 19 20 21 22 23 24 Drayton 25Ha collected collected 25 26 27 28 29 30 31

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APPENDIX B: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER 3

Table B.1 Species averages of morphological measurements and category that the species falls into. Ant species labeled as “pitfall” were present in at least 10% of pitfall traps (wet and dry season pooled) at one location in the forest (site) and were not observed removing seeds. Ant species labeled as “removed” were observed removing seeds from the soil surface at least once. Two specimens per species were measured and each measurment was taken three times. HW = head width taken across the eyes, HL = head length, EP = eye position (HW-OW) , ML = mandible length, SL = scale length, EW = eye width, EL = eye length, HF = hind femur length, WL = Weber’s length, OW = intraocular width

Measurement (mm) Ant Species Label HW HL EP ML SL EW EL HF WL OW Aphaenogaster araneoides removal 1.174 1.301 0.434 0.855 2.681 0.281 0.346 3.386 2.664 0.740 Cyphomyrmex rimosus removal 0.605 0.640 0.168 0.252 0.527 0.117 0.131 0.730 0.862 0.437 Ectatomma ruidum removal 1.695 1.700 0.493 1.269 1.703 0.384 0.472 2.385 2.780 1.202 Labidus praedator pitfalls 1.053 1.164 0.153 0.821 0.999 0.051 0.050 1.770 1.646 0.901 Leptogenys punctaticeps pitfalls 1.223 1.445 0.484 0.927 1.796 0.299 0.407 2.148 2.648 0.738 Odontomachus bauri pitfalls 2.328 2.962 0.659 1.737 2.908 0.378 0.507 3.073 3.658 1.668 Pachycondyla harpax pitfalls 1.675 1.750 0.295 1.174 1.428 0.243 0.292 1.675 2.527 1.380 Pheidole cf. cocciphaga removal 0.568 0.572 0.178 0.338 0.858 0.132 0.158 0.869 0.855 0.391 Pheidole cf. hierax pitfalls 0.668 0.629 0.146 0.361 0.760 0.115 0.154 0.813 0.803 0.522 Pheidole multispina removal 0.438 0.422 0.070 0.241 0.340 0.056 0.098 0.349 0.448 0.368 Pheidole simonsi removal 0.683 0.573 0.093 0.425 0.567 0.088 0.124 0.699 0.742 0.590 Pheidole sp. 001 pitfalls 0.699 0.674 0.170 0.460 0.951 0.123 0.156 1.062 1.017 0.529 Pheidole sp. 002 pitfalls 0.452 0.440 0.063 0.288 0.407 0.051 0.077 0.380 0.524 0.389 Pheidole sp. 007 removal 0.684 0.593 0.101 0.448 0.588 0.087 0.115 0.685 0.755 0.583 Pheidole sp. 010 pitfalls 0.574 0.528 0.073 0.373 0.500 0.062 0.091 0.563 0.708 0.501

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Table B.1 (cont.) Measurement (mm) Ant Species Label HW HL EP ML SL EW EL HF WL OW Pheidole sp. 016 pitfalls 0.453 0.413 0.062 0.294 0.392 0.048 0.080 0.423 0.523 0.392 Pheidole susannae removal 0.674 0.694 0.191 0.445 1.178 0.118 0.141 1.225 1.047 0.483 Sericomyrmex amabilis removal 1.033 0.948 0.145 0.638 0.778 0.124 0.163 1.178 1.358 0.888 Solenopsis cf. castor pitfalls 0.290 0.367 0.032 0.183 0.232 0.026 0.038 0.257 0.413 0.258 Solenopsis cf. vinsoni pitfalls 0.306 0.355 0.012 0.197 0.228 0.031 0.046 0.233 0.408 0.294 Trachymyrmex bugnioni removal 0.743 0.706 0.142 0.428 0.652 0.115 0.152 0.863 1.014 0.601 Trachymyrmex cornetzi removal 0.939 0.859 0.161 0.618 0.853 0.123 0.157 1.211 1.303 0.778 Trachymyrmex isthmicus removal 1.236 1.200 0.213 0.818 1.032 0.156 0.205 1.770 1.748 1.023 Trachymyrmex zeteki removal 1.118 1.079 0.196 0.782 0.965 0.137 0.176 1.568 1.547 0.923 Wasmannia auropunctata removal 0.435 0.448 0.097 0.198 0.385 0.072 0.105 0.403 0.458 0.338

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Table B.2. Ant species collected by season (above- and below-ground pooled) and site in the forest. D = dry season, W = wet season, R = observed removing seed species in hand-collected samples, A = collected in above-ground pitfall traps, B = collected in below-ground (subterranean) pitfall traps, * = ant species / morphospecies found in at least 10% of above- ground pitfall traps at one site

Ant species Season Site 25Ha AVA Drayton Pearson Zetek Aphaenogaster araneoides* DW RA A Azteca cf. xanthochroa sp.001 W A Camponotus cf. JTL- 004 sp.001 D A Carebara brevipilosa DW A A A Carebara urichi W B B Cyphomyrmex co. dixus D A Cyphomyrmex minutus DW A A A Cyphomyrmex rimosus D R Dolichoderus bispinosus D A Ectatomma ruidum* DW RA RA RAB RAB RA Gnamptogenys continua DW A A Gnamptogenys regularis DW A Hylomyrma dentiloba D A Hypoponera opacior W A A Labidus coecus W B AB B B B Labidus praedator* D A A Leptogenys JTL-002 D A Leptogenys punctaticeps* DW A A A Mayaponera constricta W A Megalomyrmex sp.001 W A A

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Table B.2 (cont.) Ant species Season Site 25Ha AVA Drayton Pearson Zetek Mycocepurus tardus DW A Myrmicocrypta sp.001 D A Myrmicocrypta sp.002 D A Neivamyrmex cf. iridescens W B Neivamyrmex humilis W A Neivamyrmex macrodentatus W B B Neoponera verenae DW A A A Nylanderia cf. guatemalensis D A Nylanderia sp.001 W B Odontomachus bauri* DW A A Odontomachus meinerti DW A Pachycondyla "impressa" – like D A Pachycondyla harpax* DW A AB AB A AB Pheidole cf. cocciphaga DW RA Pheidole cf. hierax* DW A AB Pheidole harrisonfordi D A A Pheidole multispina* DW A A RAB Pheidole rugiceps DW A A A A Pheidole simonsi* DW RA A Pheidole sp.001* DW A Pheidole sp.002* DW AB A Pheidole sp.004 DW AB Pheidole sp.005 DW A AB Pheidole sp.006 W B Pheidole sp.007 DW RA A Pheidole sp.008 W A

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Table B.2 (cont.) Ant species Season Site 25Ha AVA Drayton Pearson Zetek Pheidole sp.009 W A A Pheidole sp.010* W A AB Pheidole sp.015 DW A Pheidole sp.016* DW AB Pheidole sp.017 DW A A Pheidole sp.018 D A Pheidole sp.019 W A A Pheidole sp.020 D A Pheidole sp.021 DW A A Pheidole sp.023 D A Pheidole sp.024 D A Pheidole sp.031 W A Pheidole susannae D R Rogeria foreli W B Sericomyrmex amabilis DW RA RA A A RA Solenopsis cf. bicolor DW B B B AB AB Solenopsis cf. brevicornis DW A A A Solenopsis cf. castor* DW A AB A Solenopsis cf. pollux DW B A A A Solenopsis cf. vinsoni* DW AB AB A AB AB Solenopsis cf. zeteki D A A Solenopsis sp. 007 D A Solenopsis sp. 008 W A Strumigenys marginiventris DW A Trachymyrmex bugnioni DW A R Trachymyrmex cornetzi DW R R R RA Trachymyrmex isthmicus D R Trachymyrmex zeteki D R Tranopelta gilva W B B B B

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Table B.2 (cont.) Ant species Season Site 25Ha AVA Drayton Pearson Zetek Wasmannia auropunctata* DW A AB RA A RA Wasmannia rochai D A

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Figure B.1 Images of how morphological measurements were taken on ant specimens. 1 = head width (HW) taken across the eyes, 2 = head length (HL), 3 = intraocular width (OW), 4 = mandible length (ML), 5 = scape length (SL), 6 = eye width (EW), 7 = eye length (EL), 8 = hind femur length (HF), 9 = Weber’s length (WL)

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Figure B.2 Non-metric multidimensional scaling (NMDS) depicting the relationship between ant communities found in dry versus wet season pitfall traps. There were five sites sampled during the dry season and during the wet season. Ellipses represent the multivariate t distribution. 25 = 25Ha, A = AVA, D = Drayton, P = Pearson, Z = Zetek

Pitfalls Pitfalls a Wet Season a Dry Season

P D PZ Z A 0 A D NMDS 2 25

−1 25

Stress = 0.072

−1 0 1 2 NMDS 1

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Figure B.3 NMDS depicting the relationship between ant assemblages found in different subterranean traps by seed species and by controls (silica bead baited or unbaited traps) for NMDS axes 1 and 2 (A), axes 1 and 3 (B), and 2 and 3 (C). Ellipses represent the multivariate t distribution.

ApMe Beads OcPy TrBl a a a a a ApMe a Beads a OcPy a TrBl CeLo JaCo Unbaited ZaEk a a a a a CeLo a JaCo a Unbaited a ZaEk

OcPy CeLo Unbaited 1.0 CeLo OcPy ZaEk Unbaited CeLo JaCo ApMe JaCo ZaEkZaEk CeLo BeadsOcPy ApMe 0.5 TrBl ApMe JaCo ZaEk TrBl ApMe OcPy TrBl TrBl TrBl TrBl CeLo OcPy Unbaited Beads ApMeCeLo ZaEk 0 OcPy Beads Unbaited ApMeCeLo TrBl 0.0 TrBl OcPy JaCoBeads Unbaited TrBl CeLo JaCo JaCoBeadsOcPy Beads Unbaited BeadsCeLo OcPy ZaEk BeadsJaCo JaCo NMDS 2 NMDS 3 Beads −0.5 ZaEk BeadsCeLo JaCo TrBl OcPy −1 Unbaited −1.0 JaCo Unbaited ZaEk

−1.5 Stress = 0.085 Stress = 0.085 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 A NMDS 1 B NMDS 1

a ApMe a Beads a OcPy a TrBl a CeLo a JaCo a Unbaited a ZaEk

OcPy 1.0 ZaEk

JaCo CeLo ApMe CeLo 0.5 TrBl TrBl ApMe OcPyTrBl CeLo ZaEk Beads ApMeCeLo Beads OcPy 0.0 TrBl Unbaited BeadsOcPyJaCo BeadsCeLo Unbaited Beads JaCo ZaEk NMDS 2 −0.5 ZaEk JaCo TrBl OcPy Unbaited −1.0 JaCo Unbaited

−1.5 Stress = 0.085 −1.0 −0.5 0.0 0.5 C NMDS 3

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Figure B.4 Rarefaction and extrapolation curves with 95% unconditional confidence intervals for the number of ant species in subterranean traps baited with seed caches (in black) or control traps (baited with silica bead controls or unbaited traps) in red. All samples from the five sites have been pooled for this analysis. Solid lines indicate interpolation of samples collected while dashed lines represent extrapolated estimated number of species.

Associated with Seed Traps: 25

Yes

No 20

10 Estimated Number of Species

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 Number of Samples

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APPENDIX C: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER 4

Table C.1 Ant species observed removing seeds in 2013 and captured removing or on seeds or silica bead dummy seeds in 2014. 2013 data consists of records from both the dry and the wet season pooled. 2014 data includes ant species that were observed on dummy seeds (with and without extracts) and may not represent ant species that attempt seed removal.

Ant Species Tree Species (species code) 2013 2014 Apeiba membranacea Aubl. Ectatomma ruidum, Aphaenogaster araneoides, (Ape) Trachymyrmex cornetzi Azteca cf. nigro, Odontomachus bauri, Pheidole multispina, Pheidole susannae, Pheidole rugiceps Cecropia longipes Pitt. (Cec) Pheidole susannae Pheidole sp. 002, Trachymyrmex cornetzi Guazuma ulmifolia Lam. Cyphomyrmex rimosus, Cyphomyrmex minutus, (Gua) Ectatomma ruidum, Cyphomyrmex solvini, Trachymyrmex budnioni, Ectatomma ruidum, Pheidole Trachymyrmex cornetzi, simonsi, Pheidole cf. hierox, Wasmannia auropunctata Pheidole sp. 001, Sericomyrmex amabilis, Trachymyrmex bugnioni, Trachymyrmex cornetzi, Trachymyrmex zeteki, Wasmannia auropunctata Ochroma pyramidale Urb. Ectatomma ruidum, Pheidole Ectatomma ruidum, Pheidole (Och) multispina, Sericomyrmex cf. hierox, Pheidole sp. 001, amabilis, Trachymyrmex Technomyrmex fulvus, bugnioni, Trachymyrmex Trachymyrmex cornetzi, cornetzi, Trachymyrmex Wasmannia auropunctata isthmicus, Wasmannia auropunctata Trema micrantha “brown” Pheidole susannae, Ectatomma ruidum, Pheidole (L.) (Tre) Trachymyrmex cornetzi cf. hierox, Pheidole sp. 001, Trachymyrmex cornetzi

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Table C.1 (cont.) Ant Species Seed Species 2013 2014 Zanthoxylum ekmanii (Urb.) Aphaenogaster araneoides, Camponotus sericeiventris, Alain (Zan) Ectatomma ruidum, Pheidole Cyphomyrmex rimosus, cf. cocciphaga, Pheidole Ectatomma ruidum, simonsi, Pheidole sp. 007, Neoponera verenae, Pheidole susannae, Odontomachus bauri, Trachymyrmex cornetzi Pachycondyla harpax, Pheidole flavens group, Pheidole harrisonfardi, Pheidole cf. hierox, Pheidole multispina, Pheidole sp. 015, Pheidole sp. 017, Pheidole sp. 029, Pheidole sp. 030, Pheidole sp. 032, Solenopsis molesta complex, Solenopsis cf. picea, Trachymyrmex bugnioni, Trachymyrmex cornetzi, Wasmannia auropunctata

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Table C.2 Models testing for the effects of tree species identity on counts of time periods in which E. ruidum attempted to remove a seed or silica bead dummy seed. Zero-inflated and hurdle models both allowed the number of zeros to vary based on species identity. Models that did not converge are depicted with NAs. The conditional models were all the same: Count ~ tree species + (1|site/trial)

Model Type Error Distribution DF AIC dAIC GLMM Negbin2 9 333.2 0.0 GLMM Poisson 8 456.8 123.5 Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_poisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA

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Table C.3 Models testing for the effects of chemical treatment on counts of time periods in which E. ruidum attempted to remove a seed or silica bead dummy seed. Models were done individually by tree species. Zero-inflated and hurdle models both allowed the number of zeros to vary based on cache identity. Models that did not converge are depicted with NAs. The conditional models were all the same for GLMMs, Zero-inflated, and Hurdle models: Count ~ species + (1|site/trial). The conditional models for the GLM models did not include the nested random effects. GLM models were only fit if none of the other models converged.

Apeiba membranacea Model Type Error Distribution DF AIC dAIC GLM Poisson 6 18.6 0 GLM Negbin2 7 20.6 2 GLMM Poisson 8 NA NA GLMM Negbin2 9 NA NA Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_oisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA Cecropia longipes GLMM Negbin2 9 27.6 0.0 GLMM Poisson 8 NA NA Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_poisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA Guazuma ulmifolia GLMM Poisson 8 60.2 0.0 GLMM Negbin2 9 61.5 1.3 Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_poisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA Ochroma pyramidale GLMM Poisson 8 55.9 0.0 GLMM Negbin2 9 NA NA Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_poisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA

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Table C.3 (cont.) Trema micrantha “brown” Model Type Error Distribution DF AIC dAIC GLM Poisson 6 29.0 0 GLMM Poisson 8 NA NA GLMM Negbin2 9 NA NA Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_poisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA GLM Negbin2 7 NA NA Zanthoxylum ekmanii GLMM Negbin2 9 127.1 0.0 GLMM Poisson 8 134.9 7.8 Zero-inflated Poisson 14 NA NA Zero-inflated Negbin2 15 NA NA Hurdle Truncated_poisson 15 NA NA Hurdle Truncated_negbin2 15 NA NA

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Figure C.1 Laboratory colony set up for laboratory trials. Waste pile location varied with colony, but was typically in the corners of the plastic container and behind the colony chamber.

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Figure C.2 Boxplot of the percentage of time points the E. ruidum workers attempted to remove seeds or bead dummy seeds of the six-focal tree species (rows) and different treatments (columns). The line in the middle of the boxplot represents the median value, the bottom and top of the boxplot represents the 25th and 75th percentiles of the data respectively, the whiskers extend to the most extreme data point that is not an outlier, and the points represent outliers (Wickham 2009). Outliers are those that are 1.5 x the inter-quartile range which is the distance between the 25th and 75th percentiles.

100 75 50 Ape 25 ● 0 100 75 50 Cec

25 ● 0 100 75 ● 50 ● Gua 25 ● 0 100

75 ● 50 Och ●

Time Points (%) Time 25 ● ● ● 0 100 75 50 Tre 25 ● ● 0 100 ● ● 75 50 Zan ● 25 ● 0 Seeds Hexane Methanol Hexane Methanol Beads Extract Extract Control Control Treatment

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Figure C.3. Filter paper destinations by E. ruidum colony (rows) and chemical treatment (columns). Each colony was presented with a different chemical treatment on a different day with at least one day separating trials in between. Each chemical treatment consisted of five filter paper discs. Discs were originally placed in the foraging arena and the number of filter paper discs remaining in the foraging arena after an average of 70 minutes. The moved destinations consisted of the waste pile and inside the colony chamber.

Destination Foraging arena Moved 5 3 1 SAR848 5 3 1 SAR849 5 3 1 SAR850 5 3 1 SAR851 5 3 1 SAR852 5 3 1 SAR854 5 3 1 SAR869 5 3 1 SAR870 5 3 1 SAR949 5 3 1 SAR950 Filter Paper (#) Filter Paper 5 3 1 SAR951 5 3 1 SAR957 5 3 1 SAR959 5 3 1 SAR961 5 3 1 SAR962 5 3 1 SAR963 Hexane 20% Cyclohex Ethyl Ehter Control Control Control Extract Hexane 20% Cyclohex Ethyl Ehter

Treatment

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APPENDIX D: SUPPLEMENTARY TABLES AND FIGURE FOR CHAPTER 5

Table D.1 Mass of 50 Z. ekmanii seeds, used to calculate maximum emergence depth using the formula from Bond et al. (1999).

Replicate of 50 seeds Mass (mg) Mean seed mass (mg) Max emergence depth (mm) 1 840.5 16.81 70.07 2 824.4 16.49 69.61 3 831.3 16.63 69.81 4 831.4 16.63 69.81 5 862.7 17.25 70.68 6 887.6 17.75 71.35 7 838.1 16.76 69.99 8 858.8 17.18 70.57 9 818.6 16.37 69.45 10 883.8 17.68 71.25 Average 847.7 16.95 70.26

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Table D.2 Characterization of the distribution of straight-line dispersal distances for the first or second movements by E. ruidum workers based on if the distribution is normal, skewed, and displays kurtosis.

Shapiro-Wilk Movement W P Skewness Kurtosis Number One 0.82 < 0.001 1.56 2.16 Two 0.81 < 0.05 1.02 -0.57

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Table D.3 Presence of larvae (L), workers (W), and Z. ekmanii seeds (S) in different colonies and chambers of E. ruidum wax casts. Chamber depth is given in cm from the soil surface to the bottom of the chamber in parentheses. * indicates a colony that was provisioned with Z. ekmanii seeds prior to wax casting. NA refers to a chamber depth that could not be calculated based on how the wax cast broke apart during excavation.

Chamber Colony Identity 1 2 3 4 5 6 SAR843 LW (8.5) LW (10.9) LW (11.9) LW (22.7) W (43.2) — SAR844 LW (6.0) LW (8.5) LW (11.5) W (13.2) — — SAR845 LW (8.5) LW (10.0) W (11.3) W (15.3) — —

SAR846 LW (13.2) W (15.7) LW (19.2) W (21.7) W (22.7) W (25.2) SAR867 LW (7.5) W (9.5) W (11.5) — — — SAR868 LW (7.2) LW (9.7) LW (12.2) W(NA) — — SAR921* LW (8.8) WS (10.8) — — — — SAR922* W (3.8) LW (5.7) WS (6.7) LW (6.5) LW (13.2) — SAR923 LW (8.5) LW (10.5) W (17.6) — — — SAR944* LWS (5.5) LWS (7.0) — — — — SAR945* LW (8.5) LWS (11.8) — — — — SAR946* LWS (8.8) LWS (9.8) LW (18.3) — — — SAR947* LWS (5.6) LWS (9.1) LWS (15.6) — — —

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Table D.4 Mean, sample size, standard deviation (SD), and standard error (SE) of calculated bulk soil density at different seed burial depths.

Depth (cm) n Mean Soil Density (g per cm3) SD (g per m3) SE (g per cm3) 0 4 0.81 0.06 0.03 2 14 0.87 0.03 0.01 5 12 0.83 0.04 0.01 7 8 0.82 0.05 0.02 Average 38 0.82 0.04 0.01

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Table D.5 Muscle volume estimates (mm3) for an E. ruidum worker

Muscle Volume Right mandible 0.078 Left mandible 0.083 Right opener 0.018 Left opener 0.018 Right closer 0.161 Left closer 0.161

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Figure D.1 Segmented volume rendering of the dorsal (A), lateral (B), and ventral (C) views from a microCT scan for one E. ruidum individual. blue = closer muscles, yellow = opener muscles, pink = mandibles

176